1

METHODS in CLINICAL
CHEMISTRY
An accessory work to the 5th edition of

Kaplan and Pesces : Clinical Chemistry:

Theory, Analysis, Correlation*
Published by Pesce Kaplan Publishers 2009

Editors
Peter E. Hickman, MB BS, PhD, FRCPA
Methods Editor
Associate Professor
Australian National University Medical School;
Director of Chemical Pathology
The Canberra Hospital
Australian Capital Territory, Australia

Gus Koerbin, BAppSci, AFAIM

Methods Associate Editor
Principal Scientist ACT Pathology
Adjunct Professional Associate
University of Canberra
ACT Pathology
The Canberra Hospital,
Garran, Australian Capital Territory, Australia

A work of 144 Methods of Analysis describing current methodology.

*Published by Mosby, an affiliate of Elsevier; 2009

2
3

METHODS in CLINICAL
CHEMISTRY
An accessory work to the 5th edition of

Kaplan and Pesces : Clinical Chemistry:

Theory, Analysis, Correlation*
Published by Pesce Kaplan Publishers 2009

Volume I
 4

Pesce Kaplan Publishers

Methods Clinical Chemistry

All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher. Permissions may be sought directly
from Pesce Kpalan Publishers: phone: (1) 212 864 4403 or 858 278 4587 (US); e-mail:
http://www.pescekaplan.com/contact_us.htm#Feedback%20Form.

Notice

Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our knowledge, changes in (i) on state-of-the-art methodology and measurement technique, (ii) effect of
interferences, or (iii) interpretation of results may become necessary or appropriate. Readers are advised
to check the most current information in the relevant scientific literature. It is the responsibility of
the clinical laboratory practitioner, relying on their own experience and knowledge of the patient, to
determine the best procedure to take all appropriate safety precautions. To the fullest extent of the law,
neither the Publisher nor the Editors assumes any liability for any injury and/or damage to persons or
property arising out of or related to any use of the material contained in this book.
The Publisher

Previous copyrighted printed editions: 1987, electronic editions 1996, 2003

Library of Congress Cataloging-in-Publication Data

Methods Clinical chemistry [edited by] Lawrence A. Kaplan, Amadeo J. Pesce.

Bound version and electronic version.

Printed in the United States of America

 5
Contributors to Methods of Analysis
Zakaria Ahmed, PhD
Clinical Chemist
Department of Pathology and Laboratory
Medicine
Rochester General Hospital
Rochester, New York
Hassan M. E. Azzazy, PhD, DABCC, FACB
Chairman and Associate Professor
Department of Chemistry
The American University in Cairo
Cairo, Egypt
Tony Badrick, BAppSc, BSc, BA, MLitSt
(Math), MBA, PhD, FAIMS, FAACB, FQSA,
FAIM, FACB, FRCPA (Hon)
Executive ManagerLaboratories
Sullivan Nicolades Pathology
Taringa, Queensland, Australia
John Beilby, BSc(Hons), PhD, FAACB,
MHGSA, ARCPA
Principal Scientist
PathWest
Nedlands, Western Australia, Australia
Marion Black, BSc(Hons), Dip. Ed, MAACB
Senior Scientist
Clinical Biochemistry, Alfred Pathology
Service
Victoria, Australia
John R. Burnett, MB ChB, MD, PhD,
FRCPA, FAHA
Clinical Professor
PathWest Laboratory Medicine
Royal Perth Hospital
Perth, Western Australia, Australia
Kevin Carpenter, PhD, FHGSA
Principal Scientist and Head of
Department
NSW Biochemical Genetics Service
The Childrens Hospital at Westmead
Westmead, New South Wales, Australia
Kee Cheung, BSc(Hons), PhD, GradCert.
Mgt.
Manager
Pathology QueenslandPrincess Alexandra
Hospital
Woolloongabba, Queensland, Australia
William Clarke, PhD, MBA, DABCC
Director, TDM and Toxicology, Director,
CPOCT
Department of Pathology
Johns Hopkins School of Medicine
Baltimore, Maryland
Paul F. Coleman, PhD
Research Fellow
Infectious Disease Core R&D
Abbott Diagnostics
Abbott Park, Illinois
Joe DAgostino, BSc, Grad Dip FMI,
MAACB
Senior Scientist
Clinical Biochemistry Unit
Alfred Pathology Service
Alfred Hospital, Melbourne, Australia
Sheila Dawling, PhD, CChem, FRSC
Associate Professor of Pathology
Director, Toxicology
TDM Laboratory,
Associate Director
Clinical Chemistry
The Vanderbilt Clinic
Nashville, Tennessee
Joris R. Delanghe
Professor of Clinical Chemistry
Department of Clinical Chemistry
Ghent University Hospital
Gent, Belgium
Goce Dimeski, BSc
Supervising Scientist
Pathology QLD
Princess Alexandra Hospital
Woolloongabba, Brisbane, Australia
Angela Ferguson, PhD
Clinical Chemistry Fellow
Washington University School of
Medicine
Department of Pathology and Immunology
St. Louis, Missouri
Michael J. Figursk, PhD
Research Associate
University of Pennsylvania
Hospital of University of Pennsylvania
Philadelphia, Pennsylvania
John Galligan
Supervising Scientist
Pathology Queensland Central Laboratory
Royal Brisbane Hospital
Herston, Queensland, Australia
 6
Karen Golemboski, PhD, MT(ASCP)
Program Director and Chair
Clinical Laboratory Science
Bellarmine University
Louisville, Kentucky
Ronda F. Greaves, BSc, Grad Dip Ed,
MAppSc, PhD, MAACB
Senior Scientist
Complex Biochemistry Department
The Royal Childrens Hospital
RCH Laboratory Services
Parkville, Victoria, Australia
Kathryn Green, BSc(Hons), MSc(Med)
Senior Scientist
NSW Biochemical Genetics Service
The Childrens Hospital at Westmead
Westmead, New South Wales, Australia
Elizabeth M. Hall, BSc, MSc, FRCPath
Principal Clinical Scientist
Department of Clinical Biochemistry
East Kent Hospitals University
NHS Trust
Kent and Canterbury Hospital
Canterbury, Kent, United Kingdom
Peter E. Hickman, MB BS, PhD, MPH,
MAACB, FRCPA
Associate Professor
Australian National University Medical
School;
Director of Chemical Pathology
The Canberra Hospital
Australian Capital Territory, Australia
Gregory A. Hobbs, PhD, DABCC
Clinical Laboratory Science
Bellarmine University
Louisville, Kentucky
David W. Holt, BSc, PhD, DSc(Med),
CSci, EurClin Chem, FESC, FRCPath
Professor of Bioanalytics
ASI, Ltd
London, United Kingdom
David G. Hughes, BAppSci, Grad Dip Sci
Scientist
Clinical Chemistry, ACT Pathology
The Canberra Hospital
Garran, Australian Capital Territory,
Australia
Mind Jin, PhD
Temple University
Philadelphia, Pennsylvania
Atholl Johnston, BSc, MSc, PhD,
FBPharmacolS, FRCPath
Professor of Clinical Pharmacology
William Harvey Research Institute
(School of Medicine and Dentistry)
Queen Mary
University of London
London, United Kingdom
Graham Jones, MBBS, DPhil, FRCPA, FAACB
Staff Specialist in Chemical Pathology
Department of Chemical Pathology
St. Vincents Hospital
Darlinghurst, New South Wales,
Australia
Saeed A. Jortani, PhD, DABCC, FACB
Associate Professor of Pathology and
Laboratory Medicine
University of Louisville
School of Medicine
Louisville, Kentucky
Lawrence A. Kaplan, PhD, DABCC
New York, New York
Steven C. Kazmierczak, PhD, DABCC
Professor of Pathology
Director of Clinical Chemistry and
Toxicology
Oregon Health and Science University
Department of Pathology
Portland, Oregon
Sandra Klingberg, B App Sci
Supervising Scientist, Protein
Laboratory
Pathology Queensland, Central
Laboratory
Royal Brisbane Hospital
Herston, Queensland, Australia
Gus Koerbin, BAppSci, AFAIM
Principal Scientist ACT Pathology
Adjunct Professional Associate
University of Canberra
ACT Pathology
The Canberra Hospital
Garran, Australian Capital Territory,
Australia
Magdalena Korecka, PhD
Senior Research Investigator
School of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
 7
William J. Korzun, PhD, DABCC, MT(ASCP)
Associate Professor
Department of Clinical Laboratory
Sciences
Virginia Commonwealth University
Richmond, Virginia
Edmund Lamb, PhD, FRCPath
Clinical Scientist (Biochemistry) and
Head of Department
Department of Clinical Biochemistry
East Kent Hospitals
NHS Trust
Kent and Canterbury Hospital
Canterbury, Kent, United Kingdom
Stanley S. Levinson, PhD, DABCC
Professor of Pathology and Laboratory
Medicine
University of Louisville
Director of Clinical Chemistry and
Immunochemistry
Department of Veteran Affairs Medical
Center
Louisville, Kentucky
Barry Lewis, MD, FRCPA, FHGSA
Head, Department of Clinical
Biochemistry
PathWest Laboratory Medicine WA
Princess Margaret Hospital
Perth, Western Australia, Australia
Jinong Li, PhD
Clinical Chemistry Fellow
Johns Hopkins Medical Institutions
Baltimore, Maryland
Greg Maine, PhD
Manager, Global Scientific Affairs
Associate Research Fellow
Abbott Laboratories
Abbott Park, Illinois
Christopher R. McCudden, PhD, DABCC,
NRCC
Assistant Professor
Department of Pathology and Laboratory
Medicine
School of Medicine
University of North Carolina
Chapel Hill, North Carolina
Denise A. McKeown, MSci, AMRSC
Senior Analyst
St Georges, University of London
Analytical Unit
Department of Cardiac and Vascular
Sciences
London, United Kingdom
Brett McWhinney, BSc, MSc, MBA, MPhil
Supervising Scientist
HPLC Section, Department of Chemical
Pathology
Royal Brisbane Hospital
Herston, Queensland, Australia
Danni L. Meany, PhD
Clinical Chemistry Fellow
Johns Hopkins Medical Institutions
Baltimore, Maryland
James J. Miller, PhD, DABCC, FACB
Professor
University of Louisville
School of Medicine
Department of Pathology and Laboratory
Medicine
Louisville, Kentucky
Michael Milone, MD, PhD
Assistant Professor of Pathology and
Laboratory Medicine
Associate Director, Toxicology
Laboratory
School of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
Gerald J. Mizejewski, BS, MS, PhD
Senior Research Scientist
Wadsworth Center
New York State Department of Health
Albany, New York
Scott A. Muerhoff, PhD
Volwiler Research Fellow
Infectious Diseases Research and
Development
Abbott Diagnostics
Abbott Laboratories
Abbott Park, Illinois
Anthony O. Okorodudu, PhD, MBA
Professor
Director, Clinical Chemistry Division
UTMB/CMC Outreach Laboratory Services
Department of Pathology
University of Texas Medical Branch
Galveston, Texas
 8
Peter OLeary, BSc, MAACB, AFACHSE,
ARCPA, PhD
Adjunct Professor, School of Public
Health (Curtin)
Adjunct Associate Professor
School of Womens & Infants Health
(UWA),
Director, Office of Population Health
Genomics
Public Health Division
Health Department of Western Australia
Perth, Western Australia, Australia
Matthew T. Olson, MD
House Officer, Department of Pathology
Johns Hopkins Medical Institutions
Baltimore, Maryland
Felix O. Omoruyi, PhD
Fellow
Department of Pathology
Clinical Chemistry
University of Texas Medical Branch
Galveston, Texas
Mauro Panteghini, MD
Professor of Clinical Biochemistry and
Clinical Molecular Biology
University of Milan Medical School
Laboratorio Analisi
Milan, Italy
Gerardo Perotta, MPA
Interim-Coordinator, Pathology
Education
Department of Pathology and Laboratory
Medicine
University of Cincinnati College of
Medicine
Cincinnati, Ohio
Michael A. Pesce, PhD
Professor Emeritus of Pathology and
Cell Biology
Columbia University Medical Center
New York Presbyterian Hospital
New York, New York
Julia M. Potter, B Med Sc(Hons), MB BS,
PhD, FRCPA
Professor of Pathology
Australian National University Medical
School
Executive Director, ACT Pathology
The Canberra Hospital
Garran, Australian Capital Territory,
Australia
Terry Pry, PhD
Retired-Manager Scientific Affairs,
Asia-Pacific
Abbott Diagnostic Division
Abbott Laboratories
Auckland, New Zealand
Kishor Raja, BSc(Hons), MSc, PhD, CSci
Principal Clinical Scientist/Honorary
Senior Lecturer
Clinical Biochemistry Department
Kings College Hospital
London, United Kingdom
Jordan Reynolds, MD
Resident Physician
University of Cincinnati
Department of Pathology and Laboratory
Medicine
Cincinnati, Ohio
Ken Robertson, BSc, AAIMS
Senior Scientist in Charge (Research)
PathWest Laboratory Medicine
Royal Perth Hospital
Wellington St. Perth, Western
Australia, Australia
Andrea M. Rose, PhD, MBA
Senior Clinical Support Consultant
Roche Diagnostics
Indianapolis, Indiana
Enrico Rossi, PhD, MAACB
Research Biochemist
PathWest Laboratory Medicine
Nedlands, Western Australia, Australia
Randal J. Schneider, MS, PhD
Director of Clinical Chemistry and
Toxicology
ProHealth Care Laboratories
Waukesha, Wisconsin
Les Shaw, BS, PhD
Professor
University of Pennsylvania
School of Medicine
Philadelphia, Pennsylvania
Run Zhang Shi, PhD
Instructor
Assistant Director of Clinical
Chemistry and Immunology
Department of Pathology
School of Medicine
Stanford University
Stanford, California
 9

Ravinder Jit Singh, PhD John G. Toffaletti, PhD

Co-Director, Endocrine Laboratory Professor in Pathology
Organization Clinical Laboratories and Department of
Mayo Clinic Pathology
Rochester, Minnesota Duke University Medical Center
Durham, North Carolina
Patricia Slev, PhD
Assistant Professor of Pathology Susan Vickery, MSc, PhD
(Clinical) Senior Clinical Scientist
University of Utah, East Kent Hospitals University
Medical Director NHS Trust
Serologic Hepatitis and Retrovirus Kent and Canterbury Hospital
Laboratory Canterbury, Kent, United Kingdom
ARUP Laboratories
Salt Lake City, Utah Ping Wang, PhD, DABCC
Medical Director of Clinical Chemistry
Ramasamyiyer Swaminathan, MBBS, MSc, The Methodist Hospital
PhD, FRCPath Houston, Texas
Professor and Head of Department of
Chemical Pathology Gregory Ward, BSc(Hons), MSc, MAACB,
St. Thomas Hospital FAACB
London, United Kingdom Head, Biochemistry and Endocrinology
Sullivan Nicolades Pathology (Sonic
Danyal B. Syed, BSc, MA, PhD, C(ASCP), Healthcare)
CC(NRCC), DABCC, FACB Brisbane, Queensland, Australia
Laboratory Director
William F. Ryan Community Health Center Alan H. B. Wu, PhD
New York, New York Professor, Laboratory Medicine
University of California, San Francisco
Danyel H. Tacker, PhD, FACB Clinical Chemistry and Toxicology
Clinical Chemist Laboratories
Ochsner Medical CenterNew Orleans San Francisco General Hospital
New Orleans, Louisiana San Francisco, California

Jillian R. Tate, BSc(Hons), MSc Odette Youdell, BAppSci(Hons), MAACB

Senior Scientist Senior Scientist
Pathology Queensland Clinical Biochemistry
Chemical Pathology Department Alfred Pathology Service
Royal Brisbane and Womens Hospital Melbourne, Victoria
Brisbane, Queensland, Australia Australia
 10

Foreword
Foreword for the 2009 edition of Methods in Clinical Chemistry

In the mid-1980s we perceived a need for an extensive, up-to-date, compilation of methods available for
use in clinical chemistry laboratories. To meet this need, we published in 1987 Methods in Clinical
Chemistry with C.V. Mosby. This volume provided not only a review of extant methodologies, but also a
critique of each method. This enabled the authors, when appropriate, to suggest one technique as a
recommended method. Since its initial publication, Methods in Clinical Chemistry has been repeatedly
updated and made available in electronic form (CD-ROM or Internet) by Pesce Kaplan Publishers.

Like the previous version, this edition is published in parallel with the current edition of our textbook,
Clinical Chemistry: Theory, Analysis and Correlation (5th edition; Elsevier, 2010). The editors of this
work, Peter Hickman and Gus Koerbin, have assembled an international group of expert clinical chemists
from the United State, Europe, and Australia/New Zealand. We have retained the scope of previous
editions, including:
144 revised method reviews of available technologies for the analysis of each analyte,
a critique of each methodology,
analytical quality goals (when available),
recent references,
a suggested procedure for manual methods.

This edition will be available in both electronic (CD-ROM) and printed (two volumes) formats. It is our
hope that this edition will be widely used and vigorously reviewed by its users. Using new software
technology, we will provide a mechanism for input from readers for future versions of this edition. We
have created an Internet site (http://www.pescekaplan.com/) where individuals can publicly post their
comments. Eventually, the Editors will redact the suggestions into changes incorporated into Methods in
Clinical Chemistry.
 11

Preface for the 2009 edition of Methods in Clinical Chemistry

It was certainly an honor and privilege to be invited by Larry Kaplan and Amadeo Pesce to undertake the
editorial role for the 2009 edition of Methods in Clinical Chemistry. The challenge for us was to provide a
text that was contemporary, detailed but readable, and of paramount importance, a useful resource and
laboratory tool for both students and professionals as we near the end of the first decade of this
millennium.

Much in clinical chemistry has changed since the first edition was published in 1987: the breadth and
depth of our knowledge base, the development of laboratory technology, and the widespread use of the
internet and digital technology. Review of each chapter and final publication for this edition of Methods
in Clinical Chemistry has been vastly different from the original compilation, with Internet and digital
technology significantly streamlining the process. In 2003, the methods portion of the text was removed
from the 4th edition of Kaplan and Pesces Clinical Chemistry Theory, Analysis, Correlation and provided
as a CD-ROM. This edition includes 131 new and revised methods plus 13 older methods that were not
revised. Each method contains a critique of alternate methodologies; contemporary and historical,
analytical quality goals, and performance data. Methods in Clinical Chemistry is not only offered in a CD-
ROM/DVD format, but also in a two volume printed format. In addition, the methods will be accessible to
purchasers of the 5th edition of Kaplan and Pesces Clinical Chemistry Theory, Analysis, Correlation via
the publishers (Elsevier) Internet site, Evolve.

We have been very fortunate to collaborate with an outstanding team of 76 authors from all over the
world. Our clinical chemistry experts reside in 16 states of the United States, most states of Australia, and
in New Zealand, Egypt, Italy, Belgium and England, making this a truly international project. Without the
contributions of these industry leaders, this volume could not have been completed and to the team we say
a heartfelt thank you. It is somewhat clich to say that it takes a cast of thousands to produce a body of
work such as this, but without these authors and the help of the following individuals, we would not have
succeeded: Professor Julia Potter, Executive Director of ACT Pathology, Canberra, Australia for
supporting our undertaking of this project, Priscilla Delatorre for her patience, along with her formatting
and word processing skills, Nataliya Polyakov of the College of American Pathologists Surveys team for
her assistance in accessing proficiency data (usually at very short notice), Elseviers Ellen Wurm-Cutter
for her valued advice and particularly for her patience and tenacity in keeping us on schedule and to her
editorial assistant, Jennifer Hermes, for her behind-the-scenes contribution. No list of acknowledgements
would be complete without a mention of the respected and appreciated guidance, praise, constructive
criticisms and most of all encouragement that Larry Kaplan and Amadeo Pesce have provided throughout
this process, which commenced in early 2007.

We trust that this edition will be widely used and reviewed by you the reader. We invite you to use the
internet site (http://www.pescekaplan.com/) created to accept your valued input, comments and
suggestions so that future editions of Methods in Clinical Chemistry can respond to the ever changing
needs of the laboratory professional into the second decade of this millennium and beyond.

We intend to continue with this project, progressively updating methods and releasing a new version as
dictated by progress in the field.
 12

We also intend to include more international contributors, with the hope that it would become the world
methods reference. Finding appropriate authors from diverse countries has proven to be a challenge and
we ask for recommendations from our colleagues.
.

Peter Hickman
Gus Koerbin
Australia, 2009
 13

Methods
1. 25-OH-Vitamin D 24. Barbiturates* Page 205
Ravinder Jit Singh Page 17 25. Bence Jones Protein
2. 1-Antitrypsin Stanley S. Levinson Page 219
John Beilby Page 28 26. Benzodiazepines* Page 232
3. Acetaminophen 27. Beta-hCG (beta-human chorionic
Gus Koerbin, gonadotropin)
David G. Hughes, James J. Miller Page 251
Julia M. Potter Page 33 28. Beta-2-Microglobulin
4. Adrenocorticotropic Hormone (ACTH) James J. Miller Page 257
Hassan M. E. Azzazy Page 43 29. Bilirubin
5. Alanine Aminotransferase R. Swaminathan Page 261
James J. Miller Page 46 30. Blood Gas Analysis and Oxygen
6. Albumin Saturation
Kee Cheung Page 52 Goce Dimeski Page 275
7. Albumin in Urine 31. C-Reactive Protein (CRP)
Graham Jones Page 61 Odette Youdell Page 288
8. Alcohol 32. Calcium
Ping Wang Page 67 Randal J. Schneider Page 296
9. Aldolase* Page 81 33. Cancer Antigen 125 (CA 125)
10. Aldosterone Hassan M. E. Azzazy Page 304
Hassan M. E. Azzazy Page 85 34. Carbamazepine
11. Alkaline PhosphataseTotal Gus Koerbin,
Danyal B. Syed Page 90 Julia M. Potter Page 307
12. Alpha-Fetoprotein 35. Carbohydrate Antigen 15-3 (CA 15-3)
Gerald J. Mizejewski Page 101 Gregory A. Hobbs Page 317
13. Aluminum (Aluminium) 36. Carbohydrate Antigen 19-9 (CA 19-9)
Tony Badrick Page 117 Hassan M. E. Azzazy Page 320
14. Amino Acid Screening 37. Carbon Dioxide and Bicarbonate
Kevin Carpenter Page 124 William J. Korzun Page 323
15. Ammonia 38. Carcinoembryonic Antigens (CEA)
Elizabeth M. Hall Page 128 Gregory A. Hobbs Page 327
16. Amniotic Fluid Phospholipids 39. CatecholaminesPlasma
(AFPL) LS Ratio and PG Brett McWhinney Page 337
Hassan M. E. Azzazy Page 132 40. CatecholaminesUrine
17. Amylase Brett McWhinney Page 344
Ming Jin Page 150 41. Cerebrospinal Fluid (CSF) Protein
18. Angiotensin Converting Enzyme Quantitation
(ACE) Danyel H. Tacker,
Hassan M. E. Azzazy Page 157 Anthony O. Okorodudu Page 363
19. Anion Gap 42. Ceruloplasmin
Tony Badrick Page 162 Ahmed Zakaria Page 372
20. Anticonvulsive Drugs* Page 165 43. Chloride
21. Apolipoproteins A-1 and B William J. Korzun Page 377
Jillian R. Tate Page 181 44. Cholesterol
22. Aspartate Aminotransferase John R. Burnett,
James J. Miller Page 190 Ken Robertson Page 382
23. B-Type Natriuretic Peptide, 45. Cholinesterase
Danyal B. Syed Page 393
NT-proBNP, and ProBNP
Alan H.B. Wu Page 197 46. CMV (Cytomegalovirus)
Terry Pry, Greg Maine Page 405
 14

47. Copper The following analytes are in Volume

K. Raja, R. Swaminathan Page 410 II
48. Cortisol
R. Swaminathan Page 420 70. Glucose
49. Creatine Kinase John Beilby Page 651
Mauro Panteghini Page 430 71. Glycated Hemoglobin
50. Creatine Kinase Isoenzymes Andrea M. Rose Page 662
Mauro Panteghini Page 436 72. Haptoglobin
51. Creatinine Joris R. Delanghe Page 670
Edmund J. Lamb Page 440 73. Hepatitis B
52. Cyclosporin (Cyclosporine A) Paul Coleman Page 674
David W. Holt, 74. Hepatitis C Virus
Denise A. McKeown, A. Scott Muerhoff Page 680
Atholl Johnston Page 452
75. High-Density Lipoprotein (HDL)
53. D-Xylose* Page 470 Cholesterol
54. Dehydroepiandrosterone and its John R. Burnett,
sulfate (DHEA and DHEA-S) Ken Robertson Page 686
Gus Koerbin Page 481 76. Holotranscobalamin
55. Digoxin and Digitoxin Marion Black Page 698
Randal J. Schneider Page 489 77. Homocysteine
56. Drug Screen Sheila Dawling Page 703
Christopher R. McCudden Page 495 78. Homovanillic Acid
57. Estradiol Lawrence A. Kaplan Page 718
Greg Ward Page 516 79. Human Immunodeficiency Virus (HIV)
58. Estriol* Page 520 Patricia Slev Page 728
59. Ethylene Glycol 80. Immunoelectrophoresis
Ping Wang Page 535 Stanley S. Levinson Page 740
60. Fecal Electrolytes and Osmolality 81. Immunoglobulin Quantitation
Felix O. Omoruyi, Karen Golemboski Page 753
Anthony O. Okorodudu Page 544 82. Insulin and C-Peptide
61. Fecal Fat and Fat Absorption Steven C. Kazmierczak Page 762
Lawrence A. Kaplan Page 547 83. Ionized Calcium
62. Fecal Occult Blood John G. Toffaletti Page 769
R. Swaminathan Page 559 84. Iron and Iron-Binding Capacity
63. Ferritin Gerardo Perrotta,
Hassan M. E. Azzazy Page 567 Jordan Reynolds Page 781
64. Folic Acid 85. Ketones
Sheila Dawling Page 573 Lawrence A. Kaplan Page 785
65. Follicle-Stimulating Hormone (FSH) 86. Lactate Dehydrogenase and Lactate
Angela Ferguson Page 586 Dehydrogenase Isoenzymes
66. Free Thyroxine and Free Mauro Panteghini Page 793
Triiodothyronine 87. Lactic Acid
Greg Ward Page 589 Steven C. Kazmierczak Page 797
67. Gamma-Glutamyl Transferase (GGT) 88. Lead
Danyal B. Syed Page 609 Gus Koerbin Page 803
68. Gastric Fluid Analysis* Page 618 89. Lipase
69. Gentamicin and Other Ming Jin Page 814
Aminoglycosides 90. Lipoprotein (a)
Danni L. Meany, Gregory A. Hobbs Page 820
William Clarke Page 625
91. Lithium
Danni L. Meany,
William Clarke Page 823
 15

92. Luteinizing Hormone 115. Pyruvic Acid

Gregory A. Hobbs Page 828 Steven C. Kazmierczak Page 1019
93. Lysozyme* Page 834 116. Renin
94. Magnesium Greg Ward Page 1025
Steven C. Kazmierczak Page 840 117. Rheumatoid Factor
95. Maternal Fetal Screening Terry Pry Page 1030
Peter OLeary, 118. Rubella
Barry Lewis Page 846 Terry Pry, Greg Maine Page 1035
96. MetanephrinesUrine 119. Salicylates
Brett McWhinney Page 856 Gus Koerbin,
97. Methotrexate Julia M. Potter Page 1043
Michael A. Pesce Page 865 120. Serum Protein Electrophoresis
98. Methylmalonic Acid Sandra Klingberg Page 1052
Kevin Carpenter, 121. Sirolimus
Kathryn Green Page 875 Magdalena Korecka,
99. Mycophenolic Acid Michael Milone,
Michal J. Figurski, Leslie M. Shaw Page 1062
Magdalena Korecka, 122. Sodium and Potassium
Leslie M. Shaw Page 880 William J. Korzun Page 1070
100. Myoglobin 123. Steroid Hormone
Alan H.B. Wu Page 892 Receptors* Page 1078
101. Opiates* Page 899 124. Sweat Electrolytes: The Sweat Test
102. Organic Acid Screening Ronda F. Greaves Page 1104
Kevin Carpenter Page 912 125. T3 Uptake
103. Osmolality Run Zhang Shi Page 1115
Goce Dimeski Page 916 126. Tacrolimus
104. Oximetry Michael C. Milone,
Goce Dimeski Page 921 Michal Figurski,
105. Parathyroid Hormone (Parathyrin) Magda Korecka,
(PTH) Leslie M. J. Shaw Page 1120
Susan Vickery, 127. Testosterone
Edmund J. Lamb Page 934 Greg Ward, Gus Koerbin,
106. Phenylalanine* Page 939 Peter E. Hickman Page 1127
107. Phenytoin 128. Theophylline and Caffeine
Gus Koerbin Page 946 Saeed A. Jortani Page 1140
108. Phosphorus and Phosphate 129. Thyroglobulin (Tg)
Steven C. Kazmierczak Page 957 Run Zhang Shi Page 1151
109. Plasma Free Metanephrines 130. Thyroid Autoantibodies
Brett McWhinney Page 964 Run Zhang Shi Page 1158
110. Porphobilinogen Screening and 131. Thyroid-Stimulating Hormone (TSH)
Quantitation Greg Ward Page 1168
Enrico Rossi Page 974 132. Thyroxine (Total)
111. Procainamide and N- Greg Ward Page 1175
Acetylprocainamide 133. Total Serum Protein
Matthew T. Olson, Kee Cheung Page 1182
William Clarke Page 982 134. Transferrin and Carbohydrate-
112. Progesterone Deficient Transferrin
John Galligan Page 991 Sandra Klingberg Page 1191
113. Prolactin 135. Transthyretin (Prealbumin)
Sheila Dawling Page 996 Danyel H. Tacker,
114. Prostate Specific Antigen (PSA) Anthony O. Okorodudu Page 1201
Hassan M. E. Azzazy Page 1013 136. Tricyclic Antidepressants
Jinong Li, William Clarke Page 1206
 16

137. Triglycerides 141. Urine Porphyrin Quantitation

John R. Burnett, Enrico Rossi Page 1258
Ken Robertson Page 1213 142. Urine Protein, Total
138. Troponins Susan Vickery,
Jillian R. Tate, Edmund J. Lamb Page 1264
Mauro Panteghini Page 1224 143. Vitamin B12
139. Urea Joe DAgostino Page 1270
Elizabeth M. Hall Page 1246 144. Zinc
140. Uric Acid Tony Badrick Page 1276
Elizabeth M. Hall Page 1252
*Updated in the last edition.
17
25-OH-Vitamin D
25-OH-Vitamin D
Ravinder Jit Singh
Clinical significance: Refer to Chapter 33, Bone Disease, in the 5th edition of Clinical Chemistry: Theory, Analysis,
Correlation.
Chemical class: Steroid
Cholecalciferols Molecular Merck
formula Index
25-Hydroxycholecalciferol C27H44O2 1610
(25-OH-vitamin D3, calcifediol)
25-Hydroxycholecalciferol C28H46O2 1610
(25-OH-vitamin D2, ergocalciferol)
Principles of Analysis and Current Usage
Vitamin D deficiency is widely recognized, and various
treatment options are being proposed in the literature.
Vitamin D (written without the subscript) will refer to both
vitamin D2 and vitamin D3. Vitamin D itself is
biologically inert and is activated in the body through two
specific hydroxylation reactions (Figures 1 and 2). Most
evidence indicates that both natural vitamin D3 and
synthetic vitamin D2 are metabolized by the same enzyme
systems so that ingested vitamin D2 is also converted first
to 25-OH-vitamin D2 and then to 1,25-(OH)2-vitamin D2.
From the standpoint of assay development, it is critical that
any vitamin D2 metabolites present in serum be included
in the total assayed fraction. It should be noted that in
addition to 25-OH-vitamin D3 and 1,25-dihydroxyvitamin
D3, other hydroxylated metabolites of vitamin D are
known to circulate. These include 24,25-dihydroxyvitamin
D3, 25,26-dihydroxyvitamin D3 , and 1,24,25-
trihydroxyvitamin D3.
The use of methods for measuring these metabolites has
been restricted to research, and the usefulness of the
measurements is unknown; thus they will not be
considered in this discussion. Analysis of 25-OH-vitamin
D in circulation has been reported to be the best marker for
determining vitamin D deficiency [1-6].
Various methods are available for measuring circulating
concentrations of 25-OH-D. Current methods include
HPLC, RIA with low throughputtohigh throughput
automated chemiluminescence immunoassays, and liquid
chromatographytandem mass spectrometry (LC-MS/MS).
These methods have already aroused controversy [7-9].
Correlation and agreement studies between immunoassays
i
Vitamin D
Previous and current authors of this method:
First edition: Not done
Methods edition: Thomas L. Clemens
Second edition: Not updated
Third edition: Not updated
Fourth edition: Thomas L. Clemens
Fifth edition: Ravinder Jit Singh
Molecular
mass
400.62
412.62
and LC-MS/MS methods for 25-OH-D have been
reported by several investigators [7-9]. These studies
report reasonable correlations but with significant
differences, the reasons for which are not transparent
or well understood. Automated or manual competitive
immunoassays are known to have less specificity for
low-molecular-weight compounds, and immunoassays
for 25-OH-D are no exception.
The first useful techniques for measurement of 25-
OH-vitamin D were competitive protein-binding
(CPB) assays (Table 1, Method 1) [10-13]. These
procedures exploit the natural serum vitamin D
binding protein (DBP), which has a high affinity (5
108 M-1) for 25-OH-vitamin D3. The protein from
many species has similar binding characteristics,
widening the available sources of binding reagent. The
serum does not need to be from vitamin Ddeficient
animals because the endogenous vitamin D
metabolites occupy only a small number of the
binding sites.
DBP also binds other circulating vitamin D
metabolites, including 24,25-(OH)2-vitamin D and
25,26-(OH)2-vitamin D. Because of the lack of
specificity of the binding protein and the existence of
nonspecific lipid interference in serum, measurement
of 25-OH-vitamin D concentrations directly in crude
serum extracts using CPB assays results in higher
values than those observed when chromatography is
used before assay. 25-OH-vitamin D can be readily
separated from other metabolites by chromatography
on a minicolumn of silica gel. Thus in the case of 25-
OH-vitamin D assay, if a rapid index of overall
vitamin D status is desired, the use of the CPB assay
with preliminary Sep-Pak chromatography is
preferable.
In addition to CPB assays, methods based on high-
performance liquid chromatography (HPLC-UV) and
detection by ultraviolet absorbance have been used
successfully to measure 25-OH-vitamin D in serum
(Table 1, Method 2). Because of the limited sensitivity
of conventional flow-through ultraviolet detectors
(low-nanogram range), these methods generally
require greater sample volumes than CPB assays do,
but this in turn entails rigorous prepurification
18
25-OH-Vitamin D
procedures to remove the larger amounts of contaminating
lipids and proteins. In the earlier versions of HPLC
procedures, serum was equilibrated with tracer quantities
of 3H-25-OH-vitamin D3 to determine the recovery of
sterol during extraction and chromatography. The 25-OH-
vitamin D was prefractionated either by minicolumn
chromatography or by HPLC. Final resolution and
quantitation of the sterol is achieved by HPLC on
microparticulate columns of silica or C18 bonded silica.
Detection is by ultraviolet spectroscopy at 254 nm. The
concentration of sterol is then determined by relating the
peak area of unknown to a calibration curve generated by
HPLC of known amounts of authentic 25-OH-vitamin D.
The HPLC methods for 25-OH-vitamin D, though precise
and accurate, are more time consuming. When the HPLC
method is used, automation of sample injection and
column elute collection is desirable. In recent versions of
HPLC-UV methods, non-isotopic internal standards are
used, and above-mentioned limitations have been
addressed.
Although more laborious and expensive, the HPLC assays
offer certain advantages over the CPB methods [14-20].
For example, it is possible, with the appropriate columns,
to separate the natural form of 25-OH-vitamin D3 from 25-
OH-vitamin D2, a synthetic form, and thus gain a measure
of dietary versus endogenously derived vitamin D3.
Moreover, so that 25-OH-vitamin D is completely resolved
from other lipids, the HPLC methods are less susceptible
than the CPB methods to nonspecific interference. For
these reasons, the HPLC methods are increasingly
employed to measure 25-OH-vitamin D (Figures 3 and 4).
A specific radioimmunoassay for 25-OH-vitamin D has
been developed, and results indicate that this approach
allows direct measurement of 25-OH-vitamin D in serum
extracts (Table 1, Method 3). Although it has been
suggested that 25-OH-vitamin D can be reliably measured
in ethanol extracts of serum without preliminary
chromatography, comparisons of assays with and without
chromatography have clearly shown that direct assays
produce overestimation of the 25-OH-vitamin D
concentration (Figure 5) [21, 22].
Implementation of LC-MS/MS (Table 1, Method 4) has
revolutionized the use of mass spectrometry in clinical
laboratories. In large reference labs in the United States,
serum 25-OH vitamin D is now measured routinely using
LC-MS/MS, the gold standard (Figures 6 and 7) [23, 24].
Reference and Preferred Methods
It is critical to have a reference method for 25-OH vitamin
D against which commercial manufactures and clinical
labs can standardize their methods for better patient care.
In recent years, reference procedures for cholesterol have
been developed and are available as a service for
commercial manufacturers and clinical laboratories, and
this has made significant impact on the clinical practice. At
present, there exist no reference procedures for
measurement of 25-OH vitamin D.
All the methodologies described above are being used
in various clinical laboratories. It is acknowledged that
there are challenges in all of these methods, and high-
level technical expertise is required to perform the
analysis in clinical laboratories. The College of
American Pathologists (CAP) and the United
Kingdombased DEQAS (Vitamin D External Quality
Assessment Scheme) surveys provide independent
approaches to monitor the performance of laboratories
that use various methods for testing of 25-OH-D
(Table 2). The survey feedback does not assess the
accuracy of 25-OH-D measurements by laboratories
but scores laboratories for agreement within the group
using a particular method. Recent CAP data (CAP
survey, 2007 Ligands Special) indicate that clinical
laboratories using chemiluminescence immunoassays
can report a result ranging from 41 to 96 g/L for a
survey sample with a value of 75 g/L determined by
LC-MS/MS (BGS-04 in Figure 8). There could be
many reasons for these variations, including drifts in
the reagents being manufactured, but there is a clear
and urgent need for harmonization and
standardization. Considerable variation is observed in
results observed for the same sample, as demonstrated
in DEQAS data (Table 2).
NIST is developing quality-control materials (human
serum, SRM 972) that will contain 25-OH-D2, 25-OH-
D3, and the metabolite 3-epi-25-OH-D at 4 different
concentrations as characterized by LC-MS/MS. SRM
is especially important for assays for which the cross-
reactivity with these metabolites is not well defined
[9].
LC-MS/MS is becoming the technique of choice for
various reference laboratories. Laboratories which use
in-house LC-MS/MS have responsibility for many
steps of the assay. The LC-MS/MS technology for
testing of human samples is not approved by the U.S.
Food and Drug Administration (FDA), and
manufacturers of LC-MS/MS instrumentation are not
responsible for troubleshooting the assays.
Laboratories performing 25-OH-D testing by LC-
MS/MS technology have differences in their standard
operating procedures, and thus inter-laboratory CVs
are in the range of 20%. The preparation of the
reagents required for in-house LC-MS/MS assays is
conducted by individual laboratories under their
institutionally regulated standard procedures. The
complexity of the LC-MS/MS technology in its
present form demands a robust, fully automated
platform that can meet the need for throughput,
precision, and accurate testing of vitamin D and
metabolites. Multiplexed immunoassays may have the
potential of achieving accuracy and precision for
multiple vitamin D metabolites.
Specimen
Whole or heparinized blood should be collected and
the serum or plasma frozen at 80C. 25-OH-vitamin
D is stable when stored frozen at 80C.
19
25-OH-Vitamin D
Serum 25-OH-Vitamin D Reference Intervals
Recent publications have proposed that a serum 25-OH-
vitamin D concentration < 30 ng/mL be used as a cut-point
to define vitamin D deficiency [1, 5]. Latest research has
shown that deficiency of vitamin D may be associated with
susceptibility to various diseases, including cancers. For
prevention of these diseases, there are many individual
recommendations in the literature for minimum levels of
circulating 25-OH-vitamin D, but no consensus or
evidence-based-medicine guidelines have been established
to help patients and physicians. Since 2004, the Mayo
Clinic has defined 25-OH-vitamin D deficiency in patients
(using an LC-MS/MS method) based on the criteria below.
We have observed that 8.5% of the U.S. patients (n =
40,000) have < 10 ng/mL in winter, which drops to 4% in
summer. Optimum levels were present in 60% and 73% of
the population in winter and
summer, respectively.
Characteristic seasonal fluctuations are seen for serum 25-
OH-vitamin D concentration. These changes reflect the
amount of sunlight to which a person is exposed.
Concentrations of 25-OH-vitamin D are highest in late
summer and lowest in spring.
Interpretation
Upon entering the bloodstream, vitamin D is bound by a
specific transport protein, vitamin Dbinding protein
(DBP). In the liver, vitamin D is hydroxylated at the
carbon-25 position, giving rise to 25-OH-vitamin D3, the
most abundant circulating form of the vitamin. The final
hydroxylation step is catalyzed in the kidney by a 1--
hydroxylase enzyme, resulting in the production of the
biologically active form, 1,25-(OH)2-vitamin D. The
activity of the renal -hydroxylase is under tight control,
so the production of 1,25-(OH)2-vitamin D remains
constant over a wide range of substrate (25-OH-vitamin D)
concentrations. The main regulators of 1--hydroxylase
activity are calcium, parathyroid hormone, and phosphate.
Low serum calcium stimulates the secretion of parathyroid
hormone, which acts to increase the conversion of 25-OH-
vitamin D to 1,25-(OH)2-vitamin D. Hypophosphatemia
also stimulates conversion of 25-OH-vitamin D to 1,25-
(OH)2-vitamin D, but this process does not require
parathyroid hormone.
Vitamin D, through its active form 1,25-(OH)2-vitamin D,
has its most important effects on the intestine, where it
stimulates intestinal calcium and phosphate transport. It is
believed that 1,25-(OH)2-vitamin D acts at the intestinal
brush-border membrane, altering the properties of the cells
to allow greater permeability to calcium and phosphate
ions. The enhanced absorption of these ions raises their
concentration in blood to the levels necessary to permit
normal skeletal mineralization.
Vitamin D also acts directly on bone and kidney. In bone,
1,25-(OH)2-vitamin D causes bone-mineral resorption by
increasing osteoclastic resorption but probably does not
play a direct role in bone mineralization. In the kidney,
1,25-(OH)2-vitamin D decreases the excretion of both
calcium and phosphate by affecting their renal tubular
reabsorption.
Reference Intervals for 25-OH vitamin D
25-OH Vit D
concentration Clinical state
<10 ng/mL Severe deficiency*
10 ng/mL-25 ng/mL Mild to moderate
25OHD deficiency**
25 ng/mL-80 ng/mL Optimum 25OHD
levels
80 ng/mL Toxicity possible
* Could be associated with ostemalacia or rickets
**May be associated with increased risk of
osteoporosis or secondary
hyperparathyroidism
Optimum levels in the normal population
80 ng/mL is the lowest reported level associated
with toxicity in patients without primary
hyperparathyroidism and with normal
renal function
Of the circulating vitamin D metabolites, 25-OH-
vitamin D is the most abundant form and has the
longest half-life (approximately 1 to 2 weeks). Its
concentration serves as the best index of skin synthesis
and dietary intake of vitamin D. Nutritional vitamin D
deficiency, rickets or osteomalacia, is associated with
chronic low 25-OH-vitamin D levels, and subjects
who are intoxicated with vitamin D have
concentrations above 200 ng/mL. Chronic therapy
with some anticonvulsants decreases 25-OH-vitamin
D concentrations by induction of hepatic clearance.
Abnormally low concentrations of the metabolite
(resulting from malabsorption) are also observed in
patients with inflammatory bowel disease, bowel
resection, or biliary cirrhosis (Table 3) [3].
Performance Goals
25-OH-vitamin D is ordered and interpreted clinically
in light of calcium balance and homeostasis. The
quality and performance goals for calcium analysis in
the clinical labs have been revolutionized. Calcium
measurement is performed on highly automated
instruments and has a precision of less than 1%
coefficient of variation (CV) for day-to-day
performance. There is no reason to accept inferior
performance of the 25-OH-vitamin D assays in the
21st century. For better patient care, the goal should be
not only to have an accurate 25-OH-D value but also
precision for 25-OH-D testing, with a CV < 1%. We
would propose the following other goals for the 25-
OH-vitamin D assay.
Desirable interassay precision would be a 1% CV, but
the current technologies used for 25-OH-vitamin D
analysis cannot achieve this. A CV < 10% is
acceptable, but most current assays do not perform to
even this level (see Table 2). Current external quality-
20
25-OH-Vitamin D
assessment programs indicate that accuracy is also a
significant problem. Currently, commercial assays for 25-
OH-vitamin D do not perform to a satisfactory standard.
References
1 Cannell J, Hollis B, Zasloff M, Heaney R.
Diagnosis and treatment of vitamin D deficiency.
Expert Opin Pharmacother 2008;9:107-118.
2 Wicherts IS, van Schoor NM, Boeke AJP, Visser
M, Deeg DJH, Smit J, Knol DL, Lips P. Vitamin
D status predicts physical performance and its
decline in older persons. J Clin Endocrinol Metab
2007;92:2058-2065.
3 Fraser DR. The regulation of vitamin D
metabolism. Physiol Rev 1980;60:551-613.
4 Schmidt-Gayk H, Bouillon R, Roth HJ.
Measurement of vitamin D and its metabolites
(calcidiol and calcitriol) and their clinical
significance. Scand J Clin Lab Invest
1997;227:35-45.
5 Holick MF. Vitamin D deficiency. Engl J Med
2007;357:266-281.
6 Zerwekh JE. Blood biomarkers of vitamin D
status. Am J Clin Nutr 2008;87:1087S-1091S.
7 Carter GD, Carter R, Jones J, Berry J. How
accurate are assays for 25-hydroxyvitamin D?
Data from the international vitamin D external
quality assessment scheme. Clin Chem
2004;50:2195-2197.
8 Binkley N, Krueger D, Cowgill CS, Plum L, Lake
E, Hansen KE et al. Assay variation confounds
the diagnosis of hypovitaminosis D: a call for
standardization. J Clin Endocrinol Metab
2004;89:3152.
9 Souberbielle JC, Fayol V, Sault C, Lawson-Body
E, Kahan A, Cormier C. Assay-specific decision
limits for two new automated parathyroid
hormone and 25-hydroxyvitamin D assays. Clin
Chem 2005;51:395-400.
10 Belsey R, DeLuca HF, Potts JT Jr. Competitive
protein-binding assay for vitamin D and 25-OH-
vitamin D. J Clin Endocrinol Metab 1971;33:554-
557.
11 Bouillon R, Van Baelen H, DeMoore P.
Comparative study of the affinity of serum
vitamin D-binding protein. J Steroid Biochem
1980;13:1029-1034.
12 Haddad JG, Chyu KJ. Competitive protein-
binding radioassay for 25-hydroxycholecalciferol.
J Clin Endocrinol Metab 1971;33:992-995.
13 Haddad JG, Min C, Walgate J, Hahn J.
Competition by 24,25-dihydroxycholecalciferol in
the competitive protein-binding radioassay of 25-
hydroxycholecalciferol. J Clin Endocrinol Metab
1976;43:712-771.
14 Eisman JD, Shephard RM, DeLuca HF.
Determination of 25-hydroxyvitamin D2 and 25-
hydroxyvitamin D3 using high-pressure liquid
chromatography. Anal Biochem 1971;86:298.
15 Jones G. Assay of vitamin D2 and D3 and 25-
hydroxyvitamins D2 and D3 in human plasma
by high-performance liquid chromatography.
Clin Chem 1978;24:287-298.
16 Preece MA, ORiordan JLH, Lawson DEM,
Kodicek E. An assay for 25-
hydroxycholecalciferol and 25-
hydroxyergocalciferol in serum. Clin Chim
Acta 1971;54:235-243.
17 Neyestani T R, Gharavi A, Kalayi A.
Determination of serum 25-hydroxy
cholecalciferol using high-performance liquid
chromatography: a reliable tool for
assessment of vitamin D status. Int J Vitam
Nutr Res 2007;77:341-346.
18 Takeuchia A, Ishidaa Y, Sekimotoa H,
Masudaa S, Okanoa T, Nishiyamab S,
Matsudab I, Kobayashia T. Simplified
method for the determination of 25-hydroxy
and 1 alpha,25-dihydroxy metabolites of
vitamins D2 and D3 in human plasma -
Application to nutritional studies. J
Chromatogr B Biomed Appl 1997;691:313 -
319.
19 Lensmeyer GL, Wiebe DA, Binkley N,
Dresner MK. HPLC method for 25-
hydroxyvitamin D measurement: comparison
with contemporary assays. Clin Chem
2006;52:1120-1126.
20 Lensmeyer G, Wiebe D, Binkley N, Drezner
M. The authors of the article cited above
respond. Clin Chem 2006;52:2305-2306.
21 Hollis BW. Comparison of commercially
available 125I-based RIA methods for the
determination of circulating 25-hydroxy
vitamin D. Clin Chem 2000;46:1657-1661.
22 Bouillon R. Radiochemical assays for
vitamin D metabolites: technical possibilities
and clinical applications. J Steroid Biochem
1983;19:921-927.
23 Higashi T, Awada D, Shimada K.
Simultaneous determination of 25-
hydroxyvitamin D2 and 25-hydroxyvitamin
D3 in human plasma by liquid
chromatography-tandem mass spectrometry
employing derivatization with a Cookson-
type reagent. Biol Pharm Bull 2001;24:738-
743.
24 Singh RJ, Taylor RL, Reddy GS, Hollis BW,
Grebe SK. C-3 epimers can account for a
significant proportion of total circulating 25-
hydroxyvitamin D in infants, complicating
accurate measurement and interpretation of
vitamin D status. J Clin Endocrinol Metab
2006;91:3055-3061.
21
25-OH-Vitamin D

Table 1: Methods for 25-OH-Vitamin D Assay

Analyte: 25-OH-vitamin D
Method 1: Competitive protein-binding assay (CPB); employs serum or tissue-binding protein, quantitative
Principle of analysis: Competition between 25-OH-vitamin D in serum sample extract and 3H-labeled analog for
binding to tissue or serum D-binding protein; separation of bound and free labeled ligand by treatment with
dextran-coated charcoal
Comments: Preliminary chromatography essential for accurate measurement at high concentrations of 25-OH-
vitamin D
Method 2: High-performance liquid chromatography (HPLC); chromatographic, quantitative
Principle of analysis: Serum extracts are resolved on silica columns, and the concentration of 25-OH-vitamin D
is calculated by peak area (A254) integration.
Comments: Accuracy depends on efficiency of separation of 25-OH-vitamin D from interfering peaks.
Method 3: RIA and CLIA
Principle of analysis: Competition between 25-OH-vitamin D in serum sample extract and 125I-labeled or
acridinium esterlabeled analog for binding to antibody. In competitive RIA, the signal is monitored by counting
radioactivity, and assay is mostly manual. In comparison, in automated CLIA, the signal is chemiluminescence,
with moderate throughput.
Comments: Expensive; accuracy depends on standardization against gold-standard reference assay, precision
depends on consistent manufacturing and least lot-to-lot variations
Method 4: Liquid chromatographymass spectrometry (LC-MS/MS)
Principle of analysis: Non-isotopically labeled internal standard is used to account for variable recovery. Tandem
mass spec is not only sensitive but also provides specificity and does not require extensive chromatography, as
required in HPLC. Concentration of 25-OH-vitamin D is calculated by ratio of peak area/height of analyte/internal
standard.
Comments: Initially requires expensive capital investment

Table 2: Performance of 25-OH-Vitamin D Methods in Various Laboratories as

Reported to DEQAS

Sample Method n Method Mean SD CV

Automated IDS EIA 13 44.7 10.7 23.9

Chromatographic ligand-binding assay 1 48 0 0

DiaSorin liaison 37 40.7 6.4 15.8

DiaSorin liaison, total 8 41.2 5.7 13.9

DiaSorin RIA 50 44.1 6.4 14.5

HPLC 10 48.5 16.6 34.3

IDS EIA 56 49.3 9.4 19

IDS RIA 31 45.2 4.5 10

LC-MS 20 50.8 9.3 18.2

Unknown 2 57 12.5 22

All Methods 228 45.4 7.5 16.6

n, Number of laboratories using a particular method.

22
25-OH-Vitamin D

Table 3: Vitamin D Metabolite Concentrations in Disorders of Calcium Homeostasis

Condition: Nutritional rickets or osteomalacia Condition: Hypophosphatemic rickets
25-OH-D: Low 25-OH-D: Normal
1,25-(OH)2-D: Low/low-normal 1,25-(OH)2-D: Low/normal
Condition: Vitamin D-dependent rickets Condition: Primary hyperparathyroidism
a. Type I 25-OH-D: Normal
b. Type II 1,25-(OH)2-D: High/normal
25-OH-D: Condition: Pseudohyperparathyroidism
a. Normal 25-OH-D: Normal
b. Normal 1,25-(OH)2-D: Low/normal
1,25-(OH)2-D:
Condition: Sarcoidosis with hypercalcemia
a. Low 25-OH-D: Normal
b. High 1,25-(OH)2-D: High
Condition: Vitamin D intoxication
25-OH-D: High
1,25-(OH)2-D: Low/normal

Figures

Figure 1: Pathways in the metabolism of vitamin D3. Vitamin D3 ingested in diet or made in skin accumulates in liver,
where it is converted to 25-OH-vitamin D3, the major circulating form. Subsequent metabolism of 25-OH-vitamin D3 in
kidney gives rise to 1,25-(OH)2-vitamin D3, the hormonal form.
23
25-OH-Vitamin D

Figure 2: Pathways in metabolism of vitamin D2. Vitamin D2 ingested in diet or made in skin is accumulated in liver
where it is converted to 25-OH-vitamin D3, the major circulating form. Subsequent metabolism of 25-OH-vitamin D2 in
kidney gives rise to 1,25-(OH)2-vitamin D2, the hormonal form.

Figure 3: Representative HPLC-UV chromatograms.

(A), late-eluting peaks; (B), calibrator in extracted serum; (C), sample from patient with low 25(OH)D3 treated with
vitamin D2; (D), sample from patient with high concentrations of 25(OH)D3. Int. Std., internal standard; mAU,
milliabsorbance units.
24
25-OH-Vitamin D

Figure 4: HPLC-UV Chromatograms showing separation of 25(OH)-epi-D3 a recently discovered metabolite of Vit-
D. (A), elution of 25(OH)-epi-D3 (not extracted); (B), a serum extract containing 25(OH)D3 (40 g/L) and 25(OH)-epi-D3
(30 g/L); and (C), a serum extract containing 25(OH)D3 (69 g/L), 25(OH)-epi-D3 (15 g/L), and 25(OH)D2 (12 g/L).
(26)

Figure 5: Comparison of the proposed HPLC method with the Diasorin RIA [21]
25
25-OH-Vitamin D

Figure 6: 25-OH-Vitamin D Metabolites. LC-MS/MS Chromatograms-MRM ion peaks for 25-(OH)-D3 and 25-(OH)-
D2.

Figure 7: Comparison of the proposed HPLC method with LC-MS/MS. [19]

26
25-OH-Vitamin D

Figure 8: CAP survey data for 2007 on commonly used immunoassays for
25-OH-D.

The ranges of results for survey materials BGS-01 through BGS-04 are shown for laboratories using either RIA (solid
lines, n = 16) or automated chemiluminescent immunoassays (broken lines, n = 18). Single lab LC-MS/MS 25-OH-D test
results are shown (closed circles) for comparison. CLIA, chemiluminescence immunoassay.

Procedure: 25-OH-Vitamin D-Commercial Assays.

Follow the latest kit inserts supplied with the reagents
and the instrumentation.
25-OH-Vitamin D -HPLC-UV Method [19]
Reagents
The vitamin D metabolites 25(OH)D3 and 25(OH)D2 can
be obtained from Fluka Chemicals. ACS reagent-grade
acetonitrile (CH3CN), and ethyl acetate were obtained
from Fisher. Methanol (HPLC grade) was obtained from
Mallinckrodt Chemicals. Ultrapure water (18.2 M /cm)
was obtained from a MilliQ water purification system
(Millipore). The precipitation reagent contained the
internal standard laurophenone (400 g/L) in CH3CN
and was stored in an amber bottle. Strata-X (surface-
modified styrene-divinylbenzene resin) 60-mg (1 mL)
extraction cartridges were from Phenomenex. An
automated extraction instrument, the Gilson ASPEC
XL4 (Gilson Instruments), consisted of a 4-syringe
pump module and a 4-needle sampler module with four
2-way solvent ports. Areas in the sampler racks were
defined as the sample zone, reagent zone, result zone,
and a disposable extraction column (DEC) zone.
Acetonitrile was delivered via solvent ports.
Acetonitrilewater (35:65 by volume) was stored and
delivered from tubes within the reagent zone. The main
reservoir contained water. The solvent evaporator was a
Turbo VapTM LV (Caliper Life Sciences). Temperature
was set at 35C, nitrogen flow was adjusted to 10 psi on
the instrument gauge, and the typical drying time setting
was 25 min. The HPLC unit was an integrated system
with a UV3000 detector set at 275 nm, a P4000 pump set
at 1.2 mL/min, an AS2000 autosampler, and an
SCM1000 solvent system, all from Thermo Separation
Products. A silica-saturator column (250 4.6 mm [i.d.]
stainless steel column; Alltech) packed with ICN silica
gel (particle size, 63 to 100 m; MP Biochemicals) was
installed in the oven between the pump and injector and
is necessary here to prevent deterioration of the
analytical column. The guard column (12.5 4.6 mm
[i.d.]) and analytical column (250 4.6 mm [i.d.]), both
containing 5-m Stable Bond Cyanopropyl (SB-CN),
were from Agilent Technologies. All columns were
operated at 50C. The methanolwater (67:33 by
volume) used as mobile phase was filtered and degassed.
Individual calibrator stock solutions (40 mg/L) of each
metabolite are prepared in ethanol, and the concentration
was verified on a Beckman DU 7500 spectrophotometer,
27
25-OH-Vitamin D
using molar absorptivities at 265 nm (1-cm pathlength)
of 19,400 and 18,300 for 25(OH)D2 and 25(OH)D3,
respectively. From these primary stocks, dilute,
combined-stock solutions of the compounds at 10,000
g/L each in ethanol can be prepared, which are stable
for at least 1 year at 20C. Multiple working calibrators
in the range of 5 to 200 g/L for each of the 2 vitamin D
metabolites were combined in a drug-free serum pool.
The concentrations of endogenous 25(OH)D3 and
25(OH)D2 present in the pool were taken into account
when assigning the final concentration to the calibrator.
Serum calibrators were stored frozen at 20C in 10-mL
glass vials sealed with Teflon-lined caps (QuorpakTM;
Fisher Scientific) and were stable for at least 6 months.
Controls were prepared and used in the same manner.
Commercial lyophilized serum controls were custom-
prepared for us by Utak Laboratories, Inc. Reconstituted
Utak controls and thawed calibrators/controls were
stable for at least 1 month stored at 4C.
Procedure
To prepare samples, we dispensed 2 mL of precipitation
reagent with internal standard into a 13 100 mm
disposable glass test tube; we then added 1.0 mL of
serum (calibrator, control, or patient sample) to the tube
without mixing of contents to avoid balling of the
protein. The tube was allowed to sit for 5 min at room
temperature, after which it was vortex-mixed for 10 sec
to obtain a flocculent precipitate. After another 5-min
wait, the tube was vortex-mixed and centrifuged at 2000
g for 10 min. The clear supernatant was decanted into a
10 75 mm disposable glass test tube, which was then
transferred to the sample zone of the ASPEC XL4 and
protected from exposure to natural sunlight to prevent
degradation of analytes. The extraction conditions are
defined in detail in ref 21. The XL4 processed 4 samples
simultaneously and unattended in ~ 15 min. The unit
sequentially conditioned the Strata-X cartridge in the
DEC zone with 2.0 mL of CH3CN followed by 2.0 mL
of 35:65 CH3CNwater; added 1.0 mL of water to each
extract; transferred 3.5 mL of extract mixture to the
DEC; rinsed the DEC with 2.0 mL of 35:65 CH3CN
water; and eluted the Strata-X cartridge in the DEC zone
with 2.0 mL of CH3CN. The eluate was dried at 35C
under a stream of nitrogen; the dry extract was then
reconstituted with 150 L of ethyl acetateCH3CN (5:95
by volume) and vortex-mixed for 5 sec. Water (110 L)
was then added to the tube, and the contents were
vortex-mixed for 5 sec. The sample was centrifuged at
2000 g for 10 min to settle the precipitate. The clear
liquid was transferred to a glass microvial insert
positioned in an amber-colored vial. The sample was
capped and placed in the autosampler unit of the HPLC.
The extract was stable for at least 3 days at room
temperature. The processor software calculated relative
retention time for peak identification and peak-height
ratio for quantification.
25-OH-Vitamin D LC-MS/MS Method [24]
For the standard 25-OH-D method, online extraction and
HPLC chromatography of the supernatants were
performed using a TX4 Turbo Flow system (Cohesive
Technologies, Franklin, MA) with 1.0 50 mm Cyclone
extraction columns and 3.3 cm 4.6 mm, 3-m LC-18
(Supelco, St. Louis, MO) analytical columns. After
online extraction, the analytes were eluted onto the
analytical column for 90 sec with a mobile phase of
39.5% vol/vol methanol, 0.005% vol/vol formic acid.
There was a step gradient to 87% vol/vol methanol,
0.005% vol/vol formic acid for the analytical column.
The analytes then entered an API 4000 triple-quadrupole
mass spectrometer (ABI-Sciex, Toronto, Canada) and
were ionized in an atmospheric-pressure chemical-
ionization source and detected by multiple reaction
monitoring of the following ion pairs: m/z 413.0/395.3
for 25-OH-D2, m/z 401.4/383.3 for 25-OH-D3, and m/z
407.4/389.5 for 25-OH-D3-d6. The raw signals of 25-
OH-D2 and 25-OH-D3 in the calibrators, controls, and
samples were normalized to their respective internal
standard 25-OH-D3-d6 signals, and concentrations in the
samples and controls were calculated off the normalized
six-point calibration curves (0 to 200 ng/mL (0 to 500
nmol/L). Samples with concentrations that exceeded the
highest calibrator were diluted and run again. The total
25-OH-D concentrations of each control and sample
were calculated by summing the measured values of 25-
OH-D2 and 25-OH-D3.
For separation of epimers, the standard LC-18 column
was replaced with a longer 5-dinitrobenzoyl-(R)-
phenylglycine column (Chirex-PGLY and DNB 250
4.6 mm; Phenomenex, Torrance, CA), and 100 L of the
supernatant was injected. The step gradient extends only
up to 67% vol/vol methanol, 0.005% vol/vol formic acid
at an analytical column flow rate of 0.9 mL/min. The
mass spectrometer settings remained unchanged. The
concentrations of 25-OH-D2, 25-OH-D3, and 25-OH-D
were calculated as above. The concentrations of any
detected C-3 epimers of 25-OH-D2 or 25-OH-D3 were
also calculated off the normalized 25-OH-D2 and 25-
OH-D3 calibration curves, and the total 3-epi-25-OH-D
concentration is the sum of 3-epi-25-OH-D2 and 3-epi-
25-OH-D3 concentrations. Intraassay CVs were 3.8%,
2.4%, and 4.7% at 25, 54, and 140 ng/mL, respectively.
Interassay CVs were 6.4%, 6.8%, and 5.0% at 24, 52,
and 140 ng/mL respectively.
28
1-Antitrypsin
1-Antitrypsin
John Beilby
Names: 1-Antitrypsin, 1-protease inhibitor
Clinical significance: 1-Antitrypsin deficiency associated with lung and liver disease. Refer
to Chapter 52, Diseases of Genetic Origin, in the 5th edition of Clinical Chemistry: Theory, Analysis,
Correlation.
Molecular mass: 52,000 D
Chemical class: Glycoprotein
Most common allelic types: PiM, PiS, PiZ
Principles of Analysis and Current Usage
The clinical importance of the measurement of 1-
antitrypsin serum levels is to detect people with deficient
states. Severe deficiency (OMIM #107400) is clinically
under-recognized [1,2] and is associated with a
substantially increased risk for the development of
pulmonary emphysema by the 3rd and 4th decades of
life. It is also associated with the risk of hepatic disease,
cutaneous panniculitis, arterial aneurysm, bronchiectasis,
and renal disease [3]. 1-Antitrypsin is responsible for
approximately 90% of the trypsin-inhibitory capacity of
serum [4,5]. It is a member of the serine protease
inhibitor or serpin superfamily [6] and is also known as
1-protease inhibitor because of its ability to inhibit a
broad range of protease enzymes, including trypsin,
chymotrypsin, pancreatic elastase, neutral proteases of
polymorphonuclear leukocytes and macrophages, and a
number of other enzymes [7]. It is a globular
glycoprotein that is found in the 1 region of an agarose
electrophoresis pattern at pH 8.6. It has a molecular mass
of 52 kDa and consists of a 418 amino acid single
polypeptide chain with four carbohydrate side chains. 1-
Antitrypsin is found in serum and in a number of body
fluids such as saliva, tears, lymph, semen, cervical
mucus, synovial fluid, and human milk [7]. The half-life
of exogenous 1-antitrypsin in serum is approximately a
week, with catabolism taking place in the liver.
Quantitation
1-Antitrypsin concentrations in serum are measured by
immunochemical techniques. In the past, electro-
immunodiffusion and radial immunodiffusion techniques
were used. Today, automated immunoturbidimetric and
immunonephelometric methods are widely used in
laboratories. These quantitative methods determine 1-
antitrypsin concentrations in human serum or plasma by
measuring the absorbance change due to light scattering
caused by insoluble complexes that form between anti-
i confirmed by Pi (Protease Inhibitor) phenotyping [11].
Alpha1-antitrypsin
Previous and current authors of this method:
First edition: Not done
Methods edition: Gayle Jackson
Second edition: Not updated
Third edition: Steven C. Kazmierczak
Fourth edition: Gayle Jackson
Fifth edition: John Beilby
1-antitrypsin antibodies and 1-antitrypsin in the
sample. The amount of light scattering is dependent
upon the 1-antitrypsin concentration in the sample and
can be quantified by comparison with calibrators of
known 1-antitrypsin concentration.
Serum Electrophoresis
Occasionally, 1-antitrypsin deficiency can be detected
by close examination of the 1-globulin band on serum
electrophoresis gels. Since 1-antitrypsin composes
approximately 90% of the 1-globulin band, a severe
deficiency results in the almost complete absence of 1-
globulins. It should be noted that the densitometer scan
might give a normal value for the 1-globulins, even
though visual inspection clearly shows an absence of the
band. Other 1-proteins such as 1-lipoprotein may stain
to give a diffuse background, thus accounting for the
discrepancy between the visual inspection and the scan
results.
Phenotyping
1-Antitrypsin deficiency is inherited as an autosomal
co-dominant disorder, with more than 100 alleles
identified [8]. The 12.2 kb gene is located on the long
arm of chromosome 14 (14q31-32.3) and consists of
seven exons and six introns. Different phenotypes are
classified by a coding system in which the inherited
alleles are usually letters that denote the migration of the
molecule in an isoelectric pH gradient from A (for
anodal variants) to Z (for slower migrating variants). The
MM phenotype indicates individuals who are
homozygous for the normal allele, and ZZ indicates that
they are homozygous for the Z allele. The Z variant
consists of a glutamine substitution for a lysine residue
at codon 342, causing an 1-antitrypsin-deficient state
and a dysfunctional protein [9]. The S variant is due to a
substitution of valine for glutamine at codon 264,
causing intracellular degradation of the protein [10].
Low serum concentrations of 1-antitrypsin must be
The gold standard for the identification of 1-antitrypsin
variants is the phenotyping of serum samples by
isoelectric focusing on thin-layer gels in a pH gradient
[11]. This can be done on polyacrylamide or agarose
gels. In this procedure, a stable, stationary pH gradient is
obtained by the use of ampholytes which are polyamino-
29
1-Antitrypsin
polycarboxylic acids. Since 1-antitrypsin proteins have
isoelectric points in the pH region from 4.2 to 4.65,
ampholines with a pH range of 4 to 5 are used. This
technique separates the various isoforms of 1-
antitrypsin based on their migration in a pH gradient.
Each isoform migrates to the position within the pH
gradient at which the overall net charge of the protein is
zero. The separated protein bands are visualized by a
protein stain such as Coomassie blue R-250 [12]. Acid-
starch gel electrophoresis followed by crossed antigen-
antibody electrophoresis [13] has been used in the past
(Table 1, Method 6).
Isoelectric focusing is a technically challenging test and
requires a highly skilled scientist to perform the test and
interpret the results. Samples of known Pi types are not
readily available and can only be found by testing many
samples. In addition, severe deficiency is relatively
uncommon. Screening for heterozygous 1-antitrypsin
deficiency or for intermediate deficiencies does not seem
worthwhile, because these phenotypes do not correlate
statistically to respiratory problems [14].
The medically important variants are those associated
with 1-antitrypsin deficiencynamely, the S and Z
variants and the rare null (non-production) variant.
Approximately 6% of people of northern European
descent carry the S gene, and 3% to 4% carry the Z
variant [15].
Genotyping
Genotyping can largely replace the isoelectric focusing
technique. The molecular assay is readily automated
using real time PCR, and the interpretation is
straightforward, unlike the isoelectric focusing method.
The PCR assay detects the most common disease-
associated alleles, Z and S. An algorithm for
quantitation, genotyping, and phenotyping of 1-
antitrypsin has been described [16]. In this approach, 1-
antitrypsin was quantitated and genotyped when the
serum concentrations were less than 70 mg/dL (0.7g/L).
If the results were concordant, they were released with
an interpretative comment. If the quantitation and
genotype results were discrepant, isoelectric focusing
was performed, and all results were reported with an
interpretative comment. Snyder et al. [16] demonstrated
that 2% of all results were discordant. In another study,
all samples without an observed S or Z variant and a
serum concentration of less than 100 mg/dL (1.0g/L)
were phenotyped [17]. Using this technique, Bornhorst
et al. [18] identified 6.5% of patients at risk of
developing 1-antitrypsin-related symptoms owing to
the presence of rare deficiency alleles not detected by the
genotyping assay. A variation on this approach was
genotyping by direct sequencing all patient samples with
1-antitrypsinconcentrations less than 100 mg/dL
(1.0g/L) [18]. This approach identified all deficient
alleles and two new null alleles. Genotyping is quick and
easy to perform but needs to be complemented by either
phenotyping or direct sequencing so that disease
associated variants are not missed.
Reference and Preferred Methods
There is no reference method for the determination of
serum 1-antitrypsin levels. Automated
immunoturbidimetric and immunonephelometric
methods are currently used with the
immunoturbidimetric group having the best imprecision
(see performance goals below). Reference material for
serum proteins, CRM 470 was released in 1993 and has
resulted in an improvement in between-laboratory
variability of serum protein results [19].
Specimen
Serum samples are preferred, but plasma samples are
acceptable for 1-antitrypsin concentration
determination. Samples may be stored at 4C for up to 7
days prior to testing. For long-term storage or for
samples suspected of a severe deficiency of 1-
antitrypsin, storage at 70C is recommended.
Serum or plasma samples may be used for phenotyping.
Samples with 1-antitrypsin concentrations of 50% of
normal or above may be stored on a long-term basis at
70C. Storage at 20C is acceptable for 1 or 2 weeks.
Unfortunately, samples with severe 1-antitrypsin
deficiency cannot prevent normal activation of intrinsic
proteolytic enzymes and may become degraded rapidly,
even if stored at less than 20C. Bacterial
contamination or improper storage results in altered
migration rates of the bands with eventual loss of
banding [20]. Samples suspected of a severe deficiency
should be phenotyped as soon as possible after
collection.
Interferences
Some work has been published on assay interferences
for 1-antitrypsin concentration determination. However,
the results appear to be variable, depending on the
technique used and the manufacturer of the
instrumentation [21]. To identify the interferences
associated with each method, the user should refer to the
manufacturers kit insert.
1-Antitrypsin Reference Interval
In 1995, fifteen professional societies and twelve
diagnostic companies agreed on a procedure for uniform
reporting of protein reference intervals [19]. The 1-
antitrypsin reference interval was reported as 90 to 200
mg/dL (0.9 to 2.0g/L) for subjects with the MM
genotype. Age- and gender-related differences in
reference interval have been reported. Following birth,
concentrations fall during the first 6 months but rise to
adult concentrations by 1 year of age [22]. Females have
higher concentrations than men across all ages [23].
Interpretation
A number of factors have been reported to increase 1-
antitrypsin concentration, including inflammatory
disorders, malignancy, trauma, increases in estrogen
concentrations with puberty, pregnancy, or the use of the
oral contraceptive pill [23]. However, values rarely
increase more than fourfold. These factors can cause
considerable overlap in measured concentration between
30
1-Antitrypsin
mildly and moderately 1-antitrypsin-deficient subjects
and concentrations in normal subjects [7].
Severe 1-antitrypsin deficiency is a genetic disorder
that affects about 1 in 2000 to 5000 individuals [1]. The
World Health Organization guidelines recommend
screening all patients with chronic obstructive
pulmonary disease and adults and children with asthma
[24]. Any 1-antitrypsin concentration less than 100
mg/dL (1.0g/L) should be genotyped or phenotyped to
detect 1-antitrypsin variants. In the few cases where the
genotype does not agree with the 1-antitrypsin
concentration, the sample should be further investigated
by phenotyping or DNA sequencing. Serum
concentrations of 1-antitrypsin less than a protective
threshold of 50mg/dL (0.5 g/L) by nephelometry are
associated with an increasing risk of emphysema [25].
Allelic types that result in deficiencies of serum 1-
antitrypsin include PiS (mean plasma concentration,
60% to 70% of normal), PiZ (mean plasma
concentration, 10% to 15% of normal), and Pi null [26].
The ANT concentration in severely deficient subjects
rises only slightly in response to inflammatory disorders,
malignancy, trauma, and increases in estrogen
concentration.
The prevalence of the various genotypes are
approximately MM 91%, MS 6.1%, MZ 2.7%, SS 0.1%,
SZ 0.1%, ZZ 0.02% and null-null. The risk of
emphysema is 20% to 50% for the SS genotype, 80% to
100% for SZ, and 100% for the null-null genotype [1].
The null alleles do not produce 1-antitrypsin transcript,
produce truncated protein, or produce unstable proteins
that are essentially completely degraded before
secretion. The risk of emphysema is greatly increased in
a person with a severe 1-antitrypsin deficiency if they
smoke cigarettes.
1-Antitrypsin Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report show that 73%
of laboratories use immunonephelometric techniques,
and 27% use immunoturbidimetric techniques. The
imprecision of the methods as determined by the
coefficient of variation (CV%) at a mean 1-antitrypsin
concentration of 53.9 mg/dL (0.54g/L) ranged from
4.3% to 6.7% for immunonephelometric techniques and
from 2.6% to 5.8% for immunoturbidimetric techniques.
Acceptable performance criteria (CLIA-88) for
measurement of 1-antitrypsin require that laboratories
be accurate to within 3SD of the peer group mean. The
intra-individual variation of 1-antitrypsin in blood in
healthy adults has been determined to be approximately
5.9%. Desirable specifications for analytical imprecision
derived from studies of biological variation indicate an
assay imprecision of no greater than 3.0% and a total
error of no greater than 9.2% [27]. The
immunonephelometric and immunoturbidimetric
techniques are both within the desired performance
criteria as defined by CLIA-88. However, only one
immunoturbidimetric technique is within the analytical
imprecision specification if biological variability defines
required performance.
References
1. Stoller JK, Aboussouan LS. Alpha1-antitrypsin
deficiency. Lancet 2005; 365: 2225-2236.
2. Laurell CB, Eriksson, S. The electrophoretic
alpha 1-globulin pattern of serum in alpha 1
antitrypsin deficiency. Scand J Clin Lab Invest
1963; 25: 132-140.
3. Mulgrew AT, Taggart CC, McElvaney NG.
Alpha-1-antitrypsin deficiency: current
concepts. Lung 2007; 185: 191-201.
4. Crystal RG. The alpha 1-antitrypsin gene and
its deficiency states. Trends Genet 1989; 5:
411-417.
5. Lisowska-Myjak B. AAT as a diagnostic tool.
Clin Chim Acta 2005; 352: 1-13.
6. Potempa J, Korzus E, Travis J. The serpin
superfamily of proteinase inhibitors: structure,
function, and regulation. J Biol Chem 1994;
269: 15957-15960.
7. Morse JO. alpha1-antitrypsin deficiency (first
of two parts). N Engl J Med 1978; 299: 1045-
1048.
8. DeMeo DL, Silverman EK. Alpha1-antitrypsin
deficiency. 2: genetic aspects of alpha(1)-
antitrypsin deficiency: phenotypes and genetic
modifiers of emphysema risk. Thorax 2004; 59:
259-264.
9. Yoshida A, Lieberman J, Gaidulis L, Ewing C.
Molecular abnormality of human alpha1-
antitrypsin variant (Pi-ZZ) associated with
plasma activity deficiency. Proc Natl Acad Sci
USA 1976; 73: 1324-1328.
10. Owen MC, Carrell RW, Brennan SO. The
abnormality of the S variant of human alpha1-
antitrypsin. Biochim Biophys Acta 1976; 453:
257-261.
11. Stoller JK, Snider GL, Brantly ML, Fallat RJ,
Stockley RA, Turino GM et al. [American
Thoracic Society/European Respiratory Society
Statement: Standards for the diagnosis and
management of individuals with alpha1-
antitrypsin deficiency]. Pneumologie 2005; 59:
36-68.
12. Kueppers F. Determination of alpha1-
antitrypsin phenotypes by isoelectric focusing
in polyacrylamide gels. J Lab Clin Med 1976;
88: 151-155.
13. Lieberman J, Gaidulis L, Garoutte B, Mittman
C. Identification and characteristics of the
common alpha 1 -antitrypsin phenotypes. Chest
1972; 62: 557-564.
14. Morse JO, Lebowitz MD, Knudson RJ,
Burrows B. Relation of protease inhibitor
phenotypes to obstructive lung diseases in a
community. N Engl J Med 1977; 296: 1190-
1194.
15. de Serres FJ. Worldwide racial and ethnic
distribution of alpha1-antitrypsin deficiency:
31
1-Antitrypsin
summary of an analysis of published genetic
epidemiologic surveys. Chest 2002; 122: 1818-
1829.
16. Snyder MR, Katzmann JA, Butz ML, Wiley C,
Yang P, Dawson DB et al. Diagnosis of alpha1-
antitrypsin deficiency: An algorithm of
quantification, genotyping, and phenotyping.
Clin Chem 2006; 52: 2236-2242.
17. Bornhorst JA, Procter M, Meadows C,
Ashwood ER, Mao R. Evaluation of an
integrative diagnostic algorithm for the
identification of people at risk for alpha1-
antitrypsin deficiency. Am J Clin Pathol 2007;
128: 482-490.
18. Prins J, van der Meijden BB, Kraaijenhagen RJ,
Wielders JP. Inherited Chronic Obstructive
Pulmonary Disease: New Selective-Sequencing
Workup for 1-Antitrypsin Deficiency
Identifies two Previously Unidentified Null
Alleles. Clin Chem 2008; 54:101-107.
19. Dati F, Johnson AM, Whicher JT. The existing
interim consensus reference ranges and the
future approach. Clin Chem Lab Med 2001; 39:
1134-1136.
20. Ritchie RF, Smith R. Immunofixation. II.
Application to typing of alpha1-antitrypsin at
acid pH. Clin Chem 1976; 22: 1735-1737.
21. Bossuyt X, Blanckaert N. Evaluation of
interferences in rate and fixed-time
nephelometric assays of specific serum
proteins. Clin Chem 1999; 45: 62-67.
22. Ward AM, White PA, Wild G. Reference
ranges for serum alpha 1-antitrypsin. Arch Dis
Child 1985; 60: 261-262.
23. Ritchie RF, Palomaki GE, Neveux LM,
Navolotskaia O, Ledue TB, Craig WY.
Reference distributions for the positive acute
phase serum proteins, alpha1-acid glycoprotein
(orosomucoid), alpha1-antitrypsin, and
haptoglobin: a practical, simple, and clinically
relevant approach in a large cohort. J Clin Lab
Anal 2000; 14: 284-292.
24. Alpha 1-antitrypsin deficiency: memorandum
from a WHO meeting. Bull World Health
Organ 1997; 75: 397-415.
25. American Thoracic Society/European
Respiratory Society Statement: Standards for
the Diagnosis and Management of Individuals
with Alpha-1 Antitrypsin Deficiency. Am J
Respir Crit Care Med 2003; 168: 818-900.
26. Eriksson S, Elzouki AN. Alpha 1-antitrypsin
deficiency. Baillieres Clin Gastroenterol 1998;
12: 257-273.
27. Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biological variation: pros, cons
and progress. Scand J Clin Lab Invest 1999; 59:
491-500.
1-Antitrypsin: Methods Summary Table
Method 1: Electrophoresis; quantitative; estimation of
1-globulins by physical separation
Principle of analysis: Proteins separate based
on class (albumin, gamma globulins)
Comments: Serum; good screen for severe 1-
antitrypsin deficiency. The 1-globulin band is
absent on the electrophoretogram.
Method 2: Electroimmunodiffusion (EID); quantitative;
quantitation by size of immunoprecipitate in gel
Principle of analysis: Electrophoresis of 1-
antitrypsin into antibody-containing agarose
gel; height of gel pattern (rocket) proportional
to 1-antitrypsin concentration
Comments: Serum; kits not available
commercially; mainly of historical interest
Method 3: Radial immunodiffusion; quantitative;
quantitation by immunoprecipitate in gel
Principle of analysis: 1-Antitrypsin diffuses
into gel containing antibody, forming a ring-
shaped immunoprecipitate; diameter of ring
proportional to concentration
Comments: Serum; commercial plates readily
available, not widely used in diagnostic
laboratories
Method 4: Nephelometry or immunoturbidimetry;
quantitative; quantitation by immunoprecipitate
in solution
Principle of analysis: Reaction of 1-
antitrypsin with its specific antibody results in
immunoprecipitate, which has light-scattering
properties; amount of light scatter proportional
to 1- antitrypsin concentration
Comments: Serum; commercial kits readily
available
Method 5: Electrophoresis of serum on acid starch gel,
followed by antigen-antibody crossed
electrophoresis; phenotyping; physical
separation of 1-antitrypsin variants followed
by immunoprecipitin reaction in agarose
Principle of analysis: Two-step procedure :
1. Separation of 1-antitrypsin
variants on acid-starch gel by electrophoresis
2. A second electrophoresis causes the
separated variants to migrate perpendicularly to
the first separation into agarose containing
antibody to 1-antitrypsin; precipitin peaks
form; the second electrophoresis is necessary
for separation of the Z and S variants
Comments: Serum; original, standard method
for phenotyping; time consuming (1 days) and
technically difficult; mainly of historical
interest
32
1-Antitrypsin

1-Antitrypsin: Methods Summary Table (cont)

Method 6: Isoelectric focusing; separation of 1-
antitrypsin variants on agarose or
polyacrylamide gel based on surface-property
charge
Principle of analysis: 1-antitrypsin variants
are electrophoresed on polyacrylamide or
agarose gels containing ampholines with a pH
range of 4 to 5. The variants migrate under the
electric field until they reach a pH equal to their
isoelectric point.
Method 7: Genotyping for the separation of 1-
antitrypsin variants
Principle of analysis: DNA extracted from
whole blood is subject to real-time PCR
amplifying the S and Z alleles in separate tubes.
The different primer design and detection
probes have been described in several published
articles [16, 17].
33
Acetaminophen
Acetaminophen
Gus Koerbin, David G Hughes and Julia M. Potter
Name: Acetaminophen, N-acetyl-p-aminophenol, paracetamol
Clinical significance: Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C8H9NO2
Molecular weight: 151.16 D
Merck Index: 39
Chemical class: p-Aminophenol derivative
Structure:
Principles of Analysis and Current Usage
Acetaminophen did not gain much attention for therapeutic
use until 1948 when Brodie and Axelrod [1] identified it as
the active metabolite of phenacetin and acetanilide. It is
now widely used as an analgesic and antipyretic drug. It is
freely available as an over-the-counter preparation in drug
stores and supermarkets and is frequently used in suicide
attempts.
The current routine methods for clinical analysis of
acetaminophen in the United States, as shown in 2007
CAP data, are those for automated platforms. These are
either enzymatic methods with color development or
immunoassays. Previous methods, which are summarized
briefly below, including those based on the original
method of Brodie and Axelrod [1] (Table 1, Method 1), are
now superseded for routine clinical analysis and are of
historical interest only. More detail is available in the
previous edition of this text. This includes high-
performance liquid chromatography (HPLC), gas
chromatography (GC), and spectroscopic analysis, which
still have roles in industry and have been reviewed recently
[2].
Qualitative Analysis
A spot test for the detection of acetaminophen
(paracetamol) in urine is available (Table 1, Method 2).
The reaction of the conjugated metabolites of
i
Acetaminophen
Previous authors of this method:
First edition: Joseph Svirbely
Methods edition: Joseph Svirbely
Second edition: Joseph Svirbely
Third edition: Helen M. Dodds, Julia M. Potter
Fourth edition: Helen M. Dodds, Julia M. Potter
Fifth edition: Gus Koerbin, David G. Hughes, Julia M.
Potter
acetaminophen with o-cresol in ammonium hydroxide
produces an indigo product (indophenol blue) which
indicates the presence of acetaminophen.
Acetaminophen can be detected by silica gel thin-layer
chromatography (TLC) as a quenching spot under long
ultraviolet radiation. In the TLC screen,
acetaminophen has an Rf of 0.41.
EMIT methodology has been adapted [3] to produce a
rapid screening test of serum acetaminophen. In this
procedure, enzyme-labeled glucose-6-phosphate
dehydrogenase (G6PDH) acetaminophen and NAD are
diluted with Tris buffer. Serum samples are also
diluted with this same buffer. The diluted serum
samples are mixed with equal amounts of diluted,
labeled acetaminophen/NAD solution and incubated at
room temperature for 10 to 15 min. Following
incubation, 1.0 L of the reaction mixture is placed
onto filter paper, dried, and any observed fluorescence
indicates the presence of acetaminophen.
A qualitative point-of-care test for urine has been
incorporated into the Triage Tox Drug Screen panel
(Biosite, Ltd., UK), using the competitive
immunoassay ASCEND Multi-immunoassay
technology. The sample comes in contact with
lyophilized reagents, that then is applied to a solid
phase containing immobilized antibodies. The method
incorporates preset threshold concentrations for each
drug. In the absence of drug or in the presence of drug
in quantities less than the threshold concentration, no
red-colored bar is produced. Samples containing the
drug at or above the threshold concentration cause the
appearance of that bar. The quoted detection
concentration is 5mg/L. The urine samples require no
pretreatment.
34
Acetaminophen
Quantitative Analysis
Colorimetric Methods
The earliest methods were colorimetric techniques based
on the hydrolysis of acetaminophen to p-aminophenol with
addition of -naphthol for color development, which was
then measured spectrophotometrically. Applications for
either serum or urine were developed [4-6]. However,
although the p-aminophenol methods were rapid and
simple, they measured the sulfate and glucosamine
metabolites of acetaminophen, as well as parent
acetaminophen. The metabolites are clinically unimportant,
so these methods were not of great clinical use.
An alternative colorimetric technique utilized ring nitration
of the acetaminophen with nitrous acid [7]. This was
initially used in quantification in the pharmaceutical
industry and with a modification is known as the Glynn
and Kendall method [8] (Table 1, Method 3a). This method
is subject to significant interference from salicylic acid,
and many method modifications have attempted to reduce
this, ranging from mathematical corrections for known
salicylate concentration [9] to changes in the reaction
conditions [10-14].
These p-aminophenol conversion methods are rapid and
offer a degree of simplicity. However, by using harsh
chemical treatment and heat with no extraction step, the
method will measure sulfate and glucuronide conjugates of
acetaminophen, thus rendering the assay nonspecific.
Longlands and Weiner [14], in 1982, reported similar
modifications (Table 1, Method 3b). The addition of
hydrochloric acid was omitted and trichloroacetic acid
substituted. A reduction in nitrite concentration was used,
with a nitration reaction time of 2 min, and the absorbance
read at 450 nm.
Spectrophotometric Method
Most spectrophotometric methods require an extraction
procedure followed by a chemical reaction to convert
acetaminophen into a light-absorbing compound.
Liu and Oka [15] utilized ferric reduction following
extraction of acetaminophen, (Table 1, Method 4), but
although such methods are reliable and low cost, there is
potential interference from phenolic hydroxyl compounds.
More complex processes such as differential extinction, as
described by Knepil [16] and modified by Routh [17], are
based on measuring the absorbance of an alkaline solution
read against the acidic solution of identical concentration
of the sample at 290 nm (Table 1, Method 5a). This
wavelength corresponds to the isosbestic point of a
salicylate and allows quantitation of acetaminophen
without interference from salicylates.
Derivative spectroscopy is a technique where the rate of
change of absorbance with wavelength is measured. First-
derivative spectroscopy denotes this measurement in units
of absorbance per nanometer of wavelength versus
wavelength. Second-derivative spectroscopy is the
derivative of the first-derivative spectrum in units of
absorbance per nanometer squared [18]. Dingeon [19]
described a second-derivative spectroscopy method for the
determination of acetaminophen in serum (Table 1,
Method 5b). Serum samples were diluted in phosphate
buffer and after 2 min, 5 mL ethyl acetate was added,
followed by mixing and centrifugation. The organic
phase was evaporated under nitrogen, reconstituted in
methanol, and the sample scanned immediately
between 300 nm and 216 nm.
High-Performance Liquid Chromatography
A variety of HPLC assays have been developed for the
analysis of acetaminophen, such as normal phase, ion-
exchange, and reversed-phase techniques.
Most HPLC methods assaying acetaminophen use
reversed-phase liquid chromatography on a C18
column with an extraction procedure prior to injection.
A direct-injection technique was described by Manno
[20] in 1981. Equal volumes of sample and internal
standard (beta-hydroxyethyltheophylline) were mixed
and injected directly onto a Bondapak C18 column.
The mobile phase of sodium acetate and acetonitrile
eluted acetaminophen at a flow rate of 2 mL/min with
detection at 254 nm (0.01 AUFS). Both sample and
internal standard eluted within 10 min. Carryover
using this injection technique was not reported.
In 1982, Kinberger [21] published a method
describing the simultaneous determination of serum
acetaminophen, theophylline, and salicylate. The
sample and internal standard (8-chlorotheophylline)
were deproteinated with trichloroacetic acid and
extracted with methylene chloride and isopropanol.
The aqueous phase was discarded, and the supernatant
was evaporated under air. The supernatant was
reconstituted with mobile phase (methanol/acetate
buffer), centrifuged, and an aliquot injected for
analysis. The samples were analyzed using a C18
reversed-phase column with a flow rate of 1.0 mL/min
monitored at 280 nm.
The methods developed by Starkey [22] and Sood [23]
used 10-octadecylsilane-coated silica columns,
protected with guard columns of the same material.
Both assays involved a direct-injection technique.
Samples were mixed with internal standard, vortexed,
and centrifuged. In Starkeys method, proteins are not
precipitated with ZnSO4 upon addition of the internal
standard (benzoic acid). The mobile phase used was
sodium acetate and methanol, with the column eluate
monitored at 249 nm. Soods method utilized 3-
acetaminophen as the internal standard, with a mobile
phase of potassium phosphate monobasic and
methanol at a flow rate of 2.0 mL/min and measured at
254 nm. In both assays, acetaminophen and internal
standard were eluted in less than 10 min.
An HPLC method suitable for routine use in clinical
laboratories was described by Kamali and Herd [24] in
1990. The assay is based on ion-pair extraction that
eliminates the acetaminophen conjugates, glucuronide
and sulfate, preventing interference with
acetaminophen. The isocratic mobile phase consisted
35
Acetaminophen
of methanol/1% acetic acid, containing
tetrabutylammonium dihydrogen phosphate (TBA) and
potassium sulfate at a flow rate of 1.2 mL/min. Internal
standard (-hydroxyethyl theophylline) was added to the
plasma or urine samples and treated with TBA and
ammonium sulfate. The mixture was vortexed and
acetaminophen extracted with equal volumes of
chloroform and isopropanol. Following centrifugation, the
organic layer was transferred and evaporated to dryness
under nitrogen. The sample was reconstituted with mobile
phase and injected for analysis at 254 nm.
El-Mouelhi and Buszewski [25], also in 1990, described
the use of solid-phase extraction in measuring urine
acetaminophen and its metabolites.
Immunoassay Methods
The enzymatic immunoassay (EMIT) procedure is a
homogenous enzyme immunoassay based on competitive
binding, in which drug in the sample competes with
enzyme-linked drug for antibody to acetaminophen. When
the drug-linked enzyme binds to the antibody, its activity is
greatly reduced. Drug concentration in the sample can
therefore be related to enzyme activity. The enzyme label
is G6PDH, the substrate for the enzyme and NAD are
included in the reaction mixture, and conversion of NAD
to NADH is measured at 340 nm (Table 1, Method 7).
Analysis using fluorescence polarization immunoassay
(FPIA) is similar to the enzyme-linked assays. Drug in the
sample competes with tracer (fluoroscein)-labeled drug for
binding to acetaminophen antibody. The change in
polarization of the fluorescent radiation emitted by the
tracer is measured and is inversely proportional to the
amount of acetaminophen in the original sample (Table 1,
Method 8).
Enzymatic Methods
Price [26] used a commercially available kit which is based
on the enzymatic degradation of acetaminophen by a
bacterial arylacylamide amidohydrolase and where the
product is measured colorimetrically (Table 1, Method 9).
The method is similar to the colorimetric methods where
acetaminophen is converted to p-aminophenol by acid and
heat. In this assay, paracetamol is converted enzymatically
to 4-aminophenol, then coupled with o-cresol in
ammoniacal cupric sulfate to produce an indophenol dye
which is measured at 615 nm.
In 1983, Hallworth [27] modified and adapted the manual
assay for a centrifugal analyzer procedure.
Gas Chromatography
With most published GC methods, the sample must first be
extracted and derivatized to be made volatile at
temperatures inside the GC column.
Prescott [28] published a GC method utilizing flame
ionization detection. The sample containing internal
standard (p-chloroacetanilide) was extracted into ethyl
acetate and the extract evaporated. After reconstitution in
pyridine and derivatization to the trimethylsilyl
derivative, the extract was injected onto an OV-17
column (80 to 100 mesh) at 200C.
Thomas and Coldwell [29] modified Prescotts
method by using a more powerful silylating reagent
(Regisil, bis[trimethylsilyl]trifluoroacetamide) and a
more selective extractant (diethyl ether). They also
substituted an OV-1 column for Prescotts OV-17
column.
A simple and reproducible method was reported by
Thoma [30] (Table 1, Method 10). Solid ammonium
sulfate was added to an aliquot of both serum sample
and internal standard (2-acetamidophenol) and
vortexed. Methylene chloride was added to each
sample, which was then mixed and centrifuged. The
organic layer was decanted and acetic anhydride and
pyridine added. The extracts were evaporated and
reconstituted in methylene chloride prior to injection.
Hugget [31] described a rapid GC method in which the
extraction and derivatization of acetaminophen is
carried out in one step. Sample, internal standard (N-
butyryl-p-aminophenol), phosphate buffer, and
acetylating reagent (acetic anhydride, N-
methylimidazole, and chloroform) were mixed and
centrifuged. An aliquot of the chloroform layer was
injected onto a 3% (w/w) C87 hydrocarbon (Apolane-
87) on Chromosorb W HP (100 to 120 mesh) at
235C. The specimen, together with a quality-control
sample, can be analyzed in duplicate within 20 min.
Acetaminophen is detected using a flame ionization
detector with the limit of measurement of the assay
being 10 mg/L. This method has few potential sources
of interference.
Reference and Preferred Methods
There is no reference method for acetaminophen
estimation.
The spot test is an inexpensive screen for
acetaminophen in urine. The spot test will become
positive in patients with greater than 2 mg/L in the
serum. However, these methods are inherently
imprecise. Ingestion of phenacetin and benorilate, as
well as acetaminophen, may result in a positive spot
test. With the automation of acetaminophen assays on
laboratory instruments and very acceptable turnaround
times, there is little place for these qualitative
methods.
Following an overdose, when it is likely that
potentially toxic acetaminophen metabolites will be
present, treatment with the antidote, N-acetylcysteine,
should be commenced as soon as possible. In this
setting, the assay method chosen for acetaminophen
must be rapid, accurate, and precise. Consideration
must also be given to the size and manpower of the
laboratory and the available instrumentation.
36
Acetaminophen
According to survey data from the 2007 College of
American Pathologists (CAP) Participant Summary
Report, only enzymatic and immunological methods are
currently used for measurement of acetaminophen. All of
these methods are acceptable, so acetaminophen assays are
determined by the choice of instrumentation in the
laboratory.
HPLC could be considered a candidate reference method
of analysis because of its excellent sensitivity, precision,
and freedom from interference, but it is not often used as a
routine method in clinical laboratories. Although most
HPLC methods use spectrophotometric determination,
some methods employ electrochemical detection to
improve the sensitivity of the assay but are subjected to
electrical interferences and may involve lengthy, complex,
sample extractions.
Gas chromatography techniques were widely used for
quantitation of acetaminophen because of their selectivity,
small sample volumes, and the ability to measure other
drugs simultaneously. Combined gas
chromatography/mass spectroscopy has also been utilized
in assaying for acetaminophen [31] and shows enhanced
sensitivity. Both methods are time consuming and labor
intensive and not recommended for routine use at this time.
Specimen
Serum is recommended as the sample of choice. Plasma
collected into EDTA, heparin, oxalate, or citrate can be
used with most enzymatic and immunological methods. No
special handling of specimen is required. Qualitative
screening for acetaminophen in urine is not recommended
(see above). Sample stability at standard therapeutic doses
of acetaminophen for analgesia and antipyresis of 10 to 20
g/mL (65 to 130 mol/L) is at least 4 days (Koerbin
unpublished).
Interferences
Some of the automated immunoassay procedures have
been reported to suffer negative bias from hemolysis and
icterus and positive interference from lipemia [33]. The
nitration method is susceptible to interferences from
salicylate [9] and phenolic acids if patients are uremic [10].
Bilirubin has been shown to interfere with a variety of
colorimetric assays [34,35]. In the GDS Diagnostics
enzymatic assay, based on enzymatic conversion of
acetaminophen to p-aminophenol, with chromogen
activator o-cresol forming a blue-colored indophenol, an
increase in background absorbance at 600 nm caused by
the presence of bilirubin appeared to cause a false increase
in the reported acetaminophen concentration. The
magnitude of the false increase was directly proportional to
the bilirubin concentration. Electronic correction for
absorbance at a second wavelength did not adequately
compensate for the interference, which also suggests a
nonspectral mechanism. Although the false detection of
acetaminophen in hyperbilirubinemic specimens was
consistent and reproducible, the accuracy of actual
acetaminophen measurements between 50 and 150 mg/L
did not seem to be affected [35].
In a recent study by Polson [34], the effect of
hyperbilirubinemia on six commonly performed
assays representing 70% of the methods chosen by
U.S. laboratories, according to the CAP, were studied.
Immunoassays that do not require a color reaction are
less prone to interference from endogenous
substances.
This study also suggested that products and
byproducts created in colorimetric assays may show
absorbance at wavelengths near to the primary or
secondary analytical wavelengths used, causing a
positive interference. However, not all samples
showing hyperbilirubinemia exhibited these
characteristics, suggesting interference from non-
bilirubin substances.
Acetaminophen Toxicity
The clinical value of measurement of acetaminophen
lies in the detection and diagnosis of overdose (see
Interpretation). As such, it is not appropriate to quote
an optimal therapeutic range. However, standard
therapeutic doses of acetaminophen for analgesia and
antipyresis give plasma concentrations of the order of
10 to 20 g/mL (65 to 130 mol/L).
Interpretation
The mechanism of action of acetaminophen is due to
its inhibition of one of the cyclooxygenase enzymes,
COX-3 [36,37]. By inhibiting COX-3 in the
hypothalamus, prostaglandin production is reduced,
and if the temperature is raised (i.e., there is fever), the
temperature center will reset, and the temperature will
fall to normal. Likewise, COX-3 inhibition decreases
transmission in pain fibers in the spinal cord, and the
sensation of pain is decreased. Paracetamol has no
significant action on COX-1 and COX-2. This
contrasts with inhibition of cyclooxygenases 1 and 2,
through which the nonsteroidal antiinflammatory
drugs (e.g., aspirin) operate, and which also have
analgesic and some antipyretic properties.
Acetaminophen therefore has neither the
antiinflammatory nor the antithrombotic properties of
aspirin but is without the same gastrointestinal side
effects or association with Reye Syndrome.
The primary clinical use of acetaminophen
measurement is in the assessment of possible
overdose. At usual analgesic doses in an adult (500 to
1000 mg), the elimination half-life is 2 to 3 hours. At
these doses, acetaminophen is cleared by the liver, the
majority being conjugated with sulfate and
glucuronide. In acetaminophen overdose, the
conjugation pathways are saturated, and the excess
drug is metabolized by cytochrome P450. The
resultant intermediate metabolites, such as the minor
alkylating metabolite N-acetyl-p-benzo-quinone imine
(NAPQI) are highly reactive, and although they are
reduced and detoxified by reacting with hepatic
glutathione, the capacity of this system is rapidly
exceeded, and tissue necrosis results. In addition, free-
radical production is thought to contribute to the
pathology. Patients with preexisting liver disease or
37
Acetaminophen

severe nutritional deficiencies may be at greater risk of
acetaminophen toxicity if their hepatic stores of
glutathione are depleted. Patients in whom cytochrome-
P450 activity has been induced (e.g., with the use of
phenobarbital) are also at a greater risk of toxicity.
The consequent hepatotoxicity may not become apparent
for 2 to 3 days, when nonspecific symptoms develop
(gastrointestinal), and liver function tests deteriorate.
Prothrombin time (PT) including INR, is the most sensitive
test and may be significantly abnormal 18 hours after
acetaminophen overdose.
The most practical indication of the likelihood of
hepatotoxicity arising following acetaminophen overdose
is the concentration of acetaminophen in plasma/serum at a
known time after its ingestion. Treatment of acute
overdose is guided by the use of nomograms. These
nomograms are based on the observations of Prescott
[38,39] and Rumack [40] and their successful treatment
within 12 hours of ingestion of acetaminophen using N-
acetylcysteine (NAC). Evidence now suggests that
although most effective in the first 8 hours post-
ingestion, treatment with NAC provides significant
benefit regardless of timing or degree of hepatic injury
[41,42].
It is not useful to measure the serum concentration
earlier than 4 hours after ingestion of the
acetaminophen overdose; absorption is likely to be
continuing, and the distribution phase will be
incomplete. The predictive nomograms are based on
the understanding that in the normal liver following
ingestion of a therapeutic dose of acetaminophen, its
half-life is less than 4 hours. In acetaminophen
hepatotoxicity, the half-life increases [38], and the
observed incidence of hepatotoxicity is related to
serum concentrations [39]. Furthermore, early
treatment with NAC (within 8 hours of ingestion) is
more effective than later treatment [43].

Nomogram Interpretation

A Data uninterpretable if sample taken within 4 hours of ingestion. Repeat collection is recommended. Note the
minimal single hepatotoxic dose of paracetamol is 7.5 g in an adult (150 mg/kg), and if there is suspicion of a large
overdose, then treatment with NAC is recommended immediately.
B Liver damage highly likely. Treatment with NAC is recommended.
C Liver damage possible, especially in high-risk patients. These patients should be considered for treatment with
NAC and should be reviewed by a senior clinician.
D Severe liver damage unlikely. If there is doubt about the timing of ingestion or the need for treatment, treat with
NAC.
E Severe liver damage is still possible if large doses of paracetamol have been ingested. These patients should be
considered for treatment with NAC and should be reviewed by a senior clinician.
(With permission of Dr. G. Jones, St. Vincents Hospital, Sydney, Australia.)
38
Acetaminophen
NAC may act as a donor of sulfhydryl groups (cf.
glutathione), as well as acting as a radical scavenger. The
critical factors in the efficient diagnosis and treatment of
acetaminophen poisoning are:
(1) time and frequency of ingestion
(2) time of blood sampling following ingestion
(3) concentration of acetaminophen
(4) time of administration of NAC
It should be noted that the advice and use of these
predictive nomograms is valid only for a single ingestion
(not serial episodes) and does not cover ingestion of slow-
release formulations of acetaminophen [44,45].
Acetaminophen Performance Goals
With the occurrence of repeated ingestion, the serum
acetaminophen concentration is of little use other than to
confirm ingestion. Clinical treatment decisions may then
be based on estimations of intake.
Survey data from the 2007 CAP Participant Summary
Report shows imprecision values (% coefficient of
variation [CV]) for measurement of acetaminophen range
from 1.9% to 7.6% at concentrations of approximately 150
g/mL and from 1.9% to 7.3% at about 90 g/mL for
assays with > 20 participants.
Acetaminophen is not regulated under the Clinical
Laboratory Improvement Amendments of 1988 (CLIA-88)
for proficiency testing, but acceptable performance criteria
for measurement of acetaminophen require that
laboratories be accurate to within 10% of the peer-group
mean. Analytical performance goals set by the Royal
College of Pathologists of Australasia (RCPA) are 3
g/mL up to 30 g/mL and 10% > 30 g/mL (20 15
mol/L) up to 200 mol/L and 10% > 200 mol/L.
The 2007 CAP data show that there is a lack of
harmonization between commonly used acetaminophen
assays, and there are differences in concentration between
these assays of greater than 25%. These differences will
affect distribution of patients between B and C categories
of potential treatment.
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40
Acetaminophen

Tables

Table 1: Acetaminophen Methods Summary

Method 1: Azo dye formation (Brodie and Axelrod)[1]; spectrophotometric
Principle of analysis: Sample extracted into diethyl ether and back-extracted into alkali. Heat hydrolysis to p-
aminophenol that is diazotized with ammonium sulfamate and coupled with -naphthol. This red-violet color is
measured at 510 nm.
Comments: Sulfate and glucuronide conjugates render assay nonspecific; Amax = 510nm
Method 2: Spot test; qualitative, colorimetric
Principle of analysis: o-Cresol + acetaminophen indole phenol blue
Comments: Screening test; highly sensitive and relatively specific
Method 3: Nitro dye formation
a. Spectrophotometric
Principle of analysis: Samples are deproteinized with TCA, the supernatant mixed with 6NHCl and nitric acid to
form a nitrous acid derivative that is then neutralized with ammonium sulfamate. NaOH is added and the yellow
color measured at 430nm.
Comments: Salicylate and phenolic acids interfere; Amax = 430 nm
b. Spectrophotometric
Principle of analysis: A modified procedure of Glynn and Kendal [7]; trichloroacetic acid was substituted for
hydrochloric acid; a reduced nitrite concentration and a nitration reaction time of 2 min
Comments: Linear in range of 0 to 500 mg/L; HPLC more sensitive and specific; Amax = 450 nm
Method 4: Ferric reduction; spectrophotometric (simple extinction measurement)
Principle of analysis: Measures acetaminophen based on the reduction of ferric 2,4,6-tris(2-pyridyl)-S-triazine
(TPTZ)
Comments: Phenolic hydroxyl groups interfere, and TPTZ is light sensitive; Amax = 593 nm
Method 5: Spectrophotometry
a. Differential extinction measurement
Principle of analysis: Measures acetaminophen at differential absorbance peak of 290 nm, avoiding interference
with salicylate
Comments: Reading at isosbestic point of salicylate removes interference from this compound; requires narrow
band-pass spectrophotometer
b. Second derivative
Principle of analysis: Rate of change of absorbance with wavelength is measured in units of absorbance per
nanometer2
Comments: Some drugs lead to spectral interferences; Amax = 300 nm; Amin = 216 nm
Method 6: High-performance liquid chromatography (HPLC), reversed-phase; chromatographic separation
Principle of analysis: Packing octadecylsilane-bonded silica; organic solvent extraction, protein precipitation or
direct injection; mobile phase is dilute acetic acid/methanol/ethyl acetate; detection at 254 nm
Comments: Rapid and sensitive (to 1 g/mL), or improved to 1 ng/mL with more lengthy and labor intensive
extractions; ability to quantitate other drugs simultaneously
Method 7: Enzyme multiplied immunoassay (EMIT); competitive binding
Principle of analysis: Drug in patient sample competes with drug-enzyme complex for limited amount of
antibody; enzyme activity related to drug level
Comments: Available on stat basis; linear in range 10 to 200 mg/L
Method 8: Fluorescence polarization (FPIA); competitive binding
Principle of analysis: Drug in patient sample competes with fluorescein tracer for limited amount of antibody;
polarization inversely related to drug level
Comments: Requires dedicated instrument, most commonly used; linear in range 10 to 200 mg/L
Method 9: Enzymatic techniques; spectrophotometric
Principle of analysis: Acetaminophen converted to aminophenol by bacterial arylacylamidase; indophenol dye
complex formed by the reaction of aminophenol with o-cresol and ammoniacal cupric sulfate
Comments: Adaptable to automation; available on stat basis; linear in range 0 to 377.5 mg/L; Amax = 615 nm
Method 10: Gas chromatography; chromatographic separation
Principle of analysis: Extracted into methylene chloride and ammonium sulfate; converted to trimethylsilyl
derivative; injected isothermically onto column and flame ionization detectors
Comments: Replaced by HPLC and immunoassay; time consuming and labor intensive
41
Acetaminophen

Figures
Figure 1: Acetaminophen HPLC Chromatogram

Chromatogram of HPLC analysis for acetaminophen:

A, Spiked plasma: (1) acetaminophen (100 mg/L); (2) theophylline (20 mg/L); (3) -8-hydroxyethyltheophylline (3 mg/L)
(internal standard).
B, Serum patient sample: (1) acetaminophen (30 mg/L); (3) -8-hydroxyethyltheophylline (3 mg/L) (internal standard).

Procedure: High-Performance Liquid Chromatography

Principle Waters Radial Compression Module, model RCM-100
Acetaminophen is separated by reversed-phase high- Waters Radial-PAK cartridge, 8 NV C 18 4 (Nova-Pak)
performance liquid chromatography and quantitated by part #86342
measurement of peak heights relative to the internal Waters Guard-Pak Cartridge Nova-Pak C 18 part #
standard. Serum is deproteinized and extracted with 15220
chloroform; detection is by ultraviolet spectroscopy at Waters Variable Wavelength UV Detector model 481
270 nm. Hewlett-Packard Integrator model 3394
Specimen Sample Preparation:
Serum 1. Pipet 100 L of sample, control, or standards.
Reagents and Materials 2. Add 50 L of 1 M hydrochloric acid to each
1. HPLC-grade isopropanol and acetonitrile, tube.
glacial acetic acid, and chloroform. 3. Add 1 mL of the internal standard solution.
2. Stock internal standard (300 mg/L). Place 4. Vortex mix and centrifuge to 1500 rpm.
150 mg of -8-hydroxyethyltheophylline in a 5. Decant the organic layer, and place tubes in a
500-mL volumetric flask, and dilute with heating block at 60C under a gentle stream of air.
chloroform. This standard is stable indefinitely 6. Add 200 L of the mobile phase to each tube
if stored away from light. and vortex.
3. Working internal standard (3 mg/L). Place 7. Inject 50 L of sample into the HPLC system,
5 mL of stock internal standard in a 500-mL using the following chromatographic parameters:
volumetric flask with 50 mL of isopropanol, a. Chart speed: 0.5 cm/min
and make to the mark with chloroform. This is b. Ultraviolet sensitivity: 0.1 AUFS
stable indefinitely if stored away from light. (absorbance units at full scale) at 270 nm
4. Standards. Acetaminophen c. Column temperature ambient
5. Mobile phase. Add 1.36 g of sodium acetate d. Flow rate: 2.0 mL/min
and 35 mL of 20% (v/v) acetic acid to 890 mL e. Run time: Approximately 7 min
deionized water in a 1-L volumetric flask, and Elution Order; Approximate Retention Time (min)
swirl to dissolve. Filter the aqueous buffer Theobromine 2.33
using a nitrocellulose filter (such as Millipore) Acetaminophen 3.01
of 0.5-mm pore size. Filter 20 mL of Paraxanthine 3.52
isopropanol and 55 mL acetonitrile to the Theophylline 3.74
reservoir. (pH 3.8) Internal standard 4.34
Assay The retention times of caffeine and salicylate depend on
Equipment: the pH of the mobile phase.
Waters pump, model 6000A The assay is linear to at least 500 mg/L, with a lower
Waters autosampler, WISP model 712
42
Acetaminophen

limit of detection of 1 mg/L. In each sample, the peak height of acetaminophen is

A typical chromatogram is shown in Acetaminophen: compared to the internal standard from which peak
Figure 1. height ratios are calculated. Each unknown sample is
then compared to a calibrator peak height ratio of a
known concentration. The calculation is as follows:
Calculations

[Acetaminophen] = [CAL] peak height unknown/peak height of internal standard

peak height calibrator/peak height of internal standard

where [Acetaminophen] = drug concentration (mg/L),

[CAL] = calibrator concentration (mg/L)

Notes
1. When a new column is installed, check that
adequate separation of theobromine and
acetaminophen is obtained. If the separation is
inadequate, decrease the percentage of
acetonitrile in the mobile phase.
2. Salicylate may interfere, depending on the pH
of the mobile phase. To increase the retention
time of salicylate, decrease the pH. To decrease
the retention time of salicylate, increase the pH.
43
Adrenocorticotropic Hormone (ACTH)
Adrenocorticotropic Hormone (ACTH)
Hassan M.E. Azzazy
Name: Adrenocorticotropic hormone (ACTH)
Clinical Significance: Refer to Chapter 51 Adrenal Hormones and Hypertension, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation
Molecular mass: ~4500 D
Chemical class: Polypeptide (39 amino
acids)
Principles of Analysis and Current Usage
ACTH is a 39-amino-acid peptide, produced in the
anterior pituitary as part of a large precursor protein
called pro-opiomelanocortin (POMC), which contains
the sequence for several other biologically active
peptides as well. After secretion, POMC is rapidly
cleaved to its component peptides. The major stimulus
for POMC (and hence ACTH) release is
hypocortisolemia.
ACTH concentration can be determined by bioassay,
receptor assays, or immunoassays. Bioassays are based
on the ability of ACTH to stimulate steroidogenesis or
depletion of ascorbic acid in isolated adrenocortical cells
or in adrenal glands of hypophysectomized animals [1].
Bioassays are labor intensive and expensive. Receptor
assays measure only the biologically active fraction of
ACTH [2]. These assays utilize solubilized binding
proteins obtained from normal or neoplastic
adrenocortical cells. Receptor assays suffer from several
drawbacks, including lability of receptor preparations
and technical complexity.
Several radioimmunoassays (RIA) were initially
developed for ACTH measurement. Many of the RIA
methods, however, required a pre-assay extraction step
and extended incubation time because they employed
antibodies with low avidity, and the circulating
concentration of ACTH in healthy subjects is low [3,4].
Sandwich immunoradiometric assays (IRMA) have been
shown to have several advantages over the competitive
immunoassays, including improved sensitivity,
precision, and shorter incubation time [5,6]. Rosano et
i
Adrenocorticotropic Hormone (ACTH)
Previous and current authors of this method:
First edition: Not done
Methods edition: Barbara M. Goldsmith
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Hassan M.E. Azzazy
al. [7] evaluated an IRMA developed for the
measurement of ACTH in human plasma. The assay
employed goat polyclonal antibodies specific for ACTH
26-39 as capture antibodies and 125I-labeled monoclonal
antibodies specific for ACTH 1-17. Solid-phase
separation of the immune complex was achieved using
mouse anti-goat antibody-coated polystyrene beads. The
assay had a detection limit of 1.7 ng/L and total
imprecision of < 10% at ACTH concentrations between
9 and 801 ng/L. A time-resolved immunofluorometric
assay for measurement of ACTH in unextracted plasma
has also been reported [8].
Automated non-isotopic ACTH immunoassays with
short incubation times have recently been developed.
The Nichols Advantage ACTH assay utilizes acridinium
esterlabeled mouse monoclonal antibody that binds to
the C-terminal region of ACTH and a biotinylated goat
polyclonal antibody that binds to the N-terminal region.
After incubation with the patients plasma, streptavidin-
coated magnetic beads are used to separate the immune
complex. Interassay imprecision at mean ACTH values
of 17.7 and 731 ng/L were 12% and 6.1%, respectively
[9]. A second assay, the DPC IMMULITE ACTH assay,
employs monoclonal murine antibodies coated on
polystyrene beads and alkaline phosphataselabeled
polyclonal rabbit anti-ACTH antibodies. This assay also
uses a chemiluminescent substrate. Interassay
imprecision of this assay at mean ACTH concentration
of 15.7 and 811 ng/L were 11% and 2.8%, respectively.
Additionally, a single-step electrochemiluminescence
immunoassay has been developed (Elecsys ACTH
assay). The assay utilizes two murine monoclonal
antibodies. One of the antibodies is labeled with
ruthenium (detector), and the other is a biotinylated
antibody (capture) that binds to microparticles coated
with streptavidin. The reported assay detection limit is
0.5 ng/L, and the within-run and between-run
imprecision (at ACTH concentration 12 to 971 ng/L)
were 1.9% and 5%, respectively [10].
Reference and Preferred Methods
There is no reference method for ACTH. Both isotopic
and non-isotopic ACTH immunoassays are reported to
have comparable performance. Comparing results from
different ACTH immunoassays is possible only if
calibrators are clearly defined. Two standards are
44

Adrenocorticotropic Hormone (ACTH)

currently available from the National Institute of Reference Intervals

Biological Standards and Control (UK, purified human
ACTH 1-39; MRC 74/555; 6.2 IU/25 g) and the Method Specimen Reference Range
National Pituitary Agency (Baltimore, MD; synthetic (pg/mL)
a
ACTH 1-39; 4.71 IU/50 g). IRMA ACTH assays are RIA EDTA plasma
b
Newborn (1 day) 10-185
Adult (8:00 am) <120
generally preferred over RIA for investigating b
Adult (4:00-8:00 pm) <85
hypothalamic-pituitary-adrenal disorders. Because RIA a
[Endocrine Sciences. Pediatric
assays utilize polyclonal antibodies and will be able to Laboratory Services.
recognize both fragments and whole-molecule ACTH, Tarzana, CA: Endocrine
Sciences; 1988.]
they are recommended for investigating ectopic ACTH b
[Vanderbilt Pathology
production by tumors, because tumors may secrete Laboratory Services. Test
fragments as well as intact ACTH. Catalog. Nashville, TN:
Vanderbilt Pathology
Laboratory Services; 1989.]
IRMA EDTA plasma Adult (7:00-10:00 am) 9-52
Specimen [Nichols Institute Reference
ACTH is unstable in blood, easily oxidized, strongly Laboratories. Test Catalog.
adsorbs to glass surfaces, and can be rapidly degraded by San Juan Capistrano, CA:
Nichols Institute Research
plasma proteases into immunoreactive fragments. Laboratories; 1993.]
Therefore, proper care must be taken in collection, RIA Amniotic fluid 10-18 weeks: 209
handling, transportation, and storage of specimens. 26-30 weeks: 430
Preanalytical variables such as time of day at which 35-36 weeks: 162
sample is collected, stress from a poorly performed
[Tulchinsky D, Ryan KJ, eds.
venipuncture, and/or prior administration of cortisol Maternal-Fetal
must be taken into consideration. Samples for ACTH Endocrinology. Philadelphia:
tests must be drawn prior to glucocorticoid Saunders; 1980.]
administration. Morning (6:00 to 10:00 am) specimens Automated EDTA plasma Adult (AM draws) 10-60
chemiluminesc [Mayo Clinic. Test Catalog.
are preferred. Blood is usually drawn in pre-chilled ent immuno- Rochester, MN: Mayo
polystyrene EDTA tubes. Plasma is separated metric assay Clinic; 2005.]
immediately from cells in a refrigerated centrifuge and
frozen within 15 minutes. Plasma is stored frozen at
20C or preferably at 70C in new plastic vials. For Interpretation
long-term storage, aprotonin (500 kU/mL) should be Determination of ACTH in plasma is the best test to
added. Mercaptoethanol may be added to specimens to distinguish between primary and secondary adrenal
protect ACTH against oxidation. Prior to assay, thawed insufficiency. Measurement of plasma ACTH
plasma must be centrifuged to remove any fibrin clots concentration is used to assess Cushings disease,
that may interfere with the assay. adrenal tumors, ectopic ACTH-producing tumors,
Addisons disease, Nelsons syndrome, adrenal tumors,
Interferences and hypopituitarism. ACTH secretion is pulsatile and
ACTH concentration follows a nycthemeral rhythm, increases in cases of hypocortisolemia when the pituitary
with highest levels observed between 6:00 and 8:00 a.m. and hypothalamus are intact.
and lowest levels between 9:00 and 10:00 p.m. For
proper monitoring, samples should be collected at the In primary adrenal insufficiency, ACTH levels usually
same time each day (early morning preferred). exceed 100 pg/mL by RIA. It should be noted, however,
Pregnancy, menstruation, and stress may increase ACTH that ACTH level alone cannot be used to differentiate
secretion. Aminoglutethimide, amphetamine, levodopa, between control subjects and patients with central
metoclopramide, metyrapone, pyrogens, RU-486, hypoadrenalism. One study reported identical ACTH
vasopressin, hypoglycemia, and insulin have been levels in control subjects (4 to 81 pg/mL) and patients
reported to increase plasma ACTH levels. Depressed with confirmed pituitary disease (8 to 75 pg/mL) [12].
ACTH levels may be observed following administration
of dexamethasone or other corticosteroids or collection In Addisons disease (primary adrenal insufficiency),
of specimens in tubes containing heparin. elevated ACTH levels (>1000 pg/mL) are typical.
ACTH levels are also elevated in congenital adrenal
Heterophilic antibodies in serum of humans routinely hyperplasia, pituitary-dependent Cushings disease,
exposed to animals or animal serum products can react ectopic ACTH-producing tumors, and Nelsons
with the immunoglobulins included in ACTH syndrome. ACTH determinations can also help to
immunoassays, causing interference [11]. identify the cause of cortisol hypersecretion in Cushings
syndrome. ACTH levels are typically low when this is
due to lesions or hyperplasia of the adrenal cortex, and
high in cases of ectopic ACTH production or
45

Adrenocorticotropic Hormone (ACTH)

hypersecretion of ACTH by the pituitary. Low levels are 6 Zahradnik R, Brennan G, Hutchison JS, Odell
observed when adrenal insufficiency is secondary to WD. Immunoradiometric assay of corticotropin
pituitary dysfunction, adrenal carcinoma, adenoma, and with use of avidin-biotin separation. Clin Chem
hypopituitarism. 1989;35:804-7.
7 Rosano TG, Demers LM, Hillam R, Dybas MT,
Because the major stimulus to ACTH production is Leinung M. Clinical and analytical evaluation
hypocortisolemia, interpretation of ACTH concentration of an immunoradiometric assay for
requires concurrent measurement of cortisol to be made. corticotropin. Clin Chem 1995;41:1022-7.
High levels of ACTH and cortisol are observed in 8 Dobson S, White A, Hoadley M, Lovgren T,
Cushings disease and ectopic ACTH secretion. Low Ratcliffe J. Measurement of corticotropin in
levels of both hormones are observed in hypopituitarism. unextracted plasma: comparison of a time-
Elevated levels of cortisol with low ACTH may indicate resolved immunofluorometric assay and an
adrenal tumor. Elevated ACTH and low cortisol levels immunoradiometric assay, with use of the same
are observed in Addisons disease. monoclonal antibodies. Clin Chem
1987;33:1747-51.
ACTH in amniotic fluid may be of fetal origin, and low 9 Vogeser M, Engelhardt D, Jacob K.
levels may be observed in anencephalic fetuses; high Comparison of two automated
levels may indicate immaturity of hypothalamic adrenocorticotropic hormone assays. Clin Chem
regulation. Dexamethasone suppression testing may be 2000;46:1998-2000.
useful for distinguishing causes of increased ACTH 10 Verschraegen I, Anckaert E, Schiettecatte J,
levels. High-dose dexamethasone suppresses ACTH and Mees M, Garrido A, Hermsen D et al.
cortisol secretion in Cushings disease but has no effect Multicenter evaluation of a rapid
in cases of adrenal adenomas, adrenal carcinomas, and electrochemiluminescent adrenocortico-tropic
ectopic ACTH producing tumors. hormone (ACTH) immunoassay. Clin Chim
Acta 2007;380:75-80.
Performance Goals 11 Boscato LM, Stuart MC. Heterophilic
The current Clinical Laboratory Improvement antibodies: a problem for all immuno-assays.
Amendments (CLIA) performance goal for measurement Clin Chem 1988:34:27- 33.
of ACTH is for laboratories to be within 3 standard 12 Oelkers W, Diederich S, Bahr V. Diagnosis and
deviations of the peer-group mean. According to the therapy surveillance in Addisons disease: rapid
2007 College of American Pathologists (CAP) Survey, adrenocortico-tropin (ACTH) test and
coefficients of variation (CV) for all ACTH methods at measurement of plasma ACTH, renin activity,
mean values of 168.6 pg/mL (SD 20.6) and 311.3 pg/mL and aldosterone. J Clin Endocrinol Metab
(SD 45.8) were 12.2% and 14.7%, respectively. 1992;75:259-64.

References

1 Sayers G. Bioassay of ACTH using isolated

cortex cells. Applications: structure activity
relationship for ACTH and analogues, assay of
corticotrophin-releasing factor, and assay of
plasma ACTH. Ann NY Acad. Sci
1977;297:220-41.
2 Lefkowitz RJ, Roth J, Pastan I. Radio-receptor
assay of adrenocorticotropic hormone: new
approach to assay of polypeptide hormones in
plasma. Science 1970;170:633-5.
3 Gutkowska J, Julesz J, St-Louis J, Genest J.
Radioimmunoassay of corticotropin from
plasma. Clin Chem 1982;28:2229-34.
4 Arts CJ, Koppeschaar HP, Veeman W, Thijssen
JH. A direct radioimmunoassay for the
determination of adrenocortico-tropic hormone
(ACTH) and a clinical evaluation. Ann Clin
Biochem 1985; 22:247-56.
5 Dobson SH, Gray C, Smith H, Baker T,
Ratcliffe JG, White A. Selection and
optimization of monoclonal antibodies for a
two-site immunoradiometric assay for ACTH. J
Immunol Methods 1986;88:83-90.
 46
Alanine Aminotransferase

Alanine Aminotransferase
Robert L. Murray

Name: Alanine aminotransferase, ALT, L-alanine:2-oxoglutarate

aminotransferase, serum glutamate pyruvate transaminase, SGPT
Clinical significance: Refer to Chapter 27, Liver Function, in the 4th Edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Enzyme number: EC 2.6.1.2
Molecular mass: approximately 101,000 daltons
Chemical class: enzyme, protein
NRSCL reference
Method: NCCLS RS4-A

Principles of Analysis and Current Usage
Alanine aminotransferase (ALT) catalyzes the transfer of
an amino group between L-alanine and L-glutamate; the
corresponding ketoacids in this process are -
ketoglutarate and pyruvate (Figure 1). In vivo, this
reaction proceeds to the right to provide a source of
nitrogen for the urea cycle. The pyruvate thus generated is
available for entry into the citric acid cycle, whereas the
glutamate is deaminated (catalyzed by glutamate
dehydrogenase), yielding ammonia and -ketoglutarate.
The reaction is reversible, with the chemical equilibrium
favoring the formation of alanine and -ketoglutarate.
Because these products are relatively difficult to assay,
however, analytical techniques typically force the reverse
reaction, allowing quantitation of pyruvate. Two methods
of ALT analysis have enjoyed wide popularity for routine
clinical use: the Reitman-Frankel method [1], which
involves the measurement of ALT activity by conversion
of the reaction product, pyruvate, to its hydrazone (Table
1: Method 1); and the Wroblewski method [2] (Table 1:
Method 2), in which the ALT reaction is coupled to a
lactate dehydrogenase (LD) reaction. This is the most
common method in use today, and the former method is
of historical interest only.
In the LD coupled method, the pyruvate product of the
ALT reaction is reduced to lactate by nicotinamide
iALT
Previous and current authors of this method:
First edition: Robert L. Murray
Methods edition: Robert L. Murray
Second edition: Robert L. Murray
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: James J. Miller
adenine dinucleotide (NADH). The disappearance of NADH
is monitored spectrophotometrically (at 340 nm). Preferably,
the absorbance change should be monitored continuously
rather than by readings at several time points or only the end-
point.
Reference and Preferred Methods
There have been many minor modifications in the enzymatic
technique since its introduction by Wrblewski and LaDue [3-
7]. In addition, the specifications listed in the reference
method published by the International Federation of Clinical
Chemistry (IFCC) have been modified over the years, the
most recent modification in 2002 being to optimize conditions
for 37C [7,8]. The main use of the IFCC reference method is
to assay calibrators for use in routine methods. This allows
routine methods to be traceable to the IFCC reference method,
with the goal of reducing the biases between methods.
In the most recent IFCC reference method (2002) [7], 0.20 mL
of serum is preincubated for 5 minutes in 2.00 mL of a
mixture that contains all reactants except -ketoglutarate.
During this preincubation period, the added lactate
dehydrogenase (LD) rapidly converts the endogenous
pyruvate in the serum to lactate, and the pyridoxal phosphate
cofactor joins with any inactive apoenzyme to form an
increased amount of active ALT. With the addition of 0.20 mL
of -ketoglutarate, the primary reaction is initiated, and the
concentrations shown in Table 2 are reached, exclusive of the
small increases caused by the presence of endogenous material
in the serum. After steady state is reached, the rate of NADH
oxidation is monitored repeatedly at 339 nm. The rate of
change in absorbance is corrected for a reagent blank.
Most reference methods, including the current IFCC
procedure [7], have included pyridoxal phosphate. The need
for addition of this cofactor has been widely debated. The
essential nature of the cofactor has long been recognized, but
because it is usually present in human serum in adequate
amounts, many investigators do not add this component to the
reaction mixture. In the occasional patient with severe vitamin
 47
Alanine Aminotransferase
B deficiency, this could lead to a serious underestimation
of AST activity.
The presence of aminotransferases in the reagents is
possible if the LD is not carefully prepared. Good-quality
enzymes will not pose a problem; in any event, a blank
determination will identify the problem. The presence of
pyruvate is a potential source of error, since in the
presence of endogenous LD, pyruvate will be converted
to lactate, with simultaneous consumption of NADH. This
problem is circumvented by the addition of a large excess
of LD, so that endogenous pyruvate is converted during
the preincubation period, eliminating interference during
the measurement period.
Some older reference methods based on the Wrblewski
coupled enzymatic method used phosphate buffer, which
retards the recombination of added pyridoxal phosphate
with the apoenzyme. If a large amount of the inactive
enzyme is present, a falsely low activity will be observed.
Activation of apoenzyme is more efficient in Tris buffer.
However, NADH is somewhat less stable in Tris buffer
than it is in phosphate buffer. For this reason, the Tris
concentration is kept relatively low at 100 mmol/L.
Most routine methods today use the Wrblewski coupled
enzymatic method and may be traceable to the current
IFCC reference method. The American Association for
Clinical Chemistry proposed a method [3] for the small
clinical chemistry laboratory that differs from the IFCC in
that (1) a single reagent is used to avoid a two-step
addition procedure, (2) the reaction is read after 150
seconds for the following 180 seconds, and (3) pyridoxal
phosphate is not added.
The drawbacks of the dinitrophenylhydrazine method are
that (1) the pyruvate produced by the reaction results in
feedback inhibition of ALT, and thus specimens
exhibiting high activity are spuriously lowered; and (2)
any ketone in serum can react, though most do not result
in an absorbance change in the region measured.
However, acetoacetic acid and hydroxybutyric acid, both
components of ketosis, do cause false elevations.
The U.S. National Institute of Standards and Technology
(NIST) Standard Reference Material No. 909b is a
lyophilized human serum preparation intended for use in
evaluating the accuracy of routine methods. It is available
to manufacturers and laboratories for the validation of
ALT methods.
Specimen
Serum is the preferred specimen. Oxalate, heparin, and
citrate do not inhibit the enzymatic activity but may
introduce slight turbidity. Hemolyzed specimens should
be avoided, since erythrocytes contain three to five times
more ALT activity than is found in serum. ALT is stable
in serum for 3 days at room temperature or 1 week at 4C
[9]. A marked decrease in ALT activity is seen following
freeze/thaw cycles 10]. ALT has been found to be stable in
whole blood for up to 24 hours [11]. Urine has little or no
activity and is not recommended for analysis.
Interferences
There is significant ALT activity in erythrocytes and
significant hemolysis (>300 mg/dL hemoglobin) may
artifactually increase apparent ALT activity. Icteric (bilirubin
<40 mg/dL) and lipemic (triglycerides <3000 mg/dL)
specimens generally do not interfere with measurement of
ALT [12]. Metronidazole (Flagyl) may interfere with ALT
methods because of its relatively high concentration and
absorbance near 340 nm [13].
Alanine Aminotransferase Reference Interval
When analyzed at 37C by methods employing activation with
PP, the normal adult reference interval is approximately 8 to
47 U/L [14]. However, it must be noted that ALT activities are
age dependent. Healthy newborns have been reported to show
an upper reference interval of up to double the adult level.
These values decline to adult levels by approximately 3
months of age. This increased activity has been attributed to
seepage from the neonates immature hepatocytes, which have
more permeable membranes. Men have been reported to show
higher ALT values than women. Upper reference limits in
individuals 10 years of age are approximately half of those
seen in individuals at 40 years of age [15]. ALT activity peaks
at approximately the fourth to fifth decade of life and then
gradually declines.
Diurnal variations in ALT have been observed in both healthy
individuals and those with cirrhosis. Up to 45% variation may
be seen, with higher values being observed in the afternoon
[16]. Other factors that have been reported to affect ALT
include African-American race (15% higher than Caucasians),
body mass index (40 to 50% higher with high body mass
index), and exercise (20% lower in those who exercise) [17].
Ingestion of food causes no changes in measured ALT
activity.
Interpretation
In contrast to aspartate aminotransferase (AST), which is
found in both the cytoplasm and mitochondria, ALT is found
exclusively in the cytoplasm. The tissue distribution of ALT
and the ratio of ALT tissue activity to ALT plasma activity are
presented in Table 3. Based on activity per gram of wet tissue,
liver has the greatest amount of enzyme activity, with kidney
being the next most active tissue. Liver disease, in particular
hepatocyte necrosis, is the most important cause of increased
ALT activity. Because serum activities of ALT are unusually
sensitive to liver damage, increases in ALT readily occur
following moderate to excessive use of alcohol or following
exposure to a variety of hepatotoxic agents. ALT is often used
as part of a battery of enzymes to establish the presence and
extent of liver damage. The half-life of ALT is approximately
47 10 hours [18].
 48
Alanine Aminotransferase
ALT is usually higher than AST in most types of liver
disease in which the activity of both enzymes is
predominantly from the hepatocyte cytosol. When liver
necrosis is substantial, as in individuals with alcoholic
and viral hepatitis, mitochondrial AST is also released
into the blood, and AST activity is usually higher than
ALT. The ratio of AST to alanine aminotransferase
(ALT), sometimes called the De Ritis Ratio, is often used
to evaluate alcoholic liver disease [19] and severity of
liver disease in viral hepatitis [20,21]. This ratio is only
pertinent in isolated liver disease when comorbidities that
increase AST are not present.
Alanine Aminotransferase Performance Goals
The current target for total error in ALT measurements is
< 20% (CLIA). Biological variation data suggest that in
patients with stable ALT activities, total error of < 30% is
required for optimum use [22]. The interlaboratory
coefficients of variation listed for various ALT methods
in the 2007 College of American Pathologists Participant
Summary Report (C-B) are all < 5%, indicating that this
target is easily met using current ALT methods.
References
1 Reitman S, Frankel S. A colorimetric method for
the determination of serum glutamic oxalacetic
and glutamic pyruvic transaminases. Am J Clin
Pathol 1957; 28: 56-63.
2 Wrblewski F, LaDue JS. Serum glutamic-
pyruvic transaminase in cardiac and hepatic
disease. Proc Soc Exp Biol Med 1956; 91: 569-
571.
3 Butler TJ, Klotzsch SG, Osberg IM. Alanine am-
inotransferase, ALT provisional. In Faulkner
WR, Meites, S, editors: Selected methods of
clinical chemistry. Washington D.C.: American
Association for Clinical Chemistry; 1982. p. 69-
73.
4 Wilkinson JH, Baron DN, Moss DW, Walker
PG. Standardization of clinical enzyme assays: a
reference method for aspartate and alanine
transaminases. J Clin Pathol 1972; 25: 940-944.
5 Committee on Enzymes of the Scandinavian
Society for Clinical Chemistry and Clinical
Pathology. Recommended methods for the
determination of four enzymes in blood. Scand J
Clin Lab Invest 1974; 33: 291-305.
6 Enzyme Commission of the German Society for
Clinical Chemistry. Recommendations of the
German Society for Clinical Chemistry. Z Klin
Chem Klin Biochem 1972; 10: 281-91.
7 Schumann G, Bonora R, Ceriotti F, Ferard G,
Ferrero CA, Franck PFH, et al. IFCC primary
reference procedures for measurement of
catalytic activity concentrations of enzymes at
37C. International Federation of Clinical
Chemistry and Laboratory Medicine. Part 4.
Reference procedure for the measurement of
catalytic concentrations of alanine aminotransferase.
Clin Chem Lab Med 2002; 40: 718-24.
8 Bergmeyer HU, Horder M, Rej R. International
Federation of Clinical Chemistry (IFCC). Approved
recommendation on IFCC methods for the
measurement of catalytic concentrations of enzymes.
Part 3. IFCC method for alanine aminotransferase. J
Clin Chem Clin Biochem 1986; 24: 481-95.
9 Heins M, Heil W, Withold W. Storage of serum or
whole blood samples? Effects of time and
temperature on 22 serum analytes. Eur J Clin Chem
Clin Biochem 1995; 33: 231-238.
10 DiMagno EP, Corle D, O'Brien JF, Masnyk IJ, Go
VL, Aamodt R. Effect of long-term freezer storage,
thawing, and refreezing on selected constituents of
serum. Mayo Clin Proc 1989; 64: 1226-1234.
11 Ono T, Kitaguchi K, Takehara M, Shiiba M, Hayami
K. Serum-constituents analyses: effect of duration
and temperature of storage of clotted blood. Clin
Chem 1981; 27: 35-38.
12 McEnroe RJ, Burritt MF, Powers DM, Rheinheimer
DW, Wallace BH. Eds. CLSI: Interference Testing in
Clinical Chemistry, Approved Guielines Second
Edition CLSI document EP7-A2, 2005 Accessed 19
Jan 2009
13 Karlsen RL, Kristiansen G, Solberg JH. Effects of
metronidazole (Flagyl) on the determination of serum
ASAT on the SMA 12/60 Auto Analyser. Scand J
Clin Lab Invest 1983; 43: 175-177.
14 Stromme JH, Rustad P, Steensland H, Theodorsen L,
Urdal P. Reference intervals for eight enzymes in
blood of adult females and males measured in
accordance with the International Federation of
Clinical Chemistry reference system at 37oC: part of
the Nordic Reference Interval Project. Scand J Clin
Lab Invest 2004; 64: 371-384.
15 Soldin SJ, Hicks JM, editors. Pediatric reference
ranges. Washington, D.C.: AACC Press: 1995.
16 Cordoba J, O'Riordan K, Dupuis J, Borensztajin J,
Blei AT. Diurnal variation of serum alanine
transaminase activity in chronic liver disease.
Hepatol 1998; 28: 1724-1725.
17 Dufour DR. Laboratory guidelines for screening,
diagnosis and monitoring of hepatic injury.
Washington, D.C.: National Academy of Clinical
Biochemistry; 2000.
18 Price CP, Alberti KGMM. Biochemical Assessment
of Liver Function. In: Wright R, Alberti KGMM,
Karran S, Millward-Sadler GH, editors. Liver and
biliary diseasepathophysiology, diagnosis, man-
agement. London: W.B. Saunders; 1979. p. 381-416.
19 Majhi S, Baral N, Lamsal M, Mehta KD. De Ritis
ratio as diagnostic marker of alcoholic liver disease.
Nepal Med Coll J 2006; 8: 40-42.
20 Giannini E, Risso D, Botta F, Chiarbonello B, Fasoli
A, Malfatti F, et al. Validity and clinical utility of the
aspartate aminotransferase-alanine aminotransferase
ratio in assessing disease severity and prognosis in
 49
Alanine Aminotransferase

patients with hepatitis C virus-related chronic
liver disease. Arch Intern Med 2003; 163: 218-
224.
21 Giannini EG, Zaman A, Ceppa P, Mastracci L,
Risso D, Testa R. A simple approach to
noninvasively identifying significant fibrosis in
chronic hepatitis C patients in clinical practice. J Clin
Gastroenterol 2006; 40: 521-527.
22 Fraser CG. Biological variation: from principles to
practice. Washington, DC: AACC Press; 2001, p.
140.

Tables
Table 1: Methods of Alanine Aminotransferase (ALT) Analysis
Method 1: Dinitrophenylhydrazine coupling (colorimetric) (Reitman and Frankel [1]); quantitative
Principle of analysis: click here
Comments: Serum
Method 2: Enzymatic (ultraviolet monitoring) (Wrblewski and LaDue [2]); quantitative
Principle of analysis*: UV monitoring of NADH disappearance at 340 nm:

Usage: Serum; most frequently employed procedure

*Ala, Alanine; -KG, -ketoglutarate; Gl, glutamate; Lac, lactate; NAD , nicotinamide adenine dinucleotide;
+

NADH, reduced nicotinamide adenine dinucleotide; Pyr, pyruvate.

Table 2: Conditions of the 2002 IFCC ALT Reference Method

Component/Condition Concentration/Value
L-Alanine (mmol/L) 500
2-Oxoglutarate (mmol/L) 15
Buffer & concentration (mmol/L) Tris 100
pH 7.15
Pyridoxal phosphate (mmol/L) 0.1
NADH (mmol/L) 0.18
LDH (U/L) 1700
Volume fraction (v/v) 0.0833
Temperature (C) 37.0
Wave length (nm) 339
Band width (nm) 2
Light path (mm) 10
Incubation time (s) 300
Delay time (s) 90
Measurement interval (s) 180
Readings (measurement points) 6

Table 3: Tissue Distribution of ALT in Normal Human Adult Tissues

Tissue U 10-3 g of Wet Ratio of Activity in
Tissue Homogenate Tissue to That in Serum
Heart 7.1 444
Liver 44 2750
Skeletal muscle 4.8 300
Kidney 19 1188
Pancreas 2 125
Spleen 1.2 75
Lung 0.7 44
Serum 0.016 1
From Wrblewski F: Adv Clin Chem 1:313-351, 1958.
50
Alanine Aminotransferase

Figures
Figure 1: Amino transfer catalyzed by ALT.

Figure 2: Reaction of pyruvate with dinitrophenylhydrazine, as in Method 1.

Figure 3: Conversion of pyruvate to lactate with lactate dehydrogenase, as in Method 2.

51
Alanine Aminotransferase
Procedure: Kinetic Analysis of ALT [3]
Principle
The rate of the reaction in which an amino
group is transferred from L-alanine to 2-oxoglutarate
to form pyruvate and L-glutamate catalyzed by the
action of ALT is monitored by use of an indicator
reaction. The pyruvate formed is converted to lactate,
and monitoring the absorbance change at 339 nm of
the consumed NADH follows the reaction.
Reagents
1. Tris, L-alanine buffer (121.1 mmol/L Tris,
630 mmol/L L-alanine, pH 7.15). Dissolve 1.47 g of
tris(hydroxymethyl)aminomethane and 5.61 g of L-
alanine (free acid) in 80 mL of distilled water. Adjust
to pH 7.15 at 37C with 1 mol/L HCl (approximately
8.0 mL). Allow the solution to cool to the calibration
temperature, and bring to a volume of 100 mL in a
volumetric flask. This is Solution 1, stable for 3
months at 2C to 8C. Check for bacterial growth.
2. Tris/hydrochloric acid buffer (121.2
mmol/L Tris, pH 7.15). Dissolve 1.47 g of
tris(hydroxymethyl)aminomethane in 80 mL of
distilled water. Adjust to pH 7.15 at 37C with 1
mol/L HCl. Allow the solution to cool to the
calibration temperature, and bring to volume of 100
mL in a volumetric flask. This is Solution 2, stable
for 3 months at 2C to 8C. Check weekly for
bacterial growth.
3. Pyridoxal phosphate solution (6.3 mmol/L
pyridoxal phosphate). Dissolve 16.7 mg of
pyridoxal phosphate in Solution 2, and bring to 10
mL volume. This is Solution 3. Stable for 1 week at
2C to 8C when stored in a dark bottle.
4. Reduced nicotinamide adenine
dinucleotide (11.34 mmol/L NADH). Dissolve 16.1
mg of the disodium salt of NADH (or an equivalent
amount correcting for water of hydration) in 2.0 mL
of Solution 2. This is Solution 4, stable for 1 week at
2C to 8C when stored in a dark bottle.
Diluent for reagent enzymes. Dissolve 1.2 g bovine
serum albumin and 0.9 g NaCl in 100 mL of water.
Stable at least 1 month at 2C to 8 C.
5. Lactate dehydrogenase (3.57 mkat/L or
214,000 U/L). Dilute the enzyme in Diluent for
Reagent Enzymes. This is Solution 5, stable for at
least 2 days at 4C.
6. Reaction solution. Mix 10 mL of Tris-
aspartate (Solution 1) with 0.2 mL of pyridoxal
phosphate (Solution 3), 0.2 mL of NADH (solution
4), and 0.1 mL of the enzymes (Solution 5). Mix
thoroughly, and store in a dark bottle. Stable for 1
day at 2C to 8C.
7. Start reagent solution. Dissolve 407 mg of
2-oxoglutaric acid, disodium salt in 10.0 mL of
distilled water. Stable for 1 week at 2 to 8 C.
Assay
Equipment: Spectrophotometer with 2nm
band pass at 339 nm with a constant temperature
cuvette capable of maintaining a constant
temperature with less than 0.1C fluctuation. A
recording spectrophotometer is preferable.
1. Add 2 mL of reaction solution and 0.2 mL
of serum to the cuvette.
2. Mix and allow to stand for 5 minutes.
3. Add 0.2 mL of start reagent solution.
5. Mix, wait 90 s, and record the change in
absorbance for an additional 180 s. If no
recorder is available, record at least every 30
s.
6. A reagent blank is run by replacing the
sample with 9 g/L sodium chloride in step 1.
7. A sample blank should be checked by
replacing the start reagent in step 3 with 9
g/L sodium chloride. The sample blank is
not included in the calculation of AST
activity, but a rate > 1% of the ALT activity
indicates that the material is not suitable as a
calibrator. For the reagent blank of the
sample blank, replace both the start reagent
and the sample with 9 g/L sodium chloride.
Calculations
If the change in absorbance per second
(A/t) is greater than 0.0025 per second, dilute the
sample 5- to 10-fold with 9 g/L NaCl, and repeat the
measurement.
Correct for the blank reaction using the
following schema:
(A/t)A = Measured reaction rate with serum in
step 1
(A/t)B = Measured reaction rate with NaCl in
place of serum in step 1
A/tALT = (A/t)A (A/t)B and
ALT activity = 1905 x A/tALT kat/L
ALT activity in kat/L can be converted to U/L by
multiplying by 60.
52
Albumin
Albumin
Kee Cheung
Name: Albumin
Clinical significance: Refer to Chapter 31, Liver Function, in the 5th Edition of Clinical Chemistry:
Theory, Analysis, Correlation
Molecular weight: 66,248 D
Merck Index: 203
Half-life 15-19 days
Chemical class: Protein
Principles of Analysis and Current Usage
Albumin is the most abundant circulating plasma protein
i and specificity of the dye-binding methods for
(40 to 60 % of the total) . It has diverse functions, playing
important roles in the maintenance of colloid osmotic
pressure of blood, in transport of various ions, acids, and
hormones, and in nutrition. It is a globular protein with a
molecular weight of approximately 66,000 D and is unique
among major plasma proteins in containing no
carbohydrate in its single polypeptide chain of 580 amino
acids. It has a relatively low content of tryptophan and is
an anion at pH 7.4 [1]. These properties have been
exploited in the estimation of albumin in body fluids such
as plasma, CSF, interstitial fluid, urine, and amniotic fluid.
The earliest techniques for albumin analysis were based on
acid and salt precipitation of the protein (Table 1, Method
1) [2]. After precipitation with ammonium sulfate, the
albumin in the supernatant was measured by total nitrogen
analysis or by biuret reaction. Modern salt fractionation
techniques are cumbersome because of the many steps
required in the procedure and are not very specific because
of protein interactions [3]. Since they are not easily
automated, the precipitation methods are of historical
interest only.
The measurement of albumin can also be performed by a
direct determination of globulin based on tryptophan
content and calculation of the albumin content by
subtraction of globulin from total protein (Table 1, Method
2). The tryptophan content of human serum albumin is
0.2%, compared with 2% to 3% for the various globulins
[4]. Such a large difference has been used as a means for
albumin determination. In a one-reagent system, glyoxylic
acid in the presence of Ca2+ in an acid medium condenses
with the tryptophan residues in globulins to produce a
purple color that is measured at 540 nm [5]. The method
needs to be standardized with serum to compensate for the
albumin interference in the reaction. In this method, free
tryptophan does not interfere. This method has been
i
Albumin
Previous and current authors of this method:
First edition: Steven Gendler
Methods edition: Steven Gendler
Second edition: Steven Gendler
Third edition: Kee Cheung, Peter E. Hickman
Fourth edition: Not updated
Fifth edition: Kee Cheung
automated [6]. However, tryptophan-content methods
have never come into common use because of the ease
albumin.
Serum albumin can be quantitated by electrophoretic
techniques (Table 1, Method 3). The major classes of
serum protein are separated by serum protein
electrophoresis on a medium such as agarose. The
separated fractions are stained, and the percentage of
each fraction present in the samples is determined by
densitometric analysis. The concentration of albumin
is calculated by multiplication of the concentration of
total protein in the sample by the percentage of the
albumin band. This method of albumin determination
is labor intensive. In addition, there is no dye that has
been demonstrated to bind to all serum proteins
equally or to have a binding affinity that is linear with
the concentration of all serum proteins [7]. As a result,
electrophoretic procedures tend to overestimate
albumin, which is the best binder of the staining dyes.
Another problem relates to lateral diffusion of
relatively small proteins, including albumin, on
support media.
Immunological methods (Table 1, Methods 4a and 4b)
used for serum albumin quantitation include radial
immunodiffusion (RID) and electroimmunodiffusion
(EID), in which albumin either passively diffuses
(RID) or is electrophoresed (EID) onto a stationary
phase such as agarose that contains antibodies to
albumin. The precipitin lines formed by the reaction
between albumin and the antibody can be fixed and
stained. In RID, the diameter (or square of the
diameter) of the precipitin ring formed is proportional
to the albumin concentration. For EID, the height of
the rocket precipitin line is related to albumin
concentration.
The reaction between albumin and an anti-albumin
antibody can be monitored by turbidimetric or
nephelometric means (Table 1, Methods 4c and 4d).
The antibody-albumin complexes that form either
block (turbidimetry) or scatter (nephelometry) incident
light, and this can be related to the albumin
concentration. Automated nephelometric and
turbidimetric techniques have been adapted for
automated analysis as well [7,8]. However, few
53
Albumin
laboratories are currently reporting the use of
immunological techniques for the quantitation of albumin
in serum, although they are considered to be the most
specific methods. Electrophoretic and immunochemical
methods are generally used for body fluids other than
serum and plasma, in which albumin concentration is
normally low (e.g., urine and cerebrospinal fluid [CSF]).
The most widely used methods for the analysis of serum
albumin are dye-binding procedures (Table 1, Method 5).
Albumin has the ability to bind a wide variety of organic
anions, including complex dye molecules. Dye-binding
techniques are based on a shift in the absorption maximum
of the dye when bound to albumin. The shift in the
absorption maximum allows the resulting color to be
measured in the presence of excess dye, which in concert
with the high-binding affinity of albumin allows all of the
albumin molecules to take part in the reaction. A wide
variety of dyes has been employed for the measurement of
albumin, including methyl orange, 2-(4-
hydroxyazobenzene) benzoic acid (HABA), bromcresol
green (3,3,5-tetrabromo-m-cresolsulfonphthalein, or
BCG) and bromcresol purple (5,5-dibromo-o-
cresolsulfonphthalein, or BCP). BCG and BCP are most
commonly used nowadays; the others have fallen into
disuse because of undesirable interferences. Because of the
nonspecificity of the dye-binding techniques, it had been
recommended that these methods should be used for
screening purposes only [9]. However, subsequent
improvements in the reagent composition and assay
conditions have allowed the BCG and BCP methods to
become routine for the assay of albumin in serum and
plasma. Their simplicity and low cost make these two dye-
binding assays the predominant means for serum and
plasma albumin determination in modern laboratories.
Dye-binding procedures, however, have very limited
application in the determination of albumin in urine or CSF
because of low protein concentration and high
concentrations of interfering substances.
According to the 2007 College of American Pathologists
Participant Summary Report, the BCG and BCP
procedures are used in approximately equal proportions by
laboratories (43% and 57%, respectively). A handful of
laboratories report using nephelometric measurement of
albumin. New methods of albumin determination include
spectrofluorimetry [10], near-infrared spectroscopy [11],
and total internal reflected resonance light scattering [12].
CSF albumin is readily measured directly by
immunoturbidimetric methods of EID, RID, or
immunonephelometry. All these procedures are available
from commercial vendors and function in the range of
about 200 mg/L. Immunonephelometric methods have
been automated on a variety of nephelometers.
Estimation of urine albumin is important in assessing
developing renal dysfunction. Urine albumin is present in
much lower concentration (<30 mg/L) than plasma
albumin, and more sensitive techniques are required for its
measurement, such as radioimmunoassay (RIA) [13-15],
enzyme immunoassay [16-19], and immunoturbidimetric
[20,21], immunonephelometric [22], and RID techniques
[23]. Details of urine albumin methods are covered in
a separate methods section.
Reference and Preferred Methods
The IFCC Committee on plasma proteins (C-PP)
currently recommends optimized immuno-
turbidimetry/immunonephelometry as the reference
method for albumin [24], and the reference material is
BCR-470, serum proteins.
The BCG and BCP methods are currently the
preferred methods for serum and plasma albumin
determination in clinical laboratories. The BCG
method is also adapted in the dry chemistry
measurement of serum albumin. Multilayered
analytical element is coated on a clear polyester slide,
and the amount of colored complex formed is
measured by reflectance spectrophotometry.
The specificity of the BCG method is relatively good,
as shown by correlation with salt fractionation when
sera from normal patients were analyzed [25].
Probably the most promising adaptation of the BCG
reaction for albumin analysis utilizes fast reaction
readings. Gustafsson [26] reported that measuring the
absorbance of the BCG-protein complex at 629 nm at
a time shortly after mixing improves the specificity of
the assay. Interference by other proteins such as
ceruloplasmin and orosomucoid becomes significant at
times greater than 5 minutes. When BCG was
compared to EID (x axis), a regression equation of y =
0.98x + 1.83 was obtained.
Extrapolation does not appear to be necessary to
improve specificity. Non-extrapolated readings taken
at 10 to 30 seconds on a spectrophotometer [27,28], at
10 s on a reaction-rate analyzer or discrete multi-
channel system [29,30], at 0.5 to 6 s on a centrifugal
analyzer [31,32], or at 20 to 30 s on a continuous-flow
analyzer [33-35] improve the specificity of the BCG
reaction. The concentration of BCG is a factor in the
linearity of the reaction and is optimal at 60 mol/L
[30].
The BCP method has been gaining popularity as a
more specific method. It is considered to be superior
to BCG, which overestimates serum albumin,
particularly in patients with low concentrations of
albumin who may also have increased globulins or
monoclonal bands [36]. It seems to have overcome
most disadvantages seen in other dye-binding
methods. It compares well with EID (x axis), giving a
linear regression equation of y = 0.95x + 1.72 when
analysis is done on a population selected to provide a
maximum range of albumin, bilirubin, and globulin
concentrations. Sera with lipemia, hemolysis, and
drugs were also part of this tested population. BCP
does not bind to non-albumin proteins, although
ceruloplasmin and orosomucoid have not been tested
specifically. This specificity is seen without the need
of fast reaction readings. Human serum albumin Cohn
fraction V must be used as a standard, since bovine
54
Albumin
and equine serum albumindye complexes do not absorb
as strongly at 600 nm as human albumindye complexes
[37]. The BCP dye has been adapted for use in continuous-
flow analyzers, centrifugal analyzers, and random access
analyzers [37,38].
Disadvantages of the BCP methods include (1) the
technique reportedly underestimates albumin in cases of
renal insufficiency [39] and obstructive jaundice [40], (2)
BCPalbumin dye complex has a molar absorptivity about
half of that of BCGalbumin complex, thus greatly
reducing the sensitivity and precision of the BCP methods;
and (3) the BCP methods cannot be conveniently
standardized and monitored with nonhuman fluids, since
BCP does not bind strongly to bovine albumin.
Methods for urine albumin are covered in a separate
chapter.
Specimen
Serum is the specimen of choice, but heparinized plasma
can also be used if precautions are taken to prevent heparin
interferences [41]. Fasting is not required, although it may
be desirable, since marked lipemia interferes in the BCG
assay. Venostasis should be avoided when collecting
samples; hemoconcentration increases the apparent
concentrations of albumin and other plasma proteins.
Interferences
Bilirubin, slight to moderate lipemia, and salicylate do not
interfere with BCG methods. Hemoglobin decreases the
apparent albumin concentration by 1 g/L for each 100 g/L
added. Blanking does not correct this interference, and the
negative bias is therefore caused by interference with the
dye binding rather than hemoglobin color. For the BCP
method, a blank correction is required on icteric sera and
on grossly hemolyzed and grossly lipemic sera to correct
for an underestimation of albumin caused by these agents.
Heparin causes a positive interference with BCP and BCG
methods. This interference can be eliminated by the
addition of hexadimethrine bromide to a concentration of
50 mg/L in the BCP reagent [42].
Albumin Reference Interval
The existing interim consensus reference interval for
albumin in serum on the basis of the IFCC/BCR/CAP
reference material CRM 470 and valid for adult Caucasian
persons and adolescents is 35 to 52 g/L [43]. Albumin
concentrations reach these levels around 20 to 30 weeks of
gestation and remain relatively constant until at least 20
years of age and then slowly decrease with age.
Concentrations are, however, significantly lower in
preterm infants [44].
Serum albumin values are lower in the recumbent (lying
down) patient compared to ambulant ones. Some
laboratories take note of the difference between inpatients
and outpatients. For example, Walmsley and White use a
reference interval of 30 to 50 g/L for inpatients and 37 to
52 g/L for outpatients [45]. A difference of 5 g/L between
recumbent and ambulant values has been used [46].
Albumin has been found to be lower in the last two
trimesters of pregnancy [47]. Long-term fasting or
food restriction can cause a decrease in serum
albumin.
Interpretation
Albumin is synthesized by the hepatic parenchymal
cells at a rate that is dependent on colloidal osmotic
pressure and dietary protein intake [48]. The rate of
albumin synthesis is also subject to feedback
regulation determined by the plasma albumin
concentration. The half-life of albumin has been
estimated at 15 to 19 days. Traces of albumin can be
found in almost all extracellular body fluids. Little is
lost from the body by excretion. It is catabolized in
various tissues, where it is taken up by cells by
pinocytosis. Its constituent amino acids are released by
intracellular proteolysis and returned to the body pool.
Albumin has a vast capacity for ligand binding, owing
to the large number of charges on each molecule, as
well as the very large number of molecules available.
This binding property of albumin is related to its
function of transporting molecules such as bilirubin
and free fatty acids [49]. Albumin also binds many
hormones such as thyroxine, triiodothyronine, cortisol,
and aldosterone. It thus acts as a reservoir in which
these physiologically potent compounds are stored in
an active form but from which they can be readily
mobilized. This binding property extends to xenobiotic
compounds such as drugs. Albumin binds salicylate,
valproate, phenytoin, warfarin, phenylbutazone,
clofibrate, and many other drugs. Therefore, low
albumin values may explain drug toxicity at
apparently low concentrations when only the total
concentration of drug in serum is measured. In
hypoalbuminemic sera, the free (active) form of the
drug may be far greater than in sera with normal
albumin levels because of the large difference in
albumin-binding capacity between the different sera.
The increased amount of free drug may thus cause a
toxic response, a phenomenon observed in
hypoalbuminemic patients on dialysis who are treated
with phenytoin. Toxic effects of phenytoin may be
observed at serum concentrations considered
therapeutic. Some 40% of serum calcium is also bound
to albumin. A summary of the major causes of
changes in albumin concentration in serum are listed
in Table 2.
Hypoalbuminemia may also result in underestimation
of the true anion gap in the interpretation of acid-base
abnormalities [50].
In addition to its role as a binding and transport
molecule, albumin plays a large role in nutritional
status. It is argued that the protein is constructed so
that it is readily metabolized and contains all the
essential amino acids. In starvation, plasma
concentration of albumin decreases considerably more
than the levels of gamma globulins. Very low
concentrations are observed in malnutrition,
particularly in kwashiorkor (protein- and calorie-
deficient diets).
55
Albumin
Another major role of albumin is that of maintaining
colloid osmotic pressure. It accounts for 75% of the colloid
osmotic pressure of plasma. When albumin levels are
significantly decreased to about 20 g/L, edema is often
observed because of the consequent decrease of colloid
osmotic pressure. Decreases in serum albumin may occur
in response to many pathological events, thus changes in
serum albumin are very nonspecific.
Plasma albumin measurement, although important for
management and follow-up, has very little value in clinical
diagnosis. Hyperalbuminemia is usually attributable to
dehydration or hemoconcentration. Hypoalbuminemia is
usually the result of (1) hemodilution, (2) a rate of
synthesis less than the rate of albumin loss, (3) diseases
that cause a large albumin loss from urine, skin, or
intestine, or (4) increased catabolism observed in fevers,
untreated diabetes mellitus, and hyperthyroidism.
Decreased synthesis may be because of malnutrition,
malabsorption, or an inability of the liver to synthesize
albumin because of diseases such as acute or chronic
hepatitis. Low plasma albumin concentration may be a
result of large losses in the case of diseases such as
nephrotic syndrome, protein-losing enteropathy, exudative
skin lesions, or burns. Burns in particular may be
associated with severe albumin loss.
In chronic liver disease, albumin is a good indicator of
prognosis and has been used in the Child-Pugh scoring
system [51]. Currently the MELD (model for end-stage
liver disease) score [52] has taken over, and that does not
include albumin. This has been challenged recently by
Brown et al. [53], who argue that the Child-Pugh score is a
better predictor than the MELD score of survival at
specific time points, since albumin level is the most useful
predictor of survival.
There are more than 20 inherited variants of albumin
which are not associated with disease. This includes
bisalbuminemias in which two chemical types of albumin
are present. Congenital absence of albumin, or
analbuminemia, is asymptomatic except for occasional
slight edema.
Albumin Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report shows
imprecision values (% coefficient of variation) for
measurement of albumin at a concentration of 3.5 g/dL to
range from approximately 2.0% to 7.5% for the BCG
procedure and from approximately 1.5% to 2.5% for
methods employing BCP.
Acceptable performance criteria (Clinical Laboratory
Improvement Amendments [CLIA]-88) for measurement
of albumin require that laboratories be accurate to within
10% of the peer group mean. The intraindividual variation
of albumin in blood in healthy adults studied for up to 5
months has been determined to be less than 3.0% [54].
Desirable specifications for analytical imprecision derived
from studies of biological variation indicate an assay
imprecision of no greater than 1.6% and a total error
of no greater than 3.9% [55]. The two dye-binding
methods for albumin determination are therefore
within the desired performance criteria as defined by
CLIA-88 and are within (BCP) or just marginally
outside the biological variation limits (BCG).
References
1 Peters T Jr. Serum albumin. In: Putnam FW,
editor. The plasma proteins, vol. 1. New
York: Academic Press; 1975. p. 133-181.
2 Peters T Jr. Serum albumin. Adv Clin Chem
1970; 13: 37-111.
3 Henry RJ. Proteins. In: Henry RJ. Clinical
chemistry: principles and technics. New
York: Harper & Row Publishers; 1964. p.
199-226.
4 Block RJ, Weiss KW, Carroll DB, editors.
Amino acid handbook. Springfield IL:
Charles C Thomas; 1956. p. 252.
5 Goldenberg H, Drewes PA. Direct
photometric determination of globulin in
serum. Clin Chem 1971; 17: 358-362.
6 Savory J, Heintges MG, Sobel RE.
Automated procedure for simultaneously
measuring total globulin and total protein in
serum. Clin Chem 1971; 17: 301-306.
7 Blom M, Hjrne N. Immunochemical
determination of serum albumin with a
centrifugal analyser. Clin Chem 1975; 21:
195-198.
8 Dito WR. Rapid immunonephelometric
quantitation of eleven serum proteins by
centrifugal fast analyser. Am J Clin Pathol
1979; 71: 301-308.
9 International Federation of Clinical
Chemistry, Newsletter 1976 Mar 5;13.
10 Jiang CQ, Luo L. Spectrofluorimetric
determination of human serum albumin using
a doxycycline-europium probe. Anal Chim
Acta 2004; 506: 171-175.
11 Kasemsumran SY, Du YP, Murayama K,
Huehne M, Ozaki Y. Near-infrared
spectroscopic determination of human serum
albumin, gamma-globulin, and glucose in a
control serum solution with searching
combination moving window partial least
squares. Anal Chim Acta 2004; 512: 223-230.
12 Feng P, Huang CZ, Li YF. Direct
quantification of human serum albumin in
human blood serum without separation of
gamma-globulin by the total internal reflected
resonance light scattering of thorium-sodium
dodecylbenzene sulfonate at
water/tetrachloromethane interface. Anal
Biochem 2002; 308: 83-89.
13 Keen H, Chlouverakis C. An immunoassay
method for urinary albumin at low
concentrations. Lancet 1983; 2: 913-916.
14 Woo J, Floyd M, Cannon DC, Kahan B.
Radioimmunoassay for urinary albumin. Clin
Chem 1978; 24: 1464-1467.
56
Albumin

15 Jury DR, Speed JF, Dunn RJ. Urinary albumin 32 King SW, Cross RE, Savory J. Improved
radioimmunoassay using a solid phase second specificity of the bromocresol green (BCG)
antibody and solid phase iodination. Clin Chim dye method for serum albumin using an early
Acta 1985; 148: 63-67. (6 seconds) absorbance reading. Clin Chem
16 Fielding BA, Price DA, Houlton CA. Enzyme 1977; 23: 1136.
immunoassay for urinary albumin. Clin Chem 33 Cederblad G, Hickey BE, Hollender A,
1983; 29: 355-357. Akerlund G. Improved continuous-flow
17 Puri A, Casburn-Budd R, Eisen V, Slater JDH. (SMAC) determination of serum albumin.
Simpler measurement of albumin in urine or Clin Chem 1978; 24: 1191-1193.
plasma. Clin Chem 1985; 31: 1241-1242 34 Plesher CJ. Continuous-flow analysis of
serum albumin by use of the immediate
18 Feldt-Rasmussen B, Dinesen B, Deekert M.
reaction with bromcresol green. Clin Chem
Enzyme immunoassay: an improved
1978; 24: 2036-2039.
determination of urinary albumin in diabetes with
35 von Schenck H, Rebel K. Improved
incipient nephropathy. Scan J Clin Lab Invest.
continuous-flow determination of albumin
1985; 45: 539-544.
with bromcresol green. Clin Chem 1982; 28:
19 Townsend JC. A competitive immunoenzymatic
1408-1409.
assay for albumin in urine. Clin Chem 1986; 32:
36 Duly EB, Grimason S, Grimason P, Barnes
1372-1374.
G, Trinick TR. Measurement of serum
20 Harmoienen A, Vuorinen P, Jokela H.
albumin by capillary zone electrophoresis,
Turbidimetric measurement of microalbuminuria.
bromocresol green, bromocresol purple, and
Clin Chim Acta 1987; 166: 85-89.
immunoassay methods. J Clin Pathol 2003;
21 Lloyd DR, Hindle EJ, Marples J, Gatt JA. Urinary
56: 780-781.
albumin measurement by immunoturbidimetry.
37 Pinnel AE, Northam BE. New automated
Ann Clin Biochem 1987; 24: 209-210.
dye-binding method for serum albumin
22 Stamp RJ. Measurement of albumin in urine by
determination with bromcresol purple. Clin
end-point immunonephelometry. Ann Clin
Chem 1978; 24: 80-86.
Biochem 1988; 25: 442-443.
38 Haythorn P, Sheehan M. Improved
23 Watts GF, Bennett JE, Rowe DJ, Morris RW,
centrifugal analyzer assay of albumin. Clin
Gatling W, Shaw KM, Polak A. Assessment of
Chem 1979; 25: 194.
immunological methods for determining low
39 Maguire GA, Price CP. Bromocresol purple
concentrations of albumin in urine. Clin Chem
method for serum albumin gives falsely low
1986; 32: 1544-1548.
values in patients with renal insufficiency.
24 Blirup-Jensen S. Protein standardization III:
Clin Chim Acta 1986; 155: 83-88.
method optimization. Basic
40 Bush V, Reed RG. Bromocresol purple dye-
principles for quantitative determination of human
binding methods underestimate albumin that
serum proteins on
is carrying covalently bound bilirubin. Clin
automated instruments based on turbidimetry or
Chem 1987; 33: 821-823.
nephelometry. Clin Chem
41 Hallbach J, Hoffmann GE, Guder WG.
Lab Med 2001; 39:1098-1109.
Overestimation of albumin in heparinized
25 Doumas BT, Biggs HG. Determination of serum
plasma. Clin Chem 1991; 37: 566-568.
albumin. Stand Methods Clin Chem 1972; 7: 175-
42 Duggan J, Duggan PF. Albumin by
188.
bromcresol green: a case of laboratory
26 Gustafsson JEC. Improved specificity of serum
conservatism. Clin Chem 1982; 28: 1407-
albumin determination and estimation of acute
1408.
phase reactants by use of the bromocresol green
43 Dati F, Johnson AM, Whicher JT. The
reaction. Clin Chem 1976; 22: 616-622.
existing interim consensus reference ranges
27 Webster D. The immediate reaction between
and the future approach. Clin Chem Lab Med
bromocresol green and serum as a measure of
2001: 39: 1134-1136.
albumin content. Clin Chem 1977; 23: 663-665.
44 Cartlidge PHT, Rutter N. Serum albumin
28 Corcoran RM, Durnan SM. Albumin
concentrations and oedema in the newborn.
determination by a modified bromocresol green
Arch Dis Child 1986; 61: 657-660.
method. Clin Chem 1977; 23: 765-766.
45 Walmsley RN, White GH. A guide to
29 Gustafsson JEC. Automated serum albumin
diagnostic clinical chemistry. Victoria,
determination of plasma albumin by the
Australia: Blackwell Scientific Publications;
bromocresol green dye-binding method. Clin
1983. p. 242-247.
Chem 1978; 24: 369-373.
46 Jacobs DS, Kasten BL Jr, Demott WR,
30 Robertson WS. Optimizing determination of
Wolfsen WL. Laboratory test handbook with
plasma albumin by the bromocresol green dye-
DRG index. St Louis: Mosby Lexi-comp;
binding method. Clin Chem 1981; 27: 144-146.
1984. p. 32-33.
31 Izquierdo JM. Immediate reaction (0.5s) of serum
47 Tietz NW, editor. Clinical guide to laboratory
albumin with BCG in Cobas BioAnalyzer. Clin
tests. 3rd ed. Philadelphia: W.B. Saunders;
Chem 1977; 23: 1136.
1995.
57
Albumin
48 Rothschild MA, Oratz M, Schreiber SS. Albumin
synthesis. 1. N Engl J Med 1972; 286: 748-57.
AND
Rothschild MA, Oratz M, Schreiber SS. Albumin
synthesis (second of two parts). N Engl J Med
1972; 286: 816-21.
49 Natelson S, Natelson EA. Principles of applied
clinical chemistry: chemical background and
medical applications. In: Plasma Proteins in
Nutrition and Transport, vol 3. New York:
Plenum Press; 1980. p. 43-74.
50 Durward A, Mayer A, Skellett S, Taylor D, Hanna
S, Tibby SM., Murdoch IA. Hypoalbuminaemia
in critically ill children: incidence, prognosis, and
influence on the anion gap. Arch Dis Child 2003;
88: 419-422.
51 Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni
MC, Williams R. Transection of the oesophagus
for bleeding oesophageal varices. Brit J Surg
1973; 60: 646-649.
52 Kamath PS, Wiesner RH, Malinchoc M, Kremers
W, Therneau TM, Kosberg CL et al. A model to
predict survival in patients with end-stage
liver disease. Hepatology 2001; 33: 464-470.
53 Brown DB, Fundakowski CE, Lisker-
Melman M, Crippin JS, Pilgram TK,
Chapman W et al. Comparison of MELD and
Child-Pugh scores to predict survival after
chemoembolization for hepatocellular
carcinoma. J Vas Interventional Radiology
2004; 15: 1209-1218.
54 Fraser CG, Cummings ST, Wilkinson SP,
Neville RG, Knox JD, Ho O et al. Biological
variability of 26 clinical chemistry analytes in
elderly people. Clin Chem 1989; 35: 783-786.
55 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biologic variation: pros, cons
and progress, Scand. J Clin Lab Invest 1999;
59: 491-500.
58
Albumin

Tables
Table 1: Albumin Methods Summary
Method 1: Precipitation; quantitative
a. Salt fractionation
b. Solvent fractionation
c. Acid fractionation
Principle of analysis: Changes of net charge of protein result in precipitation
Comments: Historical; still used in manufacturing albumin; manual
Method 2: Tryptophan content; quantitative
Principle of analysis: Glyoxylic acid + tryptophan in globulin Purple chromogen (Amax, 540 nm); Total
protein globulin = albumin.
Comments: Correlates well with electrophoresis but requires total protein measurement; manual and automated
Method 3: Electrophoresis; quantitative
a. Moving boundary
b. Cellulose acetate
c. Cellulose acetate with elution of peak
Principle of analysis: Albumin is separated from other proteins in electrical field; percent staining of albumin
fraction multiplied by total protein value
Comments: Very labor intensive, but if albumin is eluted for measurement, very accurate; manual and automated
Method 4: Immunochemical
a. Electroimmunoassay; quantitative
Principle of Analysis: Protein migrates in electrical field through medium containing a specific antibody
Comments: Candidate reference method; somewhat labor intensive; manual
b. Radial immunodiffusion; quantitative
Principle of analysis: Protein diffuses through medium containing specific antibody
Comments: Reference method; very long incubation time; manual
c. Turbidimetry; quantitative
Principle of analysis: Antigenantibody complexes decrease light transmission more than free antigen
Comments: Reagent cost high; manual or automated
d. Nephelometry; quantitative
Principle of analysis: Antigenantibody complexes scatter light more than free antigen
Comments: Reagent cost high; automated
e. Radioimmunoassay; quantitative
Principle of analysis: Radiolabeled albumin competes with test albumin for limited amount of antibody
Comments: Primarily used for urine
f. Enzyme immunoassay; quantitative
Principle of analysis: Sandwich assay uses antibody bound to surface and peroxidase-labeled antibody
Comments: New
Method 5: Dye binding
a. Methyl orange; quantitative
Principle of analysis: Albumin binds to dye and changes spectral profile of dye
Comments: Nonspecific for albumin; manual or automated
b. HABA (2-[4-hydroxyazobenzene]benzoic acid); quantitative
Principle of Analysis: Albumin binds to dye and changes spectral profile of dye
Comments: Specific for albumin; poor sensitivity; many drug interferences; manual or automated
c. BCG (bromcresol green); quantitative
Principle of analysis: Albumin binds to dye and changes spectral profile of dye; Amax, 628 nm
Comments: Nonspecific for albumin if absorbance reading taken after 30 s; manual or automated; most often
used method
d. BCP (bromcresol purple); quantitative
Principle of analysis: Albumin binds to dye and changes spectral profile of dye; Amax, 603 nm
Comments: Specific for albumin; low sensitivity; albumins from animal sources do not bind equivalently to
human albumin; manual or automated
Method 6: Dye binding; semiquantitative
Principle of analysis: Bromphenol blue in test strip changes color from yellow to blue in presence of albumin
Comments: Nonspecific; most sensitive with albumin; most commonly used test for urine protein
59
Albumin

Table 2: Causes of Changes in Serum Albumin Concentration

Hyperalbuminemia Hypoalbuminemia

Dehydration Pregnancy
Normal Variant Reduced synthesis
- inadequate diet
- malabsorption
- liver disease
- hereditary
Increased loss
- nephrotic syndrome
- severe bleeding
- burns
Increased metabolism
- hyperthyroidism
- sepsis
- burns
- malignancy

Table 3 Albumin Reaction Conditions Using BCG

Condition Requirement
Temperature Room temperature
pH 4.20 0.05
Final concentration of reagent components BCG: 150 mmol/L
Sodium succinate buffer: 75 mmol/L
Sodium azide: 1.5 mmol/L
Brij-35: 1.2 g/L
Fraction of sample volume 0.004
Sample Serum, heparinized plasma
Linearity 10 to 60 g/L
Reaction time 30 s
Major interferences Gross hemolysis, gross lipemia
Precision (between day) CV: 2.5%-5.9 %

Figures

A. Albumin-BCG Absorption Spectra.

Dashed line, Bromcresol green reagent versus water
blank; dotted dashed line, Bromcresol green reagent and
albumin versus water blank; solid line, Bromcresol
green reagent and albumin versus reagent blank.
B. Albumin-BCP Absorption Spectra.
Absorption spectra of bromcresol purple reagent and
albumin. Dashed line, Bromcresol purple reagent versus
water blank; Dotted dashed line, Bromcresol purple
reagent and albumin versus water blank; Solid line,
Bromcresol purple reagent and albumin versus reagent
blank.
60
Albumin
Procedure: Measurement of Albumin Using
Bromcresol Green
Principle
BCG complexes with albumin, resulting in the dye
having a spectral shift. The presence of albumin
increases the absorbance at 628 nm, which is determined
spectrophotometrically (Albumin Table: Reaction
Conditions)
Specimen
Serum, heparinized plasma
Reagents and Materials
1. BCG reagent. Dissolve 0.105 g of BCG (or
0.108 g of BCG sodium salt), 8.850 g of
succinic acid, 0.100 g sodium azide, and 4 mL
Brij-35 (polyoxyethylene lauryl ether, 300 g/L)
in about 950 mL of distilled water. Adjust pH
to 4.15 to 4.25 with 6 N NaOH. Make up final
volume to 1 L with water. Store in a tightly
closed polyethylene bottle. The reagent is stable
at room temperature for at least 6 months.
2. BCG blank solution. Prepare exactly as for
BCG reagent, but add no BCG.
3. Standardization. Highly purified human serum
albumin fraction V can be used, which has been
confirmed for purity by electrophoresis and
protein concentration determined by a correctly
standardized and well-controlled biuret method.
Assay
Equipment: Any spectrophotometer may be used if it
has a band pass of less than 8 nm, an accurate
wavelength scale, and acceptable photometric accuracy
and linearity.
Procedure
1. Set spectrophotometer to zero absorbance at
628 nm with water. Read absorbance of BCG
reagent. It should be about 0.150 A. Reset
absorbance to zero again against the BCG
reagent.
2. Prepare a set of tubes labeled for standard,
control, and unknown sample.
3. Add 5.0 mL BCG reagent to each tube.
4. Add 20 L of appropriate specimen to each
tube, one at a time, and mix without delay.
5. Record absorbance at 628 nm at 30 3 s after
the addition. Observe the same time intervals
for all samples.
Calculation
Albumin in the sample (g/L) = AU / AS CS
where Cs is the concentration of albumin in the standard
in g/L.
Linearity: The method is linear from 10 to 60 g/L.
Interferences: Hyperbilirubinemia or hemolysis does not
interfere. Marked lipemia causes positive interference,
which can be corrected as follows:
Set spectrophotometer to zero absorbance at 628 nm
with BCG blank solution; add 20 L of sample to 5.0
mL of BCG blank solution; read absorbance of the
serumblank mixture (Ab); subtract Ab from absorbance
of serumreagent mixture, and calculate with this
corrected Au.
61
Albumin in Urine
Albumin in Urine
Graham Jones
Name: Albumin in urine
Clinical Significance: A marker of renal damage in diabetes and other conditions. Also a marker of
risk for cardiovascular disease. Refer to Chapter 30, Renal Function, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Common name: Microalbuminuria
Measurand
Human albumin is a single polypeptide chain of 585 amino
acids with 17 internal disulphide bonds but without
i those with increased excretion. In addition, these
carbohydrate side chains . Albumin in the circulation has
microheterogeneity due to structural flexibility, ligand
binding, and other factors. The transition of albumin from
the serum to the urine has the potential to markedly
increase the structural variability by fragmentation, internal
cleavage, oxidation, or selective tubular resorption, and
multiple forms of albumin have been identified in urine
[1,2]. This variability in albumin has the potential to lead
to standardization difficulties with assays and is known to
produce different results for individual patients using
different assays [3,4]. The development of assays for
albumin fragments detectable by HPLC but not by
standard immunoassays, the so-called immunochemical
non-reactive albumin, has raised the possibility of
improved sensitivity for detection of early renal damage
[2]. There is a need to identify a measurand which is the
most clinically relevant and analytically suitable to provide
standardized assays for urine albumin [5]. The structure of
albumin in urine and its possible effect on various assays
has been the subject of an extensive review [6].
Principles of Analysis and Current Usage (see Table 1)
Assays for albumin in urine can be divided into three main
categories: routine laboratory assays, point-of-care assays,
and other reference or developmental assays. Owing to the
issues mentioned above with regard to variability in the
structure of urine albumin, there are some systematic and
patient-specific differences between results from different
assays, depending on the measurement technology and the
antibody specificity.
The vast majority of routine laboratories use
immunoassays to quantitate albumin in urine. These may
be structured as nephelometric or turbidimetric
homogenous immunoassays or heterogenous competitive
or noncompetitive immunoassays and may use monoclonal
or polyclonal antibodies. These assays are generally
purchased from diagnostic companies and are available for
use on high-volume chemistry or immunoassay analyzers,
as well as in manual assay formats such as enzyme-linked
immunosorbent assay (ELISA) or radioimmunoassay.
These commercial assays generally provide sufficient
sensitivity to measure urine albumin down to
performance when used in remote locations [7].
i
Albumin in Urine
New method
Fifth edition: Graham Jones
concentrations found in healthy individuals, often with
limits of detection below 5 mg/L, and are able to
separate patients with normal albumin excretion from
assays have precision characteristics able to meet
biological variation criteria with claimed coefficient of
variation (CV) values for total precision of < 5%. For
determination of the albumin-creatinine ratio, there is
also the need to measure urine creatinine
concentrations. Because the presence of noncreatinine
chromogens in urine is much less than in plasma,
routine creatinine assays are generally acceptable for
this purpose. If a timed sample is received for
calculation of the albumin excretion rate, the urine
volume must be measured using appropriate scales or
a volumetric flask. Given the high prevalence of
diabetes, most laboratories need a method with high
capacity to meet the clinical needs.
There are a number of point-of-care methods available
for urine albumin measurement using different assay
formats and technologies. These may be
semiquantitative or quantitative, and some also
measure creatinine to allow calculation of the
albumin-creatinine ratio. Point-of-care testing is of
particular use in the clinical setting where a rapid
return of laboratory results is difficult [7], although in
any setting, the provision of a result for use within the
same medical consultation as the sample collection
can be beneficial. Since positive results can be referred
to a laboratory for further analysis, a point-of-care test
used as a screening test should have sufficient
sensitivity to avoid missing positive results. The use of
a high-quality quantitative point-of-care analyzer may
avoid the need for referral of positive samples. Below
are examples of point-of-care devices for
measurement of urine albumin.
The Siemens DCA Vantage analyzer (previously
known as the DCA 2000) uses a monoclonal antibody
agglutination technique for albumin, with a
simultaneous chemical creatinine assay. These tests
are performed on a single 40 L sample; results are
available in 7 minutes, using a disposable cartridge
and a portable analyzer. The system achieves good
precision, with CV < 5% for albumin and < 3% for
creatinine, with quantitation of albumin down to 5
mg/L in a laboratory evaluation [8] with similar
The Haemocue instrument is small, portable analyzer
which uses disposable cuvettes preloaded with
62
Albumin in Urine
reagents. The system uses immunoturbidimetry and
produces a result in 90 seconds from 18 L of sample. The
reporting range is 5 to 150 mg/L; however, precision was
poorer than seen in laboratory methods at all
concentrations tested, with within-run CVs of between
10% and 13% for patient samples with albumin
concentrations above 20 g/L [9,10], although overall a
good correlation with routine laboratory methods has been
achieved [10].
The Siemens Clinitek system provides semiquantitative
results for both albumin concentration and the
albumin/creatinine ratio, using a regent strip with dye-
binding techniques. The strips may be read in a small
reader device, and results higher than 20 mg/L are reported
as positive. Precision for the system cannot be easily
determined, because the results are reported in large
increments. A number of interferences are listed in the
product information, including hematuria, soaps, dyes, and
some drugs, as well as high levels of urine protein. An
evaluation of patient samples showed that approximately
12% of patients with laboratory urine albumin results
below 20 mg/L were falsely reported as elevated and 11%
of samples with low-positive lab results (20 to 55 g/L)
were reported as negative [11]. A higher false-positive rate
has been described in children [12], and it has been
recommended that low-positive results be confirmed with
laboratory testing [10].
The Roche Micral reagent strips are dipped into a urine
sample and the resultant immunologically-mediated color
formation visually compared with a semiquantitative chart.
The method has demonstrated acceptable between-user
correlation [13].The lowest positive result is a color
intensity associated with a nominal value of 20 mg/L. This
decision point has been shown to have a high false-positive
rate in two studies, indicating the need for laboratory
follow-up if available [14,15].
Measurement of urine albumin by HPLC detects different
fragments of albumin, compared to immunoassay as
described above. This leads to higher results, especially in
the low range, with consequent higher detection rates for
microalbuminuria when standard decision points are used
[16,17]. More recent work has indicated possible co-
elution of other proteins with albumin leading to
overestimation of albumin by this method [18]. The
differences between HPLC and immunoassay highlights
the need for agreed reference methods and materials.
Reference and Preferred Methods
There are currently no reference methods or reference
materials for urine albumin listed on the Joint Committee
for Traceability in Laboratory Medicine (JCTLM) database
[19]. In the absence of specific reference materials for
urine albumin, most manufacturers reference their assays
to human serum albuminfor example, using CRM470.
The preferred methods for routine use are immunoassays
for urine albumin with additional measurement of urine
creatinine to allow calculation of the albumin/creatinine
ratio. For many routine laboratories, this type of
technology has the advantages of high throughput and
performance on a routine chemistry or immunoassay
analyzer. The required key performance characteristics
are described further below.
Specimen
The amount of albumin in urine can be expressed in a
number of different formats which require different
samples [6]. These reporting formats include the
following:
Albumin Excretion Rate (AER)
Albumin excretion rate is commonly expressed as mg
per 24 hours or micrograms per minute. The former
requires a 24-hour sample, whereas the latter may use
a 24-hour sample or other timed period, such as an
overnight collection. Both samples require close
attention to start and finish times, as well as avoiding
over- and under-collection from other causes. AER is
considered to be the gold standard but is not generally
recommended for routine use because of these
collection difficulties.
Urine Albumin/Creatinine Ratio (ACR)
Expressing the albumin concentration in a spot sample
as a ratio to urine creatinine is a method to reduce the
effect of patient hydration on the albumin
concentration. ACR is measured in a spot sample,
preferably a first morning sample; a random sample is
acceptable, but daily activity may lead to false-
positive results. The units are mg/g creatinine or
mg/mmol creatinine. The use of creatinine to correct
for hydration also adds an influence of muscle mass to
the result with larger people, who produce more
creatinine, giving lower results for the same albumin
excretion. This is seen with the different decision
points for males and females recommended by some
bodies. The ACR is recommended by the American
Diabetes Association (ADA) for urine albumin testing
[20]. These recommendations also indicate that results
from at least 2 out of 3 samples over a 6-month period
are used to confirm significant changes in albumin
excretion status.
Urine Albumin Concentration (UAC)
The UAC is reported as mg/L or the equivalent g/mL
and is measured in a spot samplelike the ACR,
preferably a first morning sample, but a random
sample is acceptable. Some studies have shown
minimal difference between the sensitivity of ACR
and UAC for increased AER, so some authors
recommend the use of UAC for general purposes,
because this removes the requirement for creatinine
measurement [21].
No preservatives are usually required for urine
albumin collections, and manufacturers
recommendations should be consulted if a preservative
is required. The sample may be stored at room
temperature for up to 7 days and 1 month at 4C to
8C, according to World Health Organization (WHO)
guidelines [22]. It is possible that bacterial
contamination may affect stability at room
temperature, so earlier cooling may be preferred.
Storage at 20C causes breakdown to fragments
63
Albumin in Urine
which are measured in some assays but not others, but this
is not seen at 80C, and long-term storage is possible at
this temperature [3,23].
Interferences
Biological causes for increased urine albumin excretion
other than kidney damage include fever, exercise, heart
failure, marked hyperglycemia, and hypertension [20].
Additionally, collection shortly after ejaculation may
elevate results owing to the albumin content of semen. If
these causes are identified, repeat testing at an appropriate
time may be indicated to further evaluate positive results.
Homogenous immunoassays such as turbidimetry are at
risk of producing falsely low results due to a prozone
effect [24]. Routine procedures to identify this problem
may include (1) measurement of all samples neat and in
dilution to confirm linear dilution, (2) measurement neat
and with additional albumin added to confirm complete
recovery, or (3) testing for high total protein with a
dipstick to identify samples where excess albumin is likely
[25].
Attention should also be given to the possibility of carry-
over effects when serum and urine are run on the same
analyzer, given the > 1000-fold difference in albumin
concentrations in the two sample types.
Reference Intervals
Decision points for interpretation of urine albumin are not
based on population reference intervals but rather on
outcome-based consensus decision points. Different
professional bodies have made slightly different
recommendations, and Table 2 below is based on the data
from the American Diabetes Association nephrology
guidelines [26]. Laboratories are encouraged to adopt their
national guidelines where these are available. Of note, the
decision point of 30 mg/g creatinine is also recommended
as an indication of renal damage in the nondiabetic
population [27].
*Based on ADA Nephropathy guidelines [26].
**Albumin concentration decision point from KDOQI
[27].
Interpretation
Urine albumin is primarily measured as a marker of the
risk of development of renal damage in diabetic patients. It
is now becoming established as a marker for renal damage
in nondiabetic patients, owing to vascular disease
associated with hypertension, elevated lipids, and other
standard risk factors. An elevated urine albumin is also an
established marker of cardiovascular risk in the diabetic
and nondiabetic populations [28], and this risk may extend
down to results within the currently accepted normal
range [29]. The benefit of urine albumin in the
microalbumin range compared to measurement of total
protein is the increased sensitivity provided by albumin.
Once a result is in the macroalbumin range, the
significance is the same as frank proteinuria.
The response to the finding of an elevated urine
albumin should be increased attention to risk factors,
with the aim of reducing the risk of further damage to
the kidneys or other organs.
Performance Goals
Like many other analytes in urine, the concentration of
albumin may vary considerably from day to day. A
CV for within-subject biological variation of 36% is
listed on the Biological Variation database on the
Westgard website [30], although a recent review has
shown marked variation in the estimates for this
parameter, with the central tertile for all studies
showing a range of 28% to 47% [6]. Thus assays with
values for total analytical CVs below 7% will meet
optimal precision requirements of less than a quarter
of the within-subject variation for most estimates of
this parameter. Given that urine albumin/creatinine
ratios continue to provide information down to 10
mg/g (1.1 mg/mmol), the ability to measure albumin
down below 5 mg/L is an advantage when measuring
dilute samples. These criteria can be met by most
laboratory-based immunoassays, including
immunoturbidimetric assays that may be run on
routine chemistry analyzers. By contrast, most point-
of-care analyzers, particularly the semiquantitative
methods, are unable to provide good analytical
performance near upper limit of normal, although they
are clearly able to identify higher levels of albumin
within the microalbuminuria range.
References
1 Candiano G, Musante L, Bruschi M, Petretto
A, Santucci L, Del Boccio P et al. Repetitive
fragmentation products of albumin and alpha-
1-antitrypsin in glomerular disease associated
with nephrotic syndrome. J Am Soc Nephrol
2006:17:3139-3148.
2 Osicka TM, Comper WD. Characterization of
immunochemically nonreactive urinary
albumin. Clin Chem 2004;50:2286-2291.
3 Svridov D, Drake SK, Hortin GL. Reactivity
of urinary albumin (microalbumin) assays
with fragmented or modified albumin. Clin
Chem 2008;54:61-68.
4 Comper WD, Jerums G, Osika TM.
Differences in urinary albumin detected by
four immunoassays and high-performance
liquid chromatography. Clin Biochem
2004;37:105-111.
5 Becker GJ. Which albumin should we
measure? Kidney Int 2004;66(suppl 92):S16-
17.
6 Miller WG, Bruns DE, Hortin GL et al.
Current issues in measurement and reporting
of urinary albumin. Clin Chem 2009;55:24-
38.
7 Shephard MDS, Gill JP. An innovative
Australian point-of-care model for urine
albumin/creatinine ratio testing that supports
diabetes management in indigenous medical
64
Albumin in Urine
services and has international application. Ann
Clin Biochem 2005;42:208-215.
8 Parsons MP, Newman DJ, Newall RG, Price CP.
Validation of a point-of-care assay for the urinary
albumin/creatinine ratio. Clin Chem 1999;45:414-
417.
9 VonSchenckH.Validationofalbumindetermined
in urine with the HemoCue pointofcare
analyzer. Scand J Clin Lab Invest 2003;63:119
126.
10 Sarafidis PA, Riehle J, Bogojevic Z, Basta E,
Chugh A, Bakris GL. A comparative evaluation
of various methods for microalbuminuria
screening. Am J Nephrol 2008;28:324-329.
11 Pugia MJ, Lott JA, Luke KE, Shihabi ZK, Wians
FH, Phillips L. Comparison of instrument read
dipsticks for albumin and creatinine in urine with
visual results and quantitative methods. J Clin Lab
Analysis 1998;12:280-284.
12 Meinhardt U, Ammann RA, Fluck C, Diem P,
Mullis PE. Microalbuminuria in diabetes mellitus.
Efficacy of a new screening method in
comparison with timed overnight urine collection.
J Diabetes Complications 2003;17:254-257.
13 Mogensen CE, Viberti GC, Peheim E et al.
Multicenter evaluation of the Micral Test- II test
strip, an immunological rapid test for the
detection of microalbuminuria. Diabetes Care
1997;20:1642-1646.
14 Parikh CR, Fischer MJ, Estacio R, Schrier RW.
Rapid microalbuminuria screening in type 2
diabetes mellitus: simplified approach with Micral
test strips and specific gravity. Nephrol Dial
Transplant 2004;19:1881-1885.
15 Incerti J, Zelmaovitz T, Camargo JL, Gross JL, de
Azevedo MJ. Evaluation of tests for
microalbuminuria screening in patients with
diabetes. Nephrol Dial Transplant 2005:20:2402-
2407.
16 Brinkman JW, Bakker SJ, Gansevoort RT,
Hillege HL, Kema IP, Gans RO et al. Which
method for quantifying urinary albumin excretion
gives what outcome? A comparison of
immunonephelometry with HPLC. Kidney Int
2004;66:S69-S75
17 Polkinhorne KR, Su Q, Chadban SJ, Shaw JE,
Zimmet PZ, Atkins RC. Population prevalence of
albuminuria in the Australian Diabetes, Obesity,
and Lifestyle (AusDiab) Study:
immunonephelometry compared with high-
performance liquid chromatography. Am J
Kidney Dis 2006:47:604-613.
18 Denis Sviridov D, Meilinger B, Drake SK, Hoehn
GT, Hortin GL. Coelution of other proteins with
albumin during size-exclusion HPLC:
implications for analysis of urinary albumin. Clin
Chem 2006;52:389-397.
19 Joint Committee for Traceability in Laboratory
Medicine website. Available at
<http://www.bipm.org/en/committees/jc/jctl
m/> Accessed 05.28.2008.
20 American Diabetes Association. Standards of
medical care in diabetes. Diabetes Care
2008;31(suppl 1):S12-S54
21 Gansevoort RT, Verhave JC, Hillege HL,
Burgerhof JGM, Bakker SJL, De Zeeuw D,
De Jong PE. The validity of screening based
on spot morning urine samples to detect
subjects with microalbuminuria in the general
population. Kidney Int 2005;67:S28-S35.
22 World Health Organization. Use of
anticoagulants in diagnostic laboratory
investigations. Available at
<http://whqlibdoc.who.int/hq/2000/WHO_DI
L_00.4.pdf>
23 Parekh RS, Kao WH, Meoni LA, Ipp E,
Kimmel PL, La Page J et al. Family
Investigation of Nephropathy and Diabetes
Research Group. Reliability of urinary
albumin, total protein, and creatinine assays
after prolonged storage: the family
investigation of nephropathy and diabetes.
Clin J Am Soc Nephrol 2007;2:1156-1162.
24 Jury DR, Mikkelsen DJ, Dunn PJ. Prozone
effect and the immunoturbidimetric
measurement of albumin in urine. Clin Chem
1990;36:1518-1519.
25 Bakker AJ, Bierma-Ram A, Keidel H,
Syperda H, Zijlstra A. (Micro)albuminuria:
antigen excess detection in the Roche
Modular analyser. Clin Chem 2005;51:1070-
1071.
26 Molitch ME, DeFronzo RA, Franz MJ, Keane
WF, Mogensen CE, Parving HH, Steffes
MW. American Diabetes Association.
Nephropathy in diabetes. Diabetes Care
2004;27(Suppl 1):S79-83.
27 Levey AS, Eckardt KU, Tsukamoto Y, Levin
A, Coresh J, Rossert J et al. Definition and
classification of chronic kidney disease: a
position statement from Kidney Disease:
Improving Global Outcomes (KDIGO).
Kidney Int 2005;67:2089-2100.
28 Sarnak MJ, Levey AS, Schoolwerth AC,
Coresh J, Culleton B, Hamm LL et al. Kidney
disease as a risk factor for development of
cardiovascular disease: American Heart
Association scientific statement. Circulation.
2003;108:2154-2169.
29 Xu J, Knowler WC, Devereux RB, Yeh J,
Umans JG, Begum M et al. Albuminuria
within the normal range and risk of
cardiovascular disease and death in American
Indians: the Strong Heart Study. Am J Kid
Dis 2007;49:208-216.
30 Ricos C. Biological variation database.
Available at <www.westgard.com> Accessed
05.02.2008.
65
Albumin in Urine

Table 1: Albumin in Urine Methods Summary Table

Method1:Immunoturbidimetry
Sensitivity(mg/L):Typically<10
Principle:Antialbuminantibodiesreactwithalbumininthesampletoscatterlight,reducingtransmittedlight.
Absorbancechangecanbemeasuredatarangeofwavelengthsinthevisiblerange.Ahomogenous
immunoassay.
Usage:WidelyusedonroutinechemistryanalyzersandinHaemocuepointofcaredevice
Comments:Prozoneeffectmustbeconsidered
Method2:Immunonephelometry
Sensitivity(mg/L):Typically<10
Principle:Antialbuminantibodiesreactwithalbumininthesampletoscatterlight.Lightisdetectedatanangle
totheincidentlight.
Usage:Incommonuse
Comments:Requiresspecificanalyzerwithnephelometriccapacity
Method3:Radioimmunoassay
Sensitivity(mg/L):<5
Principle:Competitiveimmunoassay
Usage:Uncommoninroutineuse.
Comments:Thefirstmeasurementsystemforurinealbumininthemicroalbuminuricrange

Method4:HPLC(AusAMtechnologies)
Sensitivity(mg/L):<5
Principle:ZorbaxpreparativeGF250HPLCcolumn
Usage:Uncommoninroutineuse
Comments:Detectsbothimmunoandnonimmunoreactiveintactalbuminwithpossibledetectionofother
proteins
Method5:Semiquantitativedipstickdyebinding(Clinitek)
Sensitivity(mg/L):Approximately20
Principle:Bindingtohighaffinitysulfonephthaleindyewithcolorchange.Quantitationbycomparisonwith
colorchartorClinitekReaderusingreflectometry.Colourchartis10,30,80,150mg/L.Creatininecanalsobe
measuredonthesamesystem.
Usage:Pointofcaredevice
Comments:Semiquantitativeonly.Visiblehemoglobinormyoglobininsamplecanaffectresult.Coloreddrugs
ordyesmaymasktrueresponse.Lowpositiveresultsrequireconfirmation.
Method6:Semiquantitativeimmunologicaldipstick(Micral)
Sensitivity(mg/L):Approximately20
Principle:Detectionofalbuminwithalbuminenzymecomplexwithsubstratetoformcoloredpad.Comparison
withcolorchartat0,10,20,50,100mg/L.
Usage:Commoninpointofcaresetting
Comments:Usefulscreeningtest.Noequipmentrequired.Timeofexposetourineiscritical.Lowpositive
resultsrequireconfirmation.

66
Albumin in Urine

TABLE 2: Decision Points for Interpretation of Urine Albumin.*

Spot Samples Timed Samples
Albumin Albumin/Creatinine Ratio Albumin Excretion Rate
Concentration
**
mg/L mg/g or mg/mol mg/24 hours g/min
g/mg
Normal <30 <30 <2.5 (male) <30 <20
<3.5 (female)
Microalbuminuria 30-299 2.5-29 (male) 30 -299 20-199
3.5-29
(female)
Macroalbuminuria 300 30 300 200
67
Alcohol
Alcohol
Ping Wang
Name: Alcohol
Clinical significance: Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: Ethanol Methanol
C2H5OH CH3OH
Molecular mass: 46.07 32.04
Merck Index: (11th edition) 3716, 5868, 5096
Chemical class: Alcohol
Principles of Analysis and Current Usage
Ethanol, because of its extensive availability in such
products as beverages and medicines, is the most
frequently encountered toxic substance. Detection and
quantitation is often required to determine legal
impairment, evaluate patients suspected of poisoning,
determine eligibility for organ transplantation, and assess
compliance with dependency treatment programs.
Alcohol analysis is frequently requested of a laboratory
providing medical and/or forensic services. The clinical
laboratory must provide rapid and reliable results for
patients in life-threatening situations. The forensic
laboratory, while less constrained by turnaround time
requirements, is expected to provide results that are
defensible in a court of law. Today, many laboratories
performing alcohol measurements are expected to meet
both medical and legal requirements.
The term alcohol is often used to refer specifically to
ethanol but can also include the other monohydroxy
alcohols that may be ingested, methanol and isopropanol.
Therefore, in the clinical setting, an alcohol or volatile
testing procedure should detect all three substances. If a
method is used which measures only ethanol, results may
be reported with respect to ethanol, and those served by
the laboratory must be aware of this limitation.
Techniques for the determination of the alcohols include
assays that are nonspecific and semiquantitative (osmolar
and diffusion methods) and assays that are specific and
quantitative (enzymatic and chromatographic procedures)
(Table 1). (In this chapter, if the method does not
distinguish among ethanol, methanol, and isopropanol, it
will be identified as an alcohol procedure. If the technique
is specific for one of the alcohols, it will be identified as a
method for the particular substance detected [e.g.,
ethanol].)
i
Alcohol
Previous and current authors of this method:
First edition: Timothy J. Schroeder
Methods edition: Timothy J. Schroeder
Second edition: Timothy J. Schroeder
Third edition: K. Michael Parker
Fourth edition: K. Michael Parker
Fifth edition: Ping Wang
Isopropanol
C3H7OH
60.09
Early chemical methods for determination of ethanol
were often based on the oxidation of ethanol by
potassium dichromate or other oxidizing agents in a
strongly acid medium. Reduction of the dichromate
results in a color change that can be measured to
monitor the reaction. A variation of this chemical
method was introduced by Widmark. His method was
based on the simultaneous distillation and quantitative
oxidation of ethanol by dichromate. A microdiffusion
method, based on essentially the same principle, has
been developed for commercial use. In this procedure,
chromic acid reagent, contained within a sheet of glass
fiber paper, is reduced to blue-colored chromic oxide
by ethanol. Heating the sample at 80C to 120C
releases ethanol into the glass fiber sheet that is placed
directly above the sample according to the following
reaction.
Equation 1
2K2Cr2O2 (orange-yellow) + 10H2SO4 + 3C2H5OH
2Cr2(SO4)4 (blue-green) + 2K2SO4 + 3CH3COOH +
11H O + 4H+
2
Most of these chemical methods are considered
obsolete because of their poor specificity, tediousness,
and lack of adaptation to automation [1]. Furthermore,
it is important to remember that the chemical assays
are not specific for ethanol but detect volatile reducing
agents. Despite the above limitations, this type of
assay does have the advantage of being amenable to a
wide variety of specimen types, including all body
fluids and tissues.
The presence of alcohol in serum leads to an increase
in the osmolality of the serum when the osmolality is
measured by use of the freezing-point depression
technique (Table 1, Method 2). Osmometers that use
the property of vapor-pressure depression to measure
osmolality cannot be used for the purpose of
estimating alcohol concentrations, because alcohols
are volatile and contribute significantly to the vapor
pressure above a solution. This phenomenon will
result in a falsely low measurement for serum
osmolality.
68
Alcohol
In the presence of alcohol, there will be an increased gap
between the measured (by freezing-point depression) and
the calculated osmolality values. This osmolal gap
correlates reasonably well with blood alcohol
concentrations, because serum osmolality increases by
0.22 mOsm/kg H2O for each 1 mg/dL of ethanol. Using
the osmolal gap, Equation 2 can then be used to estimate
a serum ethanol concentration:
Ethanol (mg/dL) = osmolal gap 4.6
Ethanol can also be estimated according to another
variation of this equation as follows:
(Measured osmolality 290 mOsm/kg) 42.4 =
ethanol (mg/L)
The factor 42.4 is the product of the molecular mass of
ethanol (46.07 mg/mmol) and the correction for the
volume fraction of water in serum (0.92). As a variation
on this principle, the osmolar gap, which is the difference
between the measured and calculated osmolality, can be
used to estimate the concentration of an alcohol. The
difference between the measured osmolality and the
calculated osmolality is determined using the following
equation:
Calculated osmolality = 2[Na+(mmol/L)] +
glucose(mg/dL)/18 + BUN(mg/dL)/2.8
or
Calculated osmolality = 2[Na+ (mmol/L)] + glucose
(mmol/L) + urea (mmol/L)
This difference is normally 10 mOsm/kg. However, if a
low-molecular-mass toxin such as an alcohol is present,
the osmolar gap will be increased. The serum
concentration of the particular alcohol can be estimated
when only one alcohol is present using the osmolar gap
and the following relationships:
Each 1 mg/dL ethanol = 0.22 osmolality increase
Each 1 mg/dL methanol = 0.34 osmolality increase
Each 1 mg/dL isopropanol = 0.17 osmolality increase
To increase the specificity of the method, Pappas et al. [2]
proposed determining the ratio of the estimated ethanol
from the osmolar gap to the ethanol concentration
measured by a specific ethanol procedure. The ratio of
estimated ethanol to measured ethanol was helpful in
evaluating acutely intoxicated patients for the presence of
ethanol and/or another low-molecular-mass volatile.
Discrepancies in the ratio reliably predicted the presence
of another volatile.
However, use of the osmolal gap to estimate ethanol
concentrations may result in an overestimation of alcohol
concentrations by up to 30% [3]. This discrepancy has
been attributed to the atypical osmotic behavior of
ethanol, which can alter the degree of dissociation of
solutes within the specimen. Regardless of these
shortcomings, osmolal gap evaluations can be helpful in
the diagnosis of acutely intoxicated patients in emergency
situations. In addition, the osmolal gap is nonselective
with respect to the type of alcohol detected. Thus the
osmolal gap can help reveal the presence of alcohols
other than ethanol if the results of enzymatic ethanol
measurements are available.
Enzymatic methods have become the most common
means for measurement of ethanol. Alcohol
dehydrogenase (ADH), the enzyme employed in these
assays, is specific for ethanol and does not react with
acetone. However, ADH shows slight cross reactivity
with other alcohols. Relative to ethanol, the cross
reactivities are 6% for isopropanol, 1% for n-propanol,
3% for methanol, and 4% for ethylene glycol [4].
Although the high specificity of ADH for ethanol
ensures a small probability of interference, enzymatic
assays can be the source of misleading results in cases
where intoxication results from the ingestion of other
alcohols [5]. Comparison of results from enzymatic
assays with those from less specific assays (chemical
or osmometric) can help in identifying the presence of
these or other alcohols. The enzymatic ethanol assay is
based on the oxidation of alcohol to acetaldehyde with
concomitant reduction of NAD+ to NADH (Equation
3). The NADH that is produced may be measured
directly at 340 nm, or may be coupled to alternative
detection schemes involving electrochemical,
fluorometry, and colorimetric techniques. In one such
secondary reaction the NADH is coupled to a
diaphorasechromogen system producing a red-
colored solution that can be measured at 500 nm.
Equation 3:
NAD+ + alcohol alcohol dehydrogenase
NADH(340nm) + acetaldehyde + H+
Another variation on the enzymatic scheme is a
fluorometric technique. The technique is termed
radiative energy attenuation, and it measures the
degree of inhibition of the fluorescence of fluorescein
dye resulting from the production of a colored product
[6]. In the original version of this assay, the initial
reaction of ADH with ethanol is coupled with a second
reaction between NADH and the tetrazolium salt,
iodonitrotetrazolium violet dye (INT). This additional
reaction, catalyzed by diaphorase, results in the
reoxidation of NADH to NAD along with the
generation of a red colored formazan-INT. This
product has an absorbance peak at 492 nm, which
overlaps the excitation and emission spectral profile of
fluorescein included in the reaction mixture. The
decrease in fluorescence intensity is inversely related
to ethanol concentrations present in the sample. This
assay has since been reformulated to eliminate a time-
consuming probe wash step by replacing the INT with
a thiazolyl blue dye (MTT). The mechanism behind
the assay is unchanged with the reduced MTT yielding
a purple color with an absorbance at 565 nm [7].
69
Alcohol
Equation 4
There is good agreement between enzymatic methods and
chromatographic methods [8]. However, some enzymatic
methods can give falsely increased ethanol concentrations
with samples from patients with increased levels of serum
lactic acid and lactate dehydrogenase [9]. Although the
enzymatic methods are not absolutely specific for ethanol,
these methods usually meet the requirements for accuracy,
precision, and reliability. The need for rapid analysis in
emergency settings has led to the demand for more point-
of-care instrumentation. New instrumentation has been
developed that is portable, accurate, and uses specimens
such as breath and saliva that are less invasive than
traditional venipuncture. In addition, point-of-care testing
is increasingly being used in fitness-for-duty and post-
accident testing and in compliance monitoring at
detoxification and correctional centers.
Saliva is an appropriate specimen for the analysis of
ethanol, since the amount of ethanol reaching the saliva
should be equivalent to that in plasma or serum. This is
because ethanol is of low molecular mass, is highly water
soluble, and does not bind to blood proteins and therefore
freely diffuses into saliva. A number of point-of-care
saliva ethanol testing kits are commercially available.
They provide either qualitative or quantitative results, and
the majority employ the same enzymatic processes
described above. With one such test system, saliva is
collected on a sterile cotton swab provided with the kit,
and the swab is inserted into the test cartridge. The saliva
must then completely fill the reaction capillary tube in
order for the internal quality assurance device to indicate
that a proper collection was obtained. In a process that
takes approximately 2 minutes, any ethanol in the saliva
will react with the enzymes in the tube to form a colored
endpoint. The tube is fitted with a linear scale in the range
of 0 to 150 mg/dL or 0 to 350 mg/dL, depending on the
kit [10]. One limitation of this type of device, however, is
that 20 minutes must have elapsed between the cessation
of drinking and the collection of the saliva, or an
overestimation of ethanol may result. In addition, some
cross reactivity and response occurs with n-propanol and
isopropanol. For the most part, these devices have been
thoroughly tested and found to be reasonably accurate,
convenient, fast and free from such interferences as
methanol, ethylene glycol, acetone, and methyl ethyl
ketone [10].
Breath is also an appropriate noninvasive specimen for the
analysis of ethanol. A small amount of unchanged ethanol
is expired with every breath, and this amount is
proportional to the amount of ethanol in whole blood.
This is based on the tenet that ethanol in alveolar capillary
blood promptly equilibrates with alveolar air in a ratio of
2100:1 (blood:breath). In other words, 2100 mL of
expiratory air will contain the same amount of ethanol as
1 mL of whole blood [10].
Instrumentation that measures breath alcohol can be
either qualitative or quantitative. Hand-held devices
are usually qualitative in design and are used to detect
the presence of alcohol above a certain level or give a
semiquantitative estimate of the blood alcohol
concentration. Hence these devices are readily used
for roadside screening of suspected drinking drivers,
in emergency medicine, and in fitness-for-duty testing.
The principle behind these hand-held devices is
usually based on an electrochemical detection scheme.
Ethanol from the breath is oxidized by a fuel cell that
causes the production of free electrons. The electric
current generated by the free electrons is directly
proportional to the amount of ethanol oxidized by the
fuel cell [4].
The most common quantitative method for detection
of ethanol in breath is the use of an infrared detection
system. These devices are used in the measurement of
evidential breath alcohol levels. The results from such
a test can then be used as evidence in a court of law.
Measuring the infrared energy at the beginning and
end of the sample chamber quantitates the ethanol in a
breath sample. This is because the amount of ethanol
present in the breath sample is proportional to the
amount of infrared energy lost to absorption. The
wavelengths utilized are 3.4 and/or 9.5 microns,
corresponding to the CH and CO vibrational
stretching in the ethanol molecule, respectively.
Selectivity for ethanol is enhanced by the use of more
than one wavelength. The risk of misreporting an
interfering substance as ethanol was further reduced
by the introduction of an instrument that makes use of
five different infrared wavelengths to identify and
quantitate ethanol [4]. Gas chromatography with flame
ionization detection (GC-FID) is considered to be the
reference method [11].
Chromatography is not as popular as enzymatic
methods because of the need for specific expertise for
performing the analysis and the concern that the
purchase and maintenance of sophisticated
instrumentation dedicated only to a single class of
analytes is not cost effective. However, gas
chromatography offers the distinct advantage of
simultaneous detection of other alcohols and volatiles,
such as acetone, methanol, and isopropanol.
Direct or head-space injection techniques are
employed for GC analysis of volatiles. The direct
injection of blood or serum requires a thorough
manual washing of the syringe after each injection.
The addition of an autosampler to the GC system
improves throughput and assay precision (CVs) while
decreasing chances of carryover through the use of
sophisticated washing routines. Head-space
procedures inject into the GC system a vapor sample
removed from a confined space above blood in a
closed container. Head-space analysis prevents
contamination of the column and injector. Increased
sensitivity of head-space analysis can be gained by the
addition of a salt, such as sodium chloride, to the
specimen. The addition of salt results in an increase in
70
Alcohol
the concentration of the volatile substance in the vapor
phase. Both injection techniques require the sample to be
diluted with an aqueous solution containing an internal
standard to normalize variation among specimens.
Reference and Preferred Methods
The reference method for alcohol testing is gas
chromatography [11]. The choice of method for ethanol
determination depends on several factors, such as
medicolegal requirements, available instrumentation,
testing personnel expertise, and turnaround-time
limitations. In many states there are specifications
defining the methods that must be used when performing
medicolegal testing.
Diffusion methods have largely been replaced by more
specific and quantitative procedures such as the enzymatic
or gas chromatographic techniques. Diffusion methods
may still be usedonly because of their simplicity,
reagent stability (more than 1 year), and low costin
laboratories providing very basic stat screening for
volatile compounds. A test can be set up in a few minutes,
and a qualitative result can be obtained in half an hour. A
true quantitative transfer of the alcohol by diffusion
requires at least 18 hours of incubation time at room
temperature. This makes the method unsuitable as a
quantitative test. Since the technique detects all volatile
oxidizable substances, any of the simple alcohols, as well
as compounds such as ketones, would be detected.
Indirect methods based on osmometry should be used in
very limited emergency situations when more specific
methods are not available. Any condition that changes the
osmolality affects the estimation of alcohol. Significant
errors can occur in patients with diabetic hyperglycemia,
uremia, or hypernatremia. Substantially higher results are
often obtained for the estimated alcohol concentration
based on osmometry than with those measured by a
specific method [12]. The use of the estimated ethanol to
measured ethanol ratio (described above) may alert one to
the possibility of ingestion of another volatile substance.
Enzymatic methods are widely used, according to
proficiency testing surveys. Most laboratories have
automated chemistry instrumentation which can easily
and conveniently be set up to perform enzymatic ethanol
measurements. Most analyzers are capable of directly
measuring absorbance of NADH at 340 nm, and no
coupled indicator reaction is necessary. This approach,
when used on automated instruments, yields the best
precision of any of the ethanol methods. The radiative
energy attenuation method provides rapid, reliable results
using instrumentation found in many laboratories and
most toxicology sections. Since these enzymatic methods
have been designed to quantitate ethanol, other alcohols
will not be detected. Emergency drug testing will require
additional methods to detect and quantitate methanol and
isopropanol. Ingestion of these alcohols occurs with
sufficient frequency to require that toxicology laboratories
have the capacity to measure them. A quick enzymatic
assay for methanol is available for quantitative detection.
This method uses alcohol oxidase to oxidize methanol to
formaldehyde, which is then further oxidized by alcohol
dehydrogenase. The 340 nm absorbance change
associated with generation of NADH in the second
reaction is proportional to the methanol concentration.
This assay can be adapted to automatic enzymatic
platforms. Because high concentrations of ethanol
(usually > 200 mg/dL) may cause false biases in the
methanol enzymatic assay, the test result should be
interpreted together with an ethanol test result and
clinical presentation. Dipstick methods using
enzymatic systems with color-generating indicator
reactions have been useful in identifying ethanol at
high concentrations (>100 mg/dL) [13,14].
Gas chromatography remains the reference method for
alcohol testing. Proficiency testing surveys indicate
that it remains one of the commonly used methods.
Although it is specific for ethanol, the other
monohydroxy alcohols can be specifically measured in
the same analytical run. Therefore, it is often the
preferred method in clinical situations. With regard to
sample introduction, the direct-injection method
provides results (approximately 10 min analysis time)
more rapidly than the head-space technique, and
consequently it is often more suited to the demands of
emergency testing. On the other hand, when whole
blood is the specimen, problems with inlet and column
fouling and syringe plugging can rapidly develop with
the direct-injection technique. Therefore, for
processing a large volume of specimens, as in a
forensic setting, the head-space method is usually
preferred. Combination of an enzymatic method for
screening followed by confirmation by gas
chromatography provides the most accurate approach
for forensic testing.
Specimen
Specimens for the measurement of ethanol include
whole blood (venous or capillary), plasma, serum,
breath, saliva, and urine. For enzymatic methodology,
whole blood should be deproteinated prior to analysis
according to the manufacturers instructions.
Anticoagulants do not interfere with the enzymatic or
gas chromatographic ethanol methods. When gas
chromatography is used, any tissue or body fluid may
be used as specimen. Regardless of what specimen is
used, the samples must be well sealed to prevent loss
of volatiles. Other mechanisms of ethanol loss include
metabolism by microorganisms and temperature-
dependent oxidation to acetaldehyde. The addition of
sodium fluoride and refrigeration will effectively stop
the loss of ethanol by these two processes [15].
Contamination by microorganisms may also result in
false apparent increases in ethanol concentration.
Urine specimens contaminated with yeast and
containing modest amounts of glucose have been
shown to produce sizeable amounts of ethanol by the
process of fermentation [16]; for this reason,
specimens must be kept well stoppered and preferably
refrigerated to prevent loss of ethanol. It has been
shown that sealed samples of whole blood or whole
blood plus fluoride can be stored at 0C to 3C or at
room temperature (22C to 29C) without significant
71
Alcohol
loss of ethanol content over a 14-day period [17]. Several
microorganisms are capable of converting glucose to
ethanolfor example, Candida albicans. The presence of
these organisms along with glycosuria and prolonged
storage at room temperature may lead to falsely increased
ethanol results [18]. Therefore, urine specimens must be
handled in such a way to inhibit microbial production of
ethanol. A 1% solution of phenylmercuric nitrate can be
added to the urine collection container to inhibit any
microorganisms which may be present in the urine.
Interferences
The use of alcohol-containing swabs to clean the
venipuncture site has generally been discouraged, but
McIvor and Cosbey [19] have reported that alcohol swabs
led to minimal interference as measured by head-space
GC, which was unlikely to significantly affect the results.
Specimens with increased lactate and lactate
dehydrogenase concentrations can give falsely increased
ethanol results with some enzymatic assays.
Anticoagulants do not interfere with the enzymatic or GC
procedures. For gas chromatography, any tissue or body
fluid may be used. Some state regulations require that
ethanol analysis be performed on unclotted samples.
Senkowski and Thompson [20] reported that analysis
following homogenization of the clot produced results
comparable to those obtained with unclotted specimens.
Alcohol Reference Interval
Units used for reporting ethanol levels in biological
specimens depend on the purpose in which the ethanol
value will be used. Clinical laboratories generally utilize
SI units for reporting, which express ethanol in terms of
the number of molecular mass units of ethanol
(millimoles) per unit volume. However, if an ethanol
result is to be used in court proceedings, state law usually
mandates the units used for reporting. Additionally, in the
United States, most statutes require that ethanol
concentrations be expressed in terms of mass per unit
volume of whole blood [21]. Law enforcement agencies in
other countries may have different requirements.
This can cause some confusion if the specimen utilized for
analysis was plasma or serum, because these specimens
have a higher water content than whole blood and will
therefore contain more ethanol per unit volume than
whole blood. Because of the potential for error when
making conversions of ethanol content between specimen
types, some states have amended their statutes to include
per se limits for different specimen types. Ethanol, as well
as other volatiles, methanol, and n-propanol, are normally
not detectable in blood or tissues. A blood ethanol
concentration of 300 mg/dL (65.1 mmol/L) or greater may
be associated with coma, whereas values greater than 400
mg/dL (86.8 mmol/L) have been reported to be fatal.
Individuals who are chronic users of ethanol can show
even higher concentrations. In the postabsorptive phase,
ethanol is distributed approximately as follows compared
to blood (1.0): urine (1.3), saliva (1.12), breast milk (1.1),
CSF (1.14), brain (0.8), fat (0.02), and breath (1/2100).
Plasma or serum ethanol is about 1.16 times that of whole
blood, depending on the hematocrit.
Interpretation
There are both clinical and forensic applications for
the analysis of ethanol. Alcohol is a primary
depressant of the central nervous system. If alcohol is
taken in sufficient quantity, death can result from
irreversible depression of respiration. It is also
important to distinguish altered mental status due to
ethanol intoxication from other causes, especially in
cases where head trauma, low blood glucose
concentrations, or seizure are complicating the clinical
diagnosis. Because of the serious consequences of
ethanol intoxication, rapid analysis is required for
initiation of appropriate therapy. Forensically, alcohol
is monitored for the purpose of workplace drug
testing, post-accident investigations, driving
impairment, and postmortem examinations. As the
medicolegal ramifications of ethanol intoxication are
serious, the analysis must be accurate and follow strict
guidelines to ensure legal acceptance.
Medicolegal interpretation of ethanol results is usually
based on state laws. Historically, 0.1 g/dL (100
mg/dL; 21.7 mmol/L) has been used as the legal limit
for blood ethanol concentration (BEC). The trend now
is toward lower BEC limits. Most states now use a
BEC limit between 0.05 and 0.10 g/dL, with 0.08 g/dL
being the most common limit in use. In the United
States, drivers under age 21 are held to stricter
standards under zero-tolerance laws, with a BEC limit
between 0 and 0.02 g/dL. In Europe, 0.05 g/dL has
been proposed as the goal for the BEC limit. Sweden
has lowered the limit further to 0.021 g/dL [22].
Endogenous ethanol production accounts for a BEC
less than 0.3 mg/dL. Following acute ingestion, BEC
usually peaks at 0.5 to 2 hours (fasting) or 1 to 6 hours
(nonfasting). BEC < 50 mg/dL may not be
accompanied by obvious behavioral effects. As BEC
increases beyond 50 mg/dL, central nervous system
effects become more apparent, progressing from
euphoria, excitement, and confusion to stupor. At a
BEC > 150 mg/dL, most individuals are obviously
drunk. BEC > 350 mg/dL may lead to coma and death.
Chronic alcohol users may tolerate greater BEC.
Alcohol clearance is primarily by hepatic metabolism.
Between concentrations of 20 and 300 mg/dL, ethanol
metabolism follows zero-order kinetics at a rate of
approximately 10 mL of ethanol metabolized per hour,
corresponding to a decrease in blood ethanol of about
20 mg/dL/h [23]. However, alcohol metabolism varies
significantly between individuals, with alcoholics
showing up to a twofold increase. Gastric alcohol
dehydrogenase activity is less in women and alcoholic
men than in nonalcoholic men, leading to increased
ethanol bioavailability and BEC [24]. Medications
such as aspirin [25] and H2-receptor antagonists
[26,27] inhibit gastric alcohol dehydrogenase, thereby
increasing the amount of ingested ethanol reaching the
systemic circulation. Ascorbic acid has been reported
to increase ethanol clearance in healthy individuals
[28].
Methanol and isopropanol are usually not detected in
body fluids. Methanol concentrations greater than 20
72
Alcohol
mg/dL are often toxic. Patients with significant methanol
concentrations should be treated with ethanol or 4-methyl
pyrazole to inhibit the production of the toxic metabolites
formaldehyde and formic acid. Toxicity with isopropanol
usually develops at concentrations greater than 150
mg/dL. Acetone is produced as a consequence of
isopropanol metabolism and may be followed in addition
to isopropanol concentration when monitoring the
clearance of the alcohol.
Alcohol Performance Goals
Survey data from the 2007 College of American
Pathologists (CAP) Participant Summary Report show
that imprecision values (% coefficient of variation [CV])
for ethanol determinations range from approximately 4%
to 9% at a mean ethanol concentration of 40 mg/dL and
CVs of 5% to 16% at a mean ethanol concentration of 24
mg/dL. Precision for laboratories measuring ethanol by
gas chromatography is similar to laboratories that employ
enzymatic oxidation methods. Acceptable performance
criteria (CLIA-88) for measurement of ethanol require
that laboratories be accurate to within 25% of the peer-
group mean. For ethanol-free whole blood or serum
specimens, CAP has adopted an acceptable result range of
0 to 9 mg/dL.
References
1 Garriott JC, editor: Medicolegal aspects of
alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc.
2 Pappas AA, Gadsden RH, Taylor EH. Serum
osmolality in acute intoxication: A prospective
clinical study. Am J Clin Pathol 1985: 84: 74-79.
3 Bhagat CI, Beilby JP, Garcia-Webb P, Dusci LJ.
Errors in estimating ethanol concentration in
plasma by using the osmolal gap, Clin Chem
1985; 31:647-648.
4 Jones AW, Pounder DJ. Measuring blood alcohol
concentrations for clinical and forensic purposes.
In: Drug Abuse Handbook, Karch, S.B., editor,
Boca Raton, FL, 1998, CRC Press.
5 Garriott JC, editor: Medicolegal aspects of
alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc. p. 221.
6 Yost DA, Boehnlein L, Shaffer M. A novel assay
to determine ethanol in whole blood on the
Abbott TDX, Clin Chem 1984: 30: 1029A.
7 Garriott JC, editor: Medicolegal aspects of
alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc. p. 224.
8 Jortani SA, Poklis A. Emit\R ETS\R plus ethyl
alcohol assay for the determination of ethanol in
human serum and urine. J Anal Toxicol 1992:
16: 368-371.
9 Badcock NR, OReilly DA. False-positive
EMIT\R-st\T ethanol screen with post-mortem
infant plasma, Clin Chem 1992: 38: 434.
10 Jones AW. Measuring ethanol in saliva with the
QED? enzymatic test device: Comparison of
results with blood- and breath-alcohol
concentrations. J Anal Toxicol 1995: 19: 169-74.
11 Tagliaro F, Lubli G, Ghielmi S, Franchi D,
Marigo M. Chromatographic methods for blood
and alcohol determination, J Chromatogr
1992: 580: 161-190.
12 Snyder H, Williams D, Zink B,Reilly K.
Accuracy of blood ethanol determination
using serum osmolality, J Emerg Med 1992:
10: 129-133.
13 Christopher TA, Zeccardi JA. Evaluation of
the Q.E.D. saliva alcohol test: A new, rapid,
accurate device for measuring ethanol in
saliva. Ann Emerg Med 1992: 21: 1135-
1137.
14 Schwartz RH, ODonnell RM, Thorne MM,
Getson PR, Hicks JM. Evaluation of
colorimetric dipstick test to detect alcohol in
saliva: a pilot study. Ann. Emerg Med 1989:
18: 1001-1003.
15 Ellenhorn MJ, Schonwald S, Ordog G,
Wasserberger J, editors. Ellenhorns medical
toxicology: diagnosis and treatment of human
poisoning. Baltimore, MD, 1997, Williams &
Wilkins, p. 793.
16 Lough PS, Fehn R. Efficacy of 1% sodium
fluoride as a preservative in urine samples
containing glucose and Candida albicans. J
Forensic Sci 1993: 38: 266-271.
17 Winek CL, Paul LJ. Effect of short-term
storage conditions on alcohol concentrations
in blood from living human subjects, Clin
Chem 1983: 29: 1959-1960.
18 Alexander WD, Wills PD, Eldred N, Gower
R. Urinary ethanol levels and diabetes,
Lancet 1981: 1(2):789.
19 McIvor RA, Cosbey SH. Effect of using
alcoholic and non-alcoholic skin cleansing
swabs when sampling blood for alcohol
estimation using gas chromatography, Br J
Clin. Pract 1990: 44: 235-236.
20 Senkowski CM, Thompson KA. The
accuracy of blood alcohol analysis using
head-space gas chromatography when
performed on clotted samples, J Forensic Sci
1990: 35: 176-180.
21 Garriott JC, editor: Medicolegal aspects of
alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc., p. 268.
22 Jones AW. Limits of detection and
quantitation of ethanol in specimens of whole
blood from drinking drivers analyzed by
head-space gas chromatography. J Forensic
Sci 1991: 36: 1277-1279.
23 Gershman H, Steeper J. Rate of clearance of
ethanol from the blood of intoxicated patients
in the emergency department, J Emerg Med
1991: 9: 307-311.
24 Frezza M, diPadova C, Pozzato G, Terpin M,
Baraona E, Lieber CS. Higher blood alcohol
levels in women: the role of decreased gastric
alcohol dehydrogenase activity and first-pass
metabolism. N Engl J Med 1990: 322: 95-99.
25 Roine R, Gentry RT, Hernandez-Munoz R,
Baraona E, Lieber CS. Aspirin increases
blood alcohol concentrations in humans after
73
Alcohol

ingestion of ethanol. JAMA 1990: 264: 2406-

2408.
26 Caballeria J, Baraona E, Rodamilans M, Lieber
CS. Effects of cimetidine on gastric alcohol
dehydrogenase activity and blood ethanol levels.
Gastroenterol 1989: 96: 388-392.
27 DiPadova C, Roine R, Frezza M, Gentry RT,
Baraona E, Lieber CS. Effects of ranitidine on
blood alcohol levels after ethanol ingestion:
comparison with other H2-receptor antagonists.
JAMA 1992: 267: 83-86.
28 Chen MF, Boyce HW Jr, Hsu JM. Effect of
ascorbic acid on plasma alcohol clearance. J Am
Coll Nutr 1990: 9: 185-189.
29 Watts MT, McDonald OL. The effect of sodium
chloride concentration, water content, and
protein on the gas chromatographic head-space
analysis of ethanol in plasma. Am J Clin Pathol
1990: 93: 357-362.
74
Alcohol

Table 1: Alcohol Methods Summary

Methods for All Alcohols:
Method 1: Distillation-oxidation; colorimetric
Principle: Alcohol diffuses into gas phase and reacts with oxidizing agent, changing its color:
2 K2Cr2O7 + 10 H2SO4 + 3C2H5OH
(yellow-orange)
2 Cr2(SO4)4 + 2 K2SO4 + 3 CH3COOH +11 H2O + 4 H+
(blue-green)
Usage: Stat or routine; all body fluids; tissue
Comments: Nonspecific; gives reaction with all volatiles
Method 2: Osmometry; freezing-point depression
Principle: Alcohol in high concentration increases serum osmolality: (a) difference between normal and measured value is
proportional to alcohol levels, or (b) difference between measured and calculated value (osmolar gap) proportional to alcohol
concentration
Usage: Stat; serum
Comments: Nonspecific; measured osmolality increased by non-alcohols

Methods for a Specific Kind of Alcohol:

Method 3: Enzymatic:
(a) Spectrophotometric
Ethanol:
_
Principle: NAD+ + alcohol alcohol dehydrogenase NADH + H+ + acetaldehyde
Usage: Stat or routine; serum; urine
Comment: Specific for ethanol; other alcohols not readily measured
Methanol:
alcohol oxidase
Principle: Methanol + O2 formaldehyde + H2O2
H O + formaldehyde + NAD+ formaldehyde dehydrogenaseformic acid +
2 2
NADH + H+
Usage: Stat or routine; serum; urine
Comment: Specific for methanol; high ethanol concentration causes positive bias; alcohol oxidase is very labile and needs to
be prepared fresh before testing
(b) Colorimetric, dipstick
Principle:
alcohol dehydrogenase
NAD+ + alcohol NADH + H+ + acetaldehyde
diaphorase
NADH + iodonitrotetrazolium dye
NAD+ + reduced iodonitrotetrazolium dye (insoluble)
(colored)
Usage: Stat or routine; serum; urine
Comment: Specific for ethanol; other alcohols not readily measured
(c) Fluorometric
alcohol dehydrogenase
Principle: NAD+ + alcohol NADH + H+ + acetaldehyde
diaphorase
NADH + tetrazolium dye
NAD+ + reduced monotetrazolium dye (insoluble)
(fluorescein fluorescence decreased)
The reduced dye is colored, decreasing the amount of light reaching the fluorescein dye reagent and
therefore attenuating its fluorescence
Usage: Stat or routine; serum
Comment: Specific for ethanol; other alcohols not readily measured
Method 4: Gas chromatography; flame ionization detection and quantitation
Principle: Alcohol separates on chromatographic column; identification of specific alcohol by retention time; quantitation by
comparison of peak height to an internal standard. Direct injection of sample fluid onto column or head-space analysis, where
the airspace above the sample is allowed to come to equilibrium, and the air sample is chromatographed.
Usage: Stat or routine; all body fluids
Comments: Specific for all alcohols
Method 5: Infrared spectrophotometry
Principle: The amount of infrared energy lost due to absorption is proportional to the alcohol concentration. Two
wavelengths used to monitor for interference.
Usage: Forensic and compliance; breath analysis
Comments: Qualitative or quantitative analysis
75
Alcohol

Table 2: Comparison of Assay Conditions for Ethanol

Parameter Enzyme Reaction Gas Chromatography
Temperature 37C 75C
pH 8.8 7.35
Final concentration of Reduced nicotinamide adenine n-propanol: 1.2 g/L
reagent components dinucleotide: 0.6 mmol/mL (20 mmol/L)
Alcohol dehydrogenase: 50 U/mL
Tetrasodium pyrophosphate: 0.075 mol/L
Sodium semicarbazide: 0.075 mol/L
Glycine: 0.022 mol/L
Fraction of sample volume Approximately 0.008 0.33
Sample volume 0.5 mL 0.2 mL: 0.5 mL injection
Linearity 0-600 mg/dL (130 mmol/L) 0-1000 mg/dL (217 mmol/L)
Precision* 4% (automated analyzers) 7% (automated analyzers)
Time for analysis 10 min (auto analyzers) 10 min (auto analyzers)
Interferences Enzyme inhibitors; some alcohols None known
Specimen Serum, plasma, urine Whole blood, serum, plasma, urine

*Coefficient of variation; obtained from College of American Pathologists toxicology survey data

Table 3: Units for Expressing Ethanol Concentrations and Common Conversion

Factors
% g/100 %
Units g/L (wt/vol) mL mg% mg/dL ppm g/mL (wt/wt) mg/g mmol/L
g/L 1 0.1 0.1 100 100 1000 1000 0.0948 0.948 21.71
%
(wt/vol) 10 1 1 1000 1000 1E+4 1E+4 0.948 9.48 217.1
g/100
mL 10 1 1 1000 1000 1E+4 1E+4 0.948 9.48 217.1
mg% 0.01 1E-3 1E-3 1 1 10 10 9.48E-4 9.48E-3 0.2171
mg/dL 0.01 1E-3 1E-3 1 1 10 10 9.48E-4 9.48E-3 0.2171
ppm 1E-3 1E-4 1E-4 0.1 0.1 1 1 9.48E-05 9.48E-4 0.02171
g/mL 1E-3 1E-4 1E-4 0.1 0.1 1 1 9.48E-05 9.48E-4 0.02171
%
(wt/wt) 10.55 1.055 1.055 1055 1055 10548 10548 1 10 229
mg/g 1.055 10.55 10.55 105.5 105.5 1055 1055 0.1 1 22.9
mmol/ 4.61E-
L 0.0461 4.61E-3 3 4.61 4.61 46.06 46.06 4.367E-3 4.367E-2 1
76
Alcohol

Figure 1: Photograph of Conway diffusion dish showing sample well, A, and inner well, B,
containing reacted dichromate solution (blue-green).

_____________________________________________________________________

Figure 2: Chromatogram of alcohol standards obtained by direct injection of the sample into
the gas chromatograph.
Retention time in minutes is recorded next to peak. Alcohols are eluted in the following
sequence: 1.545, methanol; 1.825, acetone; 2.195, ethanol; 2.880, isopropanol; 3.970, n-propanol
(internal standard).

_____________________________________________________________________
77
Alcohol

Procedure: Gas Chromatography of Alcohols Alcohol Retention Time

Table 2 compares the assay conditions for the
two most commonly used methods. The reactions for
the enzyme method are given in Table 1. Actual
parameters for the enzyme procedure may vary,
depending on the manufacturer of the reagents and the
instrument used in the analysis.
Principle
The following direct-injection gas
chromatographic procedure can be used to separate
volatile compounds such as ethanol, methanol,
isopropanol, and acetone. After sample injection into the
heated injection port, the volatiles are separated on a
Carbowax column, detected with a flame ionization
detector, and quantitated by comparison of the peak
height ratio of the sample to internal standard, relative to
the peak height ratio of the calibration standard to
internal standard.
Specimen
Serum, plasma or whole blood may be
analyzed.
Reagents and Materials
Equipment: Gas chromatograph equipped with
a flame ionization detector and a 6 ft 2 mm (I.D.)
column packed with 60/80 Carbopack B/5% Carbowax
20M.
Reagents
1. Internal standard: Dilute 75 L n-propanol to
50 mL with deionized water. Stable when
stored in a refrigerator for up to 6 months.
2. Calibration standard: Prepare a calibration
standard of 79 mg/dL (17.1 mmol/L) by
diluting 1 mL of 100% ethanol (anhydrous
from a sealed bottle) to 1 L with deionized
water. Stable when tightly sealed and stored in
refrigerator for up to 1 month. Other dilutions
should be prepared to verify linearity of the
method (0 to 500 mg/dL; 0 to 108.5 mmol/L).
Conditions: Chromatographic parameters: injection
temperature, 225C; column temperature, 75C; detector
temperature, 250C; carrier gas (helium) flow rate, 30
mL/min; detector gas (hydrogen) flow rate, 30 mL/min;
(air) flow rate, 400 mL/min.
Assay
1. Combine 50 L of calibration standard, assay
control, or patient blood (serum) sample and
100 L of internal standard in an appropriate-
size glass vial. Cap the vial and mix.
2. Sequentially inject 0.5 L from each vial into
the chromatograph. Chromatographic run time
is approximately 6 min.
Calculation
1. Identify the ethanol peak using the retention
time:
Methanol 1.545
Acetone 1.825
Ethanol 2.195
Isopropanol 2.880
n-Propanol 3.970
(internal standard)
See Alcohol: Figure 2 for sample chromatogram.
2. Determine the peak height (in millimeters) for
the ethanol (E) and internal standard (IS) peaks
on each chromatogram.
3. Calculate peak height ratios, E/IS.
4. Calculate ethanol in samples as follows:
Ethanol (mg/dL) = ____E/IS (sample)____ 79 mg/dL
E/IS (calibration standard)
5. If an electronic integrator is used, calculate
ethanol in samples as follows:
Ethanol (mg/dL) = Factor E/IS (unknown)
where Factor = _______79 mg/dL______
E/IS (calibration standard)
Notes
1. Direct injection of the sample into the gas
chromatograph may result in inlet and column
contamination. As an alternative, analysis of
the head-space vapor above a specimen may be
performed. The following procedure can be
followed to prepare the head-space samples.
a. Place 1.0 g of sodium chloride in a
glass screw-capped tube (screw caps
should contain Teflon-lined silicone
septa) for each calibration standard,
assay control, or patient blood.
b. Add 0.5 mL of calibration standard,
control, or sample to each tube.
c. Add 0.5 mL of internal standard to
each tube, seal, and mix well.
d. Place tubes at 38C for 45 min.
e. Inject 0.5 mL of head-space vapor into
the gas chromatograph.
The same chromatographic conditions and
calculation procedure can be used for the head-
space method as with the direct-injection
technique. Preparation of calibration
standard(s) in a matrix equivalent to that in the
samples, particularly with respect to protein,
has been suggested to reduce partitioning
differences of volatiles between standard(s) and
samples [29].
2. Isopropanol, methanol, and acetone can also be
quantitated by this procedure. (refer to
retention times in Alcohol: Figure 2.) The same
peak height ratio method with n-propanol as
the internal standard is used for the calculation.
Concentration =
Peak height ratio (unknown) Standard concentration
Peak height ratio (standard)
78
Alcohol
Alcohols Gas Chromatography: Direct Injection
Important Procedural Note: Do not use injection
syringes that have been exposed to methanol as a
washing solvent. Use deionized water when rinsing
all syringes.
Principle
Most volatile compounds such as ethanol,
methanol, and isopropanol will separate from
biological fluids in the heated injection port of
a gas chromatograph. These compounds will
then be separated from each other by the GC
column and can be identified and quantitated
by comparing their peak height ratio to a
known internal standard.
Specimens of Choice
A. For nonlegal/nonforensic quantitative uses: 0.5
mL of serum collected in a red-top tube; or plasma
collected in either a heparinized or EDTA tube.
B. For nonlegal/nonforensic qualitative uses: 1
mL urine from spot aliquot collection with no
preservatives added.
C. For legal/forensic* quantitative use: 1.0 mL
whole blood collected in a gray-top NaF tube.
D. For legal/forensic* qualitative uses: 1 mL urine
from spot aliquot collection with no preservatives
added.
All specimens may be transported at
room temperature. For nonlegal cases, serum or plasma
should be separated from RBCs and may be stored at
room temperature until time of testing (up to 3 days)
then stored frozen at 20C. For legal/forensic whole
blood and all urine specimens, store at 2C to 8C prior
to testing.
*Note: For legal/forensic uses, specimen must
be accompanied by a Chain of Custody Form.
Indications
As an aid in assessing ethanol or other alcohol
abuse which may relate to acute or chronic clinical
problems (i.e., coma, hepatic damage, drug/alcohol
interactions, psychiatric disorders, acidosis); to assess
ethanol abuse for forensic purposes, including employee
monitoring and postmortem studies.
Reagents and Materials
A. n-Propanol internal standard. Using a 1.0 mL
serological pipet, deliver 0.4 mL of n-propanol to a 100
mL volumetric flask. Dilute to mark with distilled H2O.
Store in 100-mL reagent bottle at room temp. Stable for
2 months.
B. Alcohol working standards.
Note: For routine instrument calibration, a working
mixed standard will be prepared from individual
standards.
1. Ethanol standard (150 mg/dL)aqueous
Setpoint standard (Stephens Scientific Co.). Store
refrigerated at 2C to 8C. Stability as indicated by
manufacturer.
2. Isopropanol calibration standard (157
mg/dL)Using a 0.2-mL serological pipet, deliver 0.2
mL of 100% chromatoquality isopropanol (2-propanol)
to a 100-mL volumetric flask. Dilute to mark with
distilled water. Store at room temperature. Stable for 2
months.
3. Methanol calibration standard (158 mg/dL)
Same as #2 above, using chromatoquality methanol.
4. Alcohol Working Mixed Standard: Combine
100 L each of ethanol, isopropanol, and methanol
standards with 100 L of internal standard. Prepare
fresh as needed. (Total volume: 400 L.)
5. Ethanol linearity check standardsTo
respective 100 mL volumetric flasks, add 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, and 0.8 mL of 100% chromatoquality
ethanol, representing concentrations of 79, 158, 237,
316, 395, 474, 553, and 632 mg/dL of ethanol,
respectively. Dilute to mark with distilled water. Store at
room temperature. Stable for 2 months. To be used for
monthly instrument linearity check.
C. 12 75 mm glass culture tubes.
D. 100 L Eppendorf pipet.
E. Utak Volatiles Control Serum.
F. Hamilton 7002 NWG syringe.
Instruments and Parameters
A. The parameters given here are for a Shimadzu
Mini-2 gas chromatograph.
Detector: Flame ionization
Column: 5% Carbowax 20M on 60/80 Carbopack B
(Supleco); 6 ft, 2 mm ID
Temperatures: Col. 90C; Det. 240C; Inj. 240C.
Attenuation: 32
Range: 10
Nitrogen flow: 50 mL/min
Gas pressures at tank: air, 20; nitrogen, 50; hydrogen, 30
mm Hg
B. Model 3390 Hewlett Packard GC Terminal
Procedure
A. Nonforensic analytical runs will consist of an
internal control, a calibration standard if required, and
patient specimens.
1. Assess standardization by running a single
control at the beginning of each shift or after any
instrument changes (i.e., septum change, gas tank
change, etc.).
2. If control is within acceptable range, proceed to
step 4.
3. If control is out of acceptable range, repeat the
control. If it is still unacceptable, perform a
recalibration.
4. Run the patient sample. If patient sample is
positive for any alcohol, prepare a second internal
standard and sample mixture, and reinject for a duplicate
analysis. For quantitative analyses (i.e., serum or blood)
average the duplicate values for a reportable
concentration. If patient chromatogram is negative, a
single analysis is all that is necessary.
5. Run any other appropriate standard (i.e.,
methanol or isopropanol) if patient specimen indicates
need.
B. Forensic analytical runs will consist of a
calibration standard, an internal control, and the patient
specimens. Note that a standard and control must be
analyzed with each specimen or batch of specimens.
1. Assess standardization by running the 150
mg/dL setpoint calibrator. If the calibrator result is
79
Alcohol
between 142 and 158 mg/dL, proceed by running a
control. If the calibrator is outside these limits, perform
a recalibration.
2. Run the single internal control. Assess its
acceptability (will have been determined previously).
3. Run the patient/subject specimen as with
nonforensic runs, analyzing all positive samples in
duplicate.
4. Indicate type of specimen (i.e., whole blood,
urine) in the report by appending computer comments
Test performed on whole blood, or Test performed
on urine.
C. Sample Preparation
1. Add 100 L (with a 100-L Eppendorf pipet)
of internal standard solution to a glass culture tube.
2. Add 100 L (with a 100-L Eppendorf pipet)
of patient specimen, control, or appropriate standard to
the tube and mix by agitation. Analyze immediately
(within 1 min of preparation).
3. Inject 1.0 L of the mix into the GC. (Note:
Injection amounts may vary with time.) Peak height of
internal standard should be a minimum of full-scale
response. Use syringe designated for alcohols; do not
rinse with methanol.
4. Immediately press [START] on the integrator.
5. Proceed to Calculations.
Calculations and Quality Control
A. Integrated calculations
Note: For all positive patient samples, duplicate
analyses are performed. Average these duplicate results
for the reportable quantitative result for serum and/or
blood assays.
1. The integrator prints direct alcohol
concentrations in mg/dL, as well as retention times for
the alcohols of interest.
2. If only qualitative analysis is required (i.e.,
urine), compare retention times of patient sample peaks
to those of the known control. Urine concentrations at or
greater than 10 mg/dL for any of the alcohols will be
reported as Positive. Concentrations below 10 mg/dL
will be reported as None detected. Urine results will
not be reported quantitatively.
B. Manual calculations (to be used whenever
direct instrument integration is unavailable).
1. Ethanol
(a) Measure the peak height of the n-propanol I.S.
and of the ethanol in millimeters.
(b) Compute the peak height ratio (PHR) for the
150 mg/dL standard, control, and patient:
peak height of ethanol
PHR = peak height of I.S.
Compute the concentration of ethanol in the
patient and control:
PHR of patient or control 150
Concentration (mg/dL) = PHR of 150 ethanol std
2. Methanol
a. Measure peak heights as above in a for
patient, control, and 158 mg/dL methanol standard.
b. Calculate PHR as above in a. Compute
concentration of methanol in the patient and control:
PHR of patient or control 158
Concentration (mg/dL) = PHR of 158 methanol std
3. Isopropanol
a. Measure peak heights as above in a for
patient, control, and 157 mg/dL isopropanol standard.
b. Calculate PHR as above in a. Compute
concentration of isopropanol in patient and control:
PHR of patient or control 157
Concentration (mg/dL)
= PHR of 157 isopropanol std
C. Acetonereport only internally as being
positive. Do not quantitate or officially report.
D. Concentration conversions (For use when
converting to other concentration units):
1. To convert from mg/dL to g/mL, multiply by
10.
2. To convert from mg/dL to grams %, divide by
1000.
E. An acceptable run includes control values
within acceptable limits and patient duplicate analyses
within 10% of the duplicate mean. If outside of 10%,
analyze a third sample preparation, and recalculate from
the two closest values.
Interpretation
A. For ethanol, 100 mg/dL denotes legally
intoxicated. Greater than 400 mg/dL is considered
critical. Critical values for methanol and isopropanol are
30 mg/dL and 200 mg/dL, respectively. Reportable
concentrations are up to 1000 mg/dL for each alcohol.
Call all critical values to the caregiver.
B. For alcohol concentrations above 632 mg/dL,
dilute the patient specimen 1:1 (50 L plus 50 L) with
distilled H2O, add 100 L internal standard, and
analyze. Multiply calculated mg/dL result by 2.
C. Run a complete ethanol curve to establish
linearity monthly, when changing GC columns, or when
other changes dictate.
D. If methanol is detected in the patient sample,
run a blank (distilled water) taken through the
procedure, then rerun the patient. This is to rule out false
positives due to methanol from wash bottle. If methanol
appears in the blank, change water rinse.
E. Acetone will most frequently be found in
patients who are acidotic or who have ingested
isopropanol where acetone is produced as a metabolite.
Acetone detection is noted but not officially reported.
F. False-positive isopropanol levels may be a
result of improper blood drawing technique (i.e., using
an alcohol swab). A good indication that a serum
isopropanol is due to swab alcohol is that there will not
be any acetone in the sample. Also, if available, confirm
the presence of isopropanol by analyzing the patients
urine. A negative corresponding urine is also indicative
of a false-positive serum result.
G. The current method sensitivity for each alcohol
is 10 mg/dL. Negative results should be reported as < 10
mg/dL (<10).
H. Because of the relatively high threshold
concentrations for reporting alcohols (10 mg/dL), other
volatiles, although they may appear in the
chromatographic field, will generally not be detected.
Even so, if clinical histories indicate specific nonalcohol
volatiles as being possible poisons, the area supervisor
80
Alcohol

should be consulted. This would include compounds B. Add 0.5 mL of potassium dichromate
such as acetonitrile, ethyl acetate, chlorinated to the center well with a 1 mL
hydrocarbons, and formaldehyde. serological pipet.
C. Place the cover on the dish, and allow
Alcohols, Serum, Plasma, Whole Blood, Quantitative it to stand at room temperature for 2 h.
1. Process a Utak control with a known ethanol Read promptly.
concentration at the beginning of each shift for each D. Observe the color produced.
staff member who will be performing ethanol Calculations (Observations) and Quality Control
concentration measurements on that shift. A. The dichromate reagent will change
2. To a 12 75 mm tube, add 100 L of serum, color in 15 min to 2 h if ethanol or
plasma, or whole blood. other volatile reducing substances are
3. Add 100 L of n-propanol internal standard. present, with the concentration of
4. Inject 1 L into gas chromatograph. substance being proportional to time
5. Integrator will directly report ethanol of color change. Color change is from
concentrations in mg/dL. yellow, to green, to blue.
B. A yellow nonreactive color
Alcohol Spot Test (Qualitative) indicates no alcohols above the
Principle procedure threshold cutoff of 20
In a Conway microdiffusion dish, alcohols will mg/dL.
diffuse out of an aqueous specimen, be trapped, and C. A green to blue color is indicative of
form a color complex with potassium dichromate. the presence of:
Specimen Ethanol
Serum (1 mL) drawn in a red-top tube with or Isopropanol
without separator gel, whole blood drawn in a Methanol
heparinized or EDTA tube, or urine (1 mL) spot aliquot. Other reducing substances
Indications D. For all positive samples, proceed
To be used as a general adjunct alcohol with confirmatory testing using gas
screening procedure. To qualitatively assess the chromatography.
presence of ethanol, methanol, or isopropanol. E. An acceptable run includes a green-to-
Reagents and Materials blue color formation with the positive
All chemicals used should be analytical reagent control and no green-to-blue color
grade. with the negative control.
A. Conway microdiffusion dish. Interpretation and Notes
B. Potassium dichromate. A. A positive finding indicates the
Into a 1-L flask, dissolve 1.0 g of presence of a reducing substance, such
potassium dichromate in 500 mL of as alcohol, but may not be indicative
water. Add 0.1 g of silver nitrate to of toxicity or impairment.
the solution. Carefully, add 500 mL of B. Sensitivity: 20 mg/dL; report
concentrated sulfuric acid. Stir to mix, negatives as None detected.
transfer to an amber reagent bottle, C. Specificity: Nonspecific method;
and store at room temperature. Stable unable to identify specific alcohol
for 1 year. present; will react with other reducing
C. Negative control. substances.
Distilled water blank.
D. Positive control (790 g/mL).
To a 100-mL volumetric flask, add 0.1
mL of absolute 100% ethanol. Fill to
mark with distilled water. Transfer to
a reagent bottle. Store at room
temperature. Stable for 3 months.
E. 500-L Eppendorf pipet.
F. 1-mL serological pipet.
Instrument and Parameters
None
Procedure
Note: With reach run analyze a positive and
negative control.
A. Cover the bottom of the middle ring of
the microdiffusion dish with blood,
serum, or urine by adding 0.5 mL of
sample with an Eppendorf pipet.
81
Aldolase

Aldolase
Stephen M. Gendler

Name: Aldolase, ALS,

fructose-1,6-diphosphate:d-glyceraldehyde-3-phosphate lyase
Clinical significance: Refer to Chapter 36, Cardiac and muscle disease, in the 5th Edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Enzyme number: EC 4.1.2.13
Molecular mass: approximately 160,000 daltons
Chemical class: enzyme protein
Known isoenzymes: subunits A, B, and C in tetrameric form
Biochemical reaction:

Principles of Analysis and Current Usage
Aldolase (ALS) catalyzes the reversible biochemical
reaction shown above. It is present as a tetramer
composed of two of the three known subunits designated
A, B, and C. When analyzed as homotetramers, aldolase
A, B, and C have similar pH-activity profiles,
MichaelisMenten constants (Km) for fructose
diphosphate (FDP), and molecular weights. Using FDP
as substrate, the pH optima in Tris-malonate buffer [1]
and in collidine buffer [2] range from pH 7.0 to 8.0. The
Michaelis constants with FDP as substrate range from
10-6 to 3 10-6 M. The molecular weights for the
homomeric enzymes range from about 145,000 to
175,000 daltons [1]. The A homomer is found in high
concentration in skeletal muscle. The B homomer
predominates in liver, and the C homomer predominates
in brain and other tissues. The primary isoenzyme in
normal serum is the A homomer. The hybrid isoenzyme
composed of three A subunits and one C subunit is
present in a somewhat lower concentration [3]. In
aldolase analyses in current use, enzymatic activity is
determined by use of FDP as the substrate, since
aldolase cleaves FDP more rapidly than other substrates
[1]. For analytical purposes the aldolase reaction is
driven to the formation of trioses by use of hydrazine, a
trapping agent for the triosephosphates [2,4,5]. The
aldolase reaction can also be shifted to trioses by
removal of the glyceraldehyde phosphate (GAP) with the
enzyme triose-phosphate isomerase (TPI, D-
glyceraldehyde-3-phosphate:ketol isomerase, EC
5.3.1.1). This enzyme catalyzes the conversion of GAP
to dihydroxyacetone phosphate (DAP) [6]. The
ultraviolet spectrophotometric analyses use two coupled
enzyme indicator reactions, with the concomitant
i aldolase A [12] and aldolase C [13] have also been
Aldolase
Previous and current authors of this method:
First edition: Steven M Gendler
Methods edition: Steven M Gendler
Second edition: Not updated
Third edition: Not updated
Fourth edition: Steven M Gendler
Fifth edition: Not updated
formation or degradation of NADH (methods 1 and 2,
Aldolase Table: Comparison of methods for serum
aldolase (ALS) analysis). Coupled enzyme systems that
result in the appearance of NADH have been shown to
be subject to large errors caused by the presence of
endogenous interfering enzymes such as glycerol
dehydrogenase (GDH, glycerol-3-phosphate:NAD 2-
oxidoreductase, EC 1.1.1.8) or TPI. Thus the most
satisfactory design for a coupled enzymatic system is as
follows:
FDP ALS DAP + GAP
GAP TPI DAP
2 DAP + 2 NADH + 2 H+ GDH
2 glycerol 1-phosphate + 2 NAD+
In this scheme interferences can be eliminated in the
preincubation phase. Interferences can be demonstrated
by a decrease in A340 after the addition of the indicator
enzymes and sample and before the addition of FDP.
These interferences have not been characterized but may
be endogenous substrates and dehydrogenases in serum
that utilize NADH.[7] DAP or GAP contamination of
the FDP preparation can also be demonstrated by a
decrease in A340 after the addition of indicator enzymes
and FDP and before the addition of serum sample [8].
Proposed by Beisenherz et al [6] in 1953, this coupled
reaction has been optimized by Ludvigsen [9], Pinto et al
[7], and Bergmeyer and Bernt [10] and adapted to the
kinetic measurement of enzyme activity [11]. This
method has been adapted to kit form and is
commercially available (Boehringer Mannheim,
Calbiochem, and other companies).
Radioimmunoassays (RIA) for the homotetramers of
reported. RIA is not discussed further because these
assays are not widely used. In addition, the clinical
utility of these assays is as yet unproved.
82
Aldolase
Reference and Preferred Methods
A colorimetric method was published as a selected
method by the American Association for Clinical
Chemistry (AACC) in 1961 [14]. Because of the many
manipulations and the time required, this method is not
recommended.
The various modifications of the coupled enzymatic
techniques are difficult to compare, since the methods
have not been compared by one laboratory in a
controlled situation (Table 2). The Bergmeyer and Bernt
[10] method has been developed as a kit. Day-to-day
imprecision claims of 2.7% in the normal range and
1.9% in the abnormal range have been reported. For
reasons of ease of use, good reproducibility, and
adaptability to semiautomation the method of Bergmeyer
and Bernt is the method of choice.
Specimen
The anticoagulants oxalate, citrate, fluoride, heparin, and
EDTA reportedly have no effect on aldolase activity
[15]. Whether serum or plasma is used, it is essential to
remove the specimen from contact with the red blood
cells and platelets. In general, plasma is preferred over
serum because of the release of aldolase from platelets
during the clotting process. If refrigerated specimens are
not removed from the clot for 24 hours, the aldolase
level will be 12% to 46% greater than that of a properly
treated specimen [16]. The stability of the enzyme at
room temperature is reported as 5 hours to 2 days,
depending on the study. Boric acid powder, added
directly to serum to achieve a concentration of 25 g/L,
extends the stability of the enzyme at room temperature
to 6 days [17]. The enzyme is stable for at least 5 days
when kept refrigerated at 4 C.[18] The activity of
aldolase remains unchanged for at least 6 months if the
specimen is kept at -20 C [19].
Interferences
Aldolase is present in high concentrations in
erythrocytes and platelets. Because the concentration of
aldolase in red blood cells is 10 times that in serum,
hemolysis is a cause for rejection of the specimen [18].
Aldolase Reference Interval
Aldolase exhibits sex- and age-related differences. At
birth, activities are approximately twice those seen in
adults. Physical activity also influences aldolase
activities with physically active individuals showing
higher activities. Reference intervals for healthy adults
are as follows:
Males 2.61 to 5.71 U/L [16]
Females 1.98 to 5.54 U/L
Interpretation
It has been suggested that aldolase is useful in
differentiating muscle disease from other diseases [20].
Aldolase, however, is present in many tissues [21] and as
a result has not been found to be specific for muscle
disorders. In a multivariate analysis of plasma-enzyme
profiles in severe head injury, aldolase in conjunction
with aspartate aminotransferase, creatine kinase, and
lactate dehydrogenase can be used to discriminate
between survivors and nonsurvivors [22]. In contrast, in
a study performed at a university hospital, the test was
found to be worthless for the purposes ordered, including
diagnosis of polymyositis or other inflammatory
myopathies and determination of whether an elevated
creatine kinase (CK) activity was caused by skeletal
muscle damage or myocardial damage [23]. These
authors observed that the serum aldolase added little
information when CK data were available and
recommended the deletion of this test from the hospital
laboratory test repertoire. Other authors have expressed a
similar view [20].
Aldolase Performance Goals
Evaluation studies indicate total imprecision of
approximately 10% to 15% for aldolase activities in the
normal range and approximately 5% in the abnormal
range when the enzyme is measured using the method of
Bergmeyer and Bernt.
References
1 Penhoet EE, Kochman M, Rutter WJ.
Molecular and catalytic properties of aldolase
C. Biochemistry 1969; 8:4396-4402.
2 Bruns FH. Bestimmung und Eigenschaften der
Serumaldolase. Biochem Z 1954; 325: 156-162.
3 Tzvetanova E. Aldolase isoenzymes in patients
with progressive muscle dystrophy and in
human fetuses. Clin Chem 1971; 17: 926-930.
4 Sibley JA, Lehninger AL. Determination of
aldolase in animal tissues. J Biol Chem 1949;
177: 859-872.
5 Fleischer GA. Aldolase. In American
Association of Clinical Chemists: Standard
methods of clinical chemistry, New York, 1961,
Academic Press, Inc., vol. 3, pp. 14-22.
6 Beisenherz G, Boltze HJ, Bucher T et al.
Diphosphofructose-Aldolase,
Phosphoglyceraldehyde-Dehydrogenase,
Milchsaure-Dehydrogenase, Glycerophosphat-
Dehydrogenase and Pyruvat-Kinase aus
Kaninchenmuskulatur in einem Arbeitsgang. Z
Naturforsch 1953; 8b: 555-577.
7 Pinto PVC, Kaplan A, Van Dreal PA. Aldolase.
II. Spectrophotometric determination using an
ultraviolet procedure. Clin Chem 1969; 15: 349-
360.
8 Bruns FH, Bergmeyer H-U. Fructose-1,6-
diphosphate aldolase. In Bergmeyer, H.-U.,
editor: Methods of enzymatic analysis, ed. 2,
New York, 1974, Academic Press, Inc., vol. 2,
pp. 724-731.
9 Ludvigsen B. DPNH method for the estimation
of serum aldolase activity. J Lab Clin Med
1963; 61: 329-337.
10 Bergmeyer H-U, Bernt E. Fructose-1,6-
diphosphate aldolase, UV assay, manual
method. In Bergmeyer, H.-U., editor: Methods
of enzymatic analysis, ed. 2, New York, 1974,
Academic Press, Inc., pp. 1100-1105.
11 Harjanne A. The kinetic measurement of serum
Aldolase. Clin Chim Acta 1979; 92: 311-313.
83
Aldolase

12 Asaka M, Nagase K, Miyozaki T et al. Tables

Radioimmunoassay for human aldolase A. Clin
Chim Acta 1981; 117: 289-296. Table 1: Methods of serum aldolase (ALS) analysis
13 Wilson VJC, Thompson RJ. Human brain
aldolase C4 isoenzyme: purification,
radioimmunoassay, and distribution in human Method 1: NADH formation; end-point
tissues. Ann Clin Biochem 1980; 17: 114-121. spectrophotometric
14 Aldolase. In American Association of Clinical Principle of analysis:
Chemists: Standard methods of clinical FDP DAP + GAP
chemistry, New York, 1961, Academic Press, GAP TPI DAP
Inc., vol. 3, pp. 14-22.
15 Ladenson JH. Nonanalytical sources of GAP + NAD+ GAPDH
+ AsO2
variation in clinical chemistry results. In
Sonnenwirth, A.C., and Jarett, L., editors: 3-Phosphoglycerate + NADH + H
Gradwohls clinical laboratory methods and Comments: Manual; subject to large
diagnosis, ed. 8, St. Louis, 1980, The C.V. interferences
Mosby Co., vol. 1, pp. 153-154. Method 2: NADH degradation
16 Sweetin JC, Thomson WHS. Revised normal Principle of analysis:
ranges for six serum enzymes: further statistical FDP DAP + GAP
analysis and the effects of different treatments GAP TPI DAP
of blood specimens. Clin Chim Acta 1973; 48:
49-63. 2 DAP + 2 NADH + 2 H+ GDH
17 Beardslee R, Owers P. Stabilization by boric 2 Glycerol-1-phosphate + 2 NAD+
acid of aldolase activity at room temperature. Comments: Manual or automated; accurate,
Clin Chem 1976; 22: 1543-1545 (letter). precise, flexible, and easy to use
18 Demetriou JA, Drewes PA, Gin JB. Enzymes. *DAP, Dihydroxyacetone phosphate; FDP, fructose
In Henry, R.J., Cannon, D.C., and Winkelman, diphosphate; GAP, glyceraldehyde phosphate; GAPDH,
J.W., editors: Clinical chemistry: principles and glyceraldehyde phosphate dehydrogenase; GDH,
techniques, Hagerstown, MD., 1974, Harper & glycerol-3-phosphate:NAD 2-oxidoreductase; NAD+,
Row, Publishers, Inc., pp. 971-974. nicotinamide adenine dinucleotide; NADH, reduced
19 Kaldor J, Schiavone DJ. Automated procedure NAD; TPI, triosephosphate isomerase.
for serum aldolase estimation. Clin Chem 1968;
14: 735-739.
20 Jacobs DS, Masten BL Jr, Demott WR,
Wolfson WL. Laboratory test handbook with Table 2: Reaction conditions for aldolase (ALS)
DRG index, Mosby/Lexi-comp, St. Louis, analysis*
1984, The C.V. Mosby Co., pp. 33-34. Temperature: 37 C
21 Kaplan LA, Pesce AJ. Clinical chemistry: pH: 7.4
theory, analysis, and correlation. St. Louis, Final concentration of reagent components:
1984, The C.V. Mosby Co., p. 956. Collidine: 50 mmol/L
22 Bourguigrat A, Ferard G, Jung G et al. Iodoacetate: 0.27 mmol/L
Multivariate analysis of plasma enzyme profiles FDP: 3.6 mmol/L
in severe head injury. Clin Chem 1983; 29: 107- NADH: 2.7 mmol/L
109. GDH: >3 U/mL
23 Velazquez FR, Ng RH, Statland BE. Clinical TPI: >15 U/mL
utility of serum aldolase measurements. Clin LD: >9 U/mL
Chem 1985; 31: 1004. Sample volume: 200 L
24 Bergmeyer H-U. Neue Werte fur die molaren Fraction of sample volume: 0.07
Extinktion-Koeffizienten von NADH and Linearity: 27.5 U/L
NADHP zum Gebrauch im Routine- Precision (%CV): 2.7%12.4%
Laboratorium. Z Klin Chem Z Klin Biochem *From Bergmeyer, H.-V., and Bernt, E.: In Bergmeyer,
1975; 13: 507-508. H.-V., editor: Methods of enzymatic analysis,
ed. 2, New York, 1974, Academic Press, Inc.
Sources of analytical error: any specimen
with visible hemolysis will give elevated results
because of release of aldolase from cells.
Serum stored on the clot gives similar results.
LD, Lactate dehydrogenase.
84
Aldolase

Procedure: Coupled Assay of Bergmeyer and Bernt Calculations

[10] With use of the molar absorptivity () of NADH as
6300 L cm
Principle mole
FDP ALS GAP + DAP
The formula for calculation of enzyme activity is as
GAP TPI DAP
follows:
2 DAP + 2 NADH + 2 H+ GDH
2 glycerol-1-phosphate + 2 NAD+
Lactate dehydrogenase (LD) is included in the
reagent to remove the interference of endogenous
pyruvate. The oxidation of NADH is followed by
absorbance measurement at 340 nm.
where A = difference between A1 and A2 over analysis
Reagents time, T minutes
1. Buffer/substrate solution. Dissolve 220 mg of = molar absorptivity of NADH [24]
Na3FDP 8H2O or 370 mg of tricyclohexylammonium
10 6 = factor to convert concentration to micromoles
FDP 10H2O and 6.2 mg of iodoacetic acid in 90 mL of per liter
distilled water. Add 0.75 mL of collidine, mix, and For the assay described, the values are as follows:
adjust the pH to 7.4 with approximately 0.6 mL of 5 M
HCl. Dilute with distilled water to 100 mL. Final U/L = _A_ 2.76 mL _1__ 106 = A 55
concentrations are 55 mmol collidine buffer per liter at 20 min 0.2 mL 6300 2
pH 7.4, 0.3 mmol iodoacetate per liter, and 4 mmol FDP
per liter. This solution is stable for 4 weeks at 2 to 8 C. The factor of 2 indicates that for every molecule
2. NADH solution. Dissolve 25 mg of of FDP catalyzed, two molecules of NADH are
Na2NADH and 20 mg of NaHCO3 in 2 mL of distilled
converted to NAD+.
water. Final concentration is 15 mmol/L. This is stable
for 4 weeks at 2 to 8 C. Interferences
3. GDH/TPI/LD suspension. Stock solutions of Hemolysis is cause for rejection.[18]
the enzymes, as suspensions in ammonium sulfate, are Also, refrigerated serum not removed from the clot will
available from Boehringer Mannheim Biochemicals have an aldolase level 12% to 46% greater than that of a
(Indianapolis, IN). Using 3.2 mol of ammonium sulfate properly treated specimen.[16]
per liter, mix enzyme solutions so that final
concentrations are >75 U/mL of GDH per milliliter (25
C) (source: rabbit muscle), >500 U of TPI per milliliter
(25 C) (source: rabbit muscle), >233 U of LD per
milliliter (25 C) (source: rabbit muscle). This is stable
at 2 to 8 C for about 1 year. Suspend thoroughly before
use.

Assay
Equipment: 37 C constant-temperature
incubator and spectrophotometer (band pass <10 nm)
capable of reading at 340 nm.
1. Place 2.50 mL of buffer/substrate solution, 0.05
mL of NADH solution, 0.01 mL of
GDH/TPI/LD suspension, and 0.20 mL of
serum or plasma in a 3.0 mL cuvette.
2. Mix well, and incubate at 37 C for about 5
minutes.
3. Read the initial A340 (A1).
4. After exactly 20 minutes at 37 C, read the final
A340 (A2).
5. If the A (A1 - A2) is greater than 0.500, dilute
specimen five- to tenfold with isotonic saline,
and repeat the analysis using the same volumes
of reagents and specimen.
85
Aldosterone
Aldosterone
Hassan M.E. Azzazy
Name: Aldosterone
Clinical significance: Refer to Chapter 51, Adrenal Hormones and Hypertension, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass: 360.4 D
Chemical class: Mineralocorticoid
Principles of Analysis and Current Usage
Aldosterone is the most important human
mineralocorticoid; it is secreted by the zona glomerulosa of
the adrenal cortex and stimulates sodium transport across
cell membranes in the distal renal tubules. Aldosterone
plays a major role in homeostasis of sodium and potassium
and maintenance of arterial blood pressure in the sodium-
depleted state. The renin-angiotensin system regulates
aldosterone secretion. Low sodium concentration or low
blood volume causes release of renin from kidney cells,
which mediates release of aldosterone.
Aldosterone exists in picomolar concentrations in serum,
and thus sensitive assays are necessary for its
measurement. Several methods are available for
determination of aldosterone in blood and urine (Table 1).
These include immunoassays, isotope-dilution gas
chromatography/mass spectrometry ID-(GC/MS) [1], high-
performance liquid chromatography-tandem mass
spectrometry (HPLC-MS/MS) [2], and liquid
chromatography-tandem mass spectrometry (LC-MS/MS)
[3].
Radioimmunoassay (RIA) has been the most common
assay for aldosterone measurement since the early 1970s
[4]. The antibodies used were typically polyclonal, raised
in rabbits and generated against an aldosterone-3-(O-
carboxymethyloxime) bovineserum albumin conjugate
[3]. Commercial immunoassays use different approaches to
separate bound from free aldosterone: a solid-phase first
antibody, a solid-phase second antibody, an accelerated
liquid-phase second antibody, dextran-coated charcoal, or a
polyethylene glycol precipitant. RIA methods with and
without extraction steps are being employed [5,6]. The
extraction of aldosterone prior to analysis may be done into
dichloromethane, which improves the specificity of the
assay, since aldosterone concentrations in blood are in the
picomolar range. Many patients being investigated have
some degree of renal failure, and patients with chronic
renal failure have polar aldosterone metabolites present in
high concentrations in plasma; these cross-react with
aldosterone antibodies. Thus solvent extraction helps to
increase assay specificity by removing these metabolites.
Currently a large portion of RIAs for aldosterone
measurement do not involve an extraction,
i
Aldosterone
New method:
Fifth edition: Hassan M.E. Azzazy
chromatography, or prefractionation step, which are
relatively complex and time consuming (Table 1,
Methods 1 and 2) [7]. Average salivary aldosterone
values are almost a third of those in plasma, and an
RIA was also developed for aldosterone determination
in saliva [8,9].
Aldosterone measurement in urine is performed using
the same testing methodologies, but a preassay acid-
hydrolysis step is required. This is because the term
urinary aldosterone actually refers to the 18-
glucuronide conjugate of aldosterone (10% of all
urinary aldosterone metabolites), and acid hydrolysis
is required to form free aldosterone. Only 2% of
aldosterone is excreted in the free form [4].
Enzyme immunoassays are available for aldosterone,
including competitive-binding assays to monoclonal
antibodies and direct-detection assays using antibodies
conjugated to horseradish peroxidase, with the
detection limits being in the picomolar range [9,10]. A
competitive-binding chemiluminescence immunoassay
has been developed. The assay utilizes a biotinylated
mouse monoclonal anti-aldosterone antibody as the
capture reagent and acridinium ester-labeled
aldosterone as a tracer (Table 1, Method 3) [11].
Liquid chromatographytandem mass spectrometry
(LC-MS/MS) is currently being used for aldosterone
determination in both blood and urine. Current LC-
MS/MS methods use atmospheric-pressure chemical
ionization or photospray ionization along with
multiple steroid profiling [12,13]. A high-performance
liquid chromatographyatmospheric pressure chemical
ionizationtandem mass spectrometry (HPLC-APCI-
MS/MS) method was proposed as a reference method
for aldosterone quantification in serum and plasma
(Table 1, Method 4). In this method, extraction was
performed using dichloromethane/diethyl ether
containing flumethasone as internal standard. A
phenyl column was used for chromatography, and the
mobile phase was 50 mM ammonium formate (pH
7.1)/methanol (50/50, v/v). The reported accuracy of
this method ranged from 93.1% to 98.9% [2]. In other
LC-MS/MS methods, deuterated aldosterone
(aldosterone-d6) is added to samples as an internal
control. Aldosterone-d6 and endogenous aldosterone
are extracted from serum or plasma using a Strata X
cartridge, and the eluate is dried and then reconstituted
with 70/30 methanol/water containing estriol before
analysis on LC-MS/MS [13]. Recently, an LC-MS/MS
86
Aldosterone

method for aldosterone measurement with electrospray assay. Results of urinary aldosterone depend on the
ionization has been developed, with a lower limit of amount of sodium in the diet, where levels are lower
detection of 30 pmol/L and interassay coefficients of with greater intake of sodium. For adults with normal,
variation (CVs) between 4.3% and 7.5% at aldosterone low-sodium, and high-sodium diets, reported urinary
concentrations of 97 to 993 pmol/L (Table 1, Method 5) aldosterone levels are 2 to 21, 17 to 44, and 0 to 14
[12]. g/day, respectively. If the 24-hr urinary sodium
excretion is > 200 mEq/day, urinary aldosterone
Reference and Preferred Methods should be < 10 g [16,17].
ID-GC/MS is the reference method for determination of
aldosterone concentration. Although this technique
provides reliable results, methods utilizing GC/MS require
a labor-intensive and time-consuming sample Plasma/Serum Aldosterone Reference Intervals
derivatization step. Liquid chromatography tandem MS (RIA)
(LC-MS/MS) has recently been proposed to replace Age Position Reference SI Units
GC/MS to circumvent the laborious derivatization [12]. Interval (pmol/L)
Immunoassays are, however, favored for measuring (ng/dL)
aldosterone in large numbers of small-volume samples. 1-12 5-90 139-2493
Immunoassays may be problematic unless extracted months
because of low aldosterone concentration and cross- 1-2 years 7-54 194-1496
reactivity. 2-10 Supine 3-35 83-970
years
Specimen 2-10 *Upright 5-80 139-2216
Serum, plasma, and urine specimens can be used. For years
serum, venous blood is to be collected in a glass tube with 10-15 Supine 2-22 55-609
no additives; plasma can be collected in a glass or plastic years
tube with the anticoagulants heparin or EDTA. Serum or 10-15 Upright 4-48 111-1330
plasma should be stored frozen and are stable for up to years
years at 20C in an airtight container. >15 years Supine 3-16 83-443
>15 years Upright 7-30 194-831
For urine analysis, 24-hour urine collection is required, and Adrenal 200-800 5540-
the specimen should be refrigerated during collection. vein 22,160
Acetic acid (50% preferred) is added after completion of Data from Endocrine Sciences. Tarzana, CA: Pediatric
collection to achieve a pH of 2.0 to 4.0. Other allowed Laboratory Services; 1992.
preservatives include thymol and boric acid. Strong * Patient should be seated or standing for at least 2
mineral acids should not be used. Samples may be stored hours prior to collection of upright specimen.
frozen.
Interpretation
Elevated concentrations of aldosterone are observed in
Interferences primary aldosteronism due to aldosterone-secreting
Cross-reactivity with other steroids such as 5- adenomas (Conns syndrome). Primary aldosteronism
dihydroaldosterone and corticosterone depends on is characterized by suppressed renin activity and
antibody specificity, which varies in different cannot be stimulated by either sodium restriction or
immunoassays. Grossly hemolyzed or lipemic samples or treatment with a diuretic, or by demonstrating lack of
contamination of the sample or tube with 125I or other suppression of aldosterone following saline infusion or
radioisotopes would interfere with RIA. Also, administration of a mineralocorticoid. Pseudoprimary
pharmacological interference was observed in women aldosteronism is due to bilateral adrenal hyperplasia.
taking drospirenone, a synthetic progestin with anti- Sampling of adrenal venous renin and aldosterone can
mineralocorticoid activity and aldosterone-receptor be used to differentiate between adenomas and
antagonism. This drug was reported to interfere with hyperplasia as the cause of hyperaldosteronism. In
laboratory screening and confirmatory testing for hyperplasia, both adrenals secrete high levels of
diagnosing primary aldosteronism [15]. Angiotensin, aldosterone. Secondary aldosteronism may be
estrogens, laxatives, oral contraceptives, sodium observed in laxative abuse, cardiac failure, diuretic
restriction, and thiazide diuretics lead to elevated abuse, and Bartters syndrome (a rare, inherited defect
aldosterone concentration. Aminoglutethimide, ACE in the thick ascending limb of the loop of Henle).
inhibitors such as captopril, lisinopril, deoxycorticosterone,
prolonged heparin therapy, and saline decrease aldosterone Aldosterone concentration is important in the
concentration. assessment of persons with primary hypertension.
Primary hyperaldosteronism has recently become
accepted as a more frequent cause of hypertension
Reference Intervals [18]. On the other hand, low aldosterone concentration
Normal value ranges vary among different laboratories with hypertension is associated with a number of
using different assays. It is recommended that each conditions such as Turners syndrome, diabetes
laboratory establish reference ranges for their particular mellitus, acute alcoholic intoxication, and excess
87
Aldosterone
secretion of deoxycorticosterone. Low concentrations
without hypertension are observed in Addisons disease
(primary adrenal insufficiency) and in the syndrome of
hypoaldosteronism due to renin deficiency.
Measurement of aldosterone concentration by itself is of
little value, and irregularities in blood pressure and/or
disorders of sodium and potassium homeostasis are best
investigated by assessing the integrity of the renin-
angiotensin-aldosterone axis [11]. This is usually done by
measuring renin and aldosterone concentrations. Although
aldosterone measurements alone are difficult to interpret,
they may be useful for assessing response of the adrenal
cortex to stimulation. The aldosterone-to-renin ratio (ARR)
has been proposed as a screening tool for primary
aldosteronism. The clinical utility of the ARR remains
questionable because of many factors that can affect ARR
results. These include posture, time of blood collection,
and use of antihypertensive medication. A number of ARR
cutoff values have been published, owing to differences in
populations studied, collection methods, and hormone
assays used. There is no agreed-upon ARR cutoff value,
and it is unknown which population should be screened
[19,20]. The urinary aldosterone-to-active-renin ratio has
been suggested as the best independent predictor of cure of
hypertension after adrenalectomy in patients with
aldosterone-producing adenomas [21].
Performance Goals
Clinical Laboratory Improvement Amendments acceptable
performance criteria (CLIA 88) for measurement of
aldosterone require that laboratories be accurate to within
3 SD of the peer-group mean. Survey data from the
College of American Pathologists 2007 participant
summary report show imprecision values (% CV) for
extracted methods of 14.9% at a mean concentration of
50.2 g aldosterone/L and 20.3% at a mean concentration
of 14.8 g/L [22].
Within-subject and between subject biological variation
were 29.4% and 40.1% (serum) and 32.6% and 39%
(urine), respectively. Desirable specifications for analytical
imprecision derived from studies of biological variation
indicate an assay imprecision of no greater than 14.7%
(serum) and 16.3% (urine) and a bias of 12.4% (serum) and
12.7% (urine) [23].
References
1 Stckl D, Reinauer H, Thienpont LM, De
Leenheer AP. Determination of aldosterone in
human serum by isotope dilution gas
chromatography/mass spectrometry using a new
heptafluorobutyryl derivative. Biol Mass
Spectrom 1991;20:657-64.
2 Fredline VF, Taylor PJ, Dodds HM, Johnson AG.
A reference method for the analysis of aldosterone
in blood by high-performance liquid
chromatographyatmospheric pressure chemical
ionization-tandem mass spectrometry. Anal
Biochem 1997;252:308-313.
3 Cawood ML. Measurement of aldosterone in
blood. Methods Mol Biol 2006;324:177-85.
4 Jaffe BM, Behrman HR, eds. Methods of
Hormone Radioimmunoassay. New York:
Academic Press; 1979.
5 Bayard F, Beitins IZ, Kowarski A, Migeon
CJ. Measurement of aldosterone secretion
rate by radio-immunoassay. J Clin Endocrinol
Metab 1970;31:507-11.
6 Jowett TP, Slater JD, Piyasena RD, Ekins RP.
Radioimmunoassay of aldosterone in plasma
and urine: validation of a novel separation
technique and a rapid urine assay. Clin Sci
Mol Med 1973;45:607-23.
7 Stowasser M, Gordon RD. Aldosterone
assays: an urgent need for improvement. Clin
Chem 2006;52:1640-2.
8 Atherden SM, Corrie JE, Jones DB, Al-
Dujaili EA, Edwards CR. Development and
application of a direct radioimmunoassay for
aldosterone in saliva. Steroids 1985;46:845-
55.
9 Hubl W, Taubert H, Freymann E, Hofmann
F, Meissner D, Garten CD et al. A simple
solid-phase enzyme immunoassay for
aldosterone in plasma and saliva. Exp Clin
Endocrinol 1983;82:188-93.
10 Hanquez C, Rajkowski KM, Desfosses B,
Cittanova N. A competitive microtitre plate
enzyme immunoassay for plasma aldosterone
using a monoclonal antibody. J Steroid
Biochem 1988;31:939-45.
11 Schirpenbach C, Seiler L, Maser-Gluth C,
Beuschlein F, Reincke M, Bidlingmaier M.
Automated chemiluminescence-immunoassay
for aldosterone during dynamic testing:
comparison to radioimmunoassays with and
without extraction steps. Clin Chem
2006;52:1749-55.
12 Turpeinen U, Hmlinen E, Stenman UH.
Determination of aldosterone in serum by
liquid chromatography-tandem mass
spectrometry. J Chromatogr B Analyt
Technol Biomed Life Sci 2008;862:113-8.
13 Mayo Medical Laboratories Test Catalog.
Available at
<http://www.mayomedicallaboratories.com/te
st-catalog/>
14 Perschel FH, Schemer R, Seiler L, Reincke
M, Deinum J, Maser-Gluth C et al. Rapid
screening test for primary
hyperaldosteronism: ratio of plasma
aldosterone to renin concentration determined
by fully automated chemiluminesc-ence
immunoassays. Clin Chem 2004;50:1650-5.
15 Pizzolo F, Pavan C, Corrocher R, Olivieri O.
Laboratory diagnosis of primary
aldosteronism and drospirenone-
ethinylestradiol therapy. Am J Hypertens
2007;20:1334-7.
16 Bravo EL. Primary aldosteronism. Issues in
diagnosis and management. Endocrinol
Metab Clin North Am 1994;23:271-83.
17 Young WF Jr. Pheochromocytoma and
primary aldosteronism: diagnostic
88
Aldosterone

approaches. Endo-crinol Metab Clin North Am
1997;26:801-27.
18 Mulatero P, Dluhy RG, Giacchetti G, Boscaro M,
Veglio F, Stewart PM. Diagnosis of primary
aldosteronism: from screening to subtype
differentiation. Trends Endocrinol Metab
2005;16:114-9.
19 Giacchetti G, Ronconi V, Lucarelli G, Boscaro M,
Mantero F. Analysis of screening and
confirmatory tests in the diagnosis of primary
aldosteronism: need for a standardized protocol. J
Hypertens 2006;24:737-45.
20 Jansen PM, Boomsma F, van den Meiracker AH.
Aldosterone-to-renin ratio as a screening test for
primary aldosteronism: the Dutch ARRAT Study.
Neth J Med 2008;66:220-8.
21 Mourad JJ, Girerd X, Milliez P, Lopez-Sublet
M, Lejeune S, Safar ME. Urinary
aldosterone-to-active-renin ratio: a useful tool
for predicting resolution of hypertension after
adrenalectomy in patients with aldosterone-
producing adenomas. Am J Hypertens
2008;2:742-7.
22 College of American Pathologists. 2007
Survey Participant Summary Report.
Northfield, IL: CAP; 2007.
23 Rics C, Alvarez V, Cava F, Garca-Lario JV,
Hernndez A, Jimnez CV et al. Current
databases on biologic variation: pros, cons
and progress. Scand J Clin Lab Invest
1999;59:491-500.

Table 1: Characteristics of Selected Aldosterone Assays

*Method 1: DSL Active Aldosterone (non-extraction)
Sample (volume): Serum/plasma (100 L)
Detection: Polyclonal antibody, 125I tracer
Range (ng/L): 2-1600
Intraassay variability (%): 3.6-8.3
Interassay variability (%): 7.3-10.4
Reference interval (ng/L): 30-340 (serum); 30-220 (plasma)
*Method 2: Adaltis Aldosterone Maia (non-extraction)
Sample (volume): Serum/plasma (50 L)
Detection: Polyclonal antibody, 125I tracer
Range (ng/L): 6-2500
Intraassay variability (%): 3.5-5.4
Interassay variability (%): 3.6-6.4
Reference interval (ng/L): 70-350 (plasma)
*Method 3: Nichols Advantage Aldosterone [14]
Sample (volume): Serum/plasma (450 L)
Detection: Monoclonal antibody, chemiluminescence
Range (ng/L): 15-1200
Intraassay variability (%): 2.9-14.0
Interassay variability (%): 4.9-18.6
Reference interval (ng/L): 38-313 (serum)
Method 4: High-performance liquid chromatography atmospheric pressure chemical ionization
tandem mass spectrometry (HPLC-APCI-MS/MS) [2]
Sample: Serum/plasma
Limits of detection and quantification (pg/mL): 10 and 15
Linear range (pg/mL): 15-500
Assay imprecisions at 15, 20, 150, and 450 pg/mL were 18.5%, 8.8%, 10.6%, and 9.5%
Method 5: Liquid chromatographytandem mass spectrometry (LC-MS/MS) [12]
Sample (volume): Serum (500 L)
Lower limit of detection (pmol/L): 30
Linear range (pmol/L): 60-3000
Interassay variability (%): 4.3-7.5
* Commercially available immunoassays.
89
Aldosterone
90
Alkaline Phosphatase - Total

Alkaline Phosphatase - Total

Danyal B. Syed
Name: Alkaline phosphatase, ALP, AP, ortho-phosphoric monoester
phosphohydrolase (alkaline optimum)
Clinical significance: Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Enzyme number: EC 3.1.3.1
Molecular weight: Varies with tissue source of enzyme, ranges from 70,000 to 120,000 D
Chemical class: Enzyme, protein
Known enzyme forms: Bone, liver, placenta, placental-like, intestine, kidney, neutrophil, high
molecular weight, Regan (fetal), Nagao, Kasahara, hepatoma
Number of subunits: Two
Chromosomal location: Tissue-specific ALP = 2q37.1 [1] Tissue nonspecific ALP = 1p34-36
[2]
Number of mutations : Tissue nonspecific ALP (TNSALP) as of January 7, 2008 = 194 [3]
Number of polymorphisms: 12 [3]
Biochemical reaction:
R2-OH + R1-O-P-O3- ALP, pH>9 R1-OH + R2-HPO4-
Net reaction is hydrolysis if R2OH is H2O

Principles of Analysis and Current Usage
The group of nonspecific phosphatases that catalyze the
reaction shown above are collectively known as alkaline
i metal ions (such as EDTA, oxalate, citrate, cysteine, and
phosphatase (ALP)
. Phosphatases transfer a phosphate moiety from one
group to a second, forming an alcohol and a second
phosphate compound. When water is the phosphate
acceptor, inorganic orthophosphate is formed.
The earliest assays for total serum ALP measured the
release of inorganic phosphate. Newer ALP methods use
self-indicating, chromogenic substrates and rate-
enhancing buffers, leading to significant improvements
in analytical sensitivity and precision. The older ALP
assays are now of historical interest only [4-9]. These
have been extensively reviewed by McComb et al. [10],
and more recently Millan [11] has discussed (in a
general manner) various methodologies available for
assaying ALP activity. These include spectrophotometric
(including inhibition-based), amperometric,
potentiometric, electrophoretic, and high-performance
liquid chromatography (HPLC) assays. The reader is
also referred to the earlier edition of this book for critical
review of the procedures that were employed in the
clinical laboratory in the past.
The optimal reaction pH is between 9 and 10.5 and
varies with the buffer and substrate. ALP requires Mg2+
i
Alkaline Phosphatase
Previous and current authors of this method:
First edition: William C. Wenger, John A. Lott
Methods edition: Steven C. Kazmierczak, John A. Lott
Second edition: Steven C. Kazmierczak, John A. Lott
Third edition: Julie Raymond-Habecker, John A. Lott
Fourth edition: Julie Raymond-Habecker, John A. Lott
Fifth edition: Danyal B. Syed
and Zn2+ ions for stability and maximum activity. It is
inhibited by Ca2+ and inorganic phosphate,
monoethanolamine, Be2+, chelating agents of bivalent
histidine), non-alkaline pH, phenylalanine, tryptophan,
L-homoarginine, urea, iodoacetamide, and high
concentrations of Zn2+.
Reaction rates are dependent on the incubation
temperature, the tissue source of the enzyme, and the
type of substrate and buffer used. ALP is denatured
slowly at temperatures above 40C. All isoenzymes of
ALP except placental and germ cell ALPs lose their
activity rapidly at temperatures above 60C. Since the
physiological substrates of ALP are unknown, most of
the assays used in clinical laboratories for ALP employ
p-nitrophenyl phosphate (pNPP) as the substrate. The
Km value of ALP for pNPP is dependent upon a variety
of factors, including the source of the enzyme, pH and
nature of the buffer, temperature, and ionic strength. At
an alkaline pH, pNPP is colorless, but the reaction
product p-nitrophenol (pNP) is intensely yellow with a
molar absorptivity of about 18,450 Lmol-1 cm-1 at
403 nm (Figure 1).
Transphosphorylating buffers greatly increase the rate of
reaction compared to the barbital, carbonate, or glycine
buffers used in the older methods [4-6]. The most
commonly used transphosphorylating buffers are 2-
amino-2-methyl-1-propanol (AMP), diethanolamine
(DEA), Tris, and N-methyl-D-glucamine (MEG) [12].
Mannitol also acts as a transphosphorylating phosphate
acceptor and is often used as an accelerator in some
methods for ALP. In U.S. patent filing 6713275 [13],
Weisheit, Ralph and Treiber,Wolfgang describe a
method for the determination of ALP activity using
pNPP as substrate but at a wavelength of 450 10 nm
91
Alkaline Phosphatase - Total
instead of 410 5 nm in combination with a rate blank
procedure to eliminate hemoglobin interference, which
also absorbs around 415 nm.
Lack of agreement between widely used ALP methods is
common [14,15]. While this might be due to different
temperature conditions in different assays, it was
observed that AMP activates intestinal ALP more than it
does liver or bone ALP. Such findings have clinical
implications when patients have a high serum intestinal
ALP, such as patients on chronic hemodialysis or those
with liver cirrhosis [15].
Current Assay Methods for Measurement of ALP
The current methods being used in clinical laboratories
around the world are all spectroscopic methods; they
include both colorimetric and fluorometric assays. The
colorimetric methods using p-NPP as substrate are
widely used in the clinical laboratory, and the
fluorometric methods using 4-methyllumbelliferyl
phosphate [16], FDP (3,6 fluorescein diphosphate) [17],
and other such compounds as substrates have gained
popularity in research and development, especially in
ALP-conjugated ELISA assays and in the detection of
immobilized amplified products in one phase system
(DIAPOPS) [18].
There has not been much change in the current assay
methods for the measurement of ALP in the past 3
decades. The latest emphasis is on the standardization
and traceability of the assays. McComb and Bowers [19]
suggested that the variation in the reported incompatible
numerical data for ALP found in large interlaboratory
surveys could be reduced from a coefficient of variation
(CV) of 25% to 30% to a CV of as low as 2% by
expressing all numerical values on a single scale: the
International Clinical Enzyme Scale (ICES). The ICES
for ALP requires a well-defined reference system that
relates the IFCC Reference Method for ALP to
numerous stable primary and secondary ALP reference
materials. Thirteen years later at an IFCC General
Conference in Sevilla, Spain, it was decided to establish
a global reference system for the measurement of
catalytic concentrations of enzymes comprising the
following elements [20]:
Reference Measurement Procedures: Use the
existing 30C IFCC reference method as a basis for
developing a set of standard operating procedures
(SOPs) for a reaction temperature of 37C.
Network of Reference Laboratories: A group of
reference laboratories, including manufacturers
laboratories, which will provide necessary expertise
and equipment to conduct measurements following
the SOPs to a high metrological level.
Reference Materials: The existing BCR reference
materials available from International Reference
Materials and Methods (IRRMEurope) are to be
recertified by the network reference laboratories
according to the cooperation contract between IFCC
and IRMM.
The main objective of introducing reference systems for
the measurement of analytes in a clinical laboratory is to
have measurable traceability. Traceability in itself is the
most important means of achieving standardization in
the clinical laboratory field, with the goal of having
comparable results regardless of the method, the
measurement procedure (test kit) and the location of the
laboratory. International Standards Organization (ISO)
[21], the European Union [22], and CLSI all have joined
together to achieve this goal. Currently there is no IFCC
primary method available for ALP. However, most of
the instruments and kit manufacturers have incorporated
the 37C temperature for the ALP activity measurement
in their procedures as proposed. Some of the instrument
and ALP kit manufacturers, on their own, have already
begun using the calibrators and reference materials to
standardize ALP assays. Schiele et al. [23] reported an
IFCC-certified procedure for preparing a lyophilized
preparation of ALP that can be used as an enzyme
reference substance. The enzyme was partially purified
from pig kidney to an activity of 400 U/mg of protein
while largely free from contaminating enzyme activities.
The ALP was lyophilized in a matrix also containing
bovine serum albumin, MgCl2, ZnCl2, and NaCl. Vial-
to-vial catalytic concentration variability of the final
product was < 1% and the predicted annual loss of
activity was < 0.01% at 20C and < 0.04% at 4C. A
patent has been recently granted for a tissue-nonspecific
alkaline phosphatase conjugate for use as a suitable
reference or standard ALP material [24]. In this
preferred embodiment, a tissue-nonspecific ALP (tns-
AP), which can be obtained by recombinant expression
of nucleic acid coding for the AP in a prokaryotic cell,
preferably Escherichia coli, is used as an unglycosylated
tns-AP to produce the conjugate, with the dextran having
a molecular weight of 10 to 500 kDa. The claimed
advantages of this invention are its reproducibility, better
stability, and absence of risk of infection from the blood-
borne pathogens.
Reference and Preferred Methods
Currently there is no formal reference method for the
determination of ALP activity. The kinetic pNPP method
of Bowers and McComb [25,26] is a candidate reference
method for the Clinical Laboratory Standards Institute
Expert Panel on Enzymes (CLSI/EPE). It has also been
adopted by the World Health Organization for use as
Guidelines on Standard Operating Procedures for
Clinical Chemistry: Alkaline Phosphatase p-
Nitrophenol Method [27]. The method offers the
convenience and sensitivity of pNPP in AMP buffer, and
bilirubin does not interfere. With careful work, within-
day CVs of 3% to 6% have been reported [26].
The specific parameters of the assay are summarized in
Table 1. The Scandinavian Society for Clinical
Chemistry and Clinical Physiology [28] and The German
Society for Clinical Chemistry [29] have similar
methods. The American Association for Clinical
Chemistry (AACC) reference method optimizes all
reaction conditions, including temperature, pH, reagent
concentrations, and sample volume fraction [30].
Most of the clinical laboratories in the developed world
are using automated procedures with reagents,
92
Alkaline Phosphatase - Total

calibrators, and quality controls (in some cases) supplied significant concentrations of monoethanolamine (MEA),
and reaction conditions set by the manufacturers of the a potent inhibitor of ALP. Solutions of DEA can
instruments, thereby eliminating most sources of errors. deteriorate during storage, with the concomitant
formation of MEA. AMP also contains ALP inhibitors
The measured activity of ALP depends on the volume such as diamines and 5-amino-3-aza-2,2,5-
fraction of serum. Increases in activity, corrected for trimethylhexanol that inactivate the enzyme by binding
2+
dilution, have been observed when the serum fraction Zn . Some commercial pNPP substrate preparations
was decreased from 0.04 to 0.02; no further increases contain excessive amounts of pNP or inorganic
were seen below the 0.02 volume fraction. The cause of phosphate, or both, with the former producing high
this effect is unknown, but it may result from the blank absorbance and the latter inhibiting ALP. See also
dissociation of ALP aggregates at the greater dilutions. under Interpretation for interferences due to drugs.
The German Society for Clinical Chemistry proposed a Alkaline Phosphatase, Total - Reference Intervals
reference method for ALP in serum and plasma at 37C The serum reference intervals for healthy persons,
[31]. The method differs in that methylglucamine, a determined at 30C by the Bowers and McComb
substance with moderate phosphate-acceptor properties, method, are shown below [26,34]. Each laboratory must
is used as a buffer. Methylglucamine was proposed by
establish its own reference ranges either by direct data
Chromy et al. [32] and recommended by the Italian
collection or by performing correlation studies and then
Society [33] because of inhibitors in amino alcohol
buffers and the absence of same in methylglucamine. verifying the adopted reference ranges.
The reaction is started with the substrate, and linear
reaction rates prevail during prolonged measurements at ALP Reference Intervals [26]
37C. When compared to DEA, methylglucamine has a Group, ALP, Group, ALP,
favorable pK value (9.63 at 37C), has a low viscosity at Age in Years Up to U/L Age in Years
optimal buffer concentrations, and has approximately Females: Males:
equal reactivity to all human ALP isoenzymes. pNPP is Newborns 250 Newborns
used as the substrate because of the rapid enzymatic 1 to 9 350 1 to 19
hydrolysis, the high absorptivity of pNP at a pH of 10.0, 10 to 14 280 10 to 14
and a negligible isoenzyme bias. A reagent blank must 15 to 19 150 15 to 19
be used, because p-nitrophenol is generated 20 to 24 85 20 to 24
spontaneously at 37C. 25 to 34 85 25 to 34
35 to 44 95 35 to 44
Specimen 45 to 54 100 45 to 54
Blood should be drawn after a fast of at least 8 hours. 55 to 64 110 55 to 64
Serum and heparinized plasma give the same results. 65 to 74 145 65 to 74
Slight hemolysis is tolerable, but gross hemolysis should 75+ 165 75+
be avoided. Certain sample storage conditions tend to
increase serum ALP. There is a significant increase in Note: The values at 30C can be converted to values at
activity after warming of previously refrigerated or 37C by multiplying the result with a factor of
frozen sera. The ALP activity in fresh serum increases 1.344.
by up to 2% in 6 hours at 25C. Increases of up to 30% Physiological factors causing increased ALP activities
of ALP activity occur after frozen serum is thawed and include age, gender, race, body mass, food intake,
in lyophilized specimens after reconstitution. These smoking, and pregnancy.
increases may be due to the release of ALP from
complexes with lipoproteins or because the non- Interpretation
complexed enzyme has greater activity. It is best to The human ALPs (hALP) are found anchored on the cell
analyze ALP specimens the same day they are drawn. membrane by glycosylphosphatidylinositol. They are
Specimen can be drawn either in a plain red-top tube or released in the serum by the action of specific
speckled-red-top tube with gel as serum separator, phospholipases [35,36]. There are four isozymes: (1)
employing normal phlebotomy procedures. To avoid placental AP or hPLAP (human placental AP), (2) germ
contamination, do not draw tubes containing cell AP (GCAP or PLAP-like), (3) intestinal AP (IAP),
anticoagulants before the red-top/speckled-red-top tube. and (4) tissue-nonspecific AP (TNAP). Of these four,
PLAP and GCAP are the most heat stable at 65C, and
Interferences the bone AP component of TnAP the least. The
ALP is inhibited by metal-complexing anticoagulants. homology between PLAP and GCAP are about 98%,
All of EDTA, oxalate, and citrate inhibit the enzyme by whereas intestinal AP and TnAP exhibit 88% and 56%
complexing Mg2+ and Zn2+ and should not be used. homologies, respectively [37]. In nonsmoking healthy
Hemolyzed and lipemic specimens should be rejected if individuals, the PLAP and GCAP represent less than 1%
they have a high background absorbance. Bilirubin at of total AP activity in the serum. Smoking causes
concentrations of up to 20 mg/dL does not interfere. elevated levels of PLAP, which return to normal range
after 1 to 2 months of smoking cessation [38-40].
The purity of transphosphorylating buffer is of great Ectopically expressed PLAP has been associated with
importance. Sources of DEA have been found to contain cancer of the ovary, testis, lung, and colorectal tract [41].
93
Alkaline Phosphatase - Total
The PLAP-like enzyme (GCAP), on the other hand, has
been associated with testicular cancers, seminoma,
choriocarcinoma, and embryonal carcinoma. Between
25% and 66% of patients with ovarian cancer and 22%
to 89% of patients with testicular carcinoma were found
to have elevated serum levels of PLAP and PLAP-like
enzyme [38,42]. With 98% homology, these two
isozymes are difficult to assay. Specific immunoassays
have been developed for placental-like alkaline
phosphatase [43], which could be employed to monitor
tumor regression.
Serum ALP activity primarily reflects changes in bone
and liver function, even though higher ALP activities
can be found in other organs, as depicted in Table 2. One
fourth of all normal persons have low serum activities of
intestinal ALP at all times. Individuals with blood types
B and O exhibit increases in serum activity of intestinal
ALP approximately 2 hours after eating a fatty meal.
Mutations in the TnAP gene have been associated with
hypophosphatasia, a rare inherited disorder manifesting
itself in poor bone mineralization and even death at a
very young age. The status of the fetus for risk of having
missense mutations leading to severe hypophosphatasia
can be assessed by: (i) by measuring the ALP activity in
the amniotic fluid supernatant and fetal serum [44], (ii)
by using the allele specific oligonucleotide mis-sense
mutational (previously known) probes on DNA extracted
from the amniocytes (amniotic fluid cells) in the ALP
gene and look for the same mutations [45].
Electrophoretic technique is most commonly used for
differentiating among the ALP isozymes and isoforms,
but assays based on heat stability of these enzymes are
also employed. As stated earlier, serum ALP levels are
affected by many drugs, physical conditions, herbal
medicines, food intake, smoking, alcohol intake, and
pregnancy. Some 1000 drugs, herbs and physiological
conditions that affect ALP activity have been
documented [46]. The effects of some of the most
common substances are described below:
Clofibrate lowers serum ALP activity, reducing all ALP
fractions except liver; this may be caused by an
increased biliary clearance of ALP. Azathioprine lowers
ALP by an unknown mechanism. Estrogens, alone or in
combination with androgens, depress ALP activity,
whereas in other studies, estrogens and androgens
increased serum ALP activity [47,48]. The decrease seen
with estrogen therapy may result from a lowered rate of
bone turnover.
Verapamil, widely used in the treatment of cardiac
arrhythmias, angina pectoris, and essential hypertension,
increases serum ALP in hypertensive patients [49].
When verapamil was administered to hypertensive
patients for 2 months, total ALP activity increased
significantly from 166 to 191 U/L (P = 0.004), and an
increase in bone ALP in serum was subsequently
observed. These results suggest that verapamil may
affect bone metabolism that is secondary to the
enhancement of parathyroid hormone secretion.
Any drug that is hepatotoxic or induces cholestasis will
increase serum ALP, sometimes dramatically. Young
[50] lists approximately 250 drugs that increase serum
ALP. Both increases and decreases of plasma ALP are
important clinically. Table 3 lists some of the disorders
and diseases associated with abnormal serum activities
of ALP [51]. ALP is a sensitive indicator of liver
obstruction; however, ALP is also increased in bone
diseases. Frequently, ALP is measured along with other
analytes considered more specific for liver diseases, such
as 5-nucleotidase or -glutamyl transferase, to permit
differentiation between liver and bone as the source of
an increased serum ALP [51]. ALP is often interpreted
as abnormal, particularly in children and in geriatric
patients, because of the use of inappropriate reference
intervals by a laboratory.
Alkaline Phosphatase Performance Goals
Acceptable performance (accuracy) for alkaline
phosphatase assays according to the Clinical Laboratory
Improvement Amendments of 1988 (CLIA-88) is the
target value 30%, which is also the acceptability
criteria used by the College of American Pathologists
(CAP) in its surveys. A laboratory must score 80% in
each testing event for each analyte and overall in all
analytes [52].
Within-day precision of the automated methods ranges
from 2% to 6%. Long-term precision within most
laboratories is also acceptable. Over a 3-year period, a
CV of about 5% is typical for procedures using pNPP.
The inter-laboratory precision of the methods is much
poorer. In a CAP interlaboratory proficiency survey in
the past, ALP was among the least precise of the
common enzyme tests [53,54]. Reported interlaboratory
CVs ranged from 17% to 28%. The factors contributing
to poor interlaboratory precision are impure reagents,
slight variations in the reaction temperature, subtle
enzyme activation and inactivation phenomena, and
variations in conditions of specimen storage. In
comparison, the CV ranges exhibited by commonly used
instruments in a recent CAP participant summary report
[55], as depicted in Table 4, show that the
interlaboratory CVs within the same instrument group
have improved from 17% to 28% to < 2% to 13.7%.
Even among the peer groups, the interlaboratory CVs
exhibit improvement. With efforts focused on reagent
quality by the manufacturers, specified assay conditions,
and traceability to ERMs, all methods will correlate
well, and interlaboratory CVs will be reduced further to
2% as envisioned. Each laboratory must establish its
own acceptability criteria for intra-daily and inter-daily
variations based on the instrument and quality-control
materials.
References
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94
Alkaline Phosphatase - Total
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(inventors); Roche Diagnostics Operations Inc
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description.html
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total alkaline phosphatase activity in human
serum. Clin Chem. 1975;21:1988-1995.
26 Bowers GN Jr, McComb RB. Alkaline
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Washington, DC: American Association for
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27 World Health Organization Regional Office for
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http://www.searo.Who.int/EN/Section10/Sectio
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Alkaline Phosphatase - Total
28 Scandinavian Society for Clinical Chemistry
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methods for the determination of four enzymes 42
in blood. Scand J Clin Lab Invest. 1974;33:291-
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29 German Society for Clinical Chemistry.
Recommendations of the Enzyme Commission. 43
Z Klin Chem Klin Biochem. 1972;10:281-291.
30 Tietz NW, Burtis CA, Duncan P, Ervin K,
Petitclerc CJ, Rinker AD et al. A reference
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31 Working Group on Enzymes. Proposal of
standard methods for the determination of
enzyme catalytic concentrations in serum and
plasma at 37C. I. Alkaline phosphatase. Eur J
Clin Chem Clin Biochem. 1992;30:247-256. 45
32 Chromy V, Zahradnicek L, Voznicek J. Use of
N-methyl-D-glucamine as buffer in
determination of serum alkaline phosphatase
activity. Clin Chem. 1981;27:1729-1732. 46
33 Ceriotti G, Bonvicini P, Certiotti F, Franzini C,
Prencipe I, Spandrio L. Valutazione di un 47
metdod per la determinazione dellattivit della
fosfatasi alcalina (ALP) con il tampone N-
metilgucammina (MEG). Proposta del suo uso
come metodo raccomandato. Giorn It Clin Clin. 48
1984;9:167-181.
34 Munan L, Kelly A, Petitclerc C, Billon B. Atlas
of Blood Data. Prepared by the Epidemiology 49
Laboratory and the Laboratory of Clinical
Biochemistry, University of Sherbrooke,
Quebec, 1980.
35 Lehto MT, Sharon FJ. Proximity of the protein
moiety of GPI-anchored protein to the 50
membrane surface: a FRET study.
Biochemistry. 2002;41:8368-8376.
36 Low MG, Prasad ARS. A phospholipase D 51
specific for the phosphatidylinositol anchor of
cell-surface proteins is abundant in plasma.
Proc Nat Acad Sci USA. 1988;85:980-984. 52
37 Le Du MH, Stigbrand T, Taussig MJ, Menez A,
Stura EA, Crystal structure of alkaline
phosphatase from human placenta at 1.8 A
resolution, implication for a substrate
specificity. J Biol Chem. 2001;276:9158 -9165.
38 Muensch HA, Maslow WC, Azama F, Bertrand
M, Dewhurst P, Hartman B. Placental-like 53
alkaline phosphatase: re-evaluation of the tumor
marker with exclusion of smokers. Cancer.
1986; 58: 1689-1694.
39 Hirano K, Matsumoto H, Tanaka T, Hayashi,
Y, Lino S, Domar U, Stigbrand T. Specific
assays for human alkaline phosphatase 54
isozymes. Clin Chim Acta. 1987;166:265-273.
40 Williams GH, McLaughlin PJ, Johnson PM.
Tissue origin of serum placental-like alkaline
phosphatase in cigarette smokers. Clin Chim 55
Acta. 1986;155:329-333.
41 Fishman L, Miyayama H, Driscoll SG,
Fishman WH. Developmental phase-specific
alkaline phosphatase isoenzymes of human
placenta and their occurrence in human cancer.
Cancer Res. 1976;36:2268-2273.
Weissbach L, Bussar Maattz R, Mann K, The
value of tumor markers in testicular seminomas.
Results of prospective multicenter study. Eur
Urol. 1997;32:16-22.
Stinghen ST, Moura JF, Zancanella P, Rodrigues
GA, Pianovski MA, Lalli E et al. Specific
immunoassays for placental alkaline
phosphatase as a tumor marker. J Biomed
Biotech. 2006;Article ID 56087:1-8.
Warren RC, Mckenzie CF, Rodeck CH,
Moscoso G, Brock DJ, Baron L. First-trimester
diagnosis of hypophosphatasia with a
monoclonal antibody to the liver/bone/kidney
isoenzyme of alkaline phosphatase. Lancet.
1985;2(8460):856-858.
Henthorn PS, Whyte MP. Infantile
hypophosphatasia: successful prenatal
assessment by testing TnALP isoenzyme gene
mutations. Prenat Diagn. 1995;15:1001-1006.
Youngs Effect online. Available at
http://www.fxol.org
Nanji AA. Decreased activity of commonly
measured serum enzymes: causes and clinical
significance. Am J Med Technol. 1983;49:241-
245.
Posen S, Doherty E. The measurement of serum
alkaline phosphatase in clinical medicine. Adv
Clin Chem. 1981;22:163-254.
Sjd'n G, Rosenqvist M, Kriegholm E,
Nordenenstrm J, Bjrkhem I. Verapamil
increases serum alkaline phosphatase in
hypertensive patients. J Intern Med. 1990; 228:
339-342.
Young DS. Effects of Drugs on Clinical
Laboratory Tests. 3rd ed. Washington, DC:
AACC Press; 1990:3-193-25.
Lott JA, Wolf PL. Clinical Enzymology: A
Case-Oriented Approach. Chicago: Year Book
Medical; 1986:57-74.
U.S. Department of Health and Human Services.
Medicare, Medicaid and
CLIA Programs: regulations implementing the
Clinical Laboratory
Improvement Amendments of 1988 (CLIA). Final
rule. Fed Regist.
1992;57:7002-186.
Lott JA, Massion CG. Interlaboratory quality
control of enzyme analyses: the CAP
experience. In: Hamburger HA, ed. Clinical
and Analytical Concepts in Enzymology. Skoki,
IL: College of American Pathologists;
1983:233-256.
Lott JA, Tholen DW, Massion CG. Survey of
serum enzyme analyses: human tissues as a
source of enzymes. Arch Pathol Lab Med.
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College of American Pathologists. 2007
Surveys: Chemistry/Therapeutic Drug
Monitoring (C-A) Participant Summary Report.
Northfield, IL: CAP; 2007:7-8.
96
Alkaline Phosphatase - Total

Tables

Table 1: Comparison of Original Reaction

Table 2: Mean ALP Activity in Tissue Specimens
Conditions for Alkaline Phosphatase (ALP) Analysis*
(AMP Buffer System) [25]
Condition: Temperature Tissue Activity
Manual reference (Bowers and McComb) [25]: 30C (molsec-1 kg-1 of wet tissue)
German [29]: 25C Placenta 600
Scandinavian [28]: 37C Adrenal 500
AACC [30]: 30C Liver 210
Condition: pH Bone 125
Manual reference (Bowers and McComb) Spleen 125
[25]: 10.5 Lung 110
German [29]: 9.8 Intestine (whole) 80
Scandinavian [28]: 9.8 Kidney 71
AACC [26]: 10.4 Prostate 55
Condition: Final concentration of reagents Brain 35
Manual reference (Bowers and McComb) [25]: Thyroid 30
pNPP: 16 mmol/L Myocardium 25
MAP: 1.0 mol/L
Mg2+: 1.0 mmol/L
German [29]:
pNPP: 10 mmol/L
DEA: 1.0 mol/L
Mg2+: 0.5 mmol/L
Scandinavian [28]:
pNPP: 10 mmol/L
DEA: 1.0 mol/L
Mg2+: 0.5 mmol/L
AACC [26]:
pNPP: 16 mmol/L
MAP: 0.35 mol/L
Mg2+: 2.0 mmol/L
Condition: Fraction of sample volume
Manual reference (Bowers and McComb)
[25]: 0.0164
German [29]: 0.009
Scandinavian [28]: 0.009
AACC [26]: 0.196
Condition: Linearity (approximate)
Manual reference (Bowers and McComb)
[25]: To 500 U/L
German [29]: To 500 U/L
Scandinavian [28]: Linear for 10 minutes or
up to 1500 U/L
AACC [26]: To 900 U/L
Condition: Precision (in reference interval)
Manual reference (Bowers and McComb)
[25]: 3% to 6%
German [29]: 5%
Scandinavian [28]: 5%
AACC [26]: 3% to 5%
AACC, American Association for Clinical Chemistry;
German, German Society for Clinical Chemistry;
Scandinavian, Scandinavian Society for Clinical
Chemistry and Clinical Physiology.
DEA, Diethanolamine; MAP, 2-methyl-2-amino-1-
propanol; pNPP, p-nitrophenyl phosphate.
*Major interferences: EDTA, citrate, oxalate, inorganic
phosphate, calcium, and ammonium sulfate.
It has been reported that a contaminant in some lots of
DEA causes significant loss of ALP activity [25].
97
Alkaline Phosphatase - Total

Thyroiditis
Malignancy
Table 3: List of Disorders That Usually Result in Bile duct adenocarcinoma
Abnormal Serum Activities Cervical cancer
(Adapted from Reference 51) Gynecological neoplasms
Causes of Increased Serum ALP Hodgkins disease
Bone disorders Liver, primary & secondary
Fractures Lung, ectopic production
Osteomalacia Lymphoma, leukemia
Pagets disease Maxillary carcinoma
Osteoblastic lesions Multiple myeloma
Osteomyelitis Osteosarcoma
Liver diseases Ovarian cancer
Biliary atresia Pancreatic cancer
Cholestasis Prostatic cancer
Cirrhosis Renal
Fatty liver, acute Testicular
Hepatitis Urinary bladder cancer
Portal hypertension Drugs and poisons
Primary biliary cirrhosis Alcohol
Kidney disease Cholestatogenic drugs
Chronic renal failure Cimetidine
Obstruction of urinary tract Furosemide
Renal disease Halothane
Renal infarction Hepatotoxic drugs
Renal vascular hypertension Papaverine
Gastrointestinal Phenobarbital
Crohns disease Phenytoin
Duodenal ulcer
Pancreatitis, acute Miscellaneous
Peptic ulcer Cystic fibrosis
Small bowel infarction Immunoglobulin complexed ALP
Bowel ulceration Infectious mononucleosis
Splenic infarct Pulmonary infarction
Ulcerative colitis Rheumatoid arthritis
Heart disease Systemic infection
Acute myocardial infarction Unexplained, transient increase
Congestive heart failure Causes of decreased serum ALP
Hematological disorders Drugs
Pernicious anemia Clofibrate
Sickle cell anemia Sulfonamides
Thalassemia Other drugs
Endocrine disorders Miscellaneous
Acromegaly Hypophosphatasia
Diabetes mellitus Malabsorption
Diabetes, uncontrolled Malnutrition
Hyperthyroidism Zinc deficiency
Hypothyroidism
Menopause
98
Alkaline Phosphatase - Total

Table 4: CV Ranges of Currently Used Major Instruments Systems

From a 2007 College of American Pathologists (CAP) Survey Report [55]

Instruments Manufacturer # of Labs Sp # Range of Means SD range CV range

And Reagent Supplier (T) Within the Group

Abbott all instruments (37C) 200 SP # 1 374.8 U/L 6.1 1.6

(Architect, Aeroset) 200 SP # 2 35.9 U/L 1.5 4.3
201 SP # 3 139.3 U/L 2.8 2.0

Bayer (Siemens) (37C) 183 SP #1 370.6 U/L 10.7 2.9

(All Instrument models) 182 SP #2 34.9 U/L 2.3 6.6
184 SP #3 136.9 U/L 4.5 3.3

Beckman (37C) 1378 SP # 1 380.0 388.6 U/L 7.1 12.0 1.9 3.1
(All Instrument models) 1381 SP #2 30.8 31.3 U/L 1.6 2.4 5.3 7.8
1376 SP #3 137.9 139.3 U/L 3.1 5.0 2.2 3.6

Dade Behring (37C) 1357 SP #1 294.9 U/L 12.1 4.1

Dimension 1371 SP #2 39.5 U/L 5.3 13.3
1371 SP #3 116.9 U/L 7.0 6.0

Olympus (37C) 267 SP #1 357.3 U/L 15.8 4.4

268 SP #2 31.8 U/L 2.0 6.2
267 SP #3 131.0 U/L 5.7 4.3

Roche (37C) 789 SP #1 310.9 330.8 U/L 9.7 14.3 3.0 4.3
795 SP #2 28.7 35.3 U/L 1.1 3.9 2.9 13.7
794 SP #3 119.6 131.5 U/L 3.8 6.0 3.2 - 4.9

Vitros Systems (37C) 822 SP #1 323.8 338.1 U/L 10.211.8 3.1 3.5
821 SP #2 48.7 51.4 U/L 2.0 -2.2 4.1 4.3
818 SP #3 133.7 142.5 U/L 5.0 5.6 3.7 3.9

All Instruments All Models

4996 SP #1 294.9 - 388.6 U/L 6.115.8 1.64.4
5018 SP #2 28.7 50.4 U/L 1.5 - 5.3 2.9 -13.7
5011 SP #3 116.9 142.5 U/L 2.8 - 7.0 2.0 6.0
Note: For the sake of brevity, the data has been combined to reflect peer-group performance at three levels of
concentration (three CAP Proficiency Samples, CHM-03 to CHM-05). Conclusions drawn are exclusively of the author
himself and do not reflect the views of the CAP.
99
Alkaline Phosphatase - Total
Figures
Figure 1: ALP pNPP absorption spectra.
Visible-spectrum absorbance curves for substrate, p-
nitrophenyl phosphate, which has a maximum
absorbance at 311 nm at 25C in 10 mmol/L NaOH,
solid curve, and for product, p-nitrophenol, which has a
maximum absorbance at 402.5 nm at 30C in 2-amino-2-
methyl-1-propanol (pH 10.30, 30C), dot-dashed curve.
Procedure: NCCLS/EPE Reference Method
Principle
The colorless pNPP substrate is converted at alkaline pH
to the yellow pNP. The reaction is followed by
measurement of the increase in absorbance at 403 nm.
Reagents and Materials
1. AMP buffer (1.5 mol/L). Liquefy 2-methyl-2-
amino-1-propanol by warming to 30C to 35C.
Weigh 135 g of the liquid directly into a 1 L
volumetric flask, add 500 mL of distilled water,
and mix. Carefully add 190 mL of 1.0 mol/L
HCl to the flask. When the solution has cooled
to room temperature, bring to 1 L with distilled
water. Confirm that the pH at 30 C is 10.5.
This is stable for 1 month when stored in an
airtight container at 25C.
2. Magnesium acetate solution (3 mmol/L).
Dissolve 650 mg magnesium acetate 4 H2O in
1 L of water. Stable indefinitely at 4C.
3. p-Nitrophenyl phosphate solution (24.5
mmol/L). Dissolve 91 mg of disodium 2-
nitrophenyl phosphate 6H2O in 10 mL of
AMP buffer. Prepare fresh daily.
4. p-Nitrophenol spectrophotometric standard
solutions (1 mmol/L). Dissolve 139.1 mg of
pNP in 1 L of distilled water. Stable for several
months when stored in the dark. To prepare the
working standard solution, add 25 mL of the 1
mmol/L solution to 900 mL of AMP buffer, and
dilute to 1 L with distilled water. The working
standard solution is stable for at least 2 months.
Use this solution to confirm the molar
absorptivity of pNP at 403 nm.
Note: Label all reagents with name of reagents,
date of preparation, date of expiration, and
storage conditions. Any special precautions
must also be noted.
Assay
Equipment: Any recording spectrophotometer equipped
with a temperature-controlled cell compartment is
suitable for the procedure. The temperature should be
held constant at 37C 0.1C.
1. Add 50 L of specimen to 1.0 mL of
magnesium acetate solution. Thoroughly mix,
and incubate for 5 min at 37C.
2. Prewarm buffered pNPP solution to 37C, and
add 2.0 mL to the incubation mixture from step
1. Agitate thoroughly.
3. Transfer the reaction mixture to a cuvette with a
1-cm light path, and read the absorbance
change versus time at 403 nm for 2 min.
Readings can be taken immediately after
mixing.
Calculations
International units (U) of activity are expressed as
micromoles of p-nitrophenoxide formed per min.
Enzyme concentrations are expressed as international
units per liter (U/L). U/L can be calculated from the
change in absorbance by the following equation for a 1
cm light path:
U/L = A TV 106
t SV
where A = change in absorbance for time t, TV is the
total volume, SV is the specimen volume, and is the
molar absorptivity for p-nitrophenoxide (18.8 103 L
mol-1 cm-1); 106 is the factor to convert the
concentration to micromoles per liter. For the conditions
above,
U/L = A/min (3050 L/50 L) 106/18,800
or U/L = A/min 3245
The method is linear to approximately 500 U/L.
Procedure: German Society for Clinical Chemistry
Reference Method
Principle
Colorless pNPP is converted to the intensely yellow
pNP, and the phosphate group is transferred to the buffer
to form N-methyl-D-glucamine phosphate. The increase
in absorbance at 405 nm is followed.
Specimen
Refer to NCCLS document for Alkaline Phosphatase,
Total
Reagents and Materials
1. N-methyl-D-glucamine (500 mmol/L).
Dissolve 27.33 g of methylglucamine (560
mmol/L) and 1.15 g of sodium chloride (78.4
mmol/L, pH 10.6 at 20 C or 10.1 at 37C) in
about 200 mL of water. Adjust pH to 10.5 with
1 mol/L HCl, add 30 mg magnesium acetate
tetrahydrate, and bring to a total volume of 250
mL with distilled water. This is stable for at
100
Alkaline Phosphatase - Total
least 2 months at 20C if kept in a tightly
stoppered flask.
2. p-Nitrophenylphosphate (20 mmol/L).
Dissolve 4.16 g of disodium p-nitrophenyl
phosphate hexahydrate, and add water to a final
volume of exactly 50 mL. Prepared just prior to
use; it must be used within 8 hrs if stored at
20C to 25C or at the most within 1 day after
storage at 3C to 5C.
3. NaCl, (155 mmol/L). Dissolve 0.9 g of NaCl
and bring to 100 mL with distilled water.
Assay
Equipment: A spectrometer with a constant temperature
cuvette compartment is necessary. The temperature
should be held constant at 37C 0.1C.
1. Pipet 500 L of N-methyl-D-glucamine into a
cuvette, and add 10 L sample.
2. Mix thoroughly and incubate at 37C for at
least 300 sec or until the mixture has reached
this temperature.
3. Add 50 L of pNPP.
4. Mix and record the increase in absorbance after
60 sec at 405 nm.
Calculations
The reagent blank must be subtracted from the overall
change in absorbance: (A/t)overall- (A/t)blank =
(A/t)corrected. Use the corrected value in the
calculation above.
101
Alpha-Fetoprotein
Alpha-Fetoprotein
Gerald J. Mizejewski
Name: Alpha-fetoprotein, AFP
Clinical significance: Refer to Chapter 44, Pregnancy, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular weight: 69,000 D
Chemical class: Glycoprotein
Principles of Analysis and Current Usage
Introduction and Historical Background
Human alpha-fetoprotein (HAFP) is a tumor-associated
fetal mammalian glycoprotein involved with both
ontogenic and oncogenic growth [1,2]i. The fetal
protein is a 69-kDa single-polypeptide chain containing
3% to 5% carbohydrate; it exhibits a triplicate domain
structure configured by intramolecular loops dictated
by disulfide bridging [3]. In electrophoretic profile,
AFP occupies an alpha-1 anodic position running
slightly slower than albumin.
HAFP is synthesized in the yolk sac, fetal liver, and
gastrointestinal tract during pregnancy but is
reexpressed in multiple adult tumors of mixed
mesodermal/endodermal origin [4,5]. In the clinical
laboratory, HAFP has been employed both as a
postoperational tumor marker and as a gestational age-
dependent fetal defect marker demonstrating utility in
screening for neural tube defects and aneuploidies [6,7].
While maternal serum AFP (MS-AFP) concentrations
associated with neural tube fetal defects are elevated,
the chromosomal disorders are associated with much
lower levels.
Structural and Functional Aspects of AFP:
A. Structural Variants: Many molecular variants
of mammalian AFP have been reported in the scientific
literature since the 1970s (Table 2). Some of these
earlier variant forms of AFP were attributed to
carbohydrate microheterogeneity and isoforms
associated with varying isoelectric points [8,9]. Later
reports demonstrated AFP forms that were genetic
variants and lectin glycoforms demonstrable by
electrophoretic and chromatographic procedures
[10,11]. Still other variants were detected following
high-pressure liquid chromatography (HPLC) utilizing
lectin, heavy metal, and hydrophobic solid-phase
separation methodology [12]. Aberrant forms of HAFP
have been detected in the reproductive/and urinary tract
i Alpha-Fetoprotein
Previous and current authors of this method:
First edition: Not done
Methods edition: George J. Knight
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Gerald J. Mizejewski
of adult patients and in the sera of cancer patients
(breast cancer, hepatomas, etc.). Truncated forms of
HAFP (~50,000 D) have also been detected in cell
cultures comprising hepatomas, testicular embryonal
carcinomas, and breast tumors [12-14].
B. Biological Roles: Similar to albumin, serum
AFP is known to bind and transport a multitude of
ligands such as bilirubin, fatty acids, retinoids, steroids,
heavy metals, dyes, flavonoids, phytoestrogens, dioxin,
and various drugs [13,14]. Other ligands that bind to
AFP (rodent and human) include metabolic stains, L-
tryptophan, warfarin, triazine dyes, phenylbutazone,
streptomycin, phenytoin, anilinonaphthalene sulfate,
heavy metals, low-carbon-chain alcohols, and
polyunsaturated fatty acids [15]. Although the
physicochemical and structural properties of this
glycoprotein have been described, it has been the in
vitro functional roles that have been extensively
studied, especially the ligand carrier/transport functions
[12]. In addition, the ability of AFP to modulate native
and adaptive immune responses has also been pursued.
Since the first reports by Murgita et al. [16,17] in the
1970s, AFP has long been recognized as both a B- and
T- cell immunoregulatory agent. Overall, full-length
HAFP has been found immunosuppressive in both B-
and T-cell lectin stimulation, although AFP can also
induce immune cell stimulation under certain
conditions [18,19].
A multitude of studies have further established AFP as
a regulator of normal and neoplastic growth [20-24]. In
fact, it is the growth-modulating activity that
distinguishes AFP from albumin, the major blood
protein carrier/transport molecule of the albuminoid
gene family. The growth regulatory properties of AFP
have aroused investigational interest in studies of
ontogenetic and oncogenic growth in both cell cultures
and animal models. A myriad of reports have now
documented that AFP is capable of regulating growth in
ovarian, placental, uterine, hepatic, phagocytic, bone
marrow, and lymphatic cells in addition to neoplastic
cells (i.e., MCF-7 and MTW9A breast cancer)
[18,25,26]. Since the 1990s, AFP is no longer
considered only a biomarker for cancer and fetal
disorders; it is also viewed as a protein associated with
modulating cell proliferation, differentiation,
regeneration, and transformation in both ontogenetic
and oncogenic growth processes. HAFP has further
been shown to possess pro-angiogenic properties that
promote neovascularization and growth in both fetal
102
Alpha-Fetoprotein
and tumor tissues [27,28]. Finally, HAFP has recently
been shown to functionally impair dendritic cells,
causing immune dysfunction and apoptosis of antigen-
processing cells (APCs) [29].
Measurement of AFP
AFP has historically been measured by some form of
immunoassay. Although rocket electroimmunodiffusion
(Table 1, Method 2) is still occasionally used to
measure amniotic fluid AFP, this method has been
largely replaced by more sensitive immunoassays
capable of measuring the low ng/mL concentrations
required for both maternal serum AFP screening and
cancer diagnosis and monitoring. Competitive
radioimmunoassay has been widely used to quantitate
AFP, but the practice now is to measure AFP by non-
isotopic immunoassays that employ enzyme,
fluorescent, or chemiluminescent labels (Table 1).
Immunoassays employed to measure AFP are of two
major types: (1) competitive and (2) two-site solid-
phase immunometric. In a typical radioisotopic
125
competitive type (Table 1, Method 2), purified I-
labeled AFP is mixed with the sample containing AFP
and allowed to compete for a limited amount of AFP
antibody.
In the two-site immunometric assays (Table 1, Methods
2, 3, and 4), a solid phase such as magnetic particles,
plastic beads, or microtiter plates is coated with anti-
AFP antibody and incubated with patient specimen or
standards. AFP present in the sample reacts with the
anti-AFP antibody and is immobilized on the solid
phase. Unbound components of the specimen are
removed by washing of the solid phase. A second anti-
AFP antibody labeled with an enzyme, radioactive,
fluorescent, or chemiluminescent label is incubated
with the solid phase and reacts with AFP bound at a
second epitope site. Polyclonal and monoclonal
antibodies have been used in tandem for each of these
steps. Unreacted label is removed by washing of the
solid phase, and any additional reagent that is needed to
generate a signal is added. In the case of a fluorescence
or radioactive label, the bound label can be measured
directly. For enzyme labels, the solid phase must be
incubated with substrate before the detection step. For
chemiluminescence labels, reagent must be added to
generate the chemiluminescent signal. In all cases, the
amount of signal generated with the solid phase is
directly proportional to the AFP concentration of the
standard or unknown.
Reference and Preferred Methods
Because of the heterogeneity of AFP, there is no
reference method.
Standardization
Accuracy of AFP values is dependent on the source of
the calibrating standard. AFP values provided by
reference laboratories or obtained from manufactured
kits are frequently given in mass units (ng/mL), even
though no pure AFP standard in mass units is available.
Consequently, values in mass units obtained on the
same sample will vary, depending on the source of the
standard used to calibrate the assay. Three reference
preparations are available: the World Health
Organization (WHO) Reference Preparation for human
AFP (72-225), the British Standard (72-227), and the
U.S. National Reference Preparation for AFP in mid-
pregnancy maternal serum [30]. The WHO and British
standards were derived from the same original cord
serum pool and may be considered essentially
equivalent, whereas the U.S. standard has been
calibrated against the WHO standard. Laboratories
wishing to determine the relationship of their local unit
to the International Unit (IU) should obtain a reference
preparation for direct calibration of their local standard.
Interlaboratory comparison should be based only on
IU/mL, and all laboratories are encouraged to obtain the
relationship of their standard to IU either from the
manufacturer of the kit in question, or by direct
comparison. Estimates of the relationship of the IU and
mass units range from about 0.91 to 1.29 ng/IU, but 1.0
IU is usually equivalent to about 1.0 ng.
Assay Sensitivity
Currently, almost all laboratories use competitive RIA
or non-isotopic immunometric assays to measure AFP
in serum or amniotic fluid. Screening for low AFP
levels in fetal Down syndrome requires accurate
measurement of AFP down to 10 ng/mL. Cancer
screening and monitoring requires assays that can
measure values as low as 1 to 5 ng/mL.
Specimen
AFP in serum, plasma, or amniotic fluid may be
measured by any of the assays previously discussed.
Serum is preferred to plasma for AFP analysis,
although some manufacturers permit the use of plasma.
AFP may also be measured by elution from blood spots
collected on filter paper [31]. Precision of these latter
determinations is usually lower, however, because of
variability in spotting of blood, punch size, elution of
AFP, and use of various extraction buffers and is not
routinely employed for MS-AFP screening. Serum AFP
is somewhat thermostable, and samples may be shipped
at ambient temperatures after separation from red blood
cells. Specimens derived from sera are stable at room
temperature or at 4C for at least 1 week [32] and for
several months at 20C [33,34]. To avoid matrix
effects, it is absolutely essential to dilute AFP
specimens with diluent provided by the kit
manufacturer.
Storage Stability of AFP
Lantz et al. in a previous study evaluated the effect of
different sample collection, storage, and preparation
techniques on the immunoassay of serum alpha-
fetoprotein (AFP) [35]. Investigators found that
immediate freezing of serum and subsequent thawing
resulted in a significant increase in several different
analyte levels, but only small changes in AFP levels
were observed. However, there was an effect over time
on AFP concentrations, dependent on type of storage.
Frozen (0C) and refrigerated (4C) storage
temperatures were optimal. A change in AFP levels
103
Alpha-Fetoprotein
could also be influenced by the type of centrifugation
and by nonstable refrigeration frost-free cycles. Thus it
was concluded that different sample collection, storage,
and preparation techniques should be monitored in
maternal serum AFP screening programs. In a separate
study, investigators found no association between a
laboratorys AFP test volume and the reliability of
reported multiples of the median (MoM), and there was
no difference in AFP medians across geographic
medians of the United States [36].
Investigators have further examined the levels of
second-trimester maternal serum markers used in Down
syndrome screening in relation to the time between
sample collection and arrival at the laboratory [35]. All
blood samples were drawn in serum separator tubes,
centrifuged within 30 min, and stored at 4C until
shipment by air express. To examine the effect of
delayed shipment, serum marker levels (expressed as
MoM) were evaluated in the second-trimester samples
and stratified by the number of days between serum
collection and laboratory receipt. It was found that
under specified collection and shipment conditions,
second-trimester mean AFP concentrations and degrees
of measurement variance were stable for up to 9 days at
ambient temperatures. It is recommended to restrict
testing to within 6 days of draw, with the purpose of
keeping the shipping delays to a minimum. A
comparison of fresh frozen MS-AFP serums to
proficiency testing materials (PTM) found that the fresh
frozen serums did not provide a consistently different
imprecision or bias than did standard PTMs [37].
Stability of Newborn Dried Bloodspots:
Storage stability of AFP dried bloodspots was
performed to determine whether AFP was more stable
in the liquid or dry state as determined by a number of
authors (Table 4) [31,38-43]. These investigators
determined whether AFP in dried-blood spots was more
stable when stored refrigerated or at room temperature
and compared to liquid cord blood. AFP concentrations
were found to decrease 8% in cord plasma stored at
20C but to increase 3% in cord-blood spots stored at
4C. In other studies using cord bloods at room
temperature, AFP values for bloodspots (x = 41.5, SD =
29.7) and for liquid plasma correlated closely (r = 0.89,
slope = 0.71) for 40 such samples tested. In a separate
study, it was found that AFP measurement in dried
blood spots could be influenced by seasonal variation,
time in transport, and number of freeze/thaw cycles,
unlike serum AFP samples [44]. However, the overall
assessment of AFP in dried bloodspots revealed that
AFP was stable for many months when sealed in plastic
bags and frozen at 20C (see Table 4).
Assay Interferences
At the assay level, drug interference may occur to a
limited degree if AFP has been quantitated by
radioimmunoassay (RIA), enzyme immunoassay (EIA),
and chemiluminescence assay (CIA). Although little
has been reported on AFP-CIAs, AFP-RIA and EIA
drug assay interference has been previously described
in the literature (Table 3). The assay interference is
above and beyond the sensitivities of epitope cross-
reactivity with proteins such as albumin and transferrin,
as well as bilirubin. Experience indicates that AFP
measured using the non-isotopic immunometric assays
are not affected by hemoglobin up to 10 g/L,
triglycerides up to 1250 mg/dL, bilirubin up to 30
mg/dL, or protein up to 20 g/L. Freeze/thaw cycles also
do not cause appreciable changes in AFP
concentrations [45]. With the exception of heterophile
antigens, most if not all todays commercially available
CIAs display no cross-reactivity with these human
proteins, nor has any been found with anticancer drugs
such as cyclophosphamide, doxorubicin, cisplatin,
vincristine, 5-fluorouricil, and mitomycin [46,47].
However, drug inference of AFP quantitation in EIAs
has been reported for some non-cancer drugs, which
include both prescribed and over-the-counter
medications for diabetes, hypertension, inflammatory
and rheumatoid disorders, bacterial infections, and
pain/fever symptoms. However, many of these assay
flaws have since been corrected with todays improved
immunoassay platforms.
To date, most of the drug interference in AFP assays
has been eliminated with the advent and use of CIA
methodology. Such was not the case during the 1990s
when AFP assays were performed on RIA and EIA
platforms described in previous studies [48]. Results of
such earlier reports were disturbing in that commonly
used nonprescription and specifically prescribed drugs
produced reductions in AFP assay concentrations
ranging from 10% to 37% per specimen (Table 3). Such
non-cancer medications included injectable insulin
preparations whose results may have contributed to the
lower MS-AFP concentrations observed in insulin-
dependent diabetic pregnancies of that time period
[49,50]. The antibiotic drugs resulted in a 9% to 11%
reduction, antihypertensive drugs (-adrenergic
blockers) a 10% reduction, analgesic and antipyretic
drugs (aspirin) a 16% to 24% reduction, and
methyldopa a 37% to 47% reduction (Table 3). Drug
interference in clinical tests would not be unique to
AFP quantitation assays, since different drugs bind to
HAFP at similar known albumin-drug interaction sites.
Additional confounders may also be present in that
various pharmaceutical formulations of drugs may
include solubilized fillers (mannose), stabilizers, and
matrix components. Some drugs are dissolved in
aqueous alcohols, solubilizers such as DMSO, and
other buffers which could cause drug-sera interactions,
precipitations, or denaturization.
Pregnant women taking anticonvulsant drugs such as
phenytoin and valproic acid have long been known to
display elevated MS-AFP concentrations coincident
with a 1% to 2% incidence of spina bifida [51,52].
Although the reasons for this elevation remain unclear,
AFP is known to bind drugs similar to valproic acid in
a fashion resembling that of albumin binding of this
drug [46]. In a further study, an article was reported by
Einstein et al. [50,53] which linked the presence of
protease inhibitors (PIs) with results of low MS-AFP
104
Alpha-Fetoprotein
screening levels in pregnant women infected with the
human immunodeficiency virus (HIV). Attention can
be directed at the physiological properties of AFP
regarding the PIs and their biochemical substrates. It
has been known since the 1980s that human AFP binds
serum and tissue PIs (proteins), as well as small
molecular PIs such as arginine, benzamidine, and
Trasylol [54-58]. In theory, HAFP might bind to the PIs
such that AFP could serve as a protease decoy docking
protein to sequester the inhibitor and thus modulate the
proteolysis substrate rate reactions.
Alpha-Fetoprotein Reference Intervals
AFP produced by the fetus increases steadily during
pregnancy, reaching a peak concentration of 3 mg/mL
in fetal plasma at 12 to 14 weeks of gestation and
declining slowly thereafter to 50 g/mL at birth [59].
Moreover, premature newborns can show AFP levels
two- to threefold higher than term babies [60]. By the
end of the first year of life, values level off at about 5 to
8 ng/mL, a level that is maintained throughout life in
the absence of certain disease states (see AFP as a
Serum Tumor Marker below) [61].
Although there is less variability in AFP values
measured with modern kits, differences in regional
populations and assay methods can exist, and each
prenatal screening laboratory should establish its own
gestational agespecific median values. The various
options for obtaining a reliable set of medians
appropriate for your screened population have been
described above. Once medians are available, it is
customary to report the AFP test results as a multiple of
the median (MoM) to normalize for gestational age.
Each laboratory must select a MoM screening cutoff
which meets its needs.
The performances of the various present-day kits for
maternal serum AFP have been assessed in various
proficiency testing programs throughout the United
States. Such kits could include Abbott (IMX) AxSYM,
Beckman Unicel/Access, Bayer-Advia/Centaur and
Immulite. The AFP mass measurements among the
individual kits seem to largely agree, although Bayer-
Centaur MS-AFP values are sometimes slightly higher
and DPC Immulite and Beckman Unicel values slightly
lower for some specimens. Kit comparisons have also
been studied for their performance among amniotic-
fluid (AF-AFP) test samples. Again, the amniotic fluid
overall kit performance approached that observed with
the maternal serum samples. With Bayer-Centaur and
to a lesser extent Abbott-AxSYM kits, AF-AFP values
are sometimes higher, whereas Beckman Access and
DPC Immulite values are somewhat lower in all-lab
comparisons.
AFP as a Serum Tumor Marker
Cancer and Hepatic Disease:
The upper limit of the reference interval for AFP in
healthy populations has been tentatively established
(Table 8). Hunter, using a very sensitive AFP assay,
found that most healthy persons have values of 5 to 8
ng/mL or less [62]. In a study of 338 healthy adults by
Abbott Diagnostics, 99% of AFP values by an enzyme
immunoassay were under 8 ng/mL, and 100% were
below 20 ng/mL. Using cutoff values of 5 to 8.0 ng/mL
thus seems a suitable choice for general cancer
screening. However, in monitoring cancer therapy, a
series of increasing or decreasing AFP values is of
primary importance, and a value below 8 ng/mL that
shows a rise above a basal level can be of prognostic
significance.
Monoclonal and polyclonal antibodies have been used
to detect serum AFP associated with specific
malignancies. The AFP tumor marker is most useful for
monitoring response to therapy and detecting early
relapse. AFP, a marker for hepatocellular carcinoma,
has been used for screening in Asia to assess hepatic
masses in patients at particular risk for developing
hepatomas. Testing for the beta subunit of human
chorionic gonadotropin (beta-hCG) is an integral part of
the diagnosis and management of gestational
trophoblastic disease (i.e., germ cell tumors). Combined
AFP and beta-hCG testing has been found to be a
useful adjunct in the evaluation and treatment of
nonseminomatous germ cell tumors and in monitoring
the response to therapy. Combined AFP and beta-hCG
also may be useful in evaluating potential origins of
poorly differentiated metastatic testicular cancer (see
Tables 8 and 9).
AFP-Secreting Tumors:
Levels of serum AFP that exceed those seen in healthy
adults (8 ng/mL) have been reported in some patients
with benign hepatic disorders such as viral hepatitis or
cirrhosis (see Table 9). The first tumors found to
secrete AFP were the hepatocellular carcinomas
(hepatomas) in both mice and humans [1,2]. Other
tumor types later found to synthesize and secrete AFP
were testicular germ cell tumors (teratomas) and yolk
sac tumors of the ovary [4,5]. However, the AFP serum
level (500 to 900 ng/mL) secreted by germ cell tumors
did not approach the serum levels (microgram/mL)
found earlier in hepatomas. Following detection of
AFP-secreting tumor types, many other cancers have
now been classified in this category (Table 2). Aside
from the hepatoma and reproductive cancers, AFP
secretion has been linked to the gastrointestinal cancers
of endodermal origin, especially stomach and
pancreatic tumors. Less frequently, AFP synthesis and
secretion have been associated with tumors of
pineal/pituitary cysts, hepatoblastomas,
hemangioendotheliomas, hepatic bile duct carcinomas,
gallbladder carcinomas, epidermoid cysts, granulosa
cell tumors, and others (see Tables 8 and 9). AFP serum
levels have also been used as a diagnostic aid for the
differential diagnosis of seminoma versus non-
seminomatous germ cell tumors (see above). For
example, patients with germ cell tumors displaying
elevated AFP serum levels are considered to have the
non-seminomatous type [63].
105
Alpha-Fetoprotein
Interpretation
AFP produced by the fetus is transferred to the amniotic
fluid (AF) by fetal urination, and amniotic fluid levels
generally parallel those found in fetal sera, although
concentrations will be approximately 150 times lower.
AFP also appears in maternal serum as pregnancy
proceeds, by placental transfer or by diffusion across
the fetal membranes, and peaks at approximately 31 to
32 weeks of gestation. In contrast to typical second-
trimester levels of 10 to 12 g/mL in amniotic fluid,
levels in maternal serum during this same period are
only 30 to 40 ng/mL. In the second trimester, MS-AFP
increases by about 15% per week, whereas amniotic
fluid AFP declines by about 13% per week [64]. In
open lesions of the fetus such as spina bifida and
anencephaly, elevated levels of AFP are found in
amniotic fluid due to leakage of AFP into the fluid from
open lesions.
The increased concentration of AFP in amniotic fluid
results in increased MS-AFP levels as well, and
screening of maternal sera for elevated concentration of
AFP is widely used to select women at increased risk of
carrying fetuses with an open-neural-tube defect [65].
Decreased MS-AFP levels may be used to select
women at increased risk of carrying fetuses with
chromosomal defects, such as Down syndrome and
trisomy-18 [66]. Definitive diagnoses of neural tube
defects in fetuses of women with elevated serum AFP
are then achieved by measurement of the level of AFP
in amniotic fluid. Mandatory secondary diagnostic tests
for confirming affected infants include level-II
ultrasound and measurement of amniotic fluid
acetylcholinesterase [67]. Interested readers are referred
to the outstanding in-depth review of this application by
Wald and Cuckle [68].
Although AFP can be measured quite reliably, use of
maternal serum AFP levels to screen women at
increased risk of carrying fetuses with neural tube
defects was originally controversial because of concern
over misuse and misinterpretation of AFP test results;
however, these concerns are no longer valid. Successful
screening requires an integrated program involving the
laboratory, ultrasonographers, physicians, genetic
counselors, and a program coordinator. In 1984, the
U.S. Food and Drug Administration (FDA) approved a
stringent set of regulations that limited the sale,
distribution, and use of kits to centers that could
demonstrate proficiency in all aspects of the screening
process. Since that time, kits became available without
restriction, but it is strongly recommended that a
laboratory measure AFP only as separate parts of a
prenatal and/or tumor-marker screening program
because of the potential harm that could result from
incorrect interpretation of test results between cancers
versus pregnant patients.
Factors That Influence AFP Levels in Biological
Fluids.
Demographic/biological factors can also influence AFP
concentrations during perinatal and postnatal life. Some
factors that influence AFP biological fluid levels are
listed below as grouped items together with their
associated reference citations:
1. Birth Weight: Newborn AFP serum levels were
found to be correlated inversely with birth
weight. Fetal serum AFP levels are significantly
higher in babies born at 40 weeks that had a
birth weight below the population mean
compared to those above the mean. It is
recommended that AFP blood concentrations
determined for infants in the first weeks of life
be adjusted for birth weight and considered
elevated only after premature (very low) birth
weight is ruled out [69-73].
2. Gestational Age: A significant inverse linear
correlation has been reported between newborn
AFP levels and gestational age. It was
concluded that gestational age and birth weight
(size) together play a significant role in
determining newborn AFP concentrations [74-
77].
3. Parity, Gravity: No direct effect was observed
between serum AFP and parity and gravity.
However, women delivered of their second
infant showed a significant decrement in mean
birth weight of the newborn [78,79].
4. Race, Ethnicity: No differences were observed
in serum AFP of oriental, white, or Hispanic
origins, but maternal sera of blacks can average
10% higher values for a given gestational age
[78,80].
5. Maternal Body Weight: A correction formula
(~10%) for body weight is required, because
AFP is diluted out in the maternal bloodstream
in pregnant women with increased body weight
(exclusive of fetal/placental weight).
6. Diabetic Status: The insulin-dependent diabetic
patients serum assay result requires a 10%
downward correction in prenatal screening
programs. Such patients serums are usually
lower than nondiabetic patients.
7. Sex of the Fetus/Newborn: Both cord blood and
newborn serum AFP levels are known to be
considerably higher in male infants than in
females at any gestational and postnatal age [81-
84].
Abnormal AFP Levels During Pregnancy
Serum levels of gestational age-dependent AFP that fall
outside the normal limits seen in healthy pregnant
women have been reported for a multitude of
congenital malformations of the fetus/infant. The first
developmental abnormalities found associated with
abnormal AFP levels were neural tube and related
spinal defects (Table 5) [6,64,85]. Later, other types of
birth defects found to reflect aberrant/discordant AFP
levels were the chromosomal disorders (trisomies) and
various anatomical abnormalities [7,86]. The MS-AFP
concentrations (100 to 500 ng/mL) associated with fetal
defects are abnormally high; the trisomies are
accompanied by abnormally low levels. Following the
detection of AFP-associated birth defects such as spina
bifida and anencephaly, many other congenital
106
Alpha-Fetoprotein
anomalies have now been classified in the elevated
AFP category [87,88]. Abnormal levels of AFP
synthesis and secretion have been associated with three
compartments: fetal serum, amniotic fluid, and
maternal serum. Less frequently, AFP synthesis and
secretion have been observed in other biological fluids
such as urine, cerebrospinal fluid (CSF), placental
extracts, and anatomical cysts. AFP serum levels have
also been used as an ancillary aid in the diagnosis of
erythropoietic disorders (anemias), placental
disruptions, fetal death, growth restriction/retardation,
and preterm labor and birth.
Prenatal screening programs initially consisted only of
maternal serum (MS) AFP and amniotic fluid AFP,
which included ancillary acetylcholine and fetal
hemoglobin testing. During this period, neural tube
defects were the only fetal disorders for which prenatal
screening was available (Table 4). In 1992, prenatal
screening for Down syndrome using three maternal
serum markersAFP, unconjugated estriol (E3), and
human chorionic gonadotropin (hCG)was reported,
which ushered in the period of the triple test [7]. By
1996, a fourth marker, dimeric inhibin-A (DIA), was
added to the fetal defect marker (FEDM) program,
launching the quad test. The quad marker screen was
developed during the early 1990s and by 1996 provided
a means to improve the detection rate while lowering
the false-positive rate in prenatal screening for Down
syndrome (DS) and possibly other trisomies [86,89].
Neural Tube Defect Screening
It is almost universally accepted that cutoff values for
neural-tube-defect screening results are expressed as
MoM, a convention developed from the original U.K.
collaborative study [90]. The MoM selected as the
cutoff point for both serum and amniotic fluid ranges
from 2.0 to 2.5, and the exact cutoff point used by an
individual lab is determined by a trade-off between the
detection rate for neural tube defects and the false-
positive rate. It is recommended that each laboratory
establish its own medians for both maternal serum and
amniotic fluid for each gestational week. Ideally, a
minimum of 100 samples per week should be assayed
in the period from 15 to 20 weeks of gestation to
establish reliable medians. However, obtaining 100
samples for each gestational age can be difficult
because most specimens are collected at 16 to 18 weeks
of gestation, the optimum time for neural-tube-defect
screening. A more practical approach is to use the
information that the log of the median versus
gestational age is a straight line between 15 to 22 weeks
of gestation. AFP is measured on 300 to 500
consecutively screened patients (the number of
observations at each gestational week will correspond
to the distribution in the screening program), and a
median calculated derived from AFP concentrations for
each week of gestation (Tables 6.1 and 6.2). Next, a
linear regression analysis is performed of the log of the
median versus the gestational age (weighted according
to the number of observations used to calculate the
median for any given gestational age). The resulting
equation of the fitted line is then used to calculate the
median for each gestational week. Detailed information
on weighted log-linear regression of AFP values has
been published [66]. Kit manufacturers provide median
values in package inserts, and these may be used as a
general guide for expected values with that particular
kit being used. It must be cautioned, however, that
some manufacturers provide medians that are highly
erratic and deviate significantly from those expected.
Medians should rise smoothly by 15% to 20% per
gestational week in serum and fall smoothly by 11% to
14% per gestational week in amniotic fluid. Median
AFP concentrations based on over 10,000 observations
for both maternal serum and amniotic fluid are
presented in Tables 6 and 7, including median
concentrations by gestational week. In general, medians
established by most laboratories will not differ from
these results by more than 10% to 20%. These same
median values can be used to calculate MoM values to
determine the fetuss risk for having Down syndrome.
AFP levels are known to slowly rise in the aged normal
population and are usually higher in males than in
females.
In summary, AFP is present at very low levels in
healthy, nonpregnant adults (1 to 8 ng/mL).
Concentrations are increased in certain neoplasms such
as hepatocellular carcinoma [91], germ cell tumors
[60,61], and gastric and pancreatic carcinoma, reaching
levels of over 1,000,000 ng/mL in some cases. Levels
may also be elevated in diseases of hepatic origin,
including viral hepatitis and cirrhosis. An important
application of these associations is the monitoring of
progression or regression of hepatocellular carcinoma
and germ cell tumors by serial determination of AFP.
Rare cases of hereditary persistence of AFP have been
reported [92], as well as cases of AFP congenital
deficiency [93].
Alpha Fetoprotein Performance Goals
AFP assays, whether commercial kits or produced
internally, using in-house components, are among the
most stable, specific, and robust immunoassays
available. The assays employing non-isotopic labels
have shelf lives ranging from months to years.
Between-assay coefficients of variation of 5% to 10%
are readily achieved by most of the current assays in the
region of clinical usefulness, that is, in the range of 5 to
100 ng/mL, and high concentration samples can be
reliably tested by dilution.
Between-laboratory precision is excellent when results
are expressed in the IU. External quality-assessment
schemes in the United States and Britain, based on IU,
obtain between-laboratory CVs of 10% to 15%. The
College of American Pathologists proficiency surveys
also demonstrate the acceptable interlaboratory
precision achievable by two-site immunometric assays.
At a level of 21.8 ng/mL, the overall %CV for many
laboratories using this technology has achieved 6.7%.
At a level of 174.1 ng/mL, the overall %CV for
laboratories using this technology was 5.67%.
107
Alpha-Fetoprotein
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Alpha-Fetoprotein

82 Obiekwe BC, Malek N, Kitau MJ, Chard T. Suggested Website Locations:

Maternal and fetal alphafetoprotein (AFP)
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1983;146:786-789. fetoprotein-afp-test
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85 Leek AE, Ruoss CF, Kitau MJ, Chard T. r/mono/ri0ri019500.htm
Raised alpha-fetoprotein in maternal serum 7. www.pennhealth.com/health_info/Surgery/alph
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87 Thomas RL, Blakemore KJ. Evaluation of 11. pregnancy.about.com/cs/afp/a/afptesting.htm
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M. Second-trimester maternal serum alpha- info.net/slpha_feto_protien.html
fetoprotein and risk of adverse pregnancy
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111
Alpha-Fetoprotein

Table 1: Alpha-Fetoprotein Methods Summary Table

Method 1: Electroimmunodiffusion; immunoprecipitation
Principle of analysis: AFP is placed in an electric field, enabling it to migrate into an agarose gel containing
anti-AFP; extent of migration and height of precipitin line are proportional to AFP concentration.
Comments: Lacks sensitivity for serum analysis; historical, amniotic fluid only Method 2:
Radioimmunoassay; competitive binding assay
Principle of analysis: Labeled 125I-AFP competes with sample AFP for binding to anti-AFP antibodies.
Separation of bound from free ligand can be achieved by precipitation with a second antibody or by
immobilization of anti-AFP on a tube or microtiter well.
Comments: Reference method but uses radioisotopes; requires dilution to measure amniotic fluid AFP; serum
or amniotic fluid
Method 3: Immunoradiometric assay
125
Principle of analysis: Sample AFP binds to solid-phase immobilized anti-AFP antibody; I-labeled antibody
binds to immobilized AFP.
Comments: Commercial kits not available in the United States; rarely used; serum or amniotic fluid
Method 4: Immunometric assay
Principle of analysis: Same as for method 3, except the antibody is labeled with an enzyme, fluorescent, or
chemiluminescent label.
Comments: Most common assay type, measures AFP in maternal serum and amniotic fluid; suitable for cancer
monitoring

Table 2: Time Line of Aberrant Forms of Human Alpha-Fetoprotein Truncated Molecules

Resulting From Genetic Variants
Year Observer Observation Ref.
1975 Chandra Cystic fibrosis forms in serum BMJ 1:714
1975 Smith Aberrant form in CF in serum BMJ 2:392
1975 Fitzsimmons Aberrant form in CF in serum BMJ 3:544
1976 Biswas Aberrant form in CF in serum Clin Chem Acta 69:541
1976 Ramirez Aberrant from in CF in serum N Engl J Med 295:1381
1976 Norgaard-Petersen Cationic form of HAFP (electrophoresis) Clin Chem Acta 71:343 (AFP is
anionic)
1978 Marrink Cationic form* in serum Scand J Immunol* 8(Suppl 8):309
1978 Nishi Cationic form* in ELISA Scand J Immunol 8:35305
1982 Morinaga 54 Kd form in testis cancer
1983 Sarcione Breast cancer form in cytosol Cancer Res 43:3739
1983 Naketa Cationic form* in serum Oncodev Biol Med 4:101
1984 Lippes 54 Kd testicular/oviductal form Fertil Steril 59:148
1985 Sarcione 42Kd form in breast cancer cytosols Int J Cancer 35:315
1987 Sarcione Serum form in breast cancer, women Dis Markers 5:75
1987 Mizejewski Bound forms of AFP in serum CRC Press; pp.162
1990 Kronquist Aberrant pH form in Down syndrome Prenat Diagn 10:739
1990 Pavlov Aberrant form in CF in tissue Lab Delo 4:47
1999 Deutsch 52 Kd form in (liver cancer tissue) Tumour Biol 21:267
2000 Vakaria/GJM Conformationally transformed (serum) Breast Can Res Treat 63:41-52
2002 Kubota Avariant forms found in endoderm tissue J Biol Chem 277:27629
2005 Fukasawa 65 kD with missing signal sequence J Soc Gynecol Investig 12:456
(cell culture)
Data was extracted and summarized from References # 1, 4, 6, 7.
*HAFP in its native form in an anionic molecule
CF, Cystic fibrosis; ELISA, enzyme-linked immunosorbant assay.
112
Alpha-Fetoprotein

Table 3.1: Drug Interference of AFP Levels Measured by Radioimmunoassay and Enzyme
Immunoassay* - Insulin Related
Insulin Preparation Injection Type MS-AFP (ng/mL) Type of Immunoassay
Decrease RIA EIA
1. Human Recombinant-I 7%-8% X X
2. Iletin Purified Human 6%-14% X ND
3. Iletin Protamine-Zinc 8%-12% X ND
4. Iletin Purified Bovine 7%-8% X ND
5. Iletin Purified Porcine 6%-8% X X
6. Humulin L-1, R 7%-8% X X

Table 3.2: Drug Interference of AFP Levels Measured by Radioimmunoassay and Enzyme
Immunoassay* - Small Molecule Drug Related
Drug MSAFP (ng/mL) Type of Immunoassay
Decrease RIA EIA
1. Sulfasalazine 11% X ND
2. Erythromycin 9% X X
3. Propranolol 7%-10% X X
4. Methyldopa 37%-46% X X
5. Acetaminophen 16%-18% X X
6. Aspirin 19%-24% X X

EIA, Enzyme immunoassay; ND, not done; RIA, radioimmunoassay. (CIA, Chemiluminescent immunoassay was not
done.) From Goldstein PJ et al. Drug interference on AFP assays. Md Med J. 1991;40:513-516.

Table 4: The Stability of Human AFP Measured in Dried Blood Samples Reported for Years
1979 to 2000
Reference Citation Storage Results and/or Comment
Time Temperature Comment
1. Katsumata Y. Identification of fetal blood stains 1 month 25oC Stable (no
by RIA of alpha-fetoprotein. Z Rechtsmed. degradation)
1979;82:323-326.
2. Mizejewski GJ et al. Commercial RIA kit for 10 months 25oC and 4oC Stable (no
AFP measurement in newborn dried blood spots. degradation)
Clin Chem. 1982;28:1207-1210.
3. Dallaire L et al. Maternal sera AFP measured on 6 months 4oC Stable (no
dried blood spots. Prenat Diagn. 1982;2:265-271. degradation)

4. Wong et al. Studies of AFP measurement in 6 months 4oC Stable (no

dried blood spots for prenatal diagnosis. Clin degradation)
Biochem. 1982;15:170-172.
5. Mizejewski et al. AFP measured in infant dried 10-12 months 25oC and 4oC Stable (no
bloodspots. Pediatric Res. 1983;17:47-50. degradation)

6. Mizejewski et al. Birthweight and AFP in 10 months 25oC and 4oC Stable (no
newborns dried bloodspots. Pediatrics. degradation)
1984;73:736-737.
7. Augier D et al. Result of prenatal screening of Assayed 25oC Stable at time of
AFP in dried blood spots. STAT* testing
J Genet Hum. 1985;33:325-326. (Combined
with
ultrasound)
113
Alpha-Fetoprotein

8. Tsao et al. EIA for AFP on dried blood 1.0 month 37oC Stable (no
specimens. Clin Chem. 1986;32:2079-2082. degradation)
9. Fang M et al. [Measurement of Assayed 4oC Stable at time of
radioimmunoassay of alpha-fetoprotein in dried STAT* (mass 25oC testing
blood samples on paper for mass screening of screening)
hepatocellular carcinoma]. Kaku Igaku. 1986; 23:
1079-82..
10) Gonzalez C et al. Evaluation of MSAFP using 1.0 month 4oC Stable (no
dried blood samples. J Clin Chem Clin Biochem. 25oC degradation)
1988;26:79-84. 37oC
11) Verloes A et al. Non-radioactive assay of AFP Assayed 25oC Stable at time of
from dried bloodspot STAT* testing
Prenat Diagn. 1992;12:1073-1074.
12. Macri JN et al. Prenatal maternal AFP dried Prenatal 4oC Stable upon storage
blood screening. Am J Obstet Gynecol. Evaluation 25oC
1996;174:566-572.
13) Masse J. Transportation of material sera AFP Assayed STAT 0oC and 25oC, Adverse affect of
dried blood specimens. Clin Biochem. for season, seasonal variation summer transport on
2000;33:273-277. delay, and studied stability
freezing
variations

*STAT, Assayed within 1-2 days of arrival.

114
Alpha-Fetoprotein

Table 5: Prenatal Screening Timeline for Alpha-Fetoprotein Employed as a

Biomarker Alone or in Combination with Other Analytes
Year Observation/Event Reference
1972 Elevated AFP in amniotic fluid for neural tube Brock DJH et al: Lancet 2:191-
defects. Indication: potential biomarker 194.
1973 Elevated AFP in maternal serum for neural tube Leek AE et al: Lancet 2:385-386.
defects. Indication: potential biomarker
1980 Early antenatal diagnosis of ventral wall defect using Wald NJ et al: Lancet 1:368.
AFP.
1981 Amniotic fluid acetylcholinesterase diagnoses for Collaboration Study: Lancet
neural tube defects with elevated AFP levels. 2:321-323.
1984 Low maternal serum AFP levels discovered in Merkatz IR et al: Am J Obstet
prenatal Down-syndrome pregnancy samples. Gynecol 148:886-894.
1987 Combination of maternal age and AFP levels useful Cuckle HS et al: Br J Obstet
in Down syndrome pregnancies. Gynecol 94:387.
1989 Screening for Down syndrome using AFP, E3, and Wald NJ: Am J Human Genet
hCG (Triple Biomarker Test). 44:586,
1991 Low MS-AFP in congenital cardiac and Resta RG: Am J Med Genet
diaphragmatic defects. 40:129.
1992 Prenatal screen in maternal serum using multiple Haddow et al: N Engl J Med
markers for fetal distress (AFP, hCG, etc.). 327:588.
1994 Four-marker serum screening for Down syndrome Wald NJ: Prenat Diagn 14:707-
(quad test) using AFP, estriol, hCG, inhibin-A (2nd 716.
trimester)
2004 Combined (sequential) 1st and 2nd trimester Platt LD et al: 104:661-666.
screening for Down syndrome using PAPP-A, B-
hCG, followed by the AFP-triple test.
B-hCG, Beta hCG; estriol, unconjugated estriol; hCG, human chorionic gonadotrophin;
PAPP-A, pregnancy associated placental protein-A.

Table 6.1: Maternal Serum Alpha-Fetoprotein Concentrations by Gestational Week in Maternal Sera and
Amniotic Fluid (Median Values)

Gestation week 14 15 16 17 18 19 20 21 22
Maternal serum 22.2 25.5 29.4 33.6 38.6 44.5 51.0 58.5 67.2
(U/mL)
Amniotic fluid 19.2 17.4 13.8 11.0 8.8 7.0 5.5 4.4 3.5
(kU/mL)
kU/mL = 103 U/mL.

Table 6.2: Multiples of Median AFP Values in Maternal Serum

Gestational No. of Median Multiples of Median (MoM, ng/mL)

Week Samples ng/mL 2.0 MoM 2.5 MoM 3.0 MoM

15 347 14.0 62.8 78.5 94.2
16 412 36.3 72.6 90.8 108.9
17 320 41.9 83.8 104.8 125.7
18 333 48.5 97.0 121.3 145.5
19 201 56.1 112.2 140.3 168.3
20 77 64.8 129.6 162.0 194.4
*Medians are determined based on a weighted linear regression model.14
This laboratorys reportable range for AFP values in maternal serum is dependent on gestational week, as illustrated by
data obtained from 1690 serum samples at three sites. MS-AFP concentrations are reported in ng/mL.
115
Alpha-Fetoprotein

Multiples of Median (MoM, ng/mL) AFP Values in Amniotic Fluid

This laboratorys reportable range for AFP in amniotic fluid samples is dependent on gestational week, as illustrated by
data obtained from 666 amniotic fluid samples at two sites.

Table 7: AFP Values in Amniotic Fluid

Gestational No. of Median* Multiples of Median (MoM, g/mL)

Week Samples g/mL 2.0 MoM 2.5 MoM 3.0 MoM

15 92 17.3 34.6 43.3 51.9
16 138 14.4 28.8 36.0 43.2
17 152 11.9 23.8 29.8 35.7
18 133 9.9 19.8 24.8 29.7
19 103 8.1 16.2 20.3 24.3
20 48 6.7 13.4 16.8 20.1
*Medians are determined based on a weighted linear regression model.14
Note: 1 g/mL = 1000 ng/mL.
AFP concentrations are reported in g/mL

Table 8: AFP Concentrations in Benign and Malignant Disease

0- 10.1- 20.1- 500.0-

Sample No. of 10.0 20.0 500.0 1000.0 > 1000.0
Category Samples ng/mL ng/mL ng/mL ng/mL ng/mL

Apparently
Healthy Subjects 196 189 7 - - -
males 99 96 3 - - -
females 97 93 4 - - -
Testicular Cancer
seminoma 13 12 1 - - -
non-seminoma 62 30 1 23 3 5
Hepatoma
primary 16 6 - 4 - 6
secondary 5 4 1 - - -
Liver Disease
hepatitis 18 9 - 9 - -
cirrhosis 17 10 1 4 1 1
Other Cancer
ovarian 20 20 - - - -
genitourinary 15 15 - - - -
pancreatic 6 6 - - - -
In the above study, 96.4% of apparently healthy subjects had AFP values < 9.6 ng/mL, and 100%
percent of apparently healthy subjects had AFP values < 15.3 ng/mL.
116
Alpha-Fetoprotein

Table 9.1: Examples Using Alpha-Fetoprotein (AFP) as a Biomarker for Both Hepatic Disorders and
Malignant Tumors - Hepatic Disorders*
Liver Condition Number of Range of AFP Median AFP
Patients Concentrations Concentration
(ng/mL) (ng/mL)
A. Subacute hepatic necrosis 75 30 to 3300 900
B. Virus B hepatitis 101 40 to 405 350
C. Chronic active hepatitis 35 10 to 400 225
D. Alcoholic hepatitis without 40 30 to 440 75
cirrhosis
E. Primary biliary cirrhosis 35 10 to 250 50
*The tumors are presented according to tumor type and tissue of origin. The AFP was measured in serum and reported as
nanogram (ng)/mL concentrations.

Table 9.2: Examples Using Alpha-fetoprotein (AFP) as a Biomarker for both hepatic disorders and
malignant tumors - Malignant Tumors*
Tumor Type Tissue of Patient AFP Serum Level
Origin
1. Hepatocellular carcinoma Liver 35 adult males 1000 to 10,000,000
ng/mL
2. Lung tumor Lung 80-year-old male 17,000 ng/mL

3. Embryonal cell carcinoma Testicle 49-year-old male 1,700 ng/mL
4. Hepatoblastoma Liver 3-year-old female 332 ng/mL
5. Yolk sac tumor Ovary 22-year-old female 262 ng/mL
Data were extracted and compiled from the following references:
Wespsic HT. Alpha-fetoprotein: its quantitation and relationship to neoplastic disease. In: Kirkpatrick AM, Nakamura RM,
eds. Alpha-Fetoprotein Laboratory Procedures and Clinical Application. New York: Masson; 1981:115-129.
Taketa K. Multimodel application of lectin affinity electrophoresis of AFP. Electrophoresis. 1998;19:1774-1779.
Mizejewski GJ. Biological role of alpha-fetoprotein in cancer: prospects for anticancer therapy. Exp Rev Anticancer Ther.
2002;2:89-115.
*The tumors are presented according to tumor type and tissue of origin. The AFP was measured in serum and reported as
nanogram (ng)/mL concentrations.

Signifies years of age of male or female patient.

 117
Aluminum
Aluminum
Tony Badrick
Name: Aluminum, aluminium
Clinical significance: Refer to Chapter 42, Trace Metals, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Atomic symbol: Al
Atomic weight: 26.98 D
CAS No: 7429-90-5
Chemical class: Metal
Principles of Analysis and Current Usage
Aluminum is a periodic system group IIIB metal. It occurs
only in the +3 oxidation state.
It is the most abundant metal found on earth [1] and is
present in water and soil. Natural human exposure is
unavoidable, with moderate amounts entering the body via
the ingestion of food and drink and the inhalation of dust.
Aluminum does not have a known biological role. Despite
the high exposure, relatively little is absorbed, and this is
readily excreted via the kidneys. Industrial aluminum
toxicity is rare. However, in the presence of renal failure,
high levels of aluminum can accumulate in bone and
tissues. This increased absorption occurs because of (1)
oral administration of aluminum hydroxide, used as a
phosphate binder, and (2) the presence of aluminum in the
water used to prepare dialysis fluid. This retained
aluminum can lead to dementia (dialysis encephalopathy),
microcytic anemia, and bone disease (osteitis fibrosa
cystica) [1].
The current methods for the analysis of aluminum include
graphite furnace atomic absorption spectrometry
(GFAAS), flame atomic absorption spectrometry (FAAS),
electrothermal atomic absorption spectrometry (ETAAS),
neutron activation analysis (NAA), inductively coupled
plasmaatomic emission spectrometry (ICP-AES),
inductively coupled plasma-mass spectrometry (ICP-MS),
and laser microprobe mass analysis (LAMMA). Front-end
separation techniques such as chromatography are
frequently coupled with analytical methods.
i
Aluminum
Previous and current authors of this method:
First edition: Not done
Methods edition: Michael R. Wills, Sue Brown, John
Savory
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Tony Badrick
GFAAS is the most commonly used technique for the
determination of aluminum in serum, plasma, urine,
dialysate fluid, and water [2-14]. This is because GFAAS
offers the best combination of sensitivity, simplicity, and
low cost. With this technique, the sample is placed in a
graphite tube or pyrolytic platform, and in successive
steps, the temperature is raised to dry, char, and atomize
the sample into the measuring chamber, where absorption
of a specific wavelength is generated by a hollow cathode
lamp. The method of standards addition is then used to
allow matrix-matched calibration. There is usually no
preparation for water samples, and only a mixing step with
a wetting agent or dilute nitric acid for serum samples.
Instrument detection limits extend down to concentrations
of approximately 0.1 to 0.2 mol/L for serum and 0.04 to
0.12 mol/L for water, dialysate, or urine [15]. Within-run
imprecision for serum and urine are approximately 3% and
5%, with between-run imprecision of the order of 7%.
When used as a detector for high-performance liquid
chromatography (HPLC), GFAAS can analyze for species
of complexed or bound aluminum which have been
separated into fractions on the chromatography column.
The use of inductively coupled plasma (ICP) techniques is
now becoming more popular in clinical laboratories. There
are two major variants, ICPmass spectrometry (ICP-MS)
and ICPatomic emission spectrometry (ICP-AES). These
techniques offer rapid, highly sensitive, multi-element
determinationsfor example, Al, Co, Cr, Mn, Ni, and Se
[16,17]. However, the equipment comes at a relatively
high cost, with high facility costs as well (gas piping,
separate space, air-conditioning), and the technique is also
relatively complex, all of which limit its routine use in
many laboratories. Sample preparation is minimal and
involves a dilution which also reduces matrix effects that
can cause signal instability due to the presence of
dissolved solids. ICP-AES has lower sensitivity than
ETAAS or ICP-MS, but it can handle greater levels of
dissolved solids than ICP-MS and is faster than ETAAS.
However, sample volume requirements for ICP-AES will
generally be higher than for the other techniques.
The ICP-AES technique, also referred to as ICPoptical
emission spectroscopy (ICP-OES), is an excellent
 118
Aluminum
alternative to GFAAS for those laboratories possessing the
appropriate instrumentation [18-21]. ICP-AES is a multi-
elemental technique that is relatively free of chemical
interferences. The matrix problems that can exist in atomic
absorption spectrometry (AAS) are minimized in ICP-AES
because of the very high excitation temperature of the
sample [11]. The limits of detection for the ICP-AES
method have been reported to be about 0.037 and 0.148
mol/L (1 and 4 g/L) of urine and blood, respectively
[18]. Two major problems with using the ICP-AES
technique are the intense and broad emission of calcium,
which increases the aluminum background and can raise
the detection limit for this element and interference from
Titanium [19-21].
Inductively coupled plasmamass spectrometry (ICP-MS)
is a powerful technique that uses an inductively coupled
plasma as an ion source and a mass spectrometer as an ion
analyzer. It can measure the presence of > 75 elements in a
single scan and can achieve detection limits down to parts
per trillion (ppt) for many elementsconcentrations that
are two or three orders of magnitude lower than those
obtained by ICP-AES [22]. ICP-MS is more expensive
than ICP-AES and requires more highly skilled technical
operation. Aluminum concentrations in urine and saliva
were detected down to 0.1 mol/L and in blood serum to
0.004 mol/L, using ICP-MS [23]. Speciation studies have
employed ICP-MS as a detector for aluminum in tissue
fractions separated by size-exclusion chromatography
(SEC) in femur, kidney, and brain [24].
Reference and Preferred Methods
The reference method is AAS (see Method 1 below), but
ICP (Method 2) is also acceptable. The choice of method
for aluminum determination depends upon the equipment
available in the laboratory. At the time of writing this
review, about half of the participants in the College of
American Pathologists trace metals QA program were
using AAS, and approximately half were using ICP-MS.
Specimen
Aluminum is measured in serum, urine, dialysate, water,
and bone [25]. Collection of specimens for serum
aluminum analysis can be a complicating factor. Most of
the common evacuated blood collection devices used in
phlebotomy today have rubber stoppers made of aluminum
silicate. Simple puncture of the rubber stopper for blood
collection is sufficient to contaminate the sample with
aluminum sufficient to produce an abnormal concentration
of the metal. Typically, blood collected in standard
evacuated blood tubes will be contaminated by 0.74 to
2.23 mol/L of aluminum. This can be readily
demonstrated by collecting blood from a normal volunteer
into a standard evacuated phlebotomy tube. Special
evacuated blood collection tubes are required for
aluminum testing. These tubes are readily available from
commercial suppliers and should always be used. Failure
to pay attention to this issue can result in the generation of
abnormal results due to sample contamination, which can
lead to misinterpretation and misdiagnosis.
Urine specimens need not have preservatives added but
should be refrigerated to prevent bacterial growth [26].
Sample collection requirements for trace metal analysis are
available from the International Union of Pure and Applied
Chemistry [27].
Interferences
There are three general forms of interference in atomic
absorption spectrophotometry: chemical, ionization, and
matrix. Chemical interference occurs when the metal
cannot be dissociated into free atoms because of certain
complexes. This is generally overcome by the use of high
temperatures, which force dissociation, or by the addition
of another cation that competes with the complexing
interferents.
Ionization interference occurs when the atom of interest is
excited beyond the ground state and emits rather than
absorbs. This is temperature dependent and can be reduced
by adding an excess of more easily excited atoms to the
mix.
Matrix interference occurs when there is a reaction
between the atom and the matrix in the reaction cell.
Examples of these reactions include enhanced absorption
in the presence of certain solvents and the formation of
solids which produce physical interference in the light
path. Zeeman correction and continuum-source
background-correction systems are used to reduce this type
of interference.
In the case of aluminum estimation by ETAAS, various
methods used to reduce interference have been compared
[28]. The finding was that there is no significant difference
between (1) a Perkin Elmer (PE) Model 3110 AAS
equipped with a longitudinally (end) heated graphite
atomizer (HGA) and continuum-source (deuterium)
background correction; (2) a PE Model 4100ZL AAS
equipped with a transversely heated graphite atomizer
(THGA) and longitudinal Zeeman background correction;
and (3) a PE Model Z5100 AAS equipped with an HGA
and transverse Zeeman background correction.
Aluminum Reference Intervals
Serum aluminum concentrations have been reported as
0.07 to 0.56 mol/L [13]. The upper reference limit for
aluminum in a healthy, nonexposed population is reported
as 0.6 mol/L in urine [29].
Conversion factor mol/L = g/L 0.0371.
Clinical signs of aluminum toxicity occur at concentrations
> 7.4 mol/L, but levels of > 3.7 mol/L require
surveillance. A level > 2.2 mol/L warrants attention.
 119
Aluminum
Interpretation
For more than 25 years, aluminum toxicity has been
recognized as a potential hazard to patients with end-stage
renal disease (ESRD) [30]. The biological effects of
aluminum toxicity have been of considerable interest,
particularly in uremic patients, where toxins may be
involved in complex interactions with aluminum-mediated
response mechanisms.
Aluminum concentrations in blood are not a reliable
marker of aluminum absorption or organ load in dialysis
patients, only stainable aluminum at the mineralization
front reflects the histopathological changes observed in
bone. In bone, aluminum exerts three general pathological
effects: (1) inhibition of hydroxyapatite formation and
growth; (2) inhibition of bone cell proliferation; and (3)
suppression of bone cell activity. These pathological
effects of the metal lead to inhibition of bone
mineralization, decreased bone formation, and lower bone
mass. Patients with stainable bone aluminum also have a
higher volume of lamellar osteoid, a lower volume of
woven osteoid, and significantly lower numbers of both
osteoclasts and osteoblasts [31].
Aluminum Performance Goals
The analytical performance of serum aluminum
estimations in European laboratories as judged by various
external quality assurance programs in 2002 was poor,
with up to 26% of enrolled laboratories failing to achieve
agreed performance limits [32].
Recommended performance goals for serum aluminum
levels are a total error of 0.186 mol/L (5 g/L) or 12
20% of the target on an external quality assurance program
[33]. These performance goals were set on the basis of
biological variation in normal subjectsthat is, the
variation in serum aluminum levels measured biweekly for
30 weeks.
References
1 Agency for Toxic Substances and Disease
Registry, U.S. Department of Health and Human
Sources. Toxicological Profile for Aluminium.
September 2006.
http://www.atsdr.cdc.gov/toxprofiles/tp22.html.
Accessed 2 July 2007.
2 Alder JF, Samuel AJ, West TS. The single
element determination of trace metals in hair by
carbon-furnace atomic absorption spectrometry.
Anal Chim Acta 1976; 87: 313-321.
3 Alderman FR, Gitelman HJ. Improved
electrothermal determination of aluminium in
serum by atomic absorption spectroscopy. Clin
Chem 1980; 26: 258-260.
4 Bettinelli M, Baroni U, Fontana F, Poisetti P.
Evaluation of the lvov platform and matrix
modification for the determination of aluminium
in serum. Analyst 1985; 110: 19-22.
5 Bouman AA, Platenkamp AJ, Posma FD.
Determination of aluminium in human tissues by
flameless atomic absorption spectroscopy and
comparison of references values. Ann Clin
Biochem 1986; 23: 97-101.
6 Couri D, Liss L, Ebner K. Determination of
aluminium in biological samples.
Neurotoxicology 1980; 1: 17-24.
7 Gardiner PE, Stoeppler M. Optimisation of the
analytical conditions for the determination of
aluminium in human blood plasma and serum by
graphite furnace atomic absorption spectrometry.
Part 2. Assessment of the analytical method. J
Anal Atom Spectrom 1987; 2: 401-404.
8 Gorsky JE, Dietz AA. Determination of
aluminium in biological samples by atomic
absorption spectrophotometry with a graphite
furnace. Clin Chem 1978; 24: 1485-1490.
9 Guillard O, Tiphaneau K, Reiss D, Piriou A.
Improved determination of aluminium in serum
by electrothermal atomic absorption spectrometry
and zeeman background correction. Anal Lett
1984; 17: 1593-1605.
10 Keirsse H, Smeyers-Verbeke J, Verbeelen D,
Massart DL. Critical study of the speciation of
aluminium in biological fluids by size-exclusion
chromatography and electrothermal atomic
absorption spectrometry. Anal Chim Acta 1987;
196: 103-114.
11 Savory J, Wills MR. Analytical methods for
aluminium measurement. Kidney Int 1986;
29(Suppl 18): S24-S27.
van der Voet GB, de Haas EJM, de Wolff FA.
Monitoring of aluminium in whole blood, plasma,
serum, and water by a single procedure using
flameless atomic absorption spectrophotometry. J
Anal Toxicol 1985; 9: 97-100.
13 Wrobel K, Gonzalez EB, Sanz-Medel AE
Aluminium and silicon speciation in human
serum by ion-exchange high performance liquid
chromatography-electrothermal atomic absorption
spectrometry and gel electrophoresis. Analyst
1995; 120: 809-815.
14 Xu N, Majidi V, Markesbery WR, Ehmann WD.
Brain aluminium in Alzheimers disease using an
improved GFAAS method. Neurotoxicology
1992; 13: 735-743.
15 Parkinson IS, Ward MK, Kerr DN. A method for
the routine determination of aluminium in serum
and water by flameless atomic absorption
spectrometry. Clin Chim Acta 1982; 125: 125-
133.
16 Bianchi F, Maffini M, Mangia A, Marengo E,
Mucchino C. Experimental design optimization
for the ICP-AES determination of Li, Na, K, Al,
Fe, Mn, Zn in human serum. J Pharmaceutical
Biomedical Analysis 2007; 43: 659-665.
17 Dillio S, Violante N,Caimi S, Di Gregorio M,
Petrucci F, Senofonte O. Determination of trace
 120
Aluminum
metals in serum by dynamic reaction cell
inductively coupled plasma mass spectrometry.
Anal Chim Acta 2005; 573: 432-438.
18 Allain P, Mauras Y. Determination of aluminium
in blood, urine, and water by inductively coupled
plasma emission spectrometry. Anal Chem 1979;
51: 2089-2091.
19 Lichte FE, Hopper S, Osborn TW. Determination
of silicon and aluminium in biological matrices by
inductively coupled plasma emission
spectrometry. Anal Chem 1980; 52: 120-124.
20 Que Hee SS, Boyle JR. Simultaneous multi-
elemental analysis of some environmental and
biological samples by inductively coupled plasma
atomic emission spectrometry. Anal Chem 1988;
60:1033-1042.
21 Sanz-Medel A, Roza RR, Alonso RG, Vallina
AN. Atomic spectrometric methods (atomic
absorption and inductively coupled plasma atomic
emission) for the determination of aluminium at
the parts per billion level in biological fluids. J
Anal Atom Spectrom 1987; 2: 177-184.
22 Keeler R. ICP mass spectrometry shows its
mettle. Res Dev 1991; 33: 44-48.
23 Ward NI. Environmental contamination of
aluminium and other elements in North Cornwall
as a result of the Lowermoor water treatment
works incident. In: Vernet J-P, ed. Heavy metals
in the environment. Edinburgh: CEP Consultants,
1989;118-121.
24 Owen LMW, Crews HM, Bishop NJ, Massey RC.
Aluminium uptake from some foods by guinea
pigs and the characterization of aluminium in vivo
intestinal digestion by SEC-ICP-MS. Food
Chem.Toxicol 1994; 32: 697-705.
25 Tang S, Parsons PJ, Slavin W. Rapid and reliable
method for the determination of aluminium in
bone by electrothermal atomic absorption
spectrometry. Analyst 1996: 121: 195-200.
26 Bornhorst JA, Hunt JW, Urry FM, McMillin GA.
Comparison of sample preservation methods for
clinical trace element analysis by inductively
coupled plasma mass spectrometry. Am J Clin
Path 2005; 123: 578-583.
27 Cornelis R, Heinzow B, Herber RFM, Molin
Christensen J, Paulsen OM, Sabbioni E et al
Sample collection guidelines for trace elements in
blood and urine. Pure Appl Chem 1995; 67: 1575-
1608.
28 Kruger PC, Parsons PJ. Determination of serum
aluminium by electrothermal atomic absorption
spectrometry: A comparison between Zeeman and
continuum background correction systems.
Spectrochimica Acta Part B 2007; 62: 288-296.
29 Valkonen S, Aitio A. Analysis of aluminium in
serum and urine for the biomonitoring of
occupational exposure. The Science of the Total
Environment 1997; 199: 103-110.
30 Alfrey AC, LeGendre Gr, Kaehny WD. The
dialysis encephalopathy syndrome. Possible
aluminium intoxication. N Engl J Med 1976; 294:
184-188.
31 Monier-Gaugere MC, Malluche HH. Trends in
renal osteodystrophy: a survey from 1983 to 1995
in a total of 2248 patients. Nephrol Dial
Transplant 1996; 11: 111-120.
32 Taylor A, Angerer J, Claeys F, Kristiansen J,
Mazarrasa O, Menditto A, et al. Comparison of
procedures for evaluating laboratory performance
in external quality assessment schemes for lead in
blood and Aluminium in serum demonstrates the
need for the common quality specifications. Data
Supplement Clinical Chemistry Online
(http://www.clinchem.org/content/vol48/issue11/
accessed 2007-10-22).
33 Taylor A, Angerer J, Claeys F, Kristiansen J,
Mazarrasa O, Menditto A, et al. Comparison of
procedures for evaluating laboratory performance
in external quality assessment schemes for lead in
blood and Aluminium in serum demonstrates the
need for the common quality specifications. Clin
Chem 2002; 48: 2000-2007.
34 Standards Australia. Analysis of serum and
plasma for trace elements Determination of
aluminium content Graphite furnace atomic
absorption spectrophotometric method (Australian
Standard AS 4195.1-1994)
http://www.saiglobal.com/shop accessed 4 July
2007.
METHOD 1: ELECTROTHERMAL ATOMIC
ABSORPTION SPECTROSCOPY
The aluminum in serum or plasma is determined by
graphite furnace atomic absorption spectrometry. Analysis
of aluminum is routinely performed by atomic absorption
spectrometry with electrothermal atomization. The
following method is based upon the methods of Standards
Australia [34]. At the time this section was written, there
was no other standard method available.
There are a range of external quality assurance programs
available that have serum aluminum as an analyte. There
are also more specialized programs available which also
have water as a matrix [33].
Principle
Calibrators, controls, or patient specimens are mixed with
magnesium nitrate and a surfactant to prepare them for
graphite furnace atomic absorption analysis. This matrix
forms a complex with aluminum that has good thermal
stability; this allows for in situ ashing of the specimen to
vaporize aqueous and organic background materials. The
final step of analysis causes vaporization of aluminum,
which absorbs energy at the 309.3-nm line emitted from an
aluminum hollow cathode lamp. Absorbance of energy at
 121
Aluminum
this wavelength is specific for aluminum and proportional
to its concentration.
Instrumentation
A good-quality graphite furnace atomic absorption
spectrometer located in a dust-free environment is required
for analysis. The procedure requires that a Lvov platform
be placed in the graphite tube to enhance sensitivity and
accuracy, and the Zeeman background correction is
required to reduce background to a minimum.
REAGENTS
General Requirements:
All reagents shall be of analytical reagent grade quality.
Grade 1 water, as specified in ISO 3696, is used
throughout. The water should contain less than 0.01 mol
of aluminum per liter.
Solutions:
EDTA cleaning solution (2% m/V). Dissolve 2 g of
ethylenediamine N,N,N tetra-acetic acid disodium salt
(EDTA) per 100 mL of water and mix.
Diluting solution. Prepare 100 mL of a 0.2% (m/V)
solution of surfactant in water. The solution should be
shown to contain less than 0.1 mol of aluminum per liter.
NOTE: Triton X-100 has been found to be a suitable
surfactant for this application.
Human plasma or serum (for the preparation of
calibration solutions). The serum or plasma should be
shown to have less than 0.5 mol of aluminum per liter.
Nitric acid concentrated (p20 1.42 g/mL).
Nitric acid cleaning solution. Add 200 mL of nitric acid
to 800 mL of water and mix.
Standard Solutions:
Stock standard solution (50 mmol/L). Weigh 1.349
0.001g of aluminum wire (>99.9% Al), and transfer to a 1
L volumetric flask. Add 0.1g of mercurous (ll) nitrate
(Hg[NO3]2) to catalyze dissolution, 5 mL of water and 20
mL of nitric acid. When dissolution is complete, dilute to
volume with water, and store in a polyethylene bottle.
NOTES:
1. This solution has been shown to be stable for at
least 3 months.
2. Alternative standard solutions may be used in a
preparation of this stock solution, provided the
traceability to recognized national standards body
can be demonstrated.
Intermediate standard solution (200 mol/L). Pipette
4.00 mL of stock aluminum solution into a 1 liter
volumetric flask containing approximately 200 mL of
water and 10 mL of nitric acid . Dilute the contents of the
flask to volume with water. This solution should be used
within 4 hours of preparation.
APPARATUS
Glassware. Volumetric glassware of grade A or B should
be used throughout. All glassware should be cleaned with
nitric acid and rinsed thoroughly with water before use.
Use of volumetric glassware should comply with AS 2162.
Volumetric flasks should comply with AS 2164. Pipettes
should comply with AS 2166.
Plasticware. Polyethylene or PTFE containers are
preferred. Polystyrene containers have been found to be
unsuitable for diluted samples. Plasticware and automatic
sampler cups should be soaked for between 30 min and 1h
in EDTA cleaning solution, followed by rinsing with
copious quantities of water. Dry in a clean atmosphere.
Micropipettes and microsample injectors. Micropipettes
and microsample injectors should be shown to have an
accuracy of 2% and a precision of better than 2% and to
be chemically inert to the reagents used.
Atomic absorption spectrometer. The atomic absorption
spectrometer, equipped with background correction and a
graphite furnace atomizer, should be prepared in
accordance with AS 2134.2.
Signal recording. The instrument should have facility for
real-time display. If a chart recorder is used, it should have
a full-scale response time of less than 0.5 s for a chart
width of not less than 250 mm.
Purge gas. Argon is recommended for use as a purge gas.
INSTRUMENT OPERATING PARAMETERS
Analytical parameters. A variety of furnace atomizers are
available, and each manufacturers instruction manual
should provide adequate information for their operation.
Wavelength. The wavelength 309.3 nm produced by an
aluminum line source has been used in the development of
this method.
Injection Volume. To limit calibration curvature, the
volume injected should be such that the absorbance
difference between the zero concentration (C0) and the
highest concentration calibration solution (C0) is not less
than 1.7 times the absorbance difference between the zero
concentration (C0) and the mid-range concentration
calibration solution (C0). This volume should remain
constant for the calibration solutions and the test samples.
 122
Aluminum

Furnace operating parameters. The following
information is given as a guide:
(i) Dry at approximately 130C. Optimize the
drying temperature and its duration by
observing the droplet of the sample in the
atomizer during the drying cycle. The droplet
should evaporate rapidly but not boil during
this process.
(ii) Ash at approximately 1500C. Optimize the
ashing temperature and its duration so that
aluminum is not volatilized.
(iii) Atomize at approximately 2600C. Optimize
the atomization temperature and its duration
so that all aluminum is volatilized (the
absorption signal should return to the
baseline at least 1 s before the end of the
atomization cycle).
(iv) Clean the furnace at the atomizing
temperature or higher for 3 to 5 s with the gas
flow on.
(Co to C6) and then two volumes of diluting
solution and mix thoroughly.
(b) Adjust the instrument read-out to zero.
(c) Inject the injection volume of the diluted Co
solution into the furnace atomizer.
(d) Carry out the dry, ash, atomize, and clean
cycle, and record the absorbance signal (Ao)
peak area or maximum peak height.
(e) Repeat the injection, and assess the
acceptability of the absorbance signals in
accordance with the procedure outlined in AS
2134.2.
(f) Repeat steps (b) to (e) for the remaining
diluted calibrating solutions C1 to C6, and
record the absorbance signals A1 to A6.

Instrument contamination. Special attention should be Prepare a calibration graph of absorbance versus
paid to graphite components, sampler assemblies, and rinse concentration of aluminum in mol/L.
fluids.
METHOD 2: INDUCTIVELY COUPLED PLASMA
CALIBRATION [18]
Preparation of calibrating solutions. The calibrating Reagents
solutions should be prepared in serum or plasma and in A stock solution of 1000 g/mL of Al is obtained by
accordance with Table 1. dissolving 1.000g of aluminum foil (analytical-reagent
grade, E. Merck) in 20 mL of 1 + 1 sulfuric acid (Carlo-
Erba) and diluting to 1000 mL with ultra pure water and 50
Table 1: Aluminum Concentrations for mL of concentrated nitric acid (E. Merck). An intermediate
Calibration Graph solution of 100 mg/L of Al is obtained by diluting 10 mL
of the above solution to 100 mL with 1 + 20 dilute nitric
Calibration Volume of Volume Added acid.
Solutions Intermediate of Aluminum Standard solutions are obtained by direct dilution of the
Standard Plasma mol intermediate solutions or stock solutions with ultra pure
Solution or water (Milli-Q).
(mL) Serum
Absorption Measurements

mL Serum and urine are analyzed directly after dilution 1 + 1

C0 0 10.0 0.0 with ultra pure water. The calibration graph is constructed
C1 0.05 9.95 1.0 using the aqueous standards, and the instrumental
C2 0.10 9.90 2.0 conditions used are described in Table 1. Further dilution
C3 0.15 9.85 3.0 of dialysis fluids (concentrate diluted 1 + 35) or tap-water
C4 0.20 9.80 4.0 samples is not necessary.
C5 0.25 9.75 5.0
C6 0.30 9.70 6.0 Sample preparation for ICP emission analysis is as
follows. The samples are diluted 1 + 2 for serum and 2 + 3
NOTE: These calibrating solutions may be further for urine with standard 150 g/L Al solution (spike), and
dispensed into plastic tubes in smaller aliquots and stored the concentration of the metal is determined by difference
frozen at 20C for up to 6 months. after analyzing the spiked solutions by reference to a
calibration graph prepared from Al in NaCl (1100 100
Preparation of calibration graph procedure: ppm of Na+) solution. A 5 L aliquot of sample solution is
pipetted onto the graphite rod and vaporized into the
(a) To a series of plastic tubes or sample cups, plasma. If the saline content of the sample is high (e.g.,
add one volume of each calibrating solution dialysis solutions or serum), dilution (1 + 1) with water is
carried out.
 123
Aluminum

Special care has to be exercised in order to avoid possible Appendix: Abbreviations Used in the Text
contamination along the three main stages (all sources of
AAS Atomic absorption spectrometry
contamination) of analysis: sampling-storage, pre-
ETAAS Electrothermal atomic absorption spectrometry
treatment and measurement.
FAAS Flame atomic absorption spectrometry
Sampling has to be carried out in a way to ensure that GF Graphite furnace atomic absorption spectrometry
manipulations and material or utensils do not release HGA Heated graphite atomizer
aluminum. Storage of samples should be carried out in ICP-AES Inductively coupled plasmaatomic emission
special plastic vials previously treated with acid or EDTA. spectrometry
We have successfully used polystyrene tubes previously ICP-MS Inductively coupled plasmamass spectrometry
treated with 10% nitric acid for 24 h, followed by rinsing ICP-OES Inductively coupled plasmaoptical emission
with copious amounts of Milli-Q water. Regular testing for spectrometry
possible aluminum release from these tubes is performed LAMMA Laser microprobe mass analysis
for every new batch received. NAA Neutron activation analysis
PE Perkin Elmer
Preparation of the sample, reagents, and the like, is another SEC Size exclusion chromatography
important possible source of contamination. In the THGA Transversely heated graphite atomizer
proposed method, there is only a 1 + 1 dilution with water,
so the only recommendation is to use ultra-pure water
(e.g., Milli-Q water). However, regular determination of
the aluminum content by GFAAS of such water is also
advisable.

Instrumental measurement can also introduce a

contamination risk. It is recommended that all sample
manipulations, including the final measurement, should be
carried out in a Class 100 laminar-air-flow clean bench.
We have verified the importance of such clean-room
conditions for Al concentrations 10 g/L; detection
limits attained are about three times lower than those
obtained out of the clean room, and it was confirmed by
experiment that precision at concentrations of 2 to 5 g/L
is much better with complete clean-room conditions.
 124
Amino Acid Screening
Amino Acid Screening
Kevin Carpenter
Name: Amino acid screening
Clinical significance: Refer to Chapter 52, Diseases of Genetic Origin, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
Inborn errors of metabolism affecting amino acid
metabolism were among the first to be described, and from a
historical perspective, testing for inborn errors was often
limited to qualitative tests for amino acids in plasma or
i universal, but the range of techniques available to effect
urine.
While there is no doubt the amino acid disorders are now
only a small part of the inherited metabolic disease
spectrum, there is still a place for initial screening tests
which, although rarely diagnostic, may give insight into the
confirmatory investigations required. Amino acid screens
are usually run in conjunction with organic acids, and
interpretation of both requires considerable experience and
is usually confined to laboratories with a special interest in
inborn errors of metabolism.
Quantitative analysis of amino acids in physiological fluids
is a specialist investigation and outside the scope of this
chapter, but it is essential to confirm findings from
screening tests and to monitor treatment.
One of the reasons amino acid disorders figured so
prominently in the early descriptions of inborn errors of
metabolism was the availability of a suitable locating
reagent for various chromatographic techniques. Ninhydrin
(triketohydrindene hydrate), has been in use for over 90
years and reacts with primary or secondary amines through a
complex process to produce Ruhemanns purple. All -
amino acids will react with Ninhydrin at room temperature,
and heating to 105C will allow all compounds containing a
primary or secondary amine to react. Although the basic
reaction results in production of a purple color, there are
many subtle variations in the colors different amino acids
yield, which may be of value in identifying poorly separated
compounds. The amino acids proline and hydroxyproline,
for example, react in a different manner to produce a
characteristic yellow color. Ninhydrin staining may be
conveniently achieved for chromatography, simply by
running the chromatography plate or paper a second time in
a solvent containing Ninhydrin. Alternatively, spraying or
dipping in the stain dissolved in acetone can be used for
chromatography or electrophoresis on any support media.
Different stains, often used in conjunction with Ninhydrin,
can enhance detection of specific species, and platonic
i
Amino acid screening
Previous and current authors of this method:
First edition: Zulfikarali H. Verjee
Methods edition: Not updated
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Kevin Carpenter
iodide reagent can be used as an overstain after reading the
Ninhydrin stain to locate sulfur-containing amino acids,
which appear white or cream against a pink background.
Location of amino acids by Ninhydrin may be almost
separation of the amino acids in physiological fluids is wide.
There are two main categories of separation techniques:
chromatography and electrophoresis. These may be used
singly in one or two dimensions or in combination as a two-
dimensional separation.
Chromatography of amino acids was originally performed
on paper, commonly using n-butanol/acetic acid/water
solvent systems running overnight [1]. Enhanced separation
may be achieved by running in a second solvent at 90
degrees to the original, using t-butanol/methyl ethyl
ketone/ammonia solvents for the second separation.
Resolution on paper can be remarkably good, but the
medium has been largely superseded by the use of
commercially available thin-layer cellulose plates which
afford excellent and reproducible separations but may
require de-salting of urine samples prior to analysis.
Multiple samples per sheet can be applied if only a single
dimensional separation is run.
The major disadvantage of chromatographic separation is
the time required to obtain a result. Typical separations are
run overnight, and if a rapid result is required, this can be an
issue. A more rapid alternative to paper or thin-layer
chromatography is electrophoresis. Cellulose acetate
electrophoresis using a formic acid/acetic acid buffer at pH
1.9 can give reasonable separation in 20 to 30 minutes at
voltages of 200-300 V [2].
Even better separations can be achieved using paper as the
support media and operating at high voltages [3]. This
method generates considerable heat and requires
instrumentation incorporating a cooling plate and suitable
safeguards against electric shock. However, modern ceramic
cooling plates linked to a recirculating coolant reservoir all
but eliminate problems of the paper drying out during the
run, and excellent results are obtained using 2500 V, 20-
minute separation on 265 200 mm paper sheets which can
comfortably fit up to 9 urine samples per sheet.
For all electrophoresis methods, samples are applied close to
the anode end with the dibasic amino acids, ornithine,
lysine, and arginine migrating the furthest, and acidic
compounds taurine and phosphoethanolamine remaining
close to the origin. S-sulfocysteine (a marker for sulfite
oxidase deficiency) migrates slightly towards the anode, so
samples are applied a few cm above the anode position to
allow for this.
 125
Amino Acid Screening
Electrophoresis does not completely separate amino acids
with similar isoelectric points, but when used in
combination with chromatography run at 90 degrees to the
electrophoresis, the techniques are complimentary, enabling
separation of amino acids neither technique in isolation can
achieve [4]. The downside to the enhanced separation is that
only one sample per sheet can be applied, and therefore
throughput is limited.
Reference and Preferred Methods
There are no reference methods for amino acid screening.
The American College of Medical Genetics publishes
Standards and Guidelines for Clinical Genetics Laboratories
[5]. The 2006 edition states, Qualitative amino acid
analysis must reliably detect conditions in which there are
either gross or modest elevations of specific amino acids in
blood and/or urine but warns Qualitative amino acid
analysis by thin-layer chromatography (TLC) is suitable
only for the detection of gross abnormalities. As some
disorders may be missed by this method, its use for the
purpose of evaluating high-risk patients should be
discouraged.
However, TLC is widely used for initial amino acid
screening, and provided the limitations are understood, it
can still yield valuable information. For laboratories setting
up amino acid screening for the first time, TLC will give
reliable results in most hands, whereas the enhanced
resolution achieved by high-voltage electrophoresis requires
more specialized equipment and considerable skill to get
consistent results.
Specimen
Plasma or urine can be used for amino acid screens, and
many labs request both. This can be useful where increased
concentrations of particular amino acids in urine may be due
to their accumulation in blood or as a result of impaired
renal reabsorption. Comparison of the patterns obtained in
the two samples will reveal the source of the increase.
Plasma alone will not be informative for detection of renal
transport defects such as cystinuria or detection of
generalized aminoaciduria such as is seen in Fanconi
syndrome. Plasma requires deproteinization prior to
running, and urine samples may need removal of inorganic
salts by ion exchange resin if TLC is used.
Samples should be stored and transported at or below 20C
to prevent bacterial degradation. Under such conditions,
most amino acids are stable for many years. Typical
volumes required for chromatographic or electrophoretic
techniques are only a few hundred microliters. Urine
samples should be loaded or diluted according to creatinine
concentration.
Interferences
Ninhydrin will react with a number of exogenous
compounds that may interfere with identification and
interpretation. Penicillin-containing antibiotics produce a
number of Ninhydrin-positive metabolites. Other antibiotics
may also produce abnormal spots, as will acetaminophen, L-
dopa, & GABA analogues.
Diet influences the pattern of amino acids seen, with a
generalized increase in excretion postprandially and specific
increases in carnosine, anserine, and 1-methylhistidine
following white poultry meat ingestion. Catabolism will
result in a decrease in alanine concentration and an increase
in -aminoisobutyrate concentration.
Decreased levels of serine and glutamine may be seen in
bacterially contaminated urine samples.
Interpretation
Interpretation of the profiles obtained in both urine and
plasma is a highly skilled job requiring experience of what
to expect in normal and disease states, and the information
given here should be considered a very basic guide only. It
must also be noted that not all aminoacidurias result in
clinical disease [6].
In normal plasma, the most prominent amino acids are
glutamine and alanine, with lesser amounts of glycine,
valine, lysine, leucine, serine, and threonine usually visible.
The aromatic amino acids phenylalanine and tyrosine give
quite faint bands, as do the remainder of the physiological
amino acids.
In normal urine, age is an important factor in the amino acid
excretion pattern. Babies often show an immaturity of renal
reabsorption mechanisms, resulting in an increased
excretion of proline, hydroxyproline, glycine, and
sometimes cystine and lysine. Taurine is also prominent at
birth but declines rapidly. Generally heavy patterns
(nonspecific increases in all amino acids) are seen in the
neonatal period, gradually declining to adult-type patterns
during childhood and adolescence.
A normal urine pattern is dominated by glycine, with lesser
and similar-intensity bands seen for alanine, serine, and
glutamine. Lysine, cystine, and the aromatic amino acids
produce only very faint bands in the healthy individual.
Inborn errors of amino acid metabolism will usually result in
accumulation of the amino acid proximal to the defect in the
synthetic pathway in both plasma and urine. Thus
phenylketonuria gives elevated phenylalanine, nonketotic
hyperglycinemia gives elevated glycine, and maple-syrup
urine disease shows increases in valine, leucine, and
isoleucine. Urea-cycle defects will all give elevated
glutamine, but argininosuccinate synthetase deficiency will
also result in elevated citrulline, and argininosuccinate lyase
deficiency will show the presence of argininosuccinate and
its anhydride.
In all instances, interpretation should be performed in light
of the clinical condition of the patient and preferably in
conjunction with organic acid profile results.
Amino Acid Performance Goals
The key determinant of analytical acceptability for amino
acid screening is the ability to detect inherited metabolic
disease if present. Since the majority of the methods
 126
Amino Acid Screening
described here allow several samples to be run on a single
plate or sheet, the simplest way to ensure reliability may be
to run one or more positive controls on each plate. However,
since this limits the analytes being controlled to those
elevated in the control sample chosen, it may be useful to
supplement the sample with additions of other key amino
acids at concentrations typical of those found in disease. In
this way, one can be sure an elevation in a particular amino
acid can be reliably detected, and it also serves as a rough
comparator for grading the extent of the increase in a patient
sample.
References
1 Smith I, Ersser RS. Aminoacids and Related
Compounds. In: Smith I and Seakins J W T.
Chromatographic and Electrophoretic Techniques.
4 ed. London: William Heinemann Medical Books;
1976. 75-121.
2 Kohn J. Cellulose Acetate Electrophoresis. In:
Smith I. Chromatographic and Electrophoretic
Techniques. 4 ed. London: William Heinemann
Medical Books Ltd; 1976. 90-137.
3 Beale D, Smith I. High Voltage Paper
Electrophoresis. In: Smith I. Chromatographic and
Electrophoretic Techniques. 4 ed. London: William
Heinemann Medical Books Ltd; 1976. 31-65.
4 Shih VE, Mandell R, Sheinhait I. General
Metabolic Screening Tests. In: Hommes FA.
Techniques in Diagnostic Human Biochemical
Genetics.New York: Wiley-Liss; 1991. 45-68.
5 American College of Medical Genetics. Standards
and Guidelines for Clinical Genetics Laboratories.
2006 Edition.
http://www.acmg.net/Pages/ACMG_Activities/stds-
2002/f.htm Accessed: 2007-08-09
6 Shih VE. Detection of hereditary metabolic
disorders involving amino acids and organic acids.
Clin Biochem 1991; 24: 301-309.
Procedure 1: Plasma Amino Acids by Thin-Layer
Chromatography
Principle
Plasma samples are deproteinized by ethanol precipitation
and applied to cellulose TLC plates. The plates are run twice
in an n-butanol/acetone/acetic acid/water solvent, the second
run incorporating Ninhydrin into the solvent to locate the
amino acids.
Reagents
1. Absolute Ethanol
2. TLC Solvent 1 (tank 1). In a fume hood, measure
out: 21 mL of acetone, 21 mL n-butanol, 6 mL of
glacial acetic acid, and 12 mL of water. Pour into a
TLC tank and cover with a glass lid. Allow 30 min
for equilibration before use. Make fresh solutions
every 2 days.
3. Ninhydrin 0.2 % w/v. Weigh out 0.5 g of
Ninhydrin. Dissolve in 250 mL acetone. Store in a
brown stoppered bottle, stable for 1 month.
4. Locating Solvent 2 (tank 2). In a fume hood,
measure out: 21 mL of the 0.2% Ninhydrin
solution, 21 mL n-butanol, 6 mL of glacial acetic
acid, and 12 mL of water. Pour into a TLC tank,
and cover with a glass lid. Allow 30 min for
equilibration before use. Make fresh solutions
every 2 days.
5. Commercial amino acid standard mixture.
Example: Sigma A2161 diluted 1:8 with ethanol to
give equivalent of 125 mol/L concentrations in
plasma. Stored at 80C.
Equipment:
Cellulose TLC plates 10 20 cm (Merck # 5730)
TLC tanks
Micropipette for delivery of sample to TLC plate
Assay
1. Samples are deproteinized by mixing with ethanol.
In a microcentrifuge tube add 200 L of ethanol to
50 L of plasma. Centrifuge for 2 min at 10,000 g.
2. Transfer supernatant to a clean tube.
3. Mark a cellulose thin-layer plate with lines 1.5 cm
long and 1 cm apart, 1.5 cm from the bottom edge
of the plate, using a soft pencil. NOTE: Use gloves
and take care not to touch the plate; fingerprints
contain enough amino acids to stain the plate.
4. Apply 15 L of the plasma extract in 3 lots of 5
L, allowing the extract to completely dry between
applications (warm air may be used to aid drying
between applications). The sample is applied using
a 5 L positive-displacement micropipette as a
streak 1-cm long between the lines drawn on the
plate.
5. A maximum of 7 samples can be run on a single
plate. One track is reserved for the amino acid
standard mixture applied as per plasma samples.
6. Place the TLC plate in tank 1, and develop until the
solvent reaches the top of the plate (approximately
20 to 30 min).
7. Remove the plate, and allow to dry completely in a
fume hood.
8. Place the plate in tank 2, and develop until solvent
reaches the top of plate.
9. Remove the plate, and allow to dry overnight.
Color will develop at room temperature, but if an
urgent result is required, plate may be heated at
100C to aid drying and development.
10. Comparison of individual tracks with standard
mixture allows qualitative scoring of amino acids,
but gross elevations should always be followed up
by quantitative analysis in a specialist laboratory.
Procedure 2: Urine Amino Acids by High-Voltage
Electrophoresis
Principle
High-voltage electrophoresis (HVE) is a very rapid and
efficient method for separating small molecules. At pH 1.9,
the basic amino acids lysine, arginine, and histidine become
positively charged and travel rapidly towards the cathode
(), whereas the acidic amino acid cysteic acid is negatively
charged and remains near the origin close to the anode (+).
A standardized volume of urine (100 nmol creatinine) is
applied as a narrow band onto a paper sheet. Electrophoresis
is performed for 24 min at 2500 V. Staining the
electrophoretogram with Ninhydrin localizes the amino
acids. The profiles are interpreted, and then the sheet is
overstained with iodoplatinate reagent that enables
 127
Amino Acid Screening
visualization of sulfur-containing compounds, including
homocystine, methionine, cystine, and S-sulfocysteine.
NOTE: plasma may be run on the same method but must be
deproteinized before application to the paper.
Reagents
1. Formic acid/acetic acid buffer pH 1.9. Add 37.5
mL formic acid and 150 mL glacial acetic acid
slowly to approx. 1500 mL of Milli-Q water in
fume hood. Make up to 2 L with Milli-Q water.
Store in brown glass bottle at room temperature.
Stable for 1 month.
2. Ninhydrin 0.2 % w/v. Weigh out 0.5 g of
Ninhydrin. Dissolve in 250 mL acetone. Store in a
brown stoppered bottle, stable for 1 month.
3. Chloroplatinic acid, 1 g/500 mL. Weigh out 1g of
chloroplatinic acid (CAUTION: highly toxic, wear
mask and gloves). Make up to 500 mL with water.
4. Hydrochloric Acid (HCl) 6M.
5. Potassium iodide, 167 g/L. Weigh 41.75 g
potassium iodide, make up to 250 mL with water;
replace approx. every 12 months. Store in brown
glass bottle.
6. Iodoplatinate (IP) stain. Make up the stain in the
fume hood immediately prior to staining. Mix 5 mL
of chloroplatinic acid, 0.5 mL potassium iodide, 1
mL 6M HCl, and 42.5 mL acetone.
Equipment:
REQUIRED: High-voltage electrophoresis equipment
capable of running at 2500 V, with integrated
cooling system. A typical system would be:
Pharmacia HVE system:
o Hoefer PS3000 DC power supply
o Multiphor II electrophoresis unit
comprising:
o Buffer tank
o Ceramic cooling plate
o Electrode holder and electrodes
o Safety lid
o Multi Temp III cooling unit.
Electrophoresis paper, Machery Nagel MN 214,
200 265 mm
IEF Electrode strips (Pharmacia Cat. # 18-1004-
40)
Wash bottle with a fine jet
Large glass dish
Glass plate 195 mm square
Blotting paper, 445 570 mm, 135 gsm, APPM
Oven set at 100C 2C, for drying and 125C
2C for Ninhydrin stain development
Micropipette for delivery of sample
Assay
Electrophoresis
1. Calculate volume of urine in L to be applied by
dividing100 by creatinine concentration in mmol/L.
2. Turn on recirculating cooler and set to 4C
3. In lead pencil, mark out electrophoresis paper 6 cm
from one end for 9 samples. These should be 1.0
cm long, 1.5 cm from both edges, and 1.0 cm apart.
4. Apply sample as a narrow band in small volumes,
drying between applications using hot air.
5. When all samples have been applied, carefully wet
the sheet with buffer, avoiding wetting the origin
directly and allowing buffer to run up to the origin
from both sides.
6. Gently blot the sheet between a folded sheet of
filter paper and place on the ceramic cooling plate,
origin at anode end.
7. Wet the IEF electrode strips with buffer, and blot
gently before applying to the sheet 1cm from each
end.
8. Place the glass plate on the sheet to maintain good
contact with cooling plate.
9. Position IEF electrodes over electrode strips and
connect electrodes. Fit safety lid.
10. Turn on power supply, and run at constant voltage
2500 V (approximately 50-60 mA) for 25 min.
11. Turn off power supply. Remove sheet and hang to
dry in oven at 100C for 5 min.
Ninhydrin and iodoplatinate staining
1. Stain sheet by rapidly dipping through Ninhydrin
reagent in a staining tray. Dry briefly in fume hood
before transferring to oven at 120C for 3 min.
2. Interpretation of Ninhydrin staining requires
experience and knowledge of changing excretion
patterns with age. Follow-up of abnormal patterns
requires quantitation in a specialist centre.
3. If required, following interpretation of the
Ninhydrin stain, the sheet can be overstained with
iodoplatinate reagent to highlight the presence of
sulfur-containing amino acids.
4. Ensure that the staining dish and paper are
thoroughly dry, then add a volume of the IP stain.
5. Quickly and evenly dip the paper through the stain.
6. Hold the papers in the fume hood briefly to remove
any excess acetone. Paper clips are used to hold the
papers in a cylindrical shape. These cylinders are
stood on their end in a glass tank in the fume hood
overnight at room temperature. The Ninhydrin
positive bands are rapidly decolorized by the acid
present, except for pipecolic and hydroxypipecolic
acid bands that remain fluorescent purple.
7. The iodoplatinate stain will give a pale pink/peach
background with sulfur-containing amino acids,
homocystine, methionine, cystathionine, cystine,
and S-sulfocysteine, having a cream or white color.
8. Several drugs and other compounds will give
cream bands, and interpretation again requires
considerable experience.
128
Ammonia
Ammonia
Elizabeth M. Hall
Name: Ammonia
Clinical Significance: Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: NH3
Molecular mass: 17.03 D
Merck Index: 13,492
Chemical class: Amine
Reference method: None
i causes a pH change detected by a standard glass
Principles of Analysis and Current Usage
Methods for analysis of blood ammonia can be
considered under four headings: microdiffusion, ion-
selective electrode, ion-exchange, and enzymatic
methods. The pKa of the ammonium ion is 9.5, therefore
at physiological pH, nearly all ammonia is present as
ammonium ions (NH4+). Both microdiffusion methods
and ion-selective electrodes rely on alkalinization of the
sample to release ammonia gas.
Microdiffusion methods were the earliest methods used
for blood ammonia. Workers such as Conway allowed
the released ammonia to diffuse into a chamber
containing hydrochloric acid. The ammonia
concentration was determined by titration with barium
hydroxide and a pH indicator [1]. Seligson used
nesslerization to measure the ammoniamercuric
chloride and ammonia form a yellow chelate that can be
measured at 410 nm [2]. These methods were
cumbersome and required technical skill; in addition,
deamination of amino acids occurs during prolonged
incubation of plasma with alkali, leading to spuriously
high results. However, microdiffusion is the basis of two
dry chemistry methods for ammonia (Table 1, Method
1). The sample is added to borate buffer pH 9.2 to
release ammonia gas, which diffuses across a
semipermeable membrane into a layer containing a pH
indicator [3]. The color change in the pH indicator is
measured by reflectance. The dry slide method on Vitros
analyzers uses bromophenol blue, monitored at 600 nm,
to measure ammonia in plasma samples. The Ammonia
Checker is a handheld meter that uses whole blood with
bromocresol green monitored at 635 nm, with results
being available in less than 4 minutes [4].
For ammonia analysis by ion-selective electrode, plasma
is mixed with buffer pH 10.4 to liberate ammonia gas,
i
Ammonia
Previous and current authors of this method:
First edition: Nancy Gau
Methods edition: Nancy Gau
Second edition: Not updated
Third edition: Not updated
Fourth edition: Nancy Gau
Fifth edition: Elizabeth M. Hall
which diffuses across a semipermeable membrane and
electrode with silver-silver chloride reference electrode
(Table 1, Method 2) [5]. When adapted to a continuous-
flow analyzer, this method is reliable [6,7] but not
widely available.
In ion-exchange methods (Table 1, Method 3), ammonia
is isolated by adsorption onto a strong cation exchange
resin. After washing, the ammonia is eluted and
measured using the Berthelot reaction. In the Berthelot
reaction, ammonia and phenol form a blue indophenol
complex (max 630 nm) in the presence of hypochlorite.
Sodium nitroferricyanide (Na2(Fe[CN]5NO),
nitroprusside) acts as a catalyst and increases color
intensity and assay reproducibility. Microscale methods
were developed to use smaller sample volumes but
remained cumbersome and time consuming [8,9].
The most common method in use today for measurement
of plasma ammonia uses glutamate dehydrogenase
(GLDH, L-glutamate:NAD(P) oxidoreductase
(deaminating), EC1.4.1.3) with either NADH or
NADPH (Table 1, Method 4) [6].
NH4+ + 2-oxoglutarate + NADH + H+ _glutamate
dehydrogenase glutamate + NAD+ + H2O
The ammonia concentration is proportional to the
decrease in absorbance at 340 nm. Preincubation of the
sample and substrates before addition of the enzyme
avoids interference from other NADH-consuming
reactions [10]. Alternatively, the reaction may be started
by addition of coenzyme [11].
Data from the 2007 College of American Pathologists
(CAP) proficiency testing program indicate that most
laboratories in the United States use a GLDH method for
plasma ammonia measurement, with 15% using a Vitros
dry slide method. In contrast, in the United Kingdom,
18% of participants in the 2007 Wales External Quality
Assessment Scheme (n=202) use the handheld
reflectance meter, 10% use a Vitros plasma dry slide
method, and the majority use a GLDH method (A.
Thomas, personal communication).
129
Ammonia
Reference and Preferred Methods
There is no reference method for the measurement of
ammonia in biological fluids.
Blood ammonia measurement is usually required
urgently for immediate diagnosis and patient
management. Both qualitative screening methods and
quantitative methods are available. Method choice will
depend on workload and patient mix, the speed of
analysis required, and the availability of instrumentation
and technical expertise.
The preferred method for measurement of plasma
ammonia on todays laboratory automated analyzers is
either GLDH or the Vitros dry slide method.
The handheld reflectance meter has a narrow working
range (up to 285 mol/L) but is suitable for screening for
hyperammonemia by laboratories with small workloads
or at point of care. All abnormal results must be
confirmed with a quantitative method. Larger
laboratories and those serving acute pediatric units must
provide quantitative analysis for the diagnosis and
monitoring of hyperammonemia.
Specimen
Venous samples collected without stasis and avoiding
hemolysis are preferred for blood ammonia
measurement. Capillary specimens produce significantly
higher results [6,12] but may be used for rapid screening,
provided suitable reference intervals are available and
abnormal results are confirmed by laboratory analysis of
a venous sample. Plasma ammonia concentrations are
not significantly affected by diet or fasting [6], although
fasting is recommended by some [11].
Plasma from samples collected into either EDTA or
lithium heparin is suitable for most methods of ammonia
determination. However, heparin can increase
background absorbance with some buffers [11,13].
Ammonium heparin is not a suitable anticoagulant.
Ideally, each batch of sample tubes used for ammonia
sample collection should be checked for contamination
before use by adding a low standard to five tubes,
mixing well, and measuring the apparent ammonia
concentration. Tubes are acceptable if apparent ammonia
concentration increases by less than 10%. Tubes should
be stored in an airtight box [6]. Alternatively, each
sample should be accompanied by an empty tube from
the same batch, which is checked for ammonia
contamination by the analysis of low standard added to
the tube.
Plasma ammonia concentration increases during storage
of whole blood and separated plasma, owing to the
metabolic activity of erythrocytes and platelets and
deamination of plasma proteins and amino acids by
enzymes [14-16]. Samples should be transported to the
laboratory on ice and whole blood analysis or separation
of plasma performed within 15 minutes of venesection
[17]. Centrifugation must be with sufficient force to
remove platelets (>2000g) [12]. If analysis is delayed,
separated plasma may be stored at 4C for up to 1 hour.
For longer storage, freezing at 70C is recommended
[16]. Serum is unsuitable because complete clotting
takes longer than 15 minutes and because ammonia is
produced during the formation of crosslinks between
fibrin molecules [12,18].
Interferences
Contamination of the sample by ammonia from
exogenous sources or due to endogenous production
during specimen transport and handling are the most
common causes of inaccurate results. Results may be
affected by contamination of the atmosphere, water, and
laboratory equipment with ammonia. If capillary
samples are used, care must be taken to decontaminate
the skin to avoid contamination with sweat. Smokers
usually have higher plasma ammonia concentrations, and
it has been recommended that samples be collected by a
non-smoker. Within the laboratory, analysis should not
occur in proximity to a system using ammonium buffers,
and care should be taken to use deionized water and
exclude detergents. Care must also be taken to avoid loss
of ammonia from calibrators as ammonia gas.
Hemoglobin does not interfere with ammonia
determinations, but the ammonia concentration within
erythrocytes is about three times the concentration in
plasma, so hemolyzed samples are unsuitable [16].
Lipemic samples and those with high bilirubin
concentrations may be unsuitable for analysis in some
systems [11].
Endogenous production of ammonia within separated
plasma occurs due to deamination of amino acids. The
glutaminase activity of -glutamyl transferase is a major
contributor to this effect, so ammonia concentration may
be artifactually increased in samples with raised -
glutamyl transferase activity [14]. Colonization of the
laboratory analyzer with urease-positive bacteria has
also been reported to give spuriously high ammonia
results [19].
An unidentified metabolite of the antibiotic cefotaxime
may interfere in the enzymatic assay for ammonia,
giving an ammonia result of less than zero [10].
Reference Intervals and Units
Plasma ammonia concentrations are expressed in
mol/L.
Conversion factors: ammonia (g/dL) 0.714 =
ammonia (mol/L)
Ammonia Reference Intervals [20]
Plasma mol/L g/dL
Premature neonate < 200 < 143
Term neonate < 100 < 71
Infant/child < 40 < 29
Adult < 40 < 29
The higher ammonia concentrations in neonates are due
to immaturity.
130
Ammonia
Interpretation
Circulating ammonia is produced principally from the
catabolism of amino acids derived from endogenous or
dietary proteins. The gastrointestinal tract is an
important source. Usually, plasma concentrations are
maintained at low levels by urea synthesis in the liver
and by formation of glutamine. Hyperammonemia
occurs when there is disruption of the urea cycle in the
liver. This may be due to (1) inherited defects of the urea
cycle enzymes secondary to disruption of liver
metabolism or (2) bacterial production of ammonia in
the gastrointestinal or urinary tract [6,21]. The urea cycle
defects are each rare disorders but have an estimated
combined incidence of about 1:30,000, with the
commonest being ornithine transcarbamylase deficiency
(OTC). Some disorders of amino acid, organic acid, and
fatty acid metabolism may also present with
hyperammonemia. In neonates, clinically significant
hyperammonemia may occur secondary to asphyxia,
infection, and sepsis. Hyperammonemia has been
associated with parenteral nutrition, sodium valproate
therapy, acute-onset hepatic failure, and advanced
chronic liver disease [6,21]. In Reyes syndrome, acute
encephalopathy and fatty degeneration of the liver may
be associated with a viral illness or salicylate therapy
that lead to acute mitochondrial injury [6].
Ammonia is neurotoxic. Hyperammonemia has a range
of presentations from an acute catastrophic illness in
babies to episodes of lethargy and vomiting.
Measurement of ammonia should be considered in any
neonate with unexplained neurological deterioration, any
older patient with unexplained encephalopathy, and
children or adults with a history of episodes of vomiting
and lethargy or of protein avoidance, which may indicate
a mild urea-cycle defect. Increased ammonia
concentration should always be confirmed on a second
sample to exclude artifactual increases caused by poor
sample handling. Infection should always be excluded as
a cause of mild increases in ammonia in neonates.
Inherited disorders should be excluded in patients with
unexplained hyperammonemia, Reyes-like syndrome,
unexplained hepatic encephalopathy, or cyclical illness
[21]. Increased ammonia concentrations are a
nonspecific finding in liver disease, and measurements
do not aid with diagnosis, prognosis, or monitoring of
treatment [6].
Significant encephalopathy usually develops at
concentrations above 300 mol/L. Concentrations
greater than 500 mol/L usually present with coma and
convulsions and are associated with neonatal-onset
inherited metabolic diseases.
Ammonia Performance Goals
There are no studies of biological variation of ammonia
concentrations on which to base goals for analytical
performance [22]. However, with a GLDH method,
between-day precision of 3.6% can be achieved at a
concentration of 60 mol/L [11].
References
1 Conway E. Microdiffusion analysis and
volumetric error. 3rd ed. London: Crosby
Lockwood and Son Ltd; 1950.
2 Seligson D, Seligson H. A microdiffusion
method for the determination of nitrogen
liberated as ammonia. J Lab Clin Med 1951;
38: 324-330.
3 Sundberg MW, Becker RW, Esders TW,
Figueras J, Goodhue CT. An enzymic
creatinine assay and a direct ammonia assay in
coated thin films. Clin Chem 1983; 29: 645-
649.
4 Diaz J, Tornel PL, Martinez P. Reference
intervals for blood ammonia in healthy subjects,
determined by microdiffusion. Clin Chem
1995; 41: 1048.
5 Cooke RJ, Jensen RL. Micromethod for
determining plasma ammonia nitrogen with use
of an ion-selective electrode. Clin Chem 1983;
29: 867-869.
6 Green A. When and how should we measure
plasma ammonia? Ann Clin Biochem 1988; 25:
199-209.
7 Willems D, Steenssens W. Ammonia
determined in plasma with a selective electrode.
Clin Chem 1988; 34: 2372.
8 Wu J, Ash KO, Mao E. Modified micro-scale
enzymatic method for plasma ammonia in
newborn and pediatric patients; comparison
with a modified cation-exchange procedure.
Clin Chem 1978; 24: 2172-2175.
9 Oberholzer VG, Schwarz KB, Smith CH,
Dietzler DN, Hanna TL. Microscale
modification of a cation-exchange column
procedure for plasma ammonia. Clin Chem
1976; 22: 1976-1981.
10 Kirk JM. Probable cefotaxime interference in
enzymatic ammonia assay - a cautionary note.
Ann Clin Biochem 1989; 26: 195-196.
11 Roche Diagnostics. Ammonia. test information
for Cobas Integra 400/700/800. 2000.
12 Cowley DM, Nagle BA, Chalmers AH, Sinton
TJ. Effects of platelets on collection of
specimens for assay of ammonia in plasma.
Clin Chem 1985; 31: 332-333.
13 da Fonseca-Wollheim F, van Dam M.
Interference by heparin in enzymatic
determination of plasma ammonia depends on
reagent composition. Clin Chem 1992; 38:
1921-1922.
14 da Fonseca-Wollheim F. Deamidation of
glutamine by increased plasma gamma-
glutamyltransferase is a source of rapid
ammonia formation in blood and plasma
specimens. Clin Chem 1990; 36: 1479-1482.
15 da Fonseca-Wollheim F. Preanalytical increase
of ammonia in blood specimens from healthy
subjects. Clin Chem 1990; 36: 1483-1487.
16 Howanitz JH, Howanitz PJ, Skrodzki CA,
Iwanski JA. Influences of specimen processing
and storage conditions on results for plasma
ammonia. Clin Chem 1984; 30: 906-908.
131
Ammonia

17 Losty H. Appendix - Notes on the measurement

of ammonia in blood/plasma. [cited 2007, 20 Green A, Morgan I, Gray J. Neonatology and
September 25]; Available from: Clinical Biochemistry. London: ACB Venture
www.metbio.net Publications; 2003.
18 Mousli S, Wakid NW. Ammonia production 21 Losty H. Guidelines for the investigation of
during clot retraction and its use in assay of hyperammonaemia for inherited metabolic
fibrinoligase. Clin Chem 1977; 23: 1739-1743. disorders. [cited 2007, September 25];
19 Hayes LW, Swanson JR. Ammonia results Available from: www.metbio.net
increased by bacteria in aca needle tubing. Clin 22 Rics C, Garca-Lario J, Alvarez V, Cava F,
Chem 1984; 30: 1882-1883. Domenech M, Hernndez A et al. Biological
variation database and quality specifications for
imprecision, bias and total error. The 2006
update. [cited 2007, June 20]; Available from:
http://www.westgard.com/guest32.htm

Methods of Ammonia Analysis

Method 1: Microdiffusion
Principle of analysis:
NH3 gas released by increasing pH diffuses across semipermeable membrane; detection by color change of pH
indicator
Comments: Automated plasma assay or strip-based whole blood assay
Method 2: Ion-selective electrode; quantitative, EP
Principle of analysis: Diffusion of NH3 at electrode surface; pH change measured potentiometrically
Comments: Automated; good precision and accuracy
Method 3: Ion-exchange; quantitative, end point
Principle of analysis: Adsorption of ammonia followed by colorimetric analysis by Berthelot reaction:
pH 10.5
NH4+ + HOCl H2NCl + H3O+; phenol + H2NCl complex;
OH
complex + phenol indophenol blue (560 nm)
Comments: Manual; good sensitivity and accuracy
Method 4: Enzymatic; quantitative, end point
Principle of analysis:
GLDH
NH4+ + -ketoglutaric acid + NADH NAD+ + glutamic acid
Comments: Automated, good accuracy, rapid
132
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

Hassan M.E. Azzazy i

Name: Amniotic fluid phospholipidsL/S ratio and PG

Clinical significance: Refer to Chapter 44, Pregnancy, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.

Molecular formula: Depends on fatty acid moieties

Phosphatidylcholine (lecithin) (L) C40H82O9NP (dipalmitoyl-)
Sphingomyelins (S) C39H81O7N2P (palmitoyl-)
Phosphatidylinositol (PI) C40H80O14P (dipalmitoyl-)
Phosphatidylglycerol (PG) C42H83O10P (distearoyl-)
Molecular mass: Dependent on fatty acid moieties
Phosphatidylcholine (lecithin) 752 (dipalmitoyl-)
Sphingomyelin 721 (palmitoyl-)
Phosphatidylinositol about 883
Phosphatidylglycerol 779
Merck Index: 5271 (lecithin), 8590 (sphingomyelins)
Chemical class: Phospholipids
Structures: 1 Phosphoglyceride structure
2 Lecithin (Phosphatidyl choline)
3 Phosphatidyl inositol
4 Phosphatidyl glycerol
5 Sphingomyelin
1 2

3 4

i
Amniotic Fluid Phospholipids
Previous and current authors of this method:
First edition: Not done
Methods edition: Paul T. Russell
Second edition: Not updated
Third edition: Not updated
Fourth edition: Steven C. Kazmierczak
Fifth edition: Hassan M.E. Azzazy
133
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
List of Abbreviations
ANS: 8-anilino-1-naphthalenesulfonic acid
DCF: 2,7-dichlorofluorescein
FLM: Fetal lung maturity
FSI: Foam stability index
GLC: Gas-liquid chromatography
HPLC: High-performance liquid chromatography
LBC: Lamellar body count
LS ratio: lecithin sphingomyelin ratio
PG: Phosphatidyl glycerol
RDS: Respiratory distress syndrome
TLC: Thin-layer chromatography
Principles of Analysis and Current Usage
Tests for the estimation of pulmonary surfactant in
amniotic fluid fall into three general categories: (1) those
that measure the chemical constituents of surfactant, (2)
those that measure the physical properties of lung
surfactant, and (3) those that measure a variety of
chemical agents that correlate with stages of fetal
maturation.
The analysis of the chemical constituents of surfactant
entails the separation of the individual components of
the phospholipids present in amniotic fluid. Extraction
and separation, followed by quantitation, permit the
establishment of relationships such as the lecithin-to-
sphingomyelin (L/S) ratio [1], the phospholipid
profile, [2] the fatty acid composition of amniotic fluid
lecithin [3], and many others that provide an index of
fetal lung maturity [4-6]. Thin-layer chromatography
(TLC) became the most widely used technique for most
of these analyses after it was published as the method of
choice by Gluck et al. [1,7,8] in their pioneering work on
the L/S ratio.
The thin-layer chromatography system (Table 1, Method
1) is extensively used [6] and employs silica-gel
stationary phases and mobile phases consisting of either
chloroform-methanol-water or chloroform-methanol
ammonium hydroxide. TLC separations are based on the
differential adsorption of phospholipids with different
polarities. Both one-dimensional and two-dimensional
TLC systems have been used. The choice is determined
primarily by balancing the need to completely separate
the phospholipids of interestlecithin, sphingomyelin,
and phosphatidylglycerolfrom one another and the
need to separate these compounds from the other
phospholipid components of amniotic fluid.
A number of techniques can be used for the visualization
of the separated phospholipids. One can char organic
materials by spraying the TLC plates with sulfuric or
phosphoric acid and then heating to 280C, although
variations in charring temperatures can give variable
results [9]. Alternatively, ammonium sulfate can be
incorporated into the silica gel, eliminating the need for
strong acid sprays during the charring procedure [7].
Lipids are visualized under ultraviolet radiation after
spraying with rhodamine B or dichlorofluorescein.
Bismuth subnitrate reacts with the choline moiety
contained in lecithin and sphingomyelin [10]. Other
agents, such as cupric acetate and sulfuric acid with or
without dichromate, react with the fatty-acid double
bonds, yielding colored products. The intensity of the
colored spots depends on the degree of unsaturation of
the phospholipid constituent fatty acids. Iodine vapor
and molybdate ions also react with the unsaturated bonds
in the fatty acid moieties of the phospholipids.
After the phospholipids are separated by thin-layer
chromatography and identified on the chromatogram by
an indicator, the L/S ratio can be evaluated in a number
of ways.
1. The chromatogram can be evaluated by
transmission or reflection densitometry.
Compounds that are separated and charred on
thin-layer plates are quantitated
densitometrically by use of a scanner. The L/S
ratio is calculated from the recorder tracings,
which are related proportionately to the plate
concentrations of lecithin and sphingomyelin.
2. The phospholipids can be eluted from the
chromatogram and the phospholipid
quantitatively estimated by a phosphorus
determination. Phosphorus determinations may
be performed by several methods [11-15].
3. One can estimate the phospholipids on the
chromatogram by planimetry. The area of each
visualized spot is estimated by either taking the
product of the length and width of the
individual spots or using a planimeter. The
estimated area is proportional to the quantity of
material present.
Gas-liquid chromatography (GLC) (Table 1,
Method 2) has provided a means to estimate
pulmonary surfactant based on its unique fatty
acid composition. GLC involves quantification
of the palmitic acid concentration of amniotic
fluid, establishment of a ratio of palmitic acid to
stearic acid, and determination of the relative
content of palmitic acid contained in lecithin
isolated from amniotic fluid. The gas
chromatography for these fatty acid separations
has been performed on polyester columns of
diethylene glycol succinate and ethylene glycol
succinate, with flame ionization detectors.
Phosphatidylcholine has been determined as the
diacylglycerol trimethylsilyl ether derivative by
gas chromatography using a glass capillary
column [16]. Preliminary TLC is required for
this method and for some others before GLC
analysis (Table 1, Method 2) [3].
High-performance liquid chromatography (HPLC)
(Table 1, Method 3) has been used to separate
phospholipid extracted from the amniotic fluid. One
method employs diol bonded-phase columns with
gradients (2% to 15% water) of acetonitrile/H2O as the
mobile phase. Detection and quantitation are often based
upon ultraviolet absorption characteristics of double
bonds (203 nm) in the fatty acid moieties of the
phospholipids, though methods that measure saturated
phospholipid palmitate have been published [17].
134
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
In methods proposed to measure specifically the
disaturated phosphatidylcholine, total lipids are reacted
with osmium tetroxide dissolved in carbon tetrachloride
[18]. The disaturated phosphatidylcholine is then
isolated by column chromatography on neutral alumina.
A disadvantage of this method is that unsaturated lipids
are damaged and can not be analyzed further.
All the procedures that measure phospholipid
constituents of amniotic fluid require partial purification
of the compounds before chromatography. Purification
schemes usually involve one or more of the following
steps: centrifugation, extraction, acetone precipitation,
evaporation, and reconstitution. However, there is
considerable controversy about the efficacy of the first
three steps listed. Thus a large number of extraction
schemes using permutations and combinations of these
techniques are in use.
Various techniques have been developed for
measurement of lecithin [19-23], sphingomyelin [19,23],
and phosphatidylglycerol [24] using either
spectrophotometry [20-23] or radiochemical techniques
[19,24]. Radiochemical techniques, in general, have the
inherent disadvantages of high cost and waste disposal.
Enzymatic methods for lecithin, sphingomyelin [23], and
phosphatidylglycerol [25] that have many attractive
features have been published. Phospholipids are
extracted from amniotic fluid with chloroform-methanol
and, after solvent evaporation, are redissolved into a
zwitterionic detergent. Choline and glycerol are liberated
after treatment with phospholipase and are subsequently
oxidized enzymatically to generate hydrogen peroxide.
The hydrogen peroxide reacts with 4-aminoantipyrene to
produce an intense red chromogen that can be measured
at 510 nm. This approach offers the potential for rapid,
specific, and sensitive assays for key phospholipids of
pulmonary surfactant.
Methods that measure the physical properties of lung
surfactant have also attracted interest because they
measure functional lung surfactant. Besides multiple
phospholipid components, surfactant may also include a
protein component for functional integrity. These tests
measure surface tension, stable foam (bubbles),
microviscosity, and other properties [5,6]. Many of these
tests are simpler to perform and quicker to carry out than
the chemical tests and thus have strong potential for
routine laboratory use. To their disadvantage, some of
these methods require expensive equipment and include
techniques that are less familiar to most laboratory
workers than the chromatographic methods.
One of the physical methods in most common use is the
foam stability or shake test (Table 1, Method 4). This
test was devised by Clements et al. [26] and calls for
serial dilutions of amniotic fluid mixed with equal
volumes of 95% ethanol to exclude spurious surface-
active compounds. The tubes are inspected for bubbles
around the meniscus after being shaken. At the
recommended final percentage of ethanol (47.5%) the
formation of bubbles by other substances in amniotic
fluid, such as proteins, bile salts, or salts of free fatty
acids, is eliminated. Pulmonary surfactant forms stable
surface films that support bubble stability. Therefore, the
test is positive if bubbles persist.
The foam stability index (FSI) uses a mixture of
different volumes of ethanol with 0.5 mL of amniotic
fluid and provides semiquantitative evidence of
surfactant content. The index is defined as the highest
ethanol volume fraction of an amniotic fluidethanol
mixture that will permit a stable ring of bubbles at the
meniscus after vigorous shaking. An FSI value of 0.48 is
comparable to an L/S ratio of 2.0 in correlating with fetal
pulmonary maturity [27].
One can also measure surface tension directly [28-30]
using a surface balance or tensiometer (Table 1, Method
5). These measurements give good correlations with
other tests for lung surfactant [31].
The fluorescence polarization assay measures the
microviscosity of amniotic fluid lipids, which is related
to surface tension (Table 1, Method 6). A fluorescent
probe, when mixed into amniotic fluid, dissolves in the
hydrocarbon region. Its rotation in this hydrophobic
environment depends on the microviscosity of the fluid.
The greater the viscosity (that is, the more surfactant
present), the more effectively opposed is the rotation of
the probe. This results in an increased polarization of the
incident fluorescent light.
Non-chromatographic methods for assessing surfactant
activity of amniotic fluid have only been recently
developed and are rarely used. TLC remains the almost
universal technique for amniotic fluid analysis, though
there may be an increase in the use of alternative
procedures as their clinical utility is demonstrated. The
foam stability (shake) test is used as a STAT procedure
when TLC analysis is unavailable (Table 2).
One relatively new technique for assessing lung maturity
is by determination of the number of lamellar body
particles in the amniotic fluid. Lamellar bodies are
packages of phospholipids secreted by type II
pneumocytes and extruded into the lung fluid. The
majority of these particles have a volume between 2 and
20 fL. Since platelets roughly have the same size
(diameter 1 to 5 m), lamellar bodies can be counted in
automated blood-counting instrumentation using the
platelet channel. Counting lamellar bodies by using this
type of instrumentation is a rapid, simple, and cost-
effective way of rapidly assessing fetal lung maturity
(FLM) [32-34]. The performance of the lamellar body
count (LBC) and the L/S ratio for prediction of neonatal
RDS was compared in a meta-analysis [35]. The LBC
was found to perform slightly better than L/S ratio, and
the authors concluded that LBC should become the test
of first choice in the assessment of FLM. In 2001,
Neerhof et al. [36] published a standardized
methodology for LBC. and maturity was suggested by a
count of 50,000/L or more. A count below 15,000/L
indicates immaturity, and no further testing is required.
135
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
The LBC is classified as transitional if the count falls
between 15,000 and 50,000/L. Under these
circumstances, further testing such as TDx FLMII, PG
test, and/or L/S ratio is required [37]. Szallasi et al. [38]
compared LBC in amniotic fluid using four different
hematology analyzers. It was concluded that because
different hematology analyzers count lamellar bodies
differently, it would be necessary to establish analyzer-
specific LBC clinical decision limits that are confirmed
by outcome-based studies.
Reference and Preferred Methods
There is no reference method for AFPL. The method of
Gluck et al., either in its original form or in some
modified form, has been the most widely used in-house
method. Clinical interpretation and predictions have for
the most part used Glucks criteria [7]. A large number
of variations of the original procedure are in use today
because virtually every step has more than one
legitimate means for its execution. Other variations have
developed because of differences of opinion over the
need for specific steps in the procedure, such as the
acetone precipitation step. Accurate clinical correlations
are the only meaningful criteria for methodological
considerations, and so it is incumbent on each laboratory
to establish its own criteria that correlate with clinical
experience. However, those steps in the generalized
procedure that can drastically compromise the method
deserve specific mention.
A commercial one-dimensional TLC kit, Helena Fetal
Tek 200, has become available for separating lecithin
and sphingomyelin; charring is achieved using
phosphoric acid and cupric acetate. In addition, lung
maturity of unborn infants can be assessed using TDx
FLM II fluorescent polarization immunoassay that
measures the ratio of surfactants to albumin in filtered,
uncentrifuged amniotic fluid.
Technical Considerations
Centrifugation
To remove whole cells and other debris, one usually
centrifuges amniotic fluid before further processing. This
procedure has significant influence on the analysis of
amniotic fluid phospholipids and the L/S ratio [39]. Yet
this preliminary step has not been standardized.
Centrifugation speeds of 300 to 500 g for 10 minutes
pellet much of the debris but maintain phospholipid
losses within reasonable bounds.
Amniotic fluid contains phospholipids from many
sources. Some of the phospholipid is related to
surfactant, but much of it is not. When an amniotic fluid
specimen is centrifuged, even at relatively low speed
(300 g for 10 min), 5% to 10% of the phospholipids are
found in the pellet [40].
Surfactant phospholipid is present in amniotic fluid in
membranous aggregates called lamellar bodies, and
although high-speed centrifugation (33,000 g for 60
minutes or longer) is required to sediment these
structures, some sedimentation occurs during
centrifugation at only 80 g for 5 minutes [41].
Centrifugation speeds of 1000 g for 5 minutes result in a
variable loss of both total phospholipid and lamellar-
body phospholipid [42]. Some studies have shown that
the losses are often proportional for lecithin and
sphingomyelin, so the ratio remains constant (Figure 1).
However, other studies have reported that certain
centrifugation speeds can affect lecithin more than
sphingomyelin and can alter the L/S ratio [39,43-45].
The effect of centrifugal force on the L/S ratio has been
studied by Cherayil et al. [43] and is presented in Figure
1.
Centrifugation also results in the loss of
phosphatidylglycerol (PG), so that when small quantities
are present in a sample, losses can influence
interpretation of results. Losses probably occur for all
phospholipids by adsorption to the cells or cellular
debris [46].
Debris can also be removed by filtration, but losses of up
to 90% of the total phospholipid content after filtration
through Whatman No. 1 filter paper have been reported.
Filtration also can lower the L/S through
disproportionate losses of lecithin [39].
Extraction
It is important to standardize the proportion of extracting
solvent to amniotic-fluid sample used in the assay, since
the relative proportions of chloroform, methanol, and
water significantly affect the extraction of lipid [47,48].
One volume of amniotic fluid to one volume of
chloroform-methanol (1:1, v/v) is commonly used,
though chloroform-methanol (2:1, v/v) also gives good
recovery of phospholipids. After centrifugation to
facilitate phase separation, the lower organic phase is
removed with a Pasteur pipet and evaporated to moist
dryness under a stream of nitrogen. If the sample is taken
to complete dryness, the residue will not redissolve
easily in the chloroform-methanol mixture, and losses
can occur. This extraction is the starting point for the
various assays.
Two extractions of the amniotic fluid with equal volume
of fluid, methanol, and chloroform are recommended.
Although the L/S ratio remains unchanged through a
series of extractions [49], a better recovery of lipid is
obtained with more than one extraction. Since
phosphatidyl glycerol (PG) is but a minor constituent of
surfactant, complete extraction is an important factor in
the detection and quantitation of PG.
Acetone Precipitation
Most naturally occurring lecithins are insoluble in cold
acetone, and surface-active phospholipids can be
precipitated by acetone from lung fluid [50]. Glucks
method used acetone precipitation to effect a crude
isolation of surfactant phospholipids [8,50]. This step
has provoked considerable controversy. A number of
authors claim the step is unnecessary and time
consuming, but Gluck is adamant that since surface
activity resides solely in the acetone-precipitable
material (Figure 2), this step is an essential feature in
separating surface-active phospholipids from a total lipid
extract.
136
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
From 60% to 90% of the total surface-active lecithins
present in the sample are precipitated with cold acetone.
This means that 10% to 40% of the desired lecithins may
be lost. If reliable clinical correlations are to be obtained,
it must be assumed that sphingomyelin is lost
proportionately, but this may not always be the case
[51]. Some authors have reported preferential losses of
lecithin [39,52] which falsely reduce the L/S ratio. Thus
an occasional immature L/S is diagnosed when in fact
adequate surfactant is present. Other authors have not
reported losses as serious with the precipitation step
[5,53]. Despite the ongoing controversy, this step
remains an integral part of many methods in use today,
but to ensure reproducible results, one must pay strict
attention to the ice-cold temperature conditions specified
[8]. Warmer conditions will increase the solubility of
surfactant (saturated) lecithin.
Chromatography
The choice of a chromatographic system for the
separation of phospholipid components of surfactant is a
choice based on many considerations. Ideally, the
chromatographic system should separate lecithin,
sphingomyelin, and PG from the other lipids present in
amniotic fluid. It is also desirable to run standards in
parallel with the phospholipid extract if a TLC system is
employed. These optimal criteria have been difficult to
attain, and thus many of the TLC systems used for
surfactant assays have not satisfied one or the other of
these criteria. The L/S ratio determination, as introduced
by Gluck and co-workers, employed a one-dimensional,
silica-gel, thin-layer system. Subsequently, Gluck et al.
[2] introduced a two-dimensional system that offered the
opportunity to detect other components of the surfactant
matrix (notably phosphatidylinositol and
phosphatidylglycerol). The change was necessary
because the one-dimensional system used initially could
not provide sufficient resolution for the phospholipids of
interest.
It was recognized early that L/S ratios of 2:1 as
measured by TLC methods correspond to actual L/S
concentration ratios ranging from 4:1 to 6:1 [5,7]. This
discrepancy, for the most part, results from unresolved
separations by the TLC system, inadequate visualization
methods or reagents [54], and diffusion of spots,
particularly during two-dimensional TLC [55,56]. One-
dimensional TLC systems are preferable if they can
effect the separations desired because they are faster, are
less expensive, and permit standards to be run
concurrently. But often, one-dimensional systems have
not provided complete resolution of the lipid mixture. In
one instance, Painter [57] was able to attain adequate
separations on a one-dimensional system, previously
possible only by two-dimensional TLC, by treating the
plates with cupric chloride, thereby taking advantage of
the amphoteric nature of phosphatidylserine, a
phospholipid frequently not separated in the
chromatography of surfactant. Laboratory conditions
such as humidity [58] can also affect the resolutions of
TLC separations. In many instances, PG cannot be
measured on the same plate used to determine the L/S
ratio without overloading the plates with L and S
because of the low percentage of PG among total
amniotic fluid phospholipids.
Methods that use ammonium hydroxide as a constituent
in the mobile phase can have run-to-run variations
traceable to the loss of volatile ammonia from the stock
solution [59]. The ammonium hydroxide concentrations
should be within 15% of the optimum concentration, and
the working solution should be discarded at 1 week.
Visualization of Spots
In the context of the phospholipid methodology for
measurement of surfactant, there are some important
considerations. The paper by Spillman et al. [54]
compared seven of the more commonly used techniques
for detection of phospholipids after thin-layer
chromatography.
It is clear that the L/S ratio can be distorted by the
visualization process. When charring carried out under
acid conditions at high temperatures is used as the
method of detection, error can be introduced by the
difficulty of assessing the completeness of the charring
process. Similarly, when one accomplishes visualization
by staining with a lipophilic dye, it is difficult to assess
the completeness of the color development. Furthermore,
the mechanism whereby the visualization reagent
interacts with the lipid can often bias the results by
underestimating the amounts of saturated lecithin present
[54]. Reagents that react with double bonds do not give
information relating to the saturated lipid of interest.
Hence as the more saturated surfactant is produced by
the lung and extracted from amniotic fluid, detection by
double-bond reagents deviates more and more from the
true saturated lipid content. Also, the intensity of the
developed spot when treated with reagents that attack
double bonds (such as cupric acetate and sulfuric acid)
can vary widely (10-fold). One can include ammonium
sulfate in this group. This salt is used as part of the
stationary phase in certain TLC solvent systems to
eliminate the need to spray with strong acid [7].
Molybdate, 8-anilino-1-naphthalenesulfonic acid (ANS),
2,7-dichlorofluorescein (DCF), and bromthymol blue
stain interact with the lipid independently of the degree
of saturation. The color intensity of the bromthymol
blue, however, fades quickly after the plate is removed
from the ammonia vapors, and for this reason
bromthymol blue is less desirable than the other
members of this group.
Color development with molybdate and cupric acetate
requires heating for development, as the charring
techniques do. Background darkening can occur,
especially if a plate has been developed in a solvent
system that contains an unsaturated component that is
not fully removed from the plate before application of
the visualization reagent. ANS and DCF lipid complexes
are revealed under long-wavelength ultraviolet radiation,
and heating is therefore not required for spot
visualization with these reagents. DCF has a higher
background than ANS, but both of these reagents have
the advantage of not reacting chemically with lipid, thus
allowing recovery of the intact lipid material, if it is so
desired. The fluorescein indicator is less stable than
137
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
molybdic stain, and plates treated with this indicator
should be protected from light if they are kept for
extended periods for reference purposes.
If plates are heated for visualization, they should be
preheated at 110C before they are placed on the 280C
hot plate [60]. This technique is recommended because
commercial thin-layer chromatographic plates have a
tendency to shatter.
Lipid staining is facilitated when ethanol is used as
solvent for the staining reagent. Reagents dissolved in
alcohol more easily penetrate the region containing the
phospholipid, resulting in more evenly stained regions.
Exposure of the spot to the staining reagent for 2 min
ensures uniform penetration.
Detection and Quantification
For those laboratories using planimetry for measuring
the L/S ratio, lecithin and sphingomyelin spots remain
visible longer with phosphomolybdic acidstannous
chloride than with bromthymol blue. Planimetric
methods are critically discussed by Whitfield et al [61].
A review of other methods to measure the L/S ratio has
been published by Olson and Graven [62]. An L/S ratio
of 2.0 obtained by reflectance densitometry is about
equal to an L/S ratio of 4.0 determined by gravimetric
techniques (Figure 3). It is recommended that a synthetic
lecithin and sphingomyelin control be routinely used on
plates to monitor spot development to ensure that the end
point has been reached before chromatograms are
quantified [54].
The methods that propose measuring specifically the
disaturated phosphatidylcholine [18] eliminate
unsaturation with osmium tetroxide. This method is
more applicable to research than to the clinical
laboratory [55]. Special care must be taken when one is
working with osmium tetroxide.
Gas chromatographic methods have been critically
reviewed [4]. Although this methodology is complicated
and time consuming and requires expensive equipment
and higher levels of expertise, useful clinical correlations
with surfactant levels have been established [3,63,64].
Correlations of palmitic acid concentrations, palmitic
acidstearic acid ratios, fatty acid compositions of
lecithin isolated from amniotic fluid, and lecithin itself
[16] with surfactant levels have aided in successful
prediction of fetal lung status.
High-performance liquid chromatography (HPLC) offers
many practical advantages over TLC, such as greater
sensitivity, precision, accuracy, and speed. Its usefulness
has been limited because it is difficult to obtain
simultaneous lecithin, sphingomyelin, and phosphatidyl
glycerol data from an individual run. HPLC analysis of
phospholipids is also limited by the detector, which
measures fatty acid double bonds (absorption at 203 nm)
and not the specific saturated lecithin of interest. This
type of detection also gives a different response to
ultraviolet absorption for the individual phospholipids
[65].
HPLC-based methods have been published to estimate
amniotic fluid phospholipids. The method by DCosta et
al. uses an isocratic system and an ultraviolet detector to
establish an L/S ratio. L/S values above 7 correlate with
pulmonary maturity. The method by Andrews [66]
utilizes an internal standard so that individual
concentrations of all the common phospholipids can be
estimated more accurately. Gradient chromatography is
used with an ultraviolet detector. The time for each assay
is 16 min. This latter method is therefore capable of
providing an entire phospholipid profile in a short period
of time.
Of the physical methods, the shake test has been widely
used. The test is very reliable (more than 99%) when the
test is positive. Less than 1% of infants with a positive
test result develop respiratory distress syndrome (RDS).
However, approximately 50% of infants with a negative
test result develop RDS [67]. The test is inexpensive and
quick to perform and when done properly is a useful
adjunct to other methods. Its reliability requires an
uncontaminated sample of amniotic fluid. Bile salts, salts
of free fatty acids, and proteins can also exhibit surface
activity and contribute to the formation of a stable ring
of bubbles. Also, for the test to be reliable, all glassware
must be free of soap, serum, or biological fluids. The
sample must be free of blood contamination and
meconium and, if possible, should be uncentrifuged.
Clements [26] suggested a cautious centrifugation at 500
g for 5 minutes to remove red blood cells if necessary
but pointed out that a hematocrit of greater than 3%
negated the result. Also of paramount concern is the care
with which the ethanol solutions are prepared and
maintained. In the original paper, Clements [26]
classified the results as negative, intermediate, and
positive. The shake test is a valuable screening test but
does not supersede the necessity for the more specific
tests when the shake test is negative or inconclusive.
Only when foaming persists at dilutions of 1:2 and
greater should results be considered positive [4].
Surface tension measurements [28-30] give good
correlations with other tests for respiratory distress
syndrome and lung surfactant [18,26,31]. Methods that
use a tensiometer are relatively rapid but demand
meticulous attention to cleaning and maintenance of the
equipment. The foam stability index method [28] is
simple and rapid and requires no expensive equipment or
expertise. This test is sensitive to centrifugation and to
contaminants in the amniotic fluid, and patients with
hydramnios may have falsely elevated results [31].
Erythrocyte contamination is not a problem if the cells
are not hemolyzed, but plasma phospholipids do pose a
problem [28]. The final assay mixture is critical to the
results, and extremely close attention to this detail is
required [5].
The fluorescence polarization assay is reported to require
less time to perform and to give results that are less
subject to variation than the L/S ratio [68]. It has become
the most common method in use for assessing fetal lung
138
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
maturity. Results obtained with the fluorescence
polarization method are reported in artificial units (mP).
Specimen
Amniotic fluid obtained by amniocentesis is transported
to the laboratory on ice and is promptly centrifuged at
500 g for 5 minutes. However, specimens for analysis by
fluorescence polarization should not be centrifuged,
because centrifugation falsely decreases results [69]. A
3.0-mL aliquot of the supernatant is needed for
extractions.
Samples should be processed as soon as possible after
collection. Untreated amniotic fluid held at room
temperature can lose both lecithin and sphingomyelin
over a period of 48 hours. According to Wagstaff et al.
[39], after only 4.5 hours, lecithin levels are decreased
about 25%. Decreases of a small order occur with
sphingomyelin over a similar period of time. This
disproportionate loss of lecithin relative to
sphingomyelin with time results in a consistent fall in the
L/S ratio. Because there does not seem to be a significant
quantity of phospholipase activity in human amniotic
fluid, the decrease may be the result of the lipids
adhering to cellular debris in the fluid.
Centrifugation of amniotic fluid samples is necessary if
storage is contemplated. If amniotic fluid is centrifuged
at a low speed, such as 500 g for 5 minutes, immediately
after collection, it can be kept at room temperature for at
least 4 days with no apparent change in the L/S ratio
[70]. Freezing does not adversely affect the phospholipid
content [40]. Centrifuged samples stored at 20C are
stable for prolonged periods without noticeable
alteration of the L/S ratio. Thus if a delay is anticipated,
the fluid should be centrifuged and immediately
refrigerated or frozen.
Interferences
The fluorescence polarization assay is sensitive to the
effects of centrifugation [71], and contamination with
small amounts of serum or meconium could significantly
alter the determined value [72,73]. High-density
lipoproteins contained in amniotic fluid may hinder the
reliability of the method [74]. Contamination of amniotic
fluid from debris, mucus, bacteria, or cellular material
from the vagina also causes the L/S to be
uninterpretable.
Amniotic Fluid Phospholipids Reference Interval
An L/S ratio more than 2.0 is considered indicative of
fetal maturity. If a spot is seen in the PG area, the test is
considered positive. If no spot is seen, it is considered
negative. Infants of diabetic mothers may be at greater
risk of developing respiratory distress despite an L/S
ratio of more than 2.0. When using the lamellar body
count for assessing fetal lung maturity, a count of
55,000/L in a centrifuged specimen or 60,000/L in an
uncentrifuged specimen typically implies lung maturity
[32].
Interpretation
Although the number of laboratories performing the L/S
ratio test is decreasing, this test is still considered an
important method for assessing FLM. It is generally
accepted that with L/S ratios of 2 or greater there is
greatly diminished risk for RDS in the newborn. In one
study, it was found that if the L/S ratio was less than 1.5,
RD was found in 73% of the infants. If the L/S ratio was
1.5 to 2.0, RD was found in 40% of the infants. If the
L/S ratio was > 2, there was a 2% risk of RD but only a
0.9% risk if the L/S was > 2.5. This relationship does not
hold true for infants of diabetic mothers. With diabetics,
the risk for respiratory distress of the newborn is still
high, even with an L/S greater than 2. Falsely elevated
L/S ratios have been reported after intrauterine
transfusions. In a later study using an L/S ratio cutoff
value of 2.0 to indicate FLM, cited values for sensitivity
and specificity range from 80% to 85% [75]. In addition,
studies also suggest that clinical interpretation of L/S
ratios may be different between different racial groups;
African-American infants show FLM at L/S ratios of
greater than 1.2 [76].
Surfactant that contains PG has better alveolar
stabilizing properties than surfactant that has not yet
acquired PG. Thus mature lung function is also
associated with measurable quantities of PG. In the
absence of PG, the risk of respiratory distress is
significant, but development of the disease is not
inevitable [77,78]. In one study, when PG was present,
RD was found only in 8.4% of infants, but even in the
absence of PG, 20% of infants did not develop RD [79].
PG measurement proves a useful adjunct to the L/S ratio
determination when dealing with amniotic fluid that is
contaminated with blood or from diabetic mothers. PG
appears when the L/S ratio exceeds 2, and its presence
continues to increase in amniotic fluid with continuing
gestation (Figure 6). By contrast, PI
(phosphatidylinositol) closely parallels the L/S up to the
point of maturity, thereafter falling as the PG continues
to increase [77]. The presence of PG appears to provide
the final component in the biochemical maturation of the
surfactant.
The question of what PG value validly predicts FLM has
been approached both by estimation of PG
concentrations in amniotic fluid and by determination of
the proportion of phospholipid represented by PG. PG
greater than 2.1 mg/L of amniotic fluid or 10 nmol/mL
[24] correlates with fetal maturity. FLM is likewise
indicated when PG is equal to or exceeds 3% of the total
phospholipids [2].
Gluck [7] and other workers reported that the L/S ratio
was a less reliable predictor of FLM in a diabetic than in
a normal pregnancy. A delay in the L/S ratio maturation
until 36 weeks of gestation or later is not unusual.
Subsequently it was recognized that the L/S ratio may
also be unreliable with hypertension, premature rupture
of the membranes, and intrauterine growth retardation.
To address these conditions, Kulovich et al. [2,80]
incorporated the pertinent phospholipids into the lung
profile so that as many factors as possible are
considered in the prediction of fetal lung status. This
139
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
approach assumes an identical extraction of the
phospholipids. It includes an acetone precipitation step
and a two-dimensional thin-layer chromatographic
separation, followed by quantitation by reflectance
densitometry. The unambiguous, simultaneous
assessment of PG, lecithin, and sphingomyelin is the
object of the majority of the chemical methods currently
in use, although routine measurement of PG in the
nondiabetic may not be necessary.
An excessive amount of blood in the sample is an
important source of error in the L/S ratio [81].
Concentrations of blood greater than 0.05 mL per 3 mL
of amniotic fluid pose problems, particularly if the L/S is
near 2.0. The L/S of blood is near 1.8, and so fluid
contaminated with blood would falsely lower the L/S.
PG is helpful in cases of blood contamination, because
PG is not found in blood, and its presence therefore
indicates mature lung status. PG may be misleading,
however, in the patient with chorioamnionitis with
premature rupture of the membranes [82].
Amniotic Fluid Performance Goals
Standards of laboratory practice for evaluation of FLM
have been published that thoroughly covered
preanalytical, analytical, and postanalytical
considerations for FLM testing [83]. The guidelines
recommend laboratories that support obstetric patients
not to perform L/S testing if they receive < 15 requests
per week and send samples to a reference laboratory.
The current evaluation criteria for quantitative L/S ratio
is for laboratories to be within 3 standard deviations of
the peer-group mean. For PG, laboratories should be at
90% of participant consensus. In the 2007 College of
American Pathologists proficiency survey, the mean,
SD, and coefficient of variation were not calculated for
the L/S ratio in-house methods, owing to the very low
number of participating laboratories. There was a great
variation in methods and cutoffs (1.9 to 4.1 for
maturity). Interpretations, however, were consistent for
LM-04 and LM-05, but no consensus was achieved for
LM-06. For the Helena Fetal Tek 200 peer group, the
reported sample ID, mean, SD, and CV were: LM-04,
1.08, 0.09, 8.4%; LM-05, 3.54, 0.71, 20.1%; and LM-06,
1.85, 0.29, 15.8%. Again, there was great consistency on
interpretations of LM-04 and LM-05 challenges but no
consensus on LM-06 [84].
Laboratories should offer at least one test that can be
performed in less than 1 hour, such as the fluorescence
polarization immunoassay on Abbott TDx (TDx/FLM
test), the Irvine Scientific slide test for PG, or the foam
stability test (a homebrew test or Beckman Lumadex-
FSI). It was also recommended that laboratories should
perform the LBC test but report its result together with
one of the recommended rapid tests. Laboratories should
offer routine L/S testing once per day, 7 days a week.
FLM results should be communicated rapidly to the
ordering clinicians, and reports should also include
sample condition (in particular, any observed
contamination) and reference information. Laboratories
performing L/S ratio should conduct a clinical outcome
study to validate their reference intervals or compare
their methods to published clinical outcome studies. The
guidelines discouraged reporting separate reference
intervals for diabetic patients. That was based on
absence of clinical outcome study with enough RDS
cases from diabetic mothers to indicate that the L/S test
results are unreliable. Also, well-controlled diabetes
expected for diabetic pregnant women will not delay
pulmonary maturation.
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33 Lee IS, Cho YK, Min WK, Kim KS, Mok JE.
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34 DeRoche ME, Ingardia CJ, Guerette PJ, Wu
AH, LaSala CA, Mandavilli SR. The use of
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35 Wijinberger LD, Huisjes AJ, Voorbij HA,
of lamellar body count and
lecithin/sphingomyelin ratio in the prediction of
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36 Neerhof MG, Donhnal JC, Ashwood ER, Lee
IS, Anceschi MM. Lamellar body counts: a
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37 Ventolini G, Neiger R, Hood D, Belcastro C.
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38 Szallasi A, Gronowski AM, Eby CS. Lamellar
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39 Wagstaff TI, Whyley GA, Freedman G. Factors
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40 Van Voorst tot Voorst EJ. Effects of
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41 Oulton M. The role of centrifugation in the
measurement of surfactant in amniotic fluid.
Am J Obstet Gynecol 1979;135:337-43.
42 Duck-Chong CG, Brown LM, Hensley WJ.
Sedimentation of lung-derived phospholipid
during low-speed centrifugation of amniotic
fluid. Clin Chem 1971;27:1424-6.
43 Cherayil GD, Wilkinson EJ, Borkowf HI.
Amniotic fluid lecithin/sphingomyelin ratio
changes related to centrifugal force. Obstet
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141
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
44 Wilkinson EJ, Cherayil GD, Borkowf HI. L/S
ratio and the g-force factor. N Engl J Med
1977;296:286-7.
45 Lindback T, Frantz T. Effect of centrifugation
on amniotic fluid phospholipid recovery. Acta
Obstet Gynecol Scand 1975;54:101-3.
46 Hudson EA, Aimls JG. Amniotic fluid cells and
the lecithin/sphingomyelin ratio. Obstet
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47 Bligh EG, Dyer WJ. A rapid method of total
lipid extraction and purification. Can J Biochem
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48 Bhagwanani SG, Fahmy D, Turnbull AC. Quick
determination of amniotic fluid lecithin
concentration for prediction of neonatal
respiratory distress. Lancet 1972;2:66-7.
49 Sarkozi L, Kovacs HN, Fox HA, Kerenyi T.
Modified method for estimating the
phosphatidylcholine-sphingomyelin ratio in
amniotic fluid, and its use in the assessment of
fetal lung maturity. Clin Chem 1972;18:956-60.
50 Gluck L, Motoyama EK, Smits HL, Kulovich
MV. The biochemical development of surface
activity in mammalian lung. Pediatr Res
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51 Olson EB, Graven SN, Zachman RD. Amniotic
fluid lecithin to sphingomyelin ratio of 3.5 and
fetal pulmonary maturity. Pediatr Res
1975;9:65-9.
52 Armstrong D, VanWormer DE. Rapid
determination of pulmonary surfactant. Am J
Obstet Gynecol 1972;114:1083-6.
53 Parkinson CE, Harvey DR. A comparison
between the lecithin/sphingomyelin ratio and
other methods of assessing the presence of fetal
lung pulmonary surfactant in amniotic fluid. J
Obstet Gynaecol Br Commonw 1973;80:406-
11.
54 Spillman T, Cotton DB, Lynn SC Jr,
Bretaudiere JP. Influence of phospholipid
saturation on classical thin-layer
chromatographic detection methods and its
effect on amniotic fluid lecithin/sphingomyelin
ratio determinations. Clin Chem 1983;29:250-5.
55 Tsai MY. Relative merits of one- and two-
dimensional TLC of phospholipids in amniotic
fluid. Clin Chem 1981;27:1957-8.
56 Cunningham MD, McKean HE, Gillespie DH,
Greene JW. Improved prediction of fetal lung
maturity in diabetic pregnancies: a comparison
of chromatographic methods. Am J Obstet
Gynecol 1982;142:197-204.
57 Painter PC. Simultaneous measurement of
lecithin, sphingomyelin, phosphatidylglycerol,
phosphatidylinositol,
phosphatidylethanolamine, and
phosphatidylserine in amniotic fluid. Clin Chem
1980;26:1147-51.
58 Tsao FHC. The effect of humidity on the
determination of lecithin/sphingomyelin ratio
and phosphatidylglycerol by thin-layer
chromatography. Clin Chim Acta 1982;122:75-
8.
59 Glick JR Jr, Crocker CL. Concentration of
ammonium hydroxide is critical in
chromatographic solvents for amniotic fluid
phospholipids. Clin Chem 1982;28:1997-8.
60 Gross TL, Wilson MV, Kuhnert PM, Sokol RJ.
Clinical laboratory determination of
phosphatidylglycerol: one- and two-
dimensional chromatography compared. Clin
Chem1981;24:486-90.
61 Whitfield CR, Chan WH, Sproule WB, Stewart
AD. Amniotic fluid lecithin: sphingomyelin
ratio and fetal lung development. Br Med J
1972;2:85.
62 Olson EB, Graven SN. Comparison of
visualization methods used to measure the
lecithin/sphingomyelin ratio in amniotic fluid.
Clin Chem 1974;20:1408-15.
63 Szalay J, Lukacs E. Palmitic acid concentration
of amniotic fluid in diabetic pregnancy. Arch
Gynaekol 1977;222:279-83.
64 ONeal GJ, Davies IJ, Siu J. Palmitic-stearic
ratio of amniotic fluid in diabetic and non-
diabetic pregnancies and its relationship to
development of respiratory distress syndrome.
Am J Obstet Gynecol 1978;132:519-23.
65 Geurts Van Kessel WSM, Hax WMA, Demel
RA, DeGhier J. High-performance liquid
chromatographic separation and direct
ultraviolet detection of phospholipids. Biochim
Biophys Acta 1977;486:524-30.
66 Andrews AG. Estimation of amniotic fluid
phospholipids by high-performance liquid
chromatography. J Chromatogr 1984;336:139-
50.
67 Parkinson GE, Harvey D. Amniotic fluid and
pulmonary maturity. In: Sandler M, ed.
Amniotic fluid and its clinical significance.
New York: Marcel Dekker; 1981:229-252.
68 Gebhardt DOE. Relationship between the
lecithin/sphingomyelin ratio and the P value of
amniotic fluid. Am J Obstet Gynecol
1979;133:937.
69 Russell JC, Cooper CM, Ketchum CH, Torday
JS, Richardson DK, Holt JA et al. Multicenter
evaluation of TDx test for assessing fetal lung
maturity. Clin Chem 1989;35:1005-10.
70 Whitfield CR, Sproule WB. Fetal lung
maturation, Br J Hosp Med 1974;12:678-90.
71 Simon NV, Elser RC, Levisky JS, Polk DT.
Effect of centrifugation on fluorescence
polarization of amniotic fluid. Clin Chem
1981;27:930-2.
72 Blumenfeld TA, Stark RI, James LS, George
JD, Dyrenfurth I, Freda VJ et al. Determination
of fetal lung maturity by fluorescence
polarization of amniotic fluid. Am J Obstet
Gynecol 1978;130:782-7.
73 Golde SH, Vogt JF, Gabbe SG, Cabal LA.
Evaluation of the FELMA microviscosimeter in
predicting fetal lung maturity. Obstet Gynecol
1979;54:639-43.
74 Gebhardt DOE. Detection of neonatal
respiratory distress on the basis of fluorescence
142
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
polarization (microviscosity) measurements of
amniotic fluid: a word of caution [letter]. Clin
Chem 1980;26:1629.
75 Dubin SB. Assessment of fetal lung maturity by
laboratory methods. Clin Lab Med
1992;12:603-20.
76 Richardson DK, Torday JS. Racial differences
in predictive value of the
lecithin/sphingomyelin ratio. Am J Obstet
Gynecol 1994;170:1273-8.
77 Hallman M, Feldman BH, Kirkpatrick E, Gluck
L. Absence of phosphatidylglycerol (PG) in
respiratory distress syndrome in the newborn.
Pediatr Res 1977;11:714-20.
78 Hallman M, Feldman B, Gluck L. RDS: the
absence of phosphatidylglycerol in surfactant
[abstract]. Pediatr Res 1975;9:396.
79 Bent AE, Gray JH, Luther ER, Oulton M,
Peddle LJ. Phosphatidyl-glycerol determination
on amniotic fluid 10,000 g pellet in the
prediction of fetal lung maturity. Am J Obstet
Gynecol 1981;139:259-63.
80 Kulovich MV, Gluck L. The lung profile. II.
Complicated pregnancy. Am J Obstet Gynecol
1979;135:64-70.
81 Gluck L, Kulovich MV, Borer RC, Keidel WN.
The interpretation and significance of the
lecithin/sphingomyelin ratio in amniotic fluid.
Am J Obstet Gynecol 1974;120:142-55.
82 Schumacher RE, Parisi VM, Steady HM, Tsao
FHC. Bacteria causing false-positive test for
phosphatidylglycerol in amniotic fluid. Am J
Obstet Gynecol 1985;151:1067-8.
83 Ashwood ER. Standards of laboratory practice
evaluation of fetal lung maturity. Clin Chem
1997;43:211-4.
84 College of American Pathologists. 2007
Surveys: Lung Maturity. Northfield, IL: CAP;
2007.
85 Hallman M, Kulovich M, Kirkpatrick E,
Sugarman RG, Gluck L. Phosphatidylinositol
and phosphatidylglycerol in amniotic fluid:
indices of lung maturity. Am J Obstet Gynecol
1976;125:613-7.
86 Wagstaff TI, Whyley GA, Freedman G. The
measurement of the lecithin/sphingomyelin
ratio of amniotic fluid after thin-layer
chromatography. Ann Clin Biochem
1974a;11:24-7.
87 Buhi WC, Spellacy WN. Effects of blood or
meconium on the determination of the amniotic
fluid lecithin/sphingomyelin ratio. Am J Obstet
Gynecol 1975;121:321-3.
88 Torday J, Carson L, Lawson EE. Saturated
phosphatidylcholine in amniotic fluid and
prediction of the respiratory-distress syndrome.
N Engl J Med 1979;301:1013-8.
89 Oulton M, Martin TR, Faulkner GT, Stinson D,
Johnson JP. Developmental study of a lamellar
body fraction isolated from the human amniotic
fluid. Pediatr Res 1980;14:722-8.
90 Gerbie MV, Gerbie AB, Boehm J. Diagnosis of
fetal maturity by amniotic fluid phospholipids.
Am J Obstet Gynecol 1977;114:1078-82.
143
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

Chromatography
Tables (TLC)
Table 1: Methods of Amniotic Fluid Analysis Assay Ambient Ambient
Chromatographic Analysis of Phospholipids temperature
Method 1: Thin-layer chromatography (TLC)
Type of analysis: Semiquantitative Sample 1 mL 1.0, 0.75, 0.50,
Principle: Silica-gel adsorption volume 0.25, and 0.20
chromatography; visualization of phospholipids mL
by color-forming sprays, or charring
Usage: Most common Fraction of 0.50 (extraction) 0.50
Comment: Can be run as one dimension or sample
two dimensions volume
Method 2: Gas-liquid chromatography (GLC)
Type of analysis: Quantitative Final (Not applicable) 47.5% ethanol
Principle: Analysis of fatty acids on polyester concentration
columns with flame ionization detector of reagents
Usage: Rare
Comment: Measured as methyl ester Interferences Blood, meconium, Blood,
derivatives high-speed meconium,
Method 3: High-performance liquid chromatography centrifugation soap, serum,
(HPLC) before testing biological
Type of analysis: Quantitative fluids, surface
Principle: Separation of L, S, PI, PE, PS, and evaporation
PG by reversed-phase chromatography, from tubes,
detection at 203 nm movement of
Usage: Rare tubes once they
Comment: Ability to quantitate multiple have been
components simultaneously may make this an shaken, high-
important method in the future. speed
centrifugation
Measurement of Surfactant Functional Activity of sample
Method 4: Foam stability (shake test, FSI) before testing
Type of analysis: Qualitative
Principle: Measures presence of surfactant by
its ability to support foam bubbles generated by
shaking amniotic fluid with ethanol
Usage: Common as stat procedure
Comment: Problem of false-negative results
Method 5: Surface tension
Type of analysis: Quantitative
Principle: Presence of surfactant related to
surface-tension properties of amniotic fluid
Usage: Rare
Comment: Dedicated instrument, but
relatively easy to perform
Method 6: Fluorescence polarization
Type of analysis: Quantitative
Principle: Surfactant increases microviscosity
of fluid, which reduces the rotation of dissolved
fluorescent probe, resulting in an increase in
polarization of incident fluorescent light
Usage: Frequently used
Comment: Common as stat procedure;
requires dedicated instrument but relatively
easy to perform

Table 2: Comparison of Assay Conditions for

Amniotic Fluid Analysis

Thin Layer Shake Test

144
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

Figures

Figure 1. L/S (lecithin/sphingomyelin) ratio related to

centrifugal force. In amniotic fluids with an initial L/S
ratio above 2, L/S ratios decrease with increasing g force
[43].

Figure 2. Surface tensions of purified lecithin from lung

homogenates at different times of gestation in fetal
rabbit. Total lecithin extracted from lung is not surface
active. Acetone-precipitated lecithin was surface active
and present in lungs from fetuses at all gestational ages
examined. NB, Newborn [50].
145
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

Figure 4. Separation of lecithin and sphingomyelin on

thin-layer strips as described for method to determine
L/S ratio. Pt, Patient sample; STD, L and S standards.

Figure 3. Relationship between densitometry L/S ratios

and those with area measurement and by gravimetry
[81].

Figure 5. Phosphatidyl glycerol (PG) spot on silica gel

thin-layer plate according to method for PG. L, Lecithin;
PI, phosphatidyl inositol, PT, patient; S, sphingomyelin;
STD, standard (left sample, PG; middle sample, PG, L,
and S).

Figure 6. Content of phosphatidyl inositol (PI) (closed

circles) and phosphatidylglycerol (PG) (open circles) in
amniotic fluid during normal gestation. Phospholipids
146
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

were quantified by measurement of phosphorus (P)

content and expressed as percentages of total lipid
phosphorus. Means standard deviations of three to five
samples are shown for each point [85].
147
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG
Procedure: Lecithin-Sphingomyelin Ratio (L/S) by
TLC
Principle
Phospholipids are extracted from the amniotic fluid into
chloroform-methanol and then precipitated in cold
acetone. The precipitated phospholipids are separated by
thin-layer chromatography, visualized, and quantitated.
An estimate is made of the amount of lecithin and
sphingomyelin present in the amniotic fluid. This is
expressed as a ratio of lecithin to sphingomyelin (L/S)
and is used in the evaluation of FLM.
Reagents
1. Eastman Chromatograph Sheets, 6061 Silica
gel without fluorescent indicator, 20 20 cm. Cut
into 1 13 cm strips. Activate by storing in a desiccator
over Drierite for 24 hr.
2. Developing solvent. Pipet into a 50-mL round-
bottom, capped centrifuge tube 5.0 mL of methanol, 0.8
mL of distilled water, 13.0 mL of chloroform. Vortex
mix. Keep solution capped when not in use. Prepare
fresh daily.
3. Methanol. Spectral grade
4. Chloroform. Spectral grade
5. Acetone. Reagent grade
6. Bromthymol blue (417 mg/L). A water-
soluble (3,3-dibromothymol sulfone phthalein, sodium
salt) available from Aldrich Organic Chemicals
(Milwaukee, WI). Add 200 mg of bromthymol blue to
200 mL of distilled water and 32.0 mL of 1 M NaOH.
Dilute to 480 mL with distilled water. Add 5.0 g of boric
acid powder (reagent grade) and mix. This solution is
stable for 3 months.
7. Nitrogen gas. Compressed, 100%
8. 1 M NaOH (40 g/L). Place 40 g of NaOH in a
1-L volumetric flask, and slowly add about 800 mL of
distilled water to dissolve. When the NaOH is fully
dissolved, and the temperature of the solution is back to
room temperature, add distilled water to mark. Mix
thoroughly. Stable for 12 months at room temperature.
9. Standards. The lecithin, sphingomyelin, and
phosphatidylglycerol standards can be purchased from
Supelco, Inc. (Bellefonte, PA) as chloroform-methanol
solutions. The contents of each ampule are quantitatively
transferred to a volumetric flask (the ampule must be
rinsed as well) and then diluted with chloroform-
methanol (2:1, v/v) to a final concentration of 1 mg/mL.
10. Lecithin. 50 mg is diluted to 50 mL with
chloroform-methanol.
11. Sphingomyelin. 25 mg is diluted to 25 mL
with chloroform-methanol.
12. Phosphatidylglycerol. 10 mg is diluted to 10
mL with chloroform-methanol.
Assay
Equipment: centrifuge, 20 50 mm test tubes with cork
stoppers, forceps, 12-mL conical screw-capped
centrifuge tubes, 3-mL conical test tubes.
This procedure is best performed in duplicate.
1. Centrifuge 3 to 5 mL of amniotic fluid at 500 g
for 5 min at ambient temperature.
2. Pipet 1 mL of the supernatant into duplicate 12-
mL conical screw-capped test tubes. Pipet 1 mL into
each of three tubes if PG is also to be determined.
3. To each 1-mL sample of amniotic fluid, add 1
mL of methanol. Vortex mix. Add 2 mL of chloroform,
and vortex mix vigorously.
4. The tubes are centrifuged (500 g for 5 min to
facilitate phase separations). Three layers should be
seen:
a. Aqueous layer (top)
b. Protein fluff layer (middle)
c. Chloroform layer (bottom)
5. Remove as much of the chloroform layer as
possible using a disposable Pasteur transfer pipet (go
through the top two layers carefully). Place this in a 3-
mL conical tube. Evaporate to moist dryness under
nitrogen. (Note: Water bath should be below 40C.)
6. If PG is to be determined, repeat the extraction
(steps 3 to 5) for the tube for PG.
7. Wash down the lipid adhering to the sides of
each tube with 50 L of chloroform. Take to moist
dryness again.
8. Chill the 3-mL tubes with the lipid extract in ice
for 1 min. With a Pasteur pipet, add 2 drops of ice-cold
acetone while swirling the tube in the ice. Eight more
drops of ice-cold acetone is added, and the tube is iced
for another minute. The acetone is decanted. The
precipitate is washed with another 10 drops of ice-cold
acetone while the tube remains iced. The supernatant is
decanted. The precipitate is dried gently under a stream
of nitrogen.
9. The cold acetone precipitate is dissolved in 6
L of chloroform. The entire amount is then spotted 2
cm from the end of the activated TLC strip. (The PG
tube is spotted on a plate to be described later.)
10. Using forceps to handle the strips, place each
into 1.5 mL of the developing solvent in a 20 150 mm
test tube. The tube is stoppered with a cork. The strip is
chromatographed to a distance of 2 cm from the top of
the strip.
11. The strips are removed from the tubes with
forceps, air dried, and then dipped in the bromthymol
blue indicator contained in a 20 150 mm test tube.
Excess dye is blotted from the strip. The lecithin and
sphingomyelin spots appear dark yellow (Figure 4).
Visualization is made easier when the strip is briefly
placed in a 20 150 mm test tube containing
concentrated NH4OH. The ammonia vapors turn the
spots blue.
Calculation
1. Circle each spot along the outermost edges with
a pencil. Measure maximum width and length of each
spot in millimeters. Using the following calculation:
L/S ratio = _
(length of lecithin spot) (width of lecithin spot)_
(length of sphingomyelin spot) (width of sphingomyelin spot)
2. Average the values from the duplicate samples.
3. Apply L/S standards daily. Spot 30 L of
lecithin standard (10 mg/mL) on top of 15 L of
sphingomyelin standard (100 g/mL) on a fresh strip and
chromatograph.
4. Running time for L/S, 30 to 45 minutes.
148
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

Amniotic Fluid Phospholipids Reference Interval Contamination of amniotic fluid with meconium
An L/S ratio more than 2.0 is considered indicative of interferes with the interpretation of chromatograms,
fetal maturity. Infants of diabetic mothers may be at though Gerbie et al. [90] did not consider that its
greater risk of developing respiratory distress despite an presence interfered with the estimation of the L/S ratio.
L/S ratio of more than 2.0. Wagstaff et al. [39] demonstrated a consistent rise in the
L/S ratio with increasing contamination with meconium.
Procedure: Phosphatidylglycerol (PG) by TLC Chromatograms from samples contaminated with
meconium are characterized by a lysolecithin spot
Reagents immediately after the sphingomyelin spot. If
1. Chloroform. Spectral grade chromatographic separation is not good, the lysolecithin
2. Methanol. Spectral grade
spot will come to lie so close to the sphingomyelin spot
3. Glacial acetic acid. Reagent grade that the two may be confused, giving a falsely decreased
4. Solvent system. Chloroform/methanol/acetic L/S ratio [87]. Other authors report falsely high L/S
acid/water (65:25:8:4, v/v) ratios from fluid contamination with meconium. Again,
5. 2,7-Dichlorofluorescein (0.2 g/L) in ethanol. it seems prudent to rely on the presence of PG if samples
Dissolve 2 g of dye into 100 mL of ethanol. This is the
are contaminated with meconium or to assess surfactant
stock solution. Dilute 1:100 in ethanol for working
of the 10,000 g pellet [41,89].
solution. When stored at room temperature, the stock
solution is stable for 1 year and the working solution for Amniotic Fluid Phospholipids Reference Interval
3 months. If a spot is seen in the PG area, the test is considered
positive. If no spot is seen, it is considered negative.
Assay
Equipment: Silica gel 60 thin-layer plates, 5 20 cm (E. Shake Test on Amniotic Fluid: Rapid Test for
Merck, Darmstadt, Germany). Plates are activated at Surfactant
100C for 1 hr before use. Filter-paper-lined thin-layer
chromatography tank.
I. Principles of Test and Clinical Significance
1. Use the third tube prepared in step 2 of the L/S
ratio procedure. Lipid contents are dissolved in 10 L of
chloroform and spotted 2 cm from the bottom and a third A. Principle: This test depends on the
of the way in from the side of a silica-gel (5 20 cm) ability of pulmonary surface active material (surfactant)
TLC plate (activated). to generate stable foam in the presence of 95% ethanol.
It looks at a physical property of the amniotic fluid.
2. Lecithin (20 L of 1 g/L) and PG (20 L of
1 g/L) standards are spotted 2 cm from the bottom and
B. Clinical Significance: This test is a
a third of the way in from the opposite side of the same
rapid assessment of fetal lung maturity. If test result
plate.
indicates mature, RDS should not occur in infants if they
3. The plate is placed in a filter-paper-backed thin-
are delivered within 72 hr after analysis is done.
layer tank previously equilibrated with the solvent
system for at least 1 hr (replace solvent system weekly
II. Sample (Specimen)
or more often if runs become atypical).
4. The solvent system is permitted to run about 10
A. 1.5 mL of unspun amniotic fluid
cm up the plate. The plate is withdrawn from the tank
obtained by amniocentesis.
and air dried for 15 to 20 min.
5. The plate is sprayed with dichlorofluorescein
B. If test is not able to be performed right
reagent, allowed to dry, and viewed under ultraviolet
away, refrigerate sample at 2C to 8C. Then mix
radiation 15 min to 1 hr later (Figure 5).
sample well before pipetting amniotic fluid for shake
6. The visual identification of a PG spot confirms
test.
the presence of PG. This visualization is sensitive to 0.5
g and indicates the presence of PG in amniotic fluid to C. Samples that are bloody, contain
be greater than or equal to 0.5 g/mL. meconium, or are from vaginal pool collections are not
acceptable. Ascitic fluid samples from infants will give
Notes
false-positive results. These are also unacceptable
Blood contamination of amniotic fluid has some effect
specimens.
on the L/S ratio, but there is no agreement about whether
the blood increases or decreases L/S ratio [39,86,87].
III. Reagents
Samples contaminated with blood are still useful in the
prediction of fetal lung maturity if phosphatidyl glycerol
A. Normal saline (0.9% NaCl). Pour fresh
is found. PG is virtually absent from blood, meconium,
aliquot daily.
and vaginal secretions, all of which contain
phospholipids and other components that cause
B. Absolute ethanol (200 proof). Store at
inaccurate L/S ratios [41,87,88]. Alternatively, one can
room temperature.
isolate the 10,000 g pellet from the contaminated fluid
and measure fetal pulmonary surfactant from the
C. 95% ethanol. To prepare 100 mL of
lamellar bodies [41,89].
95% ethanol, add 5 mL distilled H2O to 95 mL absolute
149
Amniotic Fluid Phospholipids (AFPL): L/S Ratio and PG

ethanol. Keep tightly closed. Stable 1 month at room X. Procedure Notes

temperature. A. Comparable results are obtained using
glass tubes with an inner diameter varying from 8 to 14
IV. Equipment mm. In larger tubes, it is more difficult to detect the fine
A. 5 mL, 1 mL, and 0.5 mL volumetric pipets. bubbles.

B. 13 100 mm glass tubes and B. Evaporation decreases the stability of

corresponding plastic caps. the foam, especially in hot, dry rooms. Clean rubber
stoppers should be used to minimize evaporation.
V. Calibration N/A
C. A precipitate occasionally forms on
VI. Quality Control N/A addition of ethanol to amniotic fluid. When this happens,
the meniscus must be observed carefully to avoid
VII. Procedure and Methodology confusion between the precipitate and the foam.
A. Do not spin sample. Gently invert tube
to suspend particles. D. Ascitic fluid gives positive tests.

B. Label two 13 100 mm test tubes #1 and #2.
C. Set up the tubes in the following manner:
Tube #1:1.0 mL of unspun fluid
1.0 mL of 95% ethanol
E. This test should not be used when
meconium or blood is seen in the amniotic fluid (may
produce a false positive).

Tube #2:0.5 mL of unspun fluid

0.5 mL of normal saline
1.0 mL of 95% ethanol

Note: Use volumetric pipets.

D. Cap tubes with disposable plastic caps,

and shake vigorously for exactly 15 sec.

E. Let stand in rack for 15 min at room

temperature.

F. Examine the top of the fluid for small,

stable bubbles. Do not move the tubes. View against a
black background if hard to visualize.

G. Interpretation:

If foam persists in tubes 1 and 2 mature

If foam is in tube 1 and not tube 2borderline
No foam in tubes 1 and 2immature

VIII. Calculations (Derivation of Results)

No calculations are necessary.

Report results as immature, borderline, or
mature.

IX. Reference Value

If test result indicates mature, RDS should not

occur in an infant delivered within 72 hours after
analysis is done.
150
Amylase

Amylase
Ming Jin
Name: Amylase, -1,4-glucan 4-glucanohydrolase (AMY)
Clinical significance: Refer to Chapter 34, The Pancreas: Function and Chemical
Pathology, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Enzyme commission number: EC 3.2.1.1
Molecular weight: 54 to 62 kDa
Chemical class: Enzyme
Isoenzymes: At least seven in human tissue; salivary and
pancreatic forms in plasma

i of amylase activity by use of laser nephelometry has

Principles of Analysis and Current Usage
Amylases are enzymes that degrade complex
carbohydrate molecules into smaller components.
Amylase is produced by the exocrine pancreas and the
salivary glands to aid in the digestion of starch. Human
amylase is -amylase, which has the ability to hydrolyze
-1,4 glucosidic linkages in polysaccharides. The -1,6
linkages at the branch points remain untouched. The
result of -amylase action on polysaccharide is the
formation of dextrans, maltose, and glucose molecules.
There are two types of amylases: one is P-type, which is
produced in the pancreas, and the other is S-type, which
is present in the salivary gland.
More than 200 methods for amylase measurement have
been devised based upon different principles and
substrates. The assay conditions must be rigidly adjusted
to suit the specific requirements of amylase. The pH
optimum is 6.9 to 7.0, and calcium and chloride ions are
required cofactors. A variety of different detection
techniques are available to measure the activity of
amylase. The different approaches can be grouped
generally into three categories.
Amyloclastic Techniques (Table 1, Methods 1a and 1b)
Amyloclastic methods for amylase assay are based on
the measurement of rate hydrolysis of starch by amylase.
Turbidimetric, nephelometric, and iodometric techniques
are used to detect the rate of starch hydrolysis.
Turbidimetric technique utilizes the ability of amylase to
decrease the turbidity of a substrate suspension by
enzymatic degradation of the substrate into smaller units.
Nephelometry has also been used for amylase
measurements. Nephelometric techniques measure the
increase in light scattering due to the hydrolysis of starch
by amylase [1]. These instruments carry out rate
measurements over a short time. Recently, measurement
i
Amylase
Previous and current authors of this method:
First edition: Michael D.D. McNeely
Methods edition: Michael D.D. McNeely
Second edition: Michael D.D. McNeely
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Ming Jin
shown improvements in analytical sensitivity and
precision when compared to conventional nephelometry
[2].
Turbidimetric and light-scattering by nephelometry
measurements are carried out either kinetically or after a
fixed time interval, and the change in turbidity or light
scattering is proportional to amylase activity. These
methods are difficult to standardize because of substrate
variation, and in general they lack precision [3].
The iodometric methods are based on the ability of
iodine to form a vivid blue color after reaction with
starch. The by-products of amylase action may also form
colored substances with iodine, but their absorbance
maxima are at different wavelengths from the
characteristic starch-iodine complex. The methods are
carried out by adding iodine color reagent to the
substrate-sample mixture after an incubation period. The
greater the amount of amylase activity, the lighter will
be the color of the final solution.
The most common approach employs a fixed time
interval [4]. Substrate and sample are mixed together and
incubated for a fixed period. The iodine color solution is
then added, and a spectrophotometric measurement is
performed. The lower the final absorbance (the greater
the difference between the absorbance of the blank and
the absorbance of the sample), the higher the activity.
Amyloclastic methods are no longer widely employed,
in part because substrate variability makes
standardization difficult.
Saccharogenic Techniques (Table 1, Methods 2a and 2b)
Saccharogenic techniques depend on the measurement of
monosaccharides or disaccharides liberated by amylases
reaction with the substrate. The classical amylase
method of Somogyi is a saccharogenic technique [5].
Sample and substrate are incubated for 30 minutes, then
a serum blank and the sample reaction tube undergo
measurement for reducing substances. Since the by-
products of amylase action are reducing substances, the
enzymes action is directly proportional to the amount of
reducing substance produced.
More recently, the saccharogenic approach has been
modified for automated kinetic analysis. In these
techniques, the maltose hydrolysed by amylase from the
substrate, such as maltopentaose and maltotetraose, is
151
Amylase
converted into glucose by enzyme, which is included in
the reagent mixture. The glucose liberated by this
reaction can be measured by several enzymatic glucose
assay techniques [6].
Chromolytic Techniques (Table 1, Methods 3a, 3b, and
3c)
In recent years, commercial manufacturers have
produced a variety of intriguing and convenient amylase
methods that depend on the liberation of a dye coupled
to polysaccharides such as amylopectin. Samples are
incubated with the dyed starch substrate, and after a
fixed time, the amount of color of smaller dyed
saccharides is measured.
Dry-film procedures are based on the hydrolysis of the
dyed starch substrate. The released dyed saccharides in
the spreading layer diffuse into an underlying reagent
layer, where they are measured by reflectance
spectrophotometry. Unreacted dyed starch substrate that
is present in a spreading layer remains hidden from view
(Method 3a).
A different method depends on the release of small,
water-soluble fragments in such a way that the color can
be measured spectrophotometrically [7,8]. The substrate
used in the method is p-nitrophenyl glucosides, such as
p-nitrophenyl-maltoheptaoside (G7PNP). The substrate is
hydrolysed to produce p-nitrophenol by amylase with
coupled reaction of substrate fragments involving -
glucosidase. The reaction is monitored by measurement
of the liberated p-nitrophenol at 405 nm (Method 3b).
This method is also recommended by the International
Federation of Clinical Chemistry (IFCC) for amylase
measurement (see method procedures in Reference and
Preferred Methods).
An alternative substrate is 2-chloro-p-nitrophenyl -D-
maltotrioside (CNP-G3), which can be hydrolysed to 2-
chloro-p-nitrophenol (CNP) by amylase. The CNP is
detected spectrophotometrically and is proportional to
the amylase activity in the sample. This method does not
require glucosidases and is considered direct assay
(Method 3c).
Historically the methods with starch as substrate were
used in amylase assay. Now most the methods are used
with the well-defined substrates, and these defined
substrates have improved the reactive stoichiometry.
Chromolytic techniques use as substrates either p-
nitrophenyl-maltoheptaoside (G7PNP) or 2-chloro-p-
nitrophenyl -D-maltotrioside (CNP-G3), and
saccharogenic techniques use as substrates either
maltopentaose or maltotetraose. These are the current
methods used for amylase and are widely used on
automatic analyzers.
Reference and Preferred Methods
A primary reference procedure for amylase measurement
at 37C was established by IFCC Scientific Division,
Committee on Reference Systems for Enzymes (C-RSE)
using a chromolytic technique. The reference method is
described at the end of the section, and the detailed
reference procedure is in the Procedure Section.
Historically, the amyloclastic methods using starch as a
substrate (e.g., turbidimetric and nephelometry
techniques) and the iodometric method have been
displaced with the well-defined shorter glucosyl-chain
substrates. The use of the defined amylase substrates has
improved the reaction stoichiometry and led to more
consistent amylase measurement.
The saccharogenic methods are reliable but time
consuming and have high sample blanks and variable
substrates. A correction is required for the amount of
glucose originally in the patient sample. One can do this
by consuming all the glucose in the patients sample
before performing the complete analysis or by using a
serum blank. Such blanks may be very high, rendering
spectrophotometric measurements difficult. In general,
the reagents for these reactions are very complex and
therefore rather expensive. The reagents may also lose
their effectiveness through a compromise among the
many coupled enzyme reactions, which must go on
simultaneously. They rarely have true, zero-order
kinetics. The use of synthetic, defined substrates with the
enzymatic methods allows for reproducible substrates
and reaction products.
Chromolytic techniques can be fast, precise, consistent,
and easy to perform. The methods employing p-
nitrophenyl-modified defined substrates are more
sensitive than the saccharogenic methods because of the
higher molar absorptivity. The p-nitrophenyl
maltoheptaoside substrate has been shown to be equally
active with both salivary and pancreatic isoenzymes [9]
and to correlate very well with a saccharogenic method
with ultraviolet monitoring of the glucose reaction. This
method showed no interference by hemoglobin, lipemia,
or glucose.
The IFCC has a recommended method for amylase
measurement by chromolytic technique using the
substrate of 4,6-ethylidene(G1)-p-nitrophenyl(G7)--D-
maltoheptaoside at 30C [10]. Because most of the
procedures on the automated instruments in clinical
laboratories are performed at 37C, the amylase value of
the 30C reference method is not given a high level.
IFCC has developed the 37C reference procedures for
amylase measurement with the same substrate of 4,6-
ethylidene(G1)-p-nitrophenyl(G7)--D-maltoheptaoside
as used in the reference procedure at 30C [11].
Specimen
Either serum or heparinized plasma can be used as
sample [12]. However, one study found that heparinized
plasma samples gave significantly higher results than
serum samples when measured using dry-film
technology [13]. Whether this effect is due to the unique
properties of the dry-film technology or whether heparin
affects amylase measurements in wet chemistry
systems is not known. Because amylase has an absolute
requirement for calcium ions, chelating anticoagulants
such as citrate, oxalate, and EDTA cannot be used to
152
Amylase
collect plasma to be used for amylase measurements.
Urine specimens with no preservatives in random or
timed collections are also valid specimens.
Serum shows no loss of amylase activity for 4 days at
room temperature, for 2 weeks at 5C, for 1 year at
28C, or for 5 years at 75C. Urine specimens should
be analyzed within 12 hours at room temperature or
within 5 days at 5C, and urine should not be frozen
[10].
Interferences
Amylase assays are generally not prone to interference
from hemoglobin, bilirubin, or triglycerides. Collection
of specimens in tubes containing oxalate, citrate, or
EDTA may result in falsely decreased values due to
chelation of necessary amylase cofactors.
Amylase Reference Interval
Reference intervals of amylase differ between the
various available assays method because of differences
in substrates used and reagent preparations [14]. The
reference interval of
amylase by the IFCC recommended method at 37C is
31 to 107 U/L.
Blood amylase activity of newborns is approximately
18% of adults [12]. Mean serum amylase activity
increases from the neonatal period until adult levels are
achieved at approximately 3 to 4 years of age. There are
no significant differences between males and females in
the serum activity of amylase [12].
Interpretation
Blood total amylase increases in acute pancreatitis and
salivary gland inflammation disorders. Amylase
measurement is used primarily in the diagnosis of
pancreatitis. Following the onset of acute pancreatitis,
amylase starts to rise in 5 to 8 hours and returns to
normal on the third or fourth day. Urine amylase also
increases in acute pancreatitis, and it remains elevated
for extended periods (7 to 10 days).
The lack of specificity of total amylase measurements
has led to the development of other tests (e.g., lipase and
pancreatic-specific amylase) instead of total amylase
activity for the diagnosis of acute abdominal pain.
Studies have shown that total amylase measurement is
inferior for the diagnosis of pancreatitis when compared
to these other markers [15-18]. Reported diagnostic
efficiencies of amylase for acute pancreatitis range from
54% to 83%, while reported sensitivities and
specificities range from 46% to 90% and 73% to 96%,
respectively [15-17]. Improved methods for
measurement of lipase and pancreatic-specific amylase
have been developed [19]. If acute pancreatitis is
suspected, serum amylase should be monitored in
conjunction with lipase or pancreatic-specific amylase.
Serial measurements are generally more helpful than
single determinations in helping to establish a correct
diagnosis.
Macroamylase is the complex formed between amylase
and immunoglobulins [20]. Amylase is readily filtered
by the glomerulus and is normally found in urine, but the
large molecular mass (greater than 200 kDa) of
macroamylase prevents its filtration through the
glomerulus of the kidney and its appearance in urine.
Patients with macroamylasemia usually have increased
serum amylase activities and low or normal urine
amylase activities. No clinical symptoms are associated
with the macroamylase disorder. The clinical importance
of macroamylase lies in its potential to create confusion
during the investigation of possible pancreatitis injury,
and some cases of macroamylasemia have been found in
the investigation of pancreatitis injury.
Numerous nonpancreatic abdominal diseases, such as
appendicitis and peritonitis, can cause hyperamylasemia.
Salivary gland lesions caused by infection, irradiation,
obstruction, surgery, and tumor can increase the S-type
amylase, as well as total amylase level. Also, many
drugs have been reported to cause an increase in serum
amylase measurements [21]. The other common cause
for hyperamylasemia is renal insufficiency. Patients with
renal insufficiency can show increases in amylase
activity up to five times the upper reference interval,
especially if the creatinine clearance is less than 50
mL/min.
Amylase Performance Goals
The criteria for acceptable performance for amylase
assay by the Clinical Laboratory Improvement
Amendments (CLIA) and College of American
Pathologists (CAP) proficiency program is within 30%
of the mean value of laboratory peer groups. Amylase
assays with different methods performed well in the
clinical laboratories. The 2007 CAP
Chemistry/Therapeutic Drug Monitoring Survey (C-A)
shows that clinical laboratories use different instruments
and methods for amylase assay at 37C, and these
methods are developed by Abbott, Bayer, Beckman,
Dade Behring, Roche, and Vitro. The coefficients of
variation (CVs) of each of these methods run in variable
numbers of laboratories are from 1.7% to 9.1% at
different means. The average CV of these methods is
5.4%.
References
1 Smeaton JR, Marquardt HF. A reaction rate
nephelometer for amylase determinations. Clin
Chem 1974; 20: 896.
2 Liu TZ, Wei JS. Rapid laser nephelometric
determination of amylase activity in serum and
urine. J Formosan Med Assoc 1991; 90: 217-
220.
3 Zinterhofer L, Wardlaw S, Jatlow P, Seligson
D. Nephelometric determination of pancreatic
enzymes: I. Amylase. Clin Chim Acta 1973; 43:
5-12.
4 Caraway WT. A stable starch substrate for the
determination of amylase in serum and other
body fluids. Am J Clin Pathol 1959; 32: 97-99.
153
Amylase
5 Somogyi M. Modifications of two methods for
the assay of amylase. Clin Chem 1960; 6: 23-
35.
6 Kaufman RA, Tietz NW. Recent advances in
measurement of amylase activity: a comparative
study. Clin Chem 1980; 26: 846-853.
7 Lorentz K. -Amylase assay: Current state and
future development. J Clin Chem Clin Biochem
1979; 17: 499-504.
8 Okabe H, Uji Y, Netsu K, Noma A. Automated
measurement of amylase with 4-
nitrophenylmaltoheptaoside as a substrate and
use of a selective amylase inhibitor. Clin Chem
1984; 30: 1219-1222.
9 Rauscher E, Neumann U, Schaich E, von
Bulow S, Wahlefeld AW. Optimized conditions
for determining activity concentration of -
amylase in serum with 1,4--D-4-
nitrophenylmaltoheptaoside as substrate. Clin
Chem 1983; 31: 14-19.
10 Lorentz K. Approved recommendation on IFCC
methods for the measurement of catalytic
concentration of enzymes, Part 9: IFCC method
for -amylase (1,4-a-D-glucan4-
glucanohydrolase, EC 3.2.1.1) Clin Chem Lab
Med 1998; 36: 185-203.
11 Schumann G, Aoki R, Ferrero CA, Ehlers G,
Feard G, Gella FJ, et al. IFCC primary
reference procedures for the measurement of
catalytic activity concentrations of enzymes at
370C, Part 8: reference procedure for the
measurement of catalytic concentration of -
amylase. Clin Chem Lab Med 2006; 44: 1146-
1155.
12 Gillard BK, Simbala JA, Goodglick L.
Reference ranges for amylase isoenzymes in
serum and plasma of infants and children. Clin
Chem 1983; 29: 1119-1123.
13 Doumas BT, Hause LL, Simuncak DM.
Differences between values for plasma and
serum in tests performed in the Ektachem 700
XR analyzer, and evaluation of plasma
separator tubes (PST). Clin Chem 1989; 35:
151-153.
14 Chen CT, Dineen H, Newton JD. Specificity of
different substrates used in three amylase
assays. Clin Chem 1988; 34: 1363-1364.
15 Kazmierczak SC, Van Lente F, Hodges ED.
Diagnostic and prognostic utility of
phospholipase A activity in patients with acute
pancreatitis: comparison with amylase and
lipase. Clin Chem 1991; 37: 356-361.
16 Van Lente, F, Kazmierczak SC.
Immunologically-derived pancreatic amylase,
pancreatic lipaase, and total amylase compared
as predictors of pancreatic inflammation. Clin
Chem 1989; 35: 1542.
17 Kazmierczak SC, Van Lente F. Measuring
carboxypeptidase A activity with a centrifugal
analyzer: analytical and clinical considerations.
Clin Chem 1989; 35: 251-255.
18 Lott JA, Lu CJ. Lipase isoforms and amylase
isoenzymes: assays and application in the
diagnosis of acute pancreatitis. Clin Chem
1991; 37: 361-368.
19 Apple F, Benson P, Preese L. Lipase and
pancreatic amylase activities in tissues and in
patients with hyperamylasemia. Am J Clin
Pathol 1991; 96: 610-614.
20 Fridhandler L, Berk JE. Macroamylasemia. Adv
Clin Chem 1978; 20: 267-28.
21 Young DS, Pestaner LC, Libberman V. Effects
of drugs on clinical laboratory tests. Clin Chem
1975; 21: 255D-556D.
154
Amylase

Tables

Table 1: Methods of Amylase Analysis

Method 1: Amyloclastic
Principle of analysis:
a. Turbidimetric or nephelometric. Decrease in turbidity or scattered light (nephelometry) of a starch solution is
directly related to amylase concentration.
b. Iodometric. Degradation of starch by amylase reduces the reaction of iodine with starch; reduction in iodine-
starch product (Amax = 660 nm) is inversely related to amylase activity.
Comments:
a. Rare; with consistent substrate, automated technique acceptable
b. Rare; can be easily adapted by most laboratories

Method 2: Saccharogenic
Principle of analysis:
Glucose released from substrate is quantitated, usually by an enzymatic glucose assay procedure.
a. Maltopentaose as substrate:
Amylase
maltopentaose -------------------------------------> maltotriose + maltose
-Glucosidase
maltotriose + maltose ------------------------------> 5 glucose
Hexokinase
glucose + ATP --------------------------------------> glucose-6-phosphate + ADP
Glucose-6-Phosphate Dehydrogenase
glucose-6-phosphate + NAD+ --------------------> 6-phosphogluconate + NADH + H+

b. Maltotetraose as substrate:
Amylase
maltotetraose + H2O ------------------------------> 2 maltose
Maltose phosphorylase
maltose + phosphate ------------------------------> glucose + glucose-1-phosphate
-phosphoglucomutase
glucose-1-phosphate ------------------------------> glucose-6-phosphate

Glucose-6-phosphate dehydrogenase
glucose-6-phosphate NAD+ ---------------------> 6-phosphogluconate + NADH + H+
Comments:
Very common; can be easily adapted to many automated, discrete analyzers

Method 3: Chromolytic
Principle of analysis:
a. Liberation of a dye coupled to a polysaccharide to dyed saccharide
Amylase
dyed polysaccharide -------------------> dyed saccharides

b. Liberation of chromogen from soluble, defined substrates (4,6-ethylidene-G7PNP)

Amylase
4,6-ethylidene-G7PNP + H2O -----------------> 4,6-ethylidene-Gx + G(7x) PNP
Glycosidase
G(7x) PNP + (7x) H2O ----------------------> (7x) glucose + PNP

Where 4,6-ethylidene-G7PNP is 4,6-ethylidene(G1)-p-nitrophenyl(G7)--D-maltoheptaoside; PNP is p-

nitrophenol

c. Liberation of chromagen from soluble, defined substrates (CNP-G3)

Amylase
CNP-G3 --------------> CNP + CNP-G2 + maltotriose + glucose
Where CNP-G3 is 2-chloro-p-nitrophenyl -D-maltotrioside
Comments:
a. Common
b. Common; IFCC recommended method
c. Common
155
Amylase

Procedure: IFCC Recommendation for the
Measurement of Amylase [11]
Principal
Amylase hydrolyzes 4,6-ethylidene(G1)-p-
nitrophenyl(G7)--D-maltoheptaoside to different 4,6-
ethylidene--D-glucopyranosyl oligosaccharides and p-
nitrophenyl--D glucopyranosyl oligosaccharides, and
the latter is degraded to p-nitrophenyl by -glycosidase,
and p-nitrophenyl is detected at 405 nm. The chemical
reaction scheme is in Method 3b of the Amylase
Methods summary table.
Reagents
Determine the catalytic -glucosidase concentration
according to Appendix 1 of the reference article [11].
Reconstitute the lyophilized -glucosidase with a
volume of the Diluent for Reagent
Enzymes to obtain a catalytic concentration of the
reconstituted material of 16.9 mkat/L (1014 kU/L) at
37C.
Freeze the enzyme solution in portions of 0.25 mL at
25C.
Stability at 25C: at least 6 months.
Reaction Solution (Working Solution)
Mix 25 mL of Solution 2 with 0.25 mL of Solution 3.
Stability at 2C to 8C: 2 weeks.

1. N-2-hydroxyethylpiperazine-N-ethanesulfonic acid Starting Reagent Solution (Substrate Solution)

[HEPES] (C6H18N2O4S) 1.01 g (31.00 mmol/L) 4,6-ethylidene(G1)-4-
2. 4,6-Ethylidene(G1)-p-nitrophenyl(G7)--D- nitrophenyl(G7)-a-(1>4)-D-maltoheptaoside.
maltoheptaoside (C50H77NO38) 0.310 g (52.10 mmol/L) N-2-hydroxyethylpiperazine-N-
3. -Glucosidase (EC 3.2.1.20) ethanesulfonic acid.
4. Sodium chloride Dissolve in approximately 20 mL of water.
5. Calcium chloride dihydrate Adjust to pH (37C) 7.00 with 0.2 mol/L sodium
6. Sodium hydroxide solution (NaOH) 0.2 mol/L hydroxide solution.
7. Bovine serum albumin, fraction V, Mr = 68000 Transfer to a 25-mL volumetric flask.
8. Aqueous sodium chloride solution, 0.154 mol/L Equilibrate the volumetric flask and water to 20C.
Fill with water (20C) up to the calibration mark of the
Solution 1 volumetric flask.
6.14 g (417.5 mmol/L) calcium chloride, dihydrate. Stability at 2C to 8C: 2 weeks.
Dissolve in approximately 80 mL of water.
Transfer to a 100-mL volumetric flask. Final Concentrations of Reagents for the Measurement
Equilibrate the volumetric flask and water to 20C. of Amylase
Fill with water (20C) up to the calibration mark of the N-2-Hydroxyethylpiperazine-N-ethanesulfonic acid
volumetric flask. 50 mmol/L
Stability at 20C: 3 months. pH (37C) 7.00 0.03
4,6-Ethylidene(G1)-p-nitrophenyl(G7)--D-
Solution 2 maltoheptaoside 5 mmol/L
3.10 g (52.10 mmol/L) N-2-hydroxyethylpiperazine-N- Sodium chloride 70 mmol/L
ethanesulfonic acid. Calcium chloride 1 mmol/L
1.26 g (86.13 mmol/L) sodium chloride. -Glucosidase (37C) 135 kat/L (8100 U/L)
Dissolve in approximately 200 mL of water. Volume fraction of sample 1:31
Add 0.75 mL of Solution 1.
Adjust to pH (37C) 7.00 with 0.2 mol/L sodium Measurement Condition
hydroxide solution. Temperature 37.0C 0.1C
Transfer to a 250-mL volumetric flask. Wavelength 405 nm (1 nm)
Equilibrate the volumetric flask and water to 20C. Bandwidth <2 nm
Fill with water (20C) up to the calibration mark of the Light path length 10.00 0.01 mm
volumetric flask. Incubation time 60s s
Stability at 2C to 8C: 5 weeks. Delay time 180 s
Measurement interval 180 s
Diluent for Reagent Enzymes Reading (measurement points) >6
1.20 g bovine albumin.
0.900 g (154 mmol/L) NaCl. Measurement Procedure
Dissolve in approximately 80 mL of water. 2.000 mL Reaction Solution
Transfer to a 100-mL volumetric flask. Equilibrate to 37.0C
Equilibrate the volumetric flask and water to 20C. 0.080 mL Sample
Fill with water (20C) up to the calibration mark of the Mix thoroughly and incubate for 60 s. At the end of the
volumetric flask. incubation time, the temperature of the solution in the
Stability at 2C to 8C: at least 3 months. cuvette shall have reached 37.0C

Solution 3 0.400 mL Starting Reagent Solution

16.9 mkat/L (1014 kU/L) -glucosidase at 37C. Mix thoroughly, wait 180 s and monitor time and
absorbance for an additional 180 s
156
Amylase

Reagent and sample blank rate are also needed to

determine (see details in reference article [12])

Calculation
Amylase (kat/L) = A/t 1/ L Vt/Vs
Where A/t is absorbance change per second, is
molar absorption coefficient of p-nitrophenyl at 405nm,
L is light path length, Vt is total reaction volume, and Vs
is sample volume.

The amylase catalytic concentration in kat/L can be

converted to U/L by multiplication by the factor 60.
157
Angiotensin-Converting Enzyme (ACE)
Angiotensin-Converting Enzyme (ACE)
Hassan M.E. Azzazy
Name: Angiotensin-converting enzyme (ACE)
Clinical significance: Refer to Chapter 28, Physiology of body water and electrolytes, in 5th edition
of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass: 150-180 kDa (somatic isoform); 90-110 kDa (testes isoform)
Chemical class: Metalloprotein
Principles of Analysis and Current Usage
Angiotensin-converting enzyme, or ACE (EC 3.4.15.1),
is a zinc-metalloendopeptidase that plays an important
role in the control of blood pressure in humans through
regulation of angiotensin II and bradykinin. ACE cleaves
the C-terminal dipeptide of angiotensin I to produce the
potent vasoconstrictor angiotensin II. ACE also
inactivates bradykinin, a potent vasodilator peptide, by
sequential removal of two C-terminal dipeptides [1].
Somatic ACE isoform is attached to the cell membrane
and contains two highly homologous domains at the N-
and C-terminal regions, each possessing an active site
[2]. Testes ACE isoform is also membrane bound but
contains only a single active site corresponding to the C-
terminal somatic isoform [3]. Plasma ACE originates
from proteolytic shedding from the cell membrane [4].
ACE is present in high activity at the lung vascular
endothelial surface. ACE is also found in epithelial cells
of renal proximal tubules, the GI tract, cardiac tissues,
and in the brain [5]. The wide tissue distribution of ACE,
where other elements of the renin-angiotensin system are
not present, may suggest additional roles for ACE
besides angiotensin II production and bradykinin
inhibition [6].
Several analytical methods have been developed for
determination of ACE activity, including
spectrophotometry, fluorometry, radiometric assay,
radioimmunoassay, enzyme-linked immunosorbent
assay (ELISA), high-performance liquid
chromatography (HPLC), and capillary electrophoresis
The serum ACE concentration has been reported to
depend on the ACE gene insertion/deletion (I/D)
mutation polymorphism. The D/D genotype is associated
with a higher serum ACE concentration and may be
associated with ischemic heart disease [12]. A PCR
assay has been developed to detect the insertion itself,
which is a 287-bp alu repetitive sequence located in
intron 16 of the gene (chromosomal location of ACE
gene is 17q23) [13]. A real-time PCR assay that utilizes
fluorescent hybridization probes has been developed for
rapid genotyping of ACE gene I/D polymorphisms [14].
i Second edition: Not updated
i Fourth edition: Not updated
Angiotensin-converting enzyme (ACE)
Previous and current authors of this method:
First edition: Not done
Methods edition: Gerald Kessler
(Table 1). Most of these assays are based on the ability
of ACE to hydrolyze hippuryl-L-histidyl-L-leucine
(HHL) or furanacryloyl-L-phenylalanylglyclglycine
(FAPGG) substrates (Table 1, Methods 1, 3). The HHL
assay is based on the hydrolysis of HHL to hippuric acid
and HL. The hippuric acid formed is generally extracted
and quantified at 228 nm [7]. The FAPGG assay was
originally developed to measure ACE activity in serum
and is based on hydrolysis of FAPGG to FAP and GG
[8]. Hydrolysis of FAPGG was assessed by measuring
the decrease in absorbance at 340 nm. In addition, an
enzyme inhibitor binding assay for ACE has been
described [9] that is based on specific binding of 125I-
labeled ACE inhibitor (an analog of the ACE inhibitor
enalapril) to the active center of the enzyme (Table 1,
Method 2).
Erickson et al. [10] have reformatted a commercial
quantitative spectrophotometric CSF ACE assay for a
96-well plate. The assay is based on the ability of ACE
to hydrolyze the tripeptide N-[3-(2-furyl)acryloyl]-L-
phenylalanylglycylglycine to furylacryloylphenylalanine
and glycylglycine. The assay, which utilizes only 115 L
of CSF specimen, had a detection limit of 0.4 U/L and
upper reference value of 2.5 U/L.
A new assay has recently been described [11] for
screening of ACE inhibitory activity of potent food
sources and drugs. The assay utilizes a new substrate 3-
hydroxybutyrylglycyl-glycyl-glycine. The substrate is
cleaved by ACE and aminoacylase into amino acids and
3-hydroxybutyric acid, which is measured
Reference and Preferred Methods
There is no reference method for serum ACE. Most
assays for determining ACE activity in serum employ
HHL and FAPGG as substrates. The kinetic
spectrophotometric method is probably the most
common method. Several versions of this method have
been developed and adapted to various automated
instruments.
Third edition: Not updated
Fifth edition: Hassan M.E. Azzazy
158
Angiotensin-Converting Enzyme (ACE)
Specimen
Serum is preferred. Blood is drawn from a fasting patient
in a red-top or a serum gel tube. After centrifugation,
separated serum is stable for 1 week at 4C and can be
stored at 20C in a plastic vial for 6 months.
Interferences
The use of ACE-inhibiting antihypertensive drugs such
as captopril, cilazapril, enalapril, lisinopril, perindopril,
propranolol, ramipril, and trandolapril will decrease
ACE values. Other factors that will cause lower ACE
results include hemolysis, lipemia, EDTA, heavy metals,
Method
Spectrophotometric assay (228 nm)
Substrate: hippuryl-L-histidyl-L-leucine [15]
Fluorimetric assay
Substrate: hippuryl-L-histidyl-L-leucine and o-
phthaldialdehyde [16]
Continuous spectrophotometric assay (340 nm)
Substrate: phenylalanyl-glycyl-glycine [17]
Colorimetric assay (505 nm)
Substrate: hippuryl-L-histidyl-L-leucine with 4-amino-
antipyrine [18]
Interpretation
ACE (dipeptidyl carboxypeptidase; EC 3.4.15.1), also
known as kininase II, catalyzes the conversion of
angiotensin I to angiotensin II by cleavage of the C-
terminal dipeptide (histidyl-leucine) and also inactivates
bradykinin by cleavage of a dipeptidyl moiety [19]. ACE
is found mainly on the luminal surface of pulmonary
endothelial cells [20] but is also present in the circulation
[21].
ACE has a key role in regulation of peripheral blood
pressure and is considered a major risk factor for
cardiovascular disease [22,23].
Although increased serum activity of ACE is not specific
for sarcoidosis, ACE is used almost exclusively for
diagnosing and monitoring disease activity in sarcoidosis
[24]. ACE activity is also elevated in acute and chronic
bronchitis, pulmonary fibrosis, rheumatoid arthritis, and
Gauchers disease. Elevated ACE activities were
reported in patients with erectile dysfunction [25].
Patients with anorexia nervosa showed a low serum
ACE activity that reverted to within the normal range
following weight gain [26]. On the other hand, patients
with advanced lung neoplasms had significantly lower
levels of ACE as compared to controls [27].
ACE has also been associated with an endogenous renin-
angiotensin system in the brain [28]. Changes in ACE
and oxalate. Triiodothyronine, acetate, nitrate, fluoride,
chloride, or bromide may cause elevated values.
Reference Intervals
Reference intervals for healthy individuals vary per the
method used to measure ACE. Furthermore, reported
reference intervals also varied even when the same
method (e.g., kinetic spectrophotometry) and essentially
the same reagents were used on different instruments.
This underscores the urgent need to standardize different
ACE methods to establish common reference intervals.
Clearly, assay manufacturers can help by developing
appropriate enzyme reference materials.
Reference Intervals
19 6 (SD) kU/L
32.2 9.9 (SD) nmol/min/mL
<2 yr: 5-83 U/L
3-7 yr: 8-76 U/L
8-14 yr: 6-89 U/L
>14 yr: 8-52 U/L
<1 yr: 10.9-42.1 U/L
1-2 yr: 9.4-36 U/L
3-4 yr: 7.9-29.8 U/L
4-9 yr: 9.6-35.4 U/L
10-12 yr: 10.3-37 U/L
13-16 yr: 9-33.4 U/L
17-19 yr: 7.2-26.6 U/L
>19 yr: 6.1-21.1 U/L
activity in the brain may be caused by a variety of
neurological disorders that are reflected by changes in
ACE in the CSF [29]. Increased concentrations of ACE
have been associated with neurosarcoidosis [30] and
viral and bacterial meningitis. Low ACE levels are
reported in patients with Alzheimers disease,
Parkinsons disease, and progressive supranuclear palsy
[31].
Recent evidence indicates that ACE activity in infancy
may contribute to the link between low birth weight and
later cardiovascular disease [32]. Because angiotensin II,
the main metabolite of ACE, has been shown to have
atherogenic effects [33], increased serum ACE activity
in infancy may identify individuals with increased
cardiovascular disease later in life.
It should be noted that a homolog of ACE, termed
ACE2, was discovered in 2000 [34]. It hydrolyzes
angiotensin I to Ang-(1-9) which is converted into Ang-
(1-7) by the action of two enzymes, neutral
endopeptidase and ACE. However, ACE2 releases Ang-
(1-7) more efficiently than its catalysis of Ang-(1-9) by
cleavage of Pro-Phe bound in Ang II. Thus Ang-(1-7) is
the major biologically active product of ACE2 and is
considered to be a beneficial peptide of the RAS cascade
in the cardiovascular system. ACE2 has 42% identity
with the catalytic domain of ACE, is present in most
cardiovascular-relevant tissues, and is an ectoenzyme as
159
Angiotensin-Converting Enzyme (ACE)
ACE. Unlike ACE, ACE2 has only one catalytic site and
is insensitive to ACE inhibitors. ACE2 has vasodilatory
and antiproliferative effects of Ang-(1-7) in the heart and
kidney [35].
Performance Goals
Desirable imprecision for ACE as derived from
biological variation is 0.1% [36]. The current Clinical
Laboratory Improvement Amendments (CLIA)
performance goal for measurement of ACE is for
laboratories to be within 3 standard deviations of the
peer-group mean. According to the 2007 College of
American Pathologists (CAP) Survey, coefficients of
variation (CVs) for all ACE reagents at mean values of
48.6 (SD 6.9) and 99.2 (SD 13.1) U/L were 14.2% and
13.2%, respectively [37]. Despite the acceptable
precision of current ACE testing, standardization is
needed to reduce variations among different methods
and to develop common reference intervals.
References
1 Yang HY, Erds EG, Levin Y. A dipeptidyl
carboxypeptidase that converts angiotensin I
and inactivates bradykinin. Biochim Biophys
Acta 1970;214:374-6.
2 Soubrier F, Alhenc-Gelas F, Hubert C,
Allegrini J, John M, Tregear G, Corvol P. Two
putative active centers in human angiotensin I-
converting enzyme revealed by molecular
cloning. Proc Natl Acad Sci USA
1988;85:9386-90.
3 Ehlers MR, Fox EA, Strydom DJ, Riordan JF.
Molecular cloning of human testicular
angiotensin-converting enzyme: the testis
isozyme is identical to the C-terminal half of
endothelial angiotensin-converting enzyme.
Proc Natl Acad Sci USA 1989;86:7741-5.
4 Beldent V, Michaud A, Wei L, Chauvet MT,
Corvol P. Proteolytic release of human
angiotensin-converting enzyme. Localization of
the cleavage site. J Biol Chem 1993;268:26428-
34.
5 Zhuo J, Moeller I, Jenkins T, Chai SY, Allen
AM, Ohishi M, Mendelsohn FA. Mapping
tissue angiotensin-converting enzyme and
angiotensin AT1, AT2 and AT4 receptors. J
Hypertens 1998;16:2027-37.
6 Dzau VJ. Circulating versus local renin-
angiotensin system in cardiovascular
homeostasis. Circulation 1988;77:I4-13.
7 Cushman DW, Cheung HS. Spectrophotometric
assay and properties of the angiotensin-
converting enzyme of rabbit lung. Biochem
Pharmacol 1971;20:1637-48.
8 Harjanne A. Automated kinetic determination
of angiotensin-converting enzyme in serum.
Clin Chem 1984;30:901-2.
9 Fyhrquist F, Tikkanen I, Gronhagen-Riska C,
Hortling L, Hichens M. Inhibitor binding assay
for angiotensin-converting enzyme. Clin Chem
1984;30:696-700.
10 Erickson JA, Cousin R, Wu JT, Ashwood ER.
Quantitative spectrophotometric microplate
assay for angiotensin-converting enzyme in
cerebrospinal fluid. Clin Chem 2003;49:970-2.
11 Lam le H, Shimamura T, Sakaguchi K, Noguchi
K, Ishiyama M, Fujimura Y, Ukeda H. Assay of
angiotensin I-converting enzyme-inhibiting
activity based on the detection of 3-
hydroxybutyric acid. Anal Biochem
2007;364:104-11.
12 Lindpaintner K, Pfeffer MA, Kreutz R,
Stampfer MJ, Grodstein F, LaMotte F, Buring
J, Hennekens CH. A prospective evaluation of
an angiotensin-converting-enzyme gene
polymorphism and the risk of ischemic heart
disease. N Engl J Med 1995;332:706-11.
13 Rigat B, Hubert C, Corvol P, Soubrier F. PCR
detection of the insertion/deletion
polymorphism of the human angiotensin-
converting enzyme gene (DCP1) (dipeptidyl
carboxypeptidase 1). Nucleic Acids Res
1992;20:1433.
14 Somogyvari F, Szolnoki Z, Marki-Zay J, Fodor
L. Real-time PCR assay with fluorescent
hybridization probes for exact and rapid
genotyping of the angiotensin-converting
enzyme gene insertion/deletion polymorphism.
Clin Chem 2001;47:1728-9.
15 Ryder KW, Jay SJ, Jackson SA, Hoke SR.
Characterization of a spectrophotometric assay
for angiotensin-converting enzyme. Clin Chem
1981;27:530-4.
16 Friedland J, Silverstein E. A sensitive
fluorimetric assay for serum angiotensin-
converting enzyme. Am J Clin Pathol
1976;66:416-24.
17 Holmquist B, Bunning P, Riordan JF. A
continuous spectrophotometric assay for
angiotensin-converting enzyme. Anal Biochem
1979;95:540-8.
18 Kasahara Y, Ashihara Y. Colorimetry of
angiotensin-I converting enzyme activity in
serum. Clin Chem 1981;27:1922-5.
19 Erdos EG. Angiotensin I converting enzyme.
Circ Res 1975;36:247-55.
20 Ryan JW, Ryan US, Schultz DR, Whitaker C,
Chung A. Subcellular localization of pulmonary
angiotensin-converting enzyme (kininase II).
Biochem J 1975;146:497-9.
21 Ondetti MA, Cushman DW. Enzymes of the
renin-angiotensin system and their inhibitors.
Annu Rev Biochem 1982;51:283-308.
22 Dahlof B, Devereux RB, Kjeldsen SE, Julius S,
Beevers G, de Faire U et al. LIFE Study Group.
Cardiovascular morbidity and mortality in the
Losartan Intervention For Endpoint reduction in
hypertension study (LIFE): a randomised trial
against atenolol. Lancet 2002;359:995-1003.
23 Brunner HR, Gavras H. Angiotensin blockade
for hypertension: a promise fulfilled. Lancet
2002;359:990-2.
24 Lieberman J. Enzymes in sarcoidosis.
Angiotensin-converting-enzyme (ACE). Clin
Lab Med 1989;9:745-55.
160
Angiotensin-Converting Enzyme (ACE)
25 Hamed EA, Meki AR, Gaafar AA, Hamed SA.
Role of some vasoactive mediators in patients
with erectile dysfunction: their relationship with
angiotensin-converting enzyme and growth
hormone. Int J Impot Res 2003;15:418-25.
26 Matsubayashi S, Tamai H, Kobayashi N,
Takaichi Y, Fukata S, Hirota Y et al.
Angiotensin-converting enzyme and anorexia
nervosa. Horm Metab Res 1988;20:761-4.
27 Ashutosh K, Keighley JF. Diagnostic value of
serum angiotensin-converting enzyme activity
in lung diseases. Thorax 1976;31:552-7.
28 Phillips MI, Weyhenmeyer J, Felix D, Ganten
D, Hoffman WE. Evidence for an endogenous
brain renin-angiotensin system. Fed Proc
1979;38:2260-6.
29 Schweisfurth H, Schioberg-Schiegnitz S. Assay
and biochemical characterization of
angiotensin-I-converting enzyme in
cerebrospinal fluid. Enzyme. 1984;32:12-9.
30 Jones DB, Mitchell D, Horn DB, Edwards CR.
Cerebrospinal fluid angiotensin-converting
enzyme levels in the diagnosis of
neurosarcoidosis. Scot Med J 1991;36:144-5.
31 Zubenko GS, Volicer L, Direnfeld LK,
Freeman M, Langlais PJ, Nixon RA.
Cerebrospinal fluid levels of angiotensin-
converting enzyme in Alzheimers disease,
Parkinsons disease and progressive
supranuclear palsy. Brain Res 1985;328:215-21.
32 Forsyth JS, Reilly J, Fraser CG, Struthers AD.
Angiotensin-converting enzyme activity in
infancy is related to birth weight. Arch Dis
Child Fetal Neonatal Ed 2004;89:F442-4.
33 Scholkens BA, Landgraf W. ACE inhibition
and atherogenesis. Can J Physiol Pharmacol
2002;80:354-9.
34 Donoghue M, Hsieh F, Baronas E, Godbout K,
Gosselin M, Stagliano N et al. A novel
angiotensin-converting enzyme-related
carboxypeptidase (ACE2) converts angiotensin
I to angiotensin 1-9. Circ Res 2000;87:E1-9.
35 Burrell LM, Johnston CI, Tikellis C, Cooper
ME. ACE2, a new regulator of the renin-
angiotensin system. Trends Endocrinol Metab
2004;15:166-9.
36 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biological variation: pros, cons
and progress. Scand J Clin Lab Invest
1999;59:491-500.
37 College of American Pathologists. 2007 Survey
Participant Summary Report. Northfield, IL:
CAP; 2007.
38 Shihabi ZK, Scaro J. Liquid-chromatographic
assay of angiotensin-converting enzyme in
serum. Clin Chem 1981;27:1669-71.
39 Buttery JE, Chamberlain BR. A scheme for
determining the correct activity of the kinetic
angiotensin-converting enzyme. Clin Chem
1985;31:645-6.
40 Alves MF, Araujo MC, Juliano MA, Oliveira
EM, Krieger JE, Casarini DE et al. A
continuous fluorescent assay for the
determination of plasma and tissue angiotensin
I-converting enzyme activity. Braz J Med Biol
Res 2005;38:861-8.
41 Danilov SM, Deinum J, Balyasnikova IV, Sun
ZL, Kramers C, Hollak CE, Albrecht RF.
Detection of mutated angiotensin I-converting
enzyme by serum/plasma analysis using a pair
of monoclonal antibodies. Clin Chem
2005;51:1040-3.
161
Angiotensin-Converting Enzyme (ACE)

Table 1: Selected ACE Methods.

Method 1. Fluorometry [16]
Principle of analysis: Hydrolysis of L-Hip- L-His-L-Leu (HHL)
Detection: Measurement of the fluorescence of the ortho-phthaldialdehyde-His-Leu adduct
Comment: Hydrolysis of His-Leu by dipeptidases present in specimens may lower ACE activity by
destroying the fluorescence-emitting compound.
Method 2. Enzyme inhibition [9]
Principle of analysis: Binding of a radiolabeled specific inhibitor (compound 351A, an analog of
enalapril) to the active center of ACE
Detection: Count radioactivity
Comment: 125I-labeled ACE inhibitor (a p-hydroxybenzamidine derivative of N-(1-carboxy-3-
phenylpropyl)-L-lysyl-L-proline)
Method 3. Liquid chromatography [38]
Principle of analysis: This assay is based on separation and quantification of hippuric acid (under
conditions of reverse phase) released from HHL by serum ACE.
Detection: The column effluent is monitored at 254 nm.
Comment: This method may be performed on the same column as that used for theophylline assay,
using the same solvents and without an evaporation step.
Method 4. Kinetic spectrophotometry [39]
Principle of Analysis: Hydrolysis of N-[3-(2-furyl)acryloyl]-L-phenylalanyl-
glycylglycine (FAPGG), to N-[3-(2-furyl)acryloyl]-L-phenylalanine (FAP) and glycylglycine (GG)
Detection: Measure change in absorbance at 340 nm (or 345 nm). The change in absorbance is
converted into enzyme activity by a factor derived from the absorbance change produced by hydrolysis
of the substrate or a concentration of 1 mmol/L.
Comment: FAPGG absorbs more strongly than FAP at 340 nm; GG does not absorb at 340 nm.
Method 5. Continuous fluorescent assay [40]
Principle of analysis: Hydrolysis of internally quenched fluorescent peptides (e.g., orthoaminobenzoic
acid-Phe-Arg-Lys-2,4-dinitrophenyl-P-OH).
Detection: Continuous recording of the fluorescence (ex = 320 nm; em = 420 nm)
Comment: ACE cleaves the above peptide substrate at the Arg-Lys bond.
Method 6. Immunocapture enzyme assay (ICEA) [41]
Principle of analysis: ACE is captured by monoclonal antibodies and its activity determined by a
fluorometric method using HHL as a substrate.
Detection: Measurement of the fluorescence of the ortho-phthaldialdehyde-His-Leu adduct
Comment: This method is used for detection of mutated ACE in plasma and may be useful for
differential diagnosis of increased ACE activity.
162
Anion Gap

Anion Gap
Tony Badrick
Name: Anion Gap
Clinical significance: Refer to Chapter 28, Physiology of Body Water and Electrolytes, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Computation formula: Na+ (Cl + CO2) or (Na+ + K+) (Cl + CO2)

Principles of Analysis and Current Usage and has a typical reference interval of 7 to 16 mmol/L if
The anion gap is the difference between the measured using Formula 1, or 10 to 20 mmol/L if potassium is
cations and the measured anions and is an estimate of included in the calculation, as in Formula 2.
unmeasured anions, based on the electroneutrality of
i It can be seen from the above that an increased anion gap
total ions in solution
. The major anions and cations in plasma are: would occur in the following situations: (1) an increase
in the unmeasured anions (protein, sulphate, phosphate,
Cations mEq/L Anions mEq/L organic acids) listed above; (2) the presence of an
abnormal anion such as a drug; and (3) a decrease in the
Sodium 140 Chloride 100 unmeasured cations magnesium and calcium.
Potassium 4 Bicarbonate 27
Calcium 5 Protein 15 The anion gap may be useful clinically in the
Magnesium 1 Phosphate 2 investigation of a metabolic acidosis which can be
Sulphate 1 classified as having either an elevated anion gap or a
Organic acids 5 normal anion gap.
Total 150 150
Reference and Preferred Methods
Although we are actually looking at charge There are no reference or preferred methods; this is a
measurements in mEq/L, these are equivalent to calculation.
millimoles for the anions and cations used in the
calculation. For consistency, this terminology is used Specimen
hereafter. Note that the mEq/L of protein above does not Serum or plasma.
equate to mmol/L of protein.
If all of these anions and cations were measured, there Reference Intervals
would be no gap; however, usually only the major Reference intervals differ for different patient
ionsnamely sodium, chloride, bicarbonate, and populations. The reference interval for ambulatory
potassiumare measured. Calculations of the anion gap patients is 7 to 18 mmol/L and for hospitalized patients,
may be made in two ways: with and without potassium. 6 to 10 mmol/L. However, the reference interval was
Because potassium-concentration changes are relatively reviewed more recently by Paulson et al. [2], who
small compared to the other three analytes, it is often showed that the true mean anion gap was about 7
dropped from calculations [1]. mmol/L rather than the 12 mmol/L commonly cited [3].
In addition, they showed that there was a variance of
The anion gap is calculated as: about 7 mmol/L between instruments, making these
values instrument dependent.
(1) Measured cations (Na) measured anions (Cl + CO2)
or Interpretation
(2) Measured cations (Na + K) measured anions (Cl + There is debate about the value of the anion gap, both
CO2), clinically in the investigation of metabolic acidoses and
as a quality-control tool [1,4,5].

The main clinical utility of the anion gap is in classifying

i
Anion gap metabolic acidosis as either an increased anion gap,
Previous and current authors of this method: associated with a metabolic acidosis, or a normal gap,
First edition: W. Gregory Miller associated with hyperchloremic acidosis [6]. Some
Methods edition: P. Phillip Anderson and W. Gregory believe that the finding of an elevated anion gap is
Miller virtually synonymous with a metabolic acidosis [7] and
Second edition: P. Phillip Anderson and W. Gregory that the determination of an anion gap is mandatory
Miller whenever one is facing a suspected metabolic acidosis of
Third edition: P. Phillip Anderson and W. Gregory undetermined cause [8].
Miller
Fourth edition: P. Phillip Anderson and W. Gregory However, Gabow et al. [5] have shown that only when
Miller the anion gap is greater than 30 mmol/L is an organic
Fifth edition: Tony Badrick
163
Anion Gap
acidosis assured. Even at anion gaps of 20 to 29 mmol/L,
approximately one third of patients did not have an
organic acidosis.
Using the anion gap as a screen for lactic acidosis has
been shown to be insensitive, with normal anion gaps
present in patients with lactates as high as 9.9 mmol/L
[9].
Low anion gaps usually suggest an analytical failure,
dilution, hypoproteinemia (usually hypoalbuminemia,
since albumin represents approximately 75% of the
unmeasured anions in the anion gap), or multiple
myeloma, although the clone of immunoglobulin is
important because IgG tends to be cationic, whereas IgA
tends to be anionic at pH 7.4 [10].
Goldstein et al. [11] have shown that in the majority of
cases, low anion gaps are not reproducible and suggest a
spurious result. Samples lose CO2 while waiting to be
analyzed, thus the CO2 concentrations fall, and the anion
gap is increased. Patients who actually have negative or
low anion gaps, such as those with respiratory acidosis,
may appear to have normal anion gaps and thus could be
missed. However, since the CO2 decreases only about 4
mmol/L in the first hour in an uncapped tube [12], the
problem of missing low or negative anion gaps may not
be severe.
The anion gap is not meant to be a total quality-control
program for electrolyte analyses, but rather should be
considered a supplement to a quality-control program for
the individual analyses. In fact, it has been shown that a
combination of simple control rules used with the
individual components (sodium, chloride, and
bicarbonate) is more powerful than using anion gaps
[13].
Causes of a metabolic acidosis with an elevated anion
gap include ketoacidosis, lactic acidosis, renal failure
(due to retention of phosphoric, sulfuric, and organic
acids) and ingestions of compounds that are metabolized
to an organic acid (ethanol, methanol, salicylate,
ethylene glycol).
Metabolic acidosis with a normal anion gap occurs with
a loss of bicarbonate (diarrhea) or inability to secrete
hydrogen ion through the renal tubules (renal tubular
acidosis).
These various causes of abnormal anion gaps are
summarized in Table 1.
Anion Gap Performance Goals
The imprecision of the anion gap is determined by
summing the imprecision of the individual components
of the calculation, that is SD2anion gap = SD2Na + SD2Cl +
SD2HCO3. Thus the typical value of the SD is
approximately 2.7 mmol/L. If the true anion gap is 10
mmol/L, there is a 95% chance that the calculated value
will lie between 4.6 and 15.4 mmol/L (mean plus or
minus 2 SD).
Because of these analytical considerations, the anion gap
is too insensitive to be clinically useful in the
investigation of metabolic acidosis, and the direct
measurement of lactate and/or betahydroxybutyrate are
more useful options to follow. It also has limited value
as a quality-control tool because it is the sum of three (or
four) measurements, and error in any one of these cannot
be directly identified; an error in two or more may
cancel each other out. Use of the anion gap cannot
replace individual quality-control programs for its
component analytes.
References
1 Natelson S. On the significance of the
expression anion gap. Clin Chem 1983; 29:
282-283.
2 Paulson WD, Roberts WL, Lurie AA, Koch
DD, Butch AW, Aguanno JA. Wide variation in
serum anion gap measurements by chemistry
analyzers. Am J Clin Pathol 1998; 110: 735-
742.
3 Witte DL, Rodgers JL, Barrett DA. The anion
gap: its use in quality control. Clin Chem 1976;
22: 643-646.
4 Badrick T, Hickman PE. The anion gap: a
reappraisal. Am J Clin Pathol 1992; 98: 249-
252.
5 Gabow PA, Kaehny WD, Fennessy PV.
Diagnostic importance of an increased serum
anion gap. N Engl J Med 1980; 303: 854-858.
6 Gabow PA. Fluids and electrolytes: clinical
problems and their solutions. Boston: Little,
Brown & Co; 1983; pp. 23-42.
7 Emmett ME, Narins RG. Clinical use of the
anion gap. Medicine 1977; 56: 38-54.
8 Jacobsen D, Bredensen JE, Eide L, Ostborg J.
Anion and osmolal gaps in the diagnosis of
methanol and ethylene glycol poisoning. Acta
Med Scand 1982; 212: 17-20.
9 Iberti TJ, Leibowitz AB, Papadakos PJ, Fischer
EP. Low sensitivity of the anion gap as a screen
to detect hyperlactemia in critically ill patients.
Crit Care Med 1990; 18: 275-277.
10 Moe OW, Fuster D. Clinical acid-base
pathophysiology: disorders of plasma anion
gap. Best Practice &Research Clinical
Endocrinology & Metabolism 2003; 17: 559-
574.
11 Goldstein RJ, Lichtenstein, NS, Souder D. The
myth of the low anion gap. JAMA 1980; 96:
874-876.
12 Gambino RS, Schreiber H. The measurement of
CO2 content with the autoanalyzer. Am J Clin
Pathol 1966; 45: 406-411.
13 Cembrowski GS, Westgard JO, Kurtycz DF.
Use of anion gap of the quality control of
electrolyte analysers. Am J Clin Pathol 1983;
79: 1697-1698.
164
Table 1: Causes of Changes in the Anion Gap
Causes of Increased Anion Gap
Decreased unmeasured cations:
Hypocalcemia
Hypomagnesemia
Increased unmeasured anions:
Associated with metabolic acidosis:
Uremia (renal failure)
Ketoacidosis
Lactic acidosis
Salicylate poisoning
Ethanol
Methanol formic acid
Ethylene glycol glycolic acid
Paraldehyde
Pyroglutamate
Not necessarily associated with
metabolic acidosis:
Hyperphosphatemia
Hypersulfatemia
Large doses of antibiotics (such as
penicillin and carbenicillin
Presence of IgA myeloma protein
Treatment with lactate, citrate, or
acetate
Increase in net protein charge as in alkalosis
Increase in bromide leading to renal loss of
chloride (pyridostigmine bromide)
Anion Gap
Causes of Decreased Anion Gap
Decreased unmeasured anions:
Hypoalbuminemia
Hypophosphatemia
Increased unmeasured cations:
Hypercalcemia
Hypermagnesemia
Paraproteins
Polyclonal gamma globulins (IgG)
Drugs such as polymyxin B or lithium
Underestimation of serum sodium:
Hyperproteinemia (myeloma protein)
Hypertriglyceridemia (turbidity)
Overestimation of serum chloride:
Bromism
Turbidity (for ferric thiocyanate
method)
 165
Anticonvulsant Drugs

Anticonvulsant Drugs
i
Paul Salm
Paul J. Taylor
Julia M. Potter

Name: Valproic acid Phenobarbital Phenytoin

Clinical significance: Refer to Chapter 42, Nervous System, in the 4th Edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C8H16O2 C12H12N2O3 C15H12N2O2
Molecular weight: 144.21D 232.23D 252.26D
Merck Index: 9574 7032 7130
Chemical class: Carboxylic acid Barbiturate Hydantoin
Structure: click here click here click here

do not prepare derivatives of the anticonvulsant drugs. viref

Principles of Analysis and Current Usage 1
Most anticonvulsant drugs have specific ultraviolet
spectral characteristics (Anticonvulsant Drugs: Figure 1)
that were exploited in the initial attempts to monitor the
ii
therapeutic levels of these drugs. ref The drugs were
extracted into an organic solvent and reextracted into
aqueous solution, and the absorbance of the solution
recorded (method 1, Methods Summary Table). By use of
the appropriate solvents, the drugs could be extracted with
relative specificity. By recording the difference spectra of
the extract at two pH values, one could also increase the
specificity. The difference spectra were obtained by use of a
dual-beam spectrophotometer and placement of one solution
into the reference compartment and the other into the usual
test compartment.
A second method for measuring anticonvulsant
drugs involved extraction and separation by thin-layer
iii
chromatography. ref Quantitation was achieved by either
ultraviolet scanning of the plate or by elution of the drug
from the plate and recording of its ultraviolet absorption
(method 2, Methods Summary Table).
Gas chromatographic techniques (GC) were for
many years the primary technique used to analyze
antiepileptic drugs in biological fluids (method 3, Methods
iv
Summary Table). ref In a review by Rambec and Meijer
vref
, flame ionization detection (FID) or the more sensitive
nitrogen selective detection (NSD) systems were the most
widely used. NSD may have considerable advantage over
FID in terms of specificity and sensitivity, since almost all
antiepileptics except valproic acid contain nitrogen.
However, NSD requires more frequent and careful
maintenance. Approximately half of the gas
chromatographic methods described by Rambec and Meijer
11
This chapter was originally prepared by:
Paul Salm
Paul J. Taylor
and Julia M. Potter
However, of those procedures that use derivatization,
methylation is most commonly used. viiref In the past,
column support materials most frequently used were acid-
washed silanized materials (e.g., Gas Chrom-Q,
Chromosorb-W, and Supelcoport). Nowadays fused-silica
capillary columns are preferred because of their high
temperature stability and inertness, which not only removes
the need for derivatization, but offers high sensitivity,
selectivity, and speed. viiiref In the method described by
Volmut et al. ixref, six antiepileptic drugs may be analyzed
simultaneously by capillary gas chromatography.
High-performance liquid chromatography assays
(HPLC) require some form of sample preparation before
separation on a column (method 4, Methods Summary
Table). Sample preparation usually involves either organic
solvent extraction of the drugs or protein precipitation.
Nearly all methods use reverse-phase chromatography with
x
ultraviolet absorption spectrometry for detection. ref
Immunological procedures (methods 5a to 5i,
Methods Summary Table) developed for the measurement
of anticonvulsants include radioimmunoassay (RIA),
enzyme immunoassay (EIA), fluorometric enzyme
immunoassay (FEIA), fluorescence immunoassay (FIA),
fluorescence polarization immunoassay (FPIA),
nephelometric inhibition immunoassay (NIA), cloned
enzyme donor immunoassay (CEDIA), and dry-film
multilayer immunoassay (DFMI).
The RIA procedures (method 5a, Methods
Summary Table) involve the standard competitive binding
procedure in which there is competition between the labeled
drug with that from the sample for a limited amount of
xi
antibody. ref
The introduction of the enzyme-multiplied
immunoassay technique (EMIT) into the clinical chemistry
laboratory in the 1970s made possible the routine
monitoring of all the anticonvulsant drugs (method 5b,
Methods Summary Table). This homogeneous enzyme
immunoassay is based on the competitive protein-binding
 166
Anticonvulsant Drugs
technique, with an enzyme as the label and an antibody as
the binding protein. When the enzyme-labeled drug
becomes bound to an antibody to the drug, the activity of
the enzyme is reduced. Free drug in the sample competes
with the enzyme-labeled drug for binding to the antibody,
decreasing the amount of antibody-bound labeled drug, and
thereby decreases the antibody-induced inactivation of the
enzyme. The enzyme activity then correlates with the
concentration of the free drug.
Fluorometric enzyme immunoassay (FEIA) is a
competitive binding method that is based on the radial
partition immunoassay principle that combines solid phase
immunological techniques and radial chromatography
xii
(method 5c, Methods Summary Table). ref In FEIA the
drug of interest in the clinical sample is premixed with
enzyme-labeled conjugate and spotted on to a glass-fiber
filter paper which contains immobilized antibody at the
analysis site. These two constituents then compete for
antibody binding sites. After a suitable incubation period,
unbound label is removed from the center of the reaction
zone through radial elution by applying a wash solution.
This solution contains the substrate for the enzyme that
initiates enzymatic activity simultaneously with the wash.
The enzymatic rate of the bound fraction at the center of the
reaction zone is quantified by front surface fluorescence and
is inversely proportional to the concentration of drug present
in the sample. xiiiref
The principles of competitive protein binding to
measure levels of these drugs in serum or plasma have also
xiv
been used. ref (method 5d, Methods Summary Table).
With this method the anticonvulsant drug is labeled with a
derivative of the fluorogenic enzyme substrate umbelliferyl-
b-d-galactoside. This fluorogenic drug reagent (FDR)
remains nonfluorescent under the conditions of the assay,
until hydrolysis catalyzed by b-alactosidase yields the
fluorescent product. When antibody to the anticonvulsant
drug reacts with the FDR, the FDR is prevented from acting
as a substrate for the b-galactoside. Competitive binding
reactions are set up with a constant amount of FDR, a
limiting amount of antibody, and a patient sample
containing the anticonvulsant, as follows:
1. FDR + Antibody + Anticonvulsant
2. AntibodyFDR + Antibodynticonvulsant + FDR +
Anticonvulsant
3. FDR b-Galactosidase Umbelliferone + Galactose
The anticonvulsant in the sample competes with
the FDR for the limited number of antibody-binding sites.
The FDR not bound to antibody is hydrolyzed by -
galactosidase to produce the fluorescent product. Therefore
the fluorescence produced is proportional to the
anticonvulsant concentration in the sample. The intensity of
the fluorescence is related to the sample anticonvulsant drug
concentrations by means of a standard curve.
Fluorescence polarization immunoassay (FPIA)
uses the principle of competitive binding assay and
measures binding of tracer directly by fluorescence
polarization (method 5e, Methods Summary Table). The
fluorescent tracer consists of fluorescein covalently attached
to the drug being analyzed. When excited with polarized
light, this tracer will emit light with a degree of polarization
that is inversely proportional to its rate of rotation; the more
slowly the tracer molecule rotates, the greater the
polarization signal of the emitted light. When a specific
antibody binds to the tracer, its rotation is slowed and the
emitted light increases in polarization. It is a competitive
binding assay in which a specific fluorescein-labeled
anticonvulsant competes with endogenous drug from a
patient sample for a limited amount of antibody. The
amount of bound labeled anticonvulsant is inversely
proportional to the drug in the patient sample. The intensity
of the polarized fluorescence is proportional to the amount
of antibody-bound labeled anticonvulsant. The greater the
polarized fluorescence, the smaller the amount of drug in
the patient sample.
Another method for the measurement of phenytoin
and phenobarbital employs a labeled prosthetic group. In
this assay, a prosthetic group necessary for the activity of
the enzyme is covalently linked to the drug to be measured:
for example, phenytoin linked to flavine adenine
dinucleotide (FAD) (method 5f, Methods Summary Table).
The FAD is necessary for the enzyme apoglucose oxidase to
xv
function. ref If a limited amount of antibody to the drug is
present, the drug will bind to the antibody. In the presence
of drug in the patient sample, less FAD-labeled drug will
bind to the antibody, making more FAD available to bind to
the apoprotein. The enzyme is then activated, and the
amount of activity is proportional to the amount of drug in
the patient sample. The enzyme converts glucose and O2 to
gluconolactone and H2O2. The color reaction is generated
by use of peroxidase and a dye substrate 3,3' ,5,5'-
tetramethylbenzidine, which, when oxidized, forms a blue
colored product. In this assay, the patient serum or plasma
sample is first diluted with distilled water, and a specific
volume of the prepared specimen is placed on the reagent
area of the strip. The strip is then placed in the instrument, a
solid-phase reflectance photometer. The strip color is at 740
nm, and the results are calculated from a calibration curve.
The reaction sequence is as follows:
 167
Anticonvulsant Drugs
A rate nephelometric inhibition immunoassay is
also commonly used (method 5g, Methods Summary
Table). In this assay the anticonvulsant is covalently linked
to a protein. Drug from the patients sample competes with
this
protein-bound drug for binding to an antibody. The rate of
xvi
change of light scatter is measured by the instrument. ref
A homogeneous immunoassay technique, cloned
enzyme donor immunoassay (CEDIA), is a unique method
that utilizes genetically engineered b-galactosidase enzyme
fragments (method 5h, Methods Summary Table). The
CEDIA principle is based on the use of two inactive b-
galactosidase fragments which have been constructed using
recombinant DNA techniques. One of these is designated
the enzyme donor (ED), a small amino-terminal peptide,
and the other is the enzyme acceptor (EA), a larger protein
xvii
that has residues deleted near the amino terminus. ref The
EA and ED each on their own show no enzymatic activity;
however, active b-galactosidase forms when the fragments
recombine spontaneously. The formation of this active
enzyme can be measured spectrophotometrically by the
hydrolysis of a chromogenic substrate such as o-
nitrophenyl-b-d-galactopyranoside. A ligand molecule, such
as a drug or hormone, can be covalently linked to the ED
peptide forming a EDligand conjugate, such that the
enzyme complementation reaction is not affected. Adding a
ligand-specific antibody to the system will block or inhibit
spontaneous assembly of the enzyme by binding to the ED
ligand conjugate and therefore decrease the amount of
enzyme formed. Active enzyme formation is influenced by
the concentration of antibody available for binding to the
EDligand conjugate. In turn the concentration of available
antibody is dependent on the concentration of the analyte in
the sample that can competitively bind to the antibody.
xviiiref
The amount of enzyme created and the rate of
substrate hydrolysis is directly proportional to the sample
analyte concentration.
Another procedure is based on a combination of
multilayer film technology and competitive immunoassay
xix
(method 5i, Methods Summary Table). ref A coated
multilayer film chip is encased in a plastic test module. The
serum sample is applied to the topcoat layer in the test
module. This layer contains buffer components, surfactants
and other reagents (ie. antibody and labeled hapten drug) as
well as acting as a filter to prevent protein interference with
the immunoreaction. The sample travels from the topcoat
layer through an iron oxide screen to the signal layer. The
iron oxide serves to block excitation of the label outside the
signal layer. The signal layer contains a monoclonal
antibody specific for the drug being assayed conjugated
with a fluorescent-labeled hapten in an agarose matrix
coated onto a clear polyster film base. The sample antigen
displaces the labeled antigen from the binding sites of the
antibodies. The unbound labeled antigen then diffuses into
the upper layers where it cannot be measured. Equilibrium
occurs within 35 min, after which the fluorescent signal
from the remaining bound conjugate is measured from the
bottom of the test module by front surface fluorimetry. The
fluorescent intensity measured is inversely proportional to
the antigen concentration in the serum sample.
Reference and Preferred Methods
The ideal analytical procedure for the
anticonvulsant drugs should be as follows:
1. Both accurate and precise, affording the reliable
quantitation of the antiepileptic drugs.
2. Technically easy, so that little effort is required in
training to perform the task.
3. Rapid, to minimize delay in reporting results to
clinician. Many requests for drug quantitation arise as a
result of problems in patient management.
4. Sensitive, to allow analysis for drugs in a
microscale sample and permit simultaneous analysis of the
most commonly used drugs. Many patients with epilepsy
are on multiple-drug regimens.
Unfortunately, no method meets all these
requirements, and the decision on which procedure to adopt
depends largely on the laboratory conditions and on
personal preferences of those involved.
Spectrophotometric methods were initially used for
the measurement of anticonvulsant drug concentrations in
xx
body fluids. ref Although differential extraction
procedures were developed to overcome interference by
other substances, particularly drugs, the specificity of these
and other wet-chemistry procedures is questionable, and
the possibility of drug interferences still exists. For these
reasons, alternative modes of analysis were developed,
employing either chromatographic or immunological
procedures (Anticonvulsant Drugs: Comparison of methods
conditions).
Thin-layer chromatography procedures have been
described that semiquantitate or quantitate many
xxi
anticonvulsant drugs. ref This procedure, because of both
its lack of specificity and semiquantitative nature, is not a
very popular mode of analysis.
Gas chromatography (GC) affords reliable analysis
for anticonvulsant drugs on microscale samples. The
procedure permits the simultaneous analysis of all the
anticonvulsant drugs including valproate, and these methods
are easily automated. This simultaneous determination is an
obvious advantage as many patients with epilepsy are on
multiple drug regimens. Similar to high performance liquid
chromatography (HPLC), GC is most useful in laboratories
with a large workload. Disadvantages of GC include the
requirements of specialized equipment and considerable
expertise. GC is one of the methods of choice for the
xxii
analysis of valproic acid ref primarily because the poor
ultraviolet absorption characteristics of this compound
prevents its analysis by HPLC. Although steadily losing
ground to both HPLC and some of the competitive binding
assays, GC remains a popular method for anticonvulsant
drug analysis.
The adoption of HPLC for therapeutic drug
monitoring has been one of the most significant advances in
 168
Anticonvulsant Drugs
the clinical laboratory. The early methods used
(spectrophotometric methods, thin-layer chromatography,
and gas chromatography) have been largely replaced by
HPLC.
Some advantages of HPLC over earlier
techniques:
1. Analyte volatility and thermal stability, so essential
for gas chromatography, are not required for this type of
chromatography.
2. Relatively little sample workup is required before
analysis.
3. Characteristically, methods involving HPLC
require only a short analysis time.
4. Derivatization of the analyte is not required.
5. For laboratories with a large workload, the cost per
analysis is very low because reagent costs are low.
6. HPLC affords good sensitivity. The sensitivity
varies with the drug, but reliable quantitation at drug
concentrations as low as 1 g/mL can often be achieved.
7. HPLC methods are readily automated. Routine
analyses therefore require a minimum of technician time, a
major factor in arriving at the low cost per analysis.
8. HPLC makes possible the simultaneous analysis of
most anticonvulsant drugs in a microsample.
Some limitations of HPLC in drug analysis are as
follows:
1. Spectrophotometric, fluorometric, and
amperometric detectors are commonly used. Therefore the
analyte in question must either absorb light, fluoresce, or be
electrochemically active.
2. Equipment is expensive. Investment of capital is
only warranted if the laboratory has a substantial work load.
3. Although HPLC analysis of valproic acid has been
described these procedures require post column reactions or
precolumn derivatization.xxiiiref The methods of choice for
the analysis of valproic acid are gas chromatography,
enzyme immunoassay, or fluorescence polarization
immunoassay.
Many antiepileptic drugs can be determined by
xxiv
radioimmunoassay. ref Although extremely sensitive,
these techniques have the disadvantages of not allowing for
the simultaneous assay of drugs in patients on multiple-drug
regimens and their relatively long analysis time. They
require the availability of a liquid or gamma-ray scintillation
counter and availability and stability of suitable antisera.
Specimen
Plasma or serum may be used for the gas
chromatographic or high-performance liquid
chromatographic methods. Serum is the preferred specimen
for the immunoassays. Sera containing the anticonvulsants
discussed in this chapter can be stored at room temperature
for several hours. If frozen at -20 C , samples containing
these drugs are stable for at least 1 year. Saliva may also be
used for the measurement of some anticonvulsants. The
saliva concentration provides an estimate of the free drug
concentration for phenytoin and carbamazepine. The use of
collection tubes containing gel has been reported to decrease
the concentrations of some drugs in serum and plasma. Gel
tubes should be avoided when collecting specimens for
analysis of anticonvulsants.
Interferences
Icteric or hemolyzed specimens have less effect on
the chromatographic methods than on immunoassays.
Anticonvulsant Drugs Reference Interval
The generally accepted therapeutic ranges for the
major anticonvulsant drugs which are subject to therapeutic
drug monitoring are as follows:
Carbamazepine 412 mg/L (1751 mol/L)
Ethosuximide 40100 mg/L (280460 mol/L)
Phenobarbital 1530 mg/L (65170 mol/L)
Phenytoin 1020 mg/L (4080 mol/L)
Primidone 512 mg/L (2355 mol/L)
Valproate 50100 mg/L (350700 mol/L)
Interpretation
Therapeutic drug monitoring of anticonvulsants is
the best established of any medical therapy. The prevention
of any single seizure is important, as each episode is
potentially a life-threatening event. Epilepsy is a general
term for a group of disorders in which there are recurrent
seizures. The causes of epilepsy are numerous. In many
patients, a particular pathological brain lesion is not
identified (i.e., the seizures are idiopathic). The major
known causes of epilepsy include head injury,
cerebrovascular accidents (strokes), tumors (both primary
and secondary), infections (meningitis and encephalitis),
electrolyte disturbances, and alcohol and other drugs. The
description of a seizure determines the category or type of
epilepsy (Anticonvulsant drugs: Seizure Table). Such
definition is important, for different forms of epilepsy
respond to different anticonvulsant drugs. It should not be
overlooked that the treatment of some secondary seizures
(e.g., hypoglycemia, electrolyte disturbances, fever) is
primarily the treatment and correction of the underlying
problem.
The major drugs which are used currently in the
treatment of epilepsy can be divided into three groups with
xxv
ref All alter
regard to their mechanisms of action.
neuronal conductance of ions, particularly sodium and
calcium. Phenytoin and carbamazepine inhibit high-
frequency, repetitive neuronal firing. Phenobarbital and
valproic acid enhance the synaptic inhibition associated with
gamma-aminobutyric acid (GABA) as do the
benzodiazepines (e.g., diazepam and clonazepam).
Ethosuximide alters calcium currents in the thalamus.
Plasma/serum reference intervals for all these drugs, apart
from the benzodiazepines, have been established to assist
with their clinical use in the control of epilepsy. In the case
of benzodiazepines, there is as yet insufficient data to
warrant their routine monitoring. In the past few years the
practice of monotherapy with anticonvulsant drugs has
 169
Anticonvulsant Drugs
become increasingly accepted as desirable, i.e., a concerted
attempt should be made to control an individual patients
seizures with a single drug rather than multiple
anticonvulsants. Patients on monotherapy tend to have a
lower incidence of side effects and drug interactions, as well
as better compliance overall. However, if it becomes clear
that in an individual patient, control is not obtained with a
single agent, then combination therapy is utilized. The
combination will comprise drugs from groups with different
mechanisms of action.
The wide acceptance of the role of therapeutic drug
monitoring of anticonvulsants is due not only to the
importance of good clinical control of epilepsy, but to the
widely varying pharmacokinetics of the drugs between
individuals. The major pharmacokinetic parameters are
summarized in the Anticonvulsant drugs Table:
Pharmacokinetic parameters. The timing of the sample to be
assayed in relation to the dose is important. The most
appropriate sample for routine monitoring is the trough
concentration, i.e., that immediately prior to the next dose of
the drug. When introducing a drug or adjusting the dose, it
is not generally useful to measure serum concentrations
until the patient has reached steady state. From a practical
point of view, the steady state may be considered as being
approached at a time interval equivalent to five times the
elimination half-life, e.g., in the case of primidone
approximately 2 days, for phenytoin more than 5 days, and
for phenobarbital more than 2 weeks.
The plasma therapeutic reference intervals shown
are total concentrations. If a drug is highly protein bound, a
decrease in binding protein may potentially increase the
unbound moiety of drug. If in addition, there is rate-limited
clearance of that drug, then the unbound moiety will
increase and there will be a high likelihood of clinical
toxicity. Such is the case with phenytoin. At the same time
the total plasma concentration may remain within the
reference interval. In these circumstances, some
practitioners would also measure the free phenytoin
concentration, utilizing equilibrium dialysis or
ultracentrifugation.
Anticonvulsant Drugs Performance Goals
Survey data from the 2002 College of American
Pathologists Participant Summary Report shows
imprecision values (% coefficient of variation) for
valproate, phenobarbital, and phenytoin to range from 3.5%
to 7.5%, 3.0% to 11.0%, and 3.0% to 10.0%, respectively,
in samples with drug concentrations within the therapeutic
range.xxviref Acceptable performance criteria (CLIA-88)
for measurement of carbamazepine, phenytoin, primidone,
and valproic acid require that laboratories be accurate to
within 25% of the peer group mean, while phenobarbital
and ethosuximide results must be within 20% of the peer
group mean.
 170
Anticonvulsant Drugs

Tables
Anticonvulsant Drugs Methods Summary Table
Method 1: Extraction and ultraviolet spectroscopy
Principle of analysis: Anticonvulsant extracted free of other ultraviolet compounds; ultraviolet spectrum or the
ultraviolet difference spectra are specific for the drug
Comments: Interference with drugs and endogenous compounds
Method 2: Thin-layer chromatography (TLC)
Principle of analysis: Extracted drug separated by TLC and its Rf noted; drug quantitatively eluted and quantified by
ultraviolet spectroscopy
Comments: Difficult to separate all anticonvulsants; technically difficult to achieve reproducibility
Method 3: Gas chromatography (GC)
Principle of analysis: C18 solid phase extraction; capillary column; flame ionization detection
Comments: Can resolve all anticonvulsants simultaneously; requires simple extraction
Method 4: High performance liquid chromatography (HPLC)
Principle of analysis: Organic solvent extraction of drugs or protein precipitation of sample; anticonvulsants are
separated by reversed-phase chromatography and monitored by ultraviolet spectroscopy
Comments: Can resolve all anticonvulsants except valproate simultaneously; commonly used
Method 5: Competitive-binding assays
a. Radioimmunoassay (RIA)
Principle of analysis: Competitive binding of radioactive ligand; radio-labeled hapten competes with unknown for
antibody binding site
Comments: All problems of radioactive usage; slower than other immunoassays
b. Enzyme-multiplied immunoassay technique (EMIT)
Principle of analysis: Competitive binding with drug attached to enzyme
Comments: Can measure all drugs, but each must be done separately; commonly used
c. Fluorometric enzyme immunoassay (FEIA)
Principle of analysis: Combination of competitive binding with enzyme-labeled conjugate for antibody binding site (on
a solid-phase matrix) and radial chromatography
Comments: Measures all anticonvulsants except valproate and ethosuximide; use routine clinical chemistry analyzer
(e.g., Baxter Stratus); commonly used
d. Substrate-labeled fluorescence immunoassay (SLFIA)
Principle of analysis: Competitive binding substrate label; substrate competes with unknown antigen for antibody
binding site
Comments: Fluorescence interferes; not widely used
e. Fluorescence polarization immunoassay (FPIA)
Principle of analysis: Competitive binding with fluorescein-labeled drug; fluorescent drug competes with unknown for
antibody site
Comments: Each drug assayed individually; requires specialized instrument; most commonly used
f. Ames test strip
Principle of analysis: Antibody binding to drug labeled with prosthetic group prevents glucose oxidase activity;
competition with endogenous drug allows prosthetic group to activate enzyme; glucose oxidase is coupled to
colorimetric reaction
Comments: Designed for inexpensive instrument; reflectance spectrophotometer; rapid test
g. Rate nephelometric inhibition immunoassay
Principle of analysis: Competitive binding for hapten; hapten-protein competes with unknown for antibody binding site
Comments: Each drug individually assayed; requires specialized instrument
h. Cloned enzyme donor immunoassay (CEDIA)
Principle of analysis: Competitive binding with enzyme donor(ED)ligand conjugate for antibody binding site;
endogenous drug modulates enzymatic activity by influencing free EDligand conjugate available for complementation
Comments: Measures phenobarbital and phenytoin; each drug assayed individually; can use routine clinical chemistry
analyzers (e.g., BM Hitachi system); newer technology; not commonly used at present
i. Dry film multilayer immunoassay (OPUS)
Principle of analysis: Combination of competitive binding with a fluorescent labeled hapten in an agarose matrix and
multilayer film technology; labeled antigen competes with sample antigen for antibody binding site
Comments: Each drug assayed individually; requires specialized instrument; newer technology; not widely used at
present
 171
Anticonvulsant Drugs

Anticonvulsant Drugs: Comparison of method conditions.

Parameter: High-performance liquid chromatography
Temperature: Ambient
Sample volume: 100 L
Linearity (g/mL):
Phenytoin: 5.060.0
Phenobarbital: 7.590.0
Primidone: 2.530.0
Ethosuximide: 25.0300.0
Carbamazepine: 2.530.0
Valproic acid: ---
Precision (CV): 27
Parameter: Gas chromatography
Temperature: 60250 C
Sample volume: 500 L
Linearity (g/mL):
Phenytoin: 10.0100.0
Phenobarbital: 10.0100.0
Primidone: 10.0100.0
Ethosuximide: 10.0100.0
Carbamazepine: 10.0100.0
Valproic acid: 10.0100.0
Precision (CV): 18
Parameter: Enzyme-multiplied immunoassay technique
Temperature: 30 C
Sample volume: 50 L
Linearity (g/mL):
Phenytoin: 2.540.0
Phenobarbital: 5.080.0
Primidone: 2.520.0
Ethosuximide: 10.0150.0
Carbamazepine: 2.010.0
Valproic acid: 10.0150.0
Precision (CV): 612*
Parameter: Fluorescence polarization
Temperature: 37 C
Sample volume: 50 L
Linearity (g/mL):
Phenytoin: 2.540.0
Phenobarbital: 5.080.0
Primidone: 2.024.0
Ethosuximide: 10.0150.0
Carbamazepine: 2.020.0
Valproic acid: 12.5150.0
Precision (CV): 49*
Parameter: Fluorometric enzyme immunoassay
Temperature: 3740 C
Sample volume: 200 L
Linearity (g/mL):
Phenytoin: 2.540.0
Phenobarbital: 1.060.0
Primidone:
Ethosuximide:
Carbamazepine: 1.020.0
Valproic acid:
Precision (CV): 812*
*1990 College of American Pathologists Therapeutic Drug Monitoring Series 2 Surveys.
 172
Anticonvulsant Drugs

Anticonvulsant Drugs Table: Classification of epileptic seizures*

Type Characteristics
I. Partial (focal, local)
Electroencephalography (EEG) may reveal localized abnormality over seizure focus.
A. Simple partial Consciousness is not impaired. Includes Jacksonian motor epilepsy (confined to a
single muscle group or limb).
B. Complex partial Consciousness is impaired. Most commonly arise in the temporal lobe. Include many
different symptoms and behavior patterns.
C. Secondarily generalized Both (A) and (B) may spread to involve both hemispheres, with convulsions of all
limbs and loss of consciousness.

II. Generalized seizures (examples) Simultaneous initiation of epileptic activity in both hemispheres.
A. Absence Brief impairment of consciousness. Motor activity may not occur.
B. Myoclonic Isolated muscle jerks.
C. Clonic Rhythmic contractions, loss of consciousness.
D. Tonic-clonic (grand mal) Major convulsions, loss of consciousness, autonomic activity and prolonged
depression of central nervous system function.
* Modified from Commission on Classification and Terminology of the Internation League
against Epilepsy. Epilepsia 1981; 22:489-501.

Anticonvulsant Drugs Table, Pharmacokinetic properties.

Anticonvulsant: Carbamazepine
Volume of distribution (L/kg): 1.2
Percent protein binding: 75
Elimination half-life (h): 1217
Comments: Induction of own and other anticonvulsants metabolism; metabolite, carbamazepine-10,11-epoxide, may
contribute to adverse effects
Anticonvulsant: Ethosuximide
Volume of distribution (L/kg): 0.7
Percent protein binding: <10
Elimination half-life (h): 60
Comments:
Anticonvulsant: Phenobarbital
Volume of distribution (L/kg): 0.7
Percent protein binding: 2045
Elimination half-life (h): 70100
Comments: Predominantly hepatic clearance, approximately 25% renal; significant induction of hepatic cytochrome
P450 with increased clearance of many drugs, including other anticonvulsants
Anticonvulsant: Phenytoin
Volume of distribution (L/kg): 0.40.8
Percent protein binding: 8893
Elimination half-life (h): 726
Comments: Dose-dependent kinetics with saturable hepatic metabolism; induction as for phenobarbital; protein binding
important: NB hypoalbuminemia (e.g., renal disease)
Anticonvulsant: Primidone
Volume of distribution (L/kg): 0.6
Percent protein binding: <20
Elimination half-life (h): 1012
Comments: Metabolized to phenobarbital, but anticonvulsant in its own right; both primidone and phenobarbital should
be monitored concurrently
Anticonvulsant: Valproate
Volume of distribution (L/kg): 0.15
Percent protein binding: 8095
Elimination half-life (h): 520
Comments: Protein binding as for phenytoin; in addition, binding saturable at high concentrations, unbound valproate
concentrations increasing proportionately more than total serum concentration
 173
Anticonvulsant Drugs

Figures
Anticonvulsant drugs: Figure 1

Anticonvulsant Drugs: ultraviolet absorption spectra of 5 drugs in acetonitrile. Solid line carbamazepine (10g/mL); thick dashed
line, ethosuximide (100g/mL); dashed and dotted line, phenytoin (100g/mL); regular dashed, phenobarbital (100g/mL);
dotted, primidone, (100g/mL)

Anticonvulsant drugs: Figure 2

Permission for electronic not granted; see Methods in Clinical Chemistry Pesce AJ and Kaplan LA Eds Mosby St Louis 1987.
Assay scheme of a typical CEDIA assay.

Anticonvulsant drugs: Figure 3

Permission for electronic not granted; see Methods in Clinical Chemistry Pesce AJ and Kaplan LA Eds Mosby St Louis 1987.
Chromatograms obtained at 204 nm using tolybarb and methsuximide as the internal standards from serum standard A, level III
concentrations.
 174
Anticonvulsant Drugs
Anticonvulsant drugs: Figure 4
Chromatogram of a serum sample spiked with 50 g/ml VPA and ET, and 10 g/ml PB, PR, CZ and PT, with two internal
standards (CA and MPPH) at 20 g/ml. The serum and column were acidified prior to the extration with 0.5 M HCl. Injected
volume, 2 l of extract.
Procedure: High Performance Liquid Chromatography
xxvii
Analysis of Anticonvulsant Drugs ref
Principle
Five anticonvulsant drugs (primidone,
phenobarbital, carbamazepine, phenytoin and ethosuximide)
are separated by reversed-phase liquid chromatography and
quantitated by measurement of peak height relative to an
internal standard. Tolybarb is used as an internal standard
for the quantitation of primidone, phenobarbital,
carbamazepine, and phenytoin. Methsuximide is used as the
internal standard for the quantitation of ethosuximide as it
has a similar volatility when evaporating the extract to
dryness.
Specimen
Serum or plasma.
Reagents and Materials
1. HPLC-grade acetonitrile, methanol, isopropanol
and chloroform can be purchased from Fisher
Scientific (Winnipeg, Manitoba, Canada).
2. Standards. The pure drugs can be purchased from
Sigma Chemical Co., St. Louis, MO (phenytoin),
Aldrich Chemical Co., Milwaukee, WI (tolybarb,
5-ethyl-5-p-tolybarbituric acid), BDH Chemicals,
Toronto, Ontario, Canada, (phenobarbital), Ayerst
Laboratories, Motreal, Quebec, Canada
(primidone), Ciba-Geigy Canada Ltd.,
Mississauga, Ontario (carbamazepine), and Parke
Davis Canada Inc., Scarborough, Ontario
(ethosuximide and methsuximide).
3. Phosphate buffer.
a. 10 mmol/L potassium phosphate buffer
(pH 6.3) is prepared by dissolving 2.72 g
of KH2PO4 in 1900 mL of distilled water.
The pH is adjusted to 6.3 with about 2 mL
of 1 M NaOH, then the volume brought to
2 L.
b. 1 mmol/L potassium phosphate buffer
(pH 6.8) is prepared by dissolving 13.6 g
of KH2PO4 in 90 mL of distilled water.
The pH is adjusted to 6.8 with about 3 mL
of 10 M NaOH, then the volume is
brought to 100 mL.
4. Stock internal standards are prepared by
dissolving 30 mg of tolybarb and 20 mg of
methsuximide in 20 mL of methanol.
5. Solvent extraction solution is prepared by
combining 500 L of the stock internal standard
solution with 500 mL of 5% isopropanol in
chloroform.
6. Mobile phase is prepared by combining 1300 mL
of phosphate buffer (pH 6.3) with 350 mL of
methanol and 350 mL of acetonitrile. The mixture
is degassed by stirring in a magnetic mixer for 30
min, then filtered by suction through a 0.45-m
pore filter (Millipore Corp., Bedford, MA). The
apparent pH of the mobile phase is 7.0.
Assay
1. Equipment. The liquid chromatographic system
consists of a Series 2 pump, a model LC-75
variable wavelength detector, a R-100 strip chart
recorder (all from PerkinElmer Corp., Norwalk,
 175
Anticonvulsant Drugs
CT), and a model 7105 injector (Rheodyne Inc.,
Cotati, CA). A 5-m particle size, 250 4.6 mm
Supelcosil LC-1 analytical column and a 5-m
particle size, 20 4.6 mm Supelguard column
(Supelco, Bellefonte, PA) are used to effect the
drug separations.
2. Sample preparation.
a. Add 100 L of 1 mmol/L phosphate buffer (pH
6.8) and 1.5 mL of solvent extracting solution
(containing internal standard) to 100 L of serum
or plasma.
b. Vortex mix the sample for 30 sec and centrifuge
briefly to separate the two phases.
c. Aspirate the aqueous phase (top layer) to waste
and transfer the organic phase (lower layer) to a
clean tube.
d. The organic layer is evaporated at room
temperature with a dried air stream. The samples
are removed from the air stream within 2 min of
reaching dryness to avoid loss of ethosuximide and
its internal standard due to their high volatility.
e. Reconstitute the residue with 100 L of mobile
phase.
3. Chromatographic parameters.
a. Flow rate 2.0 mL/min.
b. Wavelength 204 nm.
c. Sensitivity 0.08 AUFS(absorbance units at full
scale).
d. Column temperature ambient.
e. Injection volume 4 L.
f. Run time 7 min.
A typical chromatogram is shown in the Figure 4:
Anticonvulsant Drugs HPLC Elution Pattern.
Calculations
In each sample, peak heights (PH) of each drug are
compared to the internal standard from which peak height
ratios (PHR) are calculated. Each unknown sample PHR is
compared to a calibrator PHR of known concentration. The
calculation of drug concentration is as follows.
[DRUG] = [CAL] PH of sample/ PH of internal standard
PH of calibrator/ PH of internal
standard
where [DRUG] = drug concentration (mg/L)
[CAL] = calibrator concentration (mg/L)
Bibliography
See popups in the text
Procedure: Antiepileptic Drugs Including Valproate by
xxviii
ref
Capillary Gas Chromatography
Principle
All six drugs (valproic acid, ethosuximide,
phenobarbital, primidone, carbamazepine, and phenytoin)
are extracted from acidified serum samples by a simple
solid-phase (C18) extraction process. Prior to evaporation,
basic methanol is added to the eluates from extraction
columns to convert acid drugs to their potassium salts, in
order to reduce the loss of volatile drugs. The residue is then
redissolved in acidic methanol and 2 L is injected into the
gas chromatograph equipped with a split capillary inlet
system and a flame ionization detector.
Specimen
Serum.
Reagents and Materials
1. Six antiepileptic drugs (SPOFA, Prague,
Czechoslovakia and VEB Arzneimittelwerk,
Dresden, Germany).
2. Stock standard solution of the six anticonvulsant
drugs is prepared in analytical reagent grade
methanol to a concentration of 1 g/L.
3. Stock internal standard is prepared by dissolving
40 mg of MPPH [5-(4-methylphenyl)-5-
phenylhydantoin] in 20 mL of methanol.
4. 0.5 M HCl solution in water. Dilute 4.2 mL of
concentrated HCl to 100 mL with H2O.
5. 0.05 M KOH in methanol. Add 0.281 g of KOH
to 80 mL of methanol and bring to 100 mL with
methanol.
6. 0.05 M HCl in methanol. Dilute 42 L of
concentrated HCl to 100 mL of methanol.
Assay
Equipment: A HP-5790A gas chromatograph
(Hewlett-Packard, Avondale, PA) equipped with a split
injection port (ratio 1:20) using an empty glass liner and
flame ionisation detection. A fused-silica capillary column
packed with cross-linked 5% phenylmethylsilicone gum
phase HP-5 (Hewlett-Packard), 25 m 0.20 mm ID, 0.33
m film thickness is used to separate the drugs of interest.
Sample preparation
1. Add 100 L of 0.5 M HCl and 10 L of internal
standard solution (MPPH) to 500 L of serum and
thoroughly vortex mix.
2. Precondition the extraction column (a 2 mL
polyethylene syringe packed with 200 mg of
Silipor C18) with a 1 mL wash of methanol
followed by a 1 mL wash of 0.5 M HCl.
3. Pour mixture (from step 1) onto the column and
allow the mixture to flow through. Then rinse the
column with two 1-mL volumes of H2O and dry by
applying a vacuum.
4. The drugs are then eluted from the column with 1
mL of methanol. A 50- L aliquot of 0.05 M KOH
is added to the methanol solution. The solvent is
then evaporated under nitrogen at 40 C in a
 176
Anticonvulsant Drugs

heating block. The residue is reconstituted in 50 L Specimens may be transported at room temperature.
of 0.05 M HCl in methanol. Separate serum component and store at room
5. Chromatographic parameters: temperature, up to 24 h, until time of analysis, or
a. Injection temperature 240 C prepare ultrafiltrate (Section VI.A), then freeze at -
b. Detector temperature 300 C 20C until time of testing, marking the sample as
c. Dual ramp column temperature program such.
(60250 C) (NOTE: Typical laboratory room temperature ranges
d. Nitrogen inlet pressure 100 kPa between 22 and 25 C. If temperature is >25 C,
e. Injection volume 2 L separated serum should be refrigerated at 28 C.
Grossly hemolyzed, lipemic, or icteric specimens are
Calculation unacceptable samples for routine analysis.
1. A single-point calibration:
III. Indications
Peak Height Ratio (PHR) = Peak Height of Drug
For the monitoring of phenytoin for therapeutic drug
Peak Height of Internal Standard
monitoring (TDM) purposes, as an aid in setting the
dosing regimen, and to assess toxicity, when total
[Drug] = PHR of Drug [Standard]
phenytoin drug analysis does not provide adequate
PHR of Standard
information.
where [Drug] = concentration of drug (mg/L)
[Standard] = concentration of standard (mg/L) IV. Reagents and Material
A. Bio-Rad Control Materials (Level 1 and 2) Store
at 4 C. Stable as labeled.
Bibliography B. 100-L and 1-mL Eppendorf pipets.
See popups in text C. Amicon CentriFree Micropartition Filters
(Amicon).
Free Phenytoin
Abbott TDx/FLx D. Sample Cartridges (Abbott).
E. Sample Cuvettes (Abbott).
I. Principle F. TDx Reagent Pack Adapter for FLx
The concentration of free, or unbound, phenytoin in G. Calibration and Routine Carousels (Abbott).
samples can be determined using fluorescence H. Buffer (Abbott); store at room temperature.
polarization immunoassay. After a filtration step, a The following Abbott Laboratories Phenytoin Assay
clear supernatant containing the drug is analyzed. Kit Materials:
Unlabeled drug in the sample competes with the tracer I. Phenytoin Reagent Pack* (Batch Assay).
labeled with fluorescein for limited antibody sites. J. Free Phenytoin Calibrators*.
The tracer, when excited by linearly polarized light,
emits fluorescence with a degree of polarization
*Store at 4 C. Stable for 1 month after opening.
inversely related to its rate of rotation. If the patient
contains high concentrations of the drug, the
polarization value is low. If the patient sample V. Instrument and parameters.
contains a low concentration of the drug, the
polarization value is high. Abbott TDx/FLx; however, any of the automated drug
analyzers which have a linear range to 0.5 g /mL are
acceptable

II. Specimens of Choice VI. Procedure

Serum (2 mL) drawn in a red-top Vacutainer tube
without separator gel. (NOTE: Gel tubes are not Each routine run will generally consist of Bio-
recommended, because of the potential to decrease Rad Level 1 and 2, along with patients,
drug concentration during storage. This is negated processed in singlecate.
somewhat by always aliquoting serum into a separate
pour-off tube immediately.) Recommended drawing Controls and patients require Amicon filtering;
time is the trough level, immediately prior to the next calibrators do not.
scheduled dose of drug, or during the acute phase of
toxicity.
 177
Anticonvulsant Drugs

A. Pretreatment (for Control and Patient
Specimens)
When conducting ultrafiltration prior to
measurement of free phenytoin, check that the
room temperature at that time does not exceed
25 C. If the temperature is >25 C, contact the
area supervisor. If the sample is ultrafiltered at
room temperature of 3037 C protein binding
will be affected and a different reference range
may need to be applied:
12 g/mL at 25 C.
1.53 g/mL at 37 C
1. Free phenytoin calibrators, controls, and
patient specimens should be mixed by
gentle inversion. Avoid foaming; if this
should occur, allow sample to sit until
foam dissipates.
2. Number an Amicon fFilter for each
specimen to be tested and place in a
suitable rack. A maximum of 20
specimens can be run in one batch.
3. Pipet 1 mL of control or patient sample to
be assayed into its corresponding Amicon
filter.
4. Cap the Amicon filter sample resevoir.
5. Place filter into a fixed-angle centrifuge
and centrifuge samples for 25 min.
6. After centrifugation, transfer 100 L of
ultrafiltrate into appropriate sample wells
and place in a sample carousel.
B. Calibration (no Amicon filtering required)
1. Performing the Calibration
Assays should be calibrated whenever:
A new assay is introduced
An assay activation (new reagent
pool) is issued
The memory circuit board (Board
#2) is replaced
Assay control values fall outside of
the acceptable range specified in the
specific assay section and the
control is not suspect.
Assays may require recalibration (when
indicated by unacceptable control data)
whenever:
A new lot number of reagent is used
A new lot of buffer is used
Any dispense component is replaced
Any instrument calibration
procedure is performed
A wide variation in room
temperature is experienced
The TDx/FLx System stores a calibration
curve for each assay. The random access
calibration curves are separate and distinct
from the calibration curves stored for the
batch assays. Therefore, prior to running
an assay, the instrument must be
calibrated for that assay in the mode of
operation being run.
Notes and Interpretations
A. Optimal therapeutic concentrations of free
phenytoin will generally be between 1 and 2
g/ml. Frequency and severity of toxic effects
due to drug therapy will increase as the free
phenytoin level exceeds 3.0 g/mL. Results
may be reported up to 10 g/mL (refer to
"Alert" values information.)
B. Specificity: No known interfering substances.
C. Sensitivity: 0.5 g/mL; report negatives as <0.5
g/mL.
IX. reference
Abbott Laboratories' TDx and TDx/FLx Operations Manual.

i
This chapter was originally prepared by:
Paul Salm
Paul J. Taylor
and Julia M. Potter
ii
Plaa, G.L. and Hine, C.H.:
A method for the simultaneous determination of phenobarbital and diphenylhydantoin in blood, J. Lab. Clin. Med. 47:649-
657, 1956.
Svensmark, O., and Kristensen, P.:
Determination of diphenylhydantoin and phenobarbital in small amounts of serum, J. Lab. Clin. Med. 61:501-507, 1963.
iii

Pippenger, C.E., Scott, J.E., and Gillen, H.W.:

Thin-layer chromatography of anticonvulsant drugs, Clin. Chem. 15:255-260, 1969.
 178
Anticonvulsant Drugs

Huisman, J.W.:
The estimation of some important anticonvulsant drugs in serum, Clin. Chim. Acta 13:323-328, 1966.
iv
Rambeck, B., and Meijer, J.W.A.:
Gas chromatographic methods for the determination of antiepileptic drugs: A systematic review, Ther. Drug Monit. 2:385-
396, 1980.
v
Rambeck, B., and Meijer, J.W.A.:
Gas chromatographic methods for the determination of antiepileptic drugs: A systematic review, Ther. Drug Monit. 2:385-
396, 1980.
vi
Meijer, J.W.A.:
Simultaneous quantitative determination of anti-epileptic drugs, including carbamazepine, in body fluids, Epilepsia
12:341-352, 1971.
Gardner-Thorpe, C., Parsonage, M.J.H., Smethurst, P.F., and Toothill, C.:
A comprehensive gas chromatographic scheme for the estimation of antiepileptic drugs, Clin. Chim. Acta 36:223-230,
1972.
Toseland, P.A., Albani, M., and Gauchel, F.D.:
Organic nitrogen selective detector used in gas-chromatographic determination of some anticonvulsant and barbiturate
drugs in plasma and tissues, Clin. Chem. 21:98-103, 1975.
Rambeck, B., Meijer, J.W.A.:
Comprehensive method for the determination of anti-epileptic drugs using a novel extraction technique and a fully
automated gas chromatograph, Arzneim. Forsch. 29:99-103, 1979.
vii
Rambeck, B., and Meijer, J.W.A.:
Gas chromatographic methods for the determination of antiepileptic drugs: A systematic review, Ther. Drug Monit. 2:385-
396, 1980.
viii
Plotczyk, L.L.:
Application of fused-silica capillary gas chromatography to the analysis of underivatized drugs, J. Chromatog. 240:349-
360, 1982.
ix
Volmut, J., Matisova, E., and Pham Thi Ha.: Simultaneous determination of six antiepileptic drugs by capillary gas
chromatography, J. Chromatogr 527:428-435, 1990.
x
Meatherall, R., and Ford, D.:
Isocratic liquid chromatographic determination of theophylline, acetaminophen, chloramphenicol, caffeine,
anticonvulsants, and barbiturates in serum, Ther. Drug Monit. 10:101-115, 1988.
Riedmann, M., Rambeck, B., and Meijer, J.W.A.:
Quantitative simultaneous determination of eight common antiepileptic drugs and metabolites by liquid chromatography,
Ther. Drug Monit. 3:397-413, 1981.
Soldin, S.J., and Gilbert Hill, J.:
Rapid micromethod for measuring anticonvulsant drugs in serum by high-performance liquid chromatography, Clin.
Chem. 22:856-859, 1976.
Christofides, J.A., and Fry, D.E.:
Measurement of anticonvulsants in serum by reverse-phase ion-pair liquid chromatography, Clin. Chem. 26:499-501,
1980.
Kabra, P.M., Koo, H.Y., and Marton, L.J.:
Simultaneous liquid-chromatographic determination of 12 common sedatives and hypnotics in serum, Clin. Chem.
24:657-662, 1978.
Kabra, P.M., Stafford, B.E., and Marton, L.J.:
Simultaneous measurement of phenobarbital, phenytoin, primidone, ethosuximide, and carbamazepine in serum by high-
pressure liquid chromatography, Clin. Chem. 23:1284-1288, 1977.
Ou, C.N., and Rognerud, C.L.:
Simultaneous measurement of ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine, and their bioactive
metabolites by liquid chromatography, Clin. Chem. 30:1667-1670, 1984.
Adams, R.F., and Vandemark, F.L.:
Simultaneous high-pressure liquid-chromatographic determination of some anticonvulsants in serum, 22:25-31, 1976.
Szabo, G.K., and Browne, T.R.:
 179
Anticonvulsant Drugs

Improved isocratic liquid-chromatographic simultaneous measurement of phenytoin, phenobarbital, primidone,

carbamazepine, ethosuximide, and N-desmethylmethsuximide in serum, 28:100-104, 1982.
xi
Cook, C.E., Christensen. H.D., Amerson, E.W., et al.: Radioimmunoassay of anticonvulsant drugs: phenytoin, phenobarbital,
and primidone. In Kellaway, P., and Petersen, I.S., editors: Quantitative analytical studies in epilepsy, New York, 1976, Raven
Press.
xii
Price, C.P., and Newman, D.J. editors:
Radial partition immunoassay. In Principles and practice of immunoassay, New York, 1991, Stockton Press.
xiii
Geigel, J.L., Brotherton, M.M., Cronin, P., et al:
Radial partition immunoassay, Clin. Chem. 28:1894-1898, 1982.
xiv

Wong, R.C., Burd, J.F., and Carrico, R.J., et al:

Substrate-labeled fluorescent immunoassay for phenytoin in human serum, Clin. Chem. 25:686-691, 1979.
xv
Tybach, R.:
Adaptation of prosthatic-group label homogenenous immunoassay to reagent strip format, Clin. Chem. 27:1499-1504,
1981.
xvi
Nishikawa, T., Kubo, H., and Saito, M.:
Competitive nephelometric immunoassay method for antiepileptic drugs in patient blood, J. Immunol. Methods 29:85-89,
1979.
xvii
Henderson, D.R., Friedman, S.B., Harris, J.D., et al.:
CEDIA, a new homogeneous immunoassay system, Clin. Chem. 32:1637-1641, 1986.
xviii
Engel, W.D., Khanna, P.L.:
CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique, J. Immunol. Methods 150:99-102, 1992.
xix
Jandreski, M.A., Shah, J.C., Gardincius, J., and Bermes Jr., E.W.: Clinical exaluation of five therapeutic drugs using dry
film multilayer technology on the OPUS immunoassay system, J. Clin. Lab. Anal. 5:415-421, 1991.
xx
Plaa, G.L. and Hine, C.H.:
A method for the simultaneous determination of phenobarbital and diphenylhydantoin in blood, J. Lab. Clin. Med. 47:649-
657, 1956.
Svensmark, O., and Kristensen, P.:
Determination of diphenylhydantoin and phenobarbital in small amounts of serum, J. Lab. Clin. Med. 61:501-507, 1963.
xxi

Comparison of method conditions).

Pippenger, C.E., Scott, J.E., and Gillen, H.W.:
Thin-layer chromatography of anticonvulsant drugs, Clin. Chem. 15:255-260, 1969.
Huisman, J.W.:
The estimation of some important anticonvulsant drugs in serum, Clin. Chim. Acta 13:323-328, 1966.
xxii

Willox, S., and Foote, S.E.:

Simple method for measuring valproate (Epilim) in biological fluids, J. Chromatogr. 151:67-70, 1978.
Jakobs, C., Bohasch, M., and Hanefeld, F.:
New direct micromethod for determination of valproic acid in serum by gas chromatography, J. Chromatogr. 146:494-
497, 1978.
Berry, D.J., and Clarke, L.A.:
Determination of valproic acid (dipropylacetic acid) in plasma by gas-liquid chromatography, J. Chromatogr. 156:301-
307, 1978.
Freeman, D.J., and Rawall, N.:
Extraction of underivatized valproic acid from serum before gas chromatography, Clin. Chem. 26:674-675, 1980.
xxiii
Fairinotte R. Pfaff, MC and Manhuzier G. Simultaneous determination of phenobarbital and valproic acid using high
performance liquid chromatography. Ann. Biol. Clin. 36:347-353, 1978. Gupta RN, Keane PM, and Gupta ML, Valproic acid in
plasma as determined by liquid chromatography. Clin Chem 25:1984-1985, 1979.
xxiv

Cook, C.E., Christensen. H.D., Amerson, E.W., et al.: Radioimmunoassay of anticonvulsant drugs: phenytoin, phenobarbital, and
primidone, In Kellaway, P., and Petersen, I.S., editors: Quantitative analytical studies in epilepsy, New York, 1976, Raven Press.
xxv
Albers, G.A., and Peroutka, S.J.:
Neurologic disorders. In K.L. Melmon, H.F. Morrelli, B.B. Hoffman, D.W. Nierenberg, editors: Melmon and Morrellis
clinical pharmacology: Basic principles in therapeutics, ed. 3, New York, 1992, McGraw-Hill Inc. Health Professions Division.
 180
Anticonvulsant Drugs

xxvi
All conclusions and interpretations in the Infobase with respect to the College of American Pathologists data base are those of
the author of this section or the editors of the Infobase, and not those of the College.
xxvii
Meatherall, R., and Ford, D.:
Isocratic liquid chromatographic determination of theophylline, acetaminophen, chloramphenicol, caffeine,
anticonvulsants, and barbiturates in serum, Ther. Drug Monit. 10:101-115, 1988.
xxviii
Volmut, J., Matisova, E., and Pham Thi Ha.:
Simultaneous determination of six antiepileptic drugs by capillary gas chromatography, J. Chromatogr. 547:428-435,
1990.
181
Apolipoproteins A-1 and B
Apolipoproteins A-1 and B
Jillian R. Tate
Name: Apolipoproteins A-1 (apo A-1) and B-100 (apo B)
Clinical significance: Predictors of cardiovascular disease risk. Refer to Chapter 37, Coronary Artery
Disease: Lipid Metabolism, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass: 28,300 D (apo A-1); 549,000 D (apo B-100)
Chemical class: Proteins
Reference method: HPLC-MS (apo A-1); no reference method available for apo B
Principles of Analysis and Current Usagei
Apo A-1 and B-100 are measured by immunochemical
methods. The original measurements were by gel
techniques, including radial immunodiffusion (RID) and
rocket or electroimmunoassay (EIA), and by
radioimmunoassay (RIA). Whereas the gel methods
were difficult to automate and used large amounts of
apolipoprotein antiserum, the advantage of RIA was its
increased analytical sensitivity, precision, and lesser use
of antiserum. RIA, however, is difficult to automate, and
reagents have a limited shelf life [1-3].
More recent methods include enzyme-linked
immunosorbent assay (ELISA), particle-concentration
fluorescence immunoassay (PCFIA), and
immunonephelometric (INA) and immunoturbidimetric
(ITA) assays, all of which use mono-specific antibodies
in the liquid phase. Dedicated protein analyzers using
end-point or rate INA or centrifugal analyzers using ITA
methods became available in the 1980s and 1990s in
routine clinical laboratories. In todays laboratories,
high-throughput, fully automated chemistry analyzers
measure a range of proteins by ITA or particle-enhanced
ITA, including the measurement of apo A-1 and apo B-
100. The precision of the tests performed on automated
analyzers is high. By ITA methods, total imprecision is
between 1.6% and 3.5% CV in the apo A-1 measuring
range 47 to 209 mg/dL (0.47 to 2.09 g/L) and 1.1% to
6.3% CV in the apo B measuring range 43 to 150 mg/dL
(0.43 to 1.50 g/L). By INA, total imprecision is between
3.3% and 6.0% CV in the apo A-1 measuring range 58 to
167 mg/dL (0.58 to 1.67 g/L), and 2.4% to 6.0% CV in
the apo B measuring range 58 to 186 mg/dL (0.58 to
1.86 g/L) [1,4].
Immunoturbidimetric Assays
In end-point ITA methods for apo A-1 and B-100,
diluted sample is mixed and incubated with reagent 1
containing a polymer (e.g., polyethylene glycol [PEG]),
ii
Apolipoproteins A-1 and B
Previous and current authors of this method:
First edition: Not done
Methods edition: Suman T. Patel, Howard A.I. Newman
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Jillian R. Tate
then specific polyclonal goat or sheep anti-apo A-1 or
apo B antiserum (reagent 2) is added to the cuvette, and
the reaction proceeds, forming insoluble antigen-
antibody complexes. The resultant turbidity is measured
in a photometric system as an increase in absorption at
340 nm over a defined period of time with subtraction of
the sample blank reading. Very small changes in
turbidity against a higher background scatter are
precisely measured using the photometer of modern
analyzers (Tables 1a and 1b, Methods 1 through 3).
Immunonephelometric Methods
In end-point INA methods for apo A-1 and B-100,
diluted sample is added to buffer containing a
precipitation enhancer (e.g., PEG), then specific
polyclonal rabbit anti-apo A-1 or apo B antiserum is
added to the cuvette, and the reaction proceeds, forming
soluble antigen-antibody complexes. The immuno-
particles scatter light from an infrared LED, and after a
fixed time the scattered light, which is measured at an
angle to the incident beam of light, is converted into
protein concentration using a standard curve and V-Lin
Integral modeling (Tables 1a and 1b, Method 4).
In peak-rate INA methods for apo A-1 and apo B-100,
the antigen-antibody reaction is monitored continuously
as the reagents (buffer and polyclonal goat anti-apo A-1
or apo B antibody) and diluted sample are mixed in the
cuvette. The immune complexes scatter light from a
laser beam (670 nm), and the rate of change in
absorbance measured at an angle of 90 degrees over a
fixed time is determined. Apo A-1 and apo B
concentration are determined by comparison against a
calibration curve (Tables 1a and 1b, Method 5).
Reference and Preferred Methods
Reference Materials and Methods
The candidate primary reference measurement procedure
for apo A-1 quantification is an enzymatic hydrolysis
isotope dilutionmass spectrometric method [5]. The
primary standard used is a hydrolyzed peptide unique to
apo A-1 that was purified by reversed-phase HPLC and
its mass determined by amino acid analysis. The method
was validated by assessing the concentration of apo A-1
in the European Community Bureau of Reference (BCR)
lyophilized Certified Reference Material (CRM 393).
For apo B-100, no primary reference method is
available.
182
Apolipoproteins A-1 and B
Use of reference materials in a physical state equivalent
to that of patient samples is necessary to reduce the
variation in values among laboratories and between
different measurement systems. Primary standards for
apo A-1 and B-100 (i.e., pure proteins) are not suitable
for manufacturers assay systems because they show
instability and/or matrix effects. Pure apo B-100 is
particularly unsuited as a primary standard because of
self-association, sensitivity to oxidation, and irreversible
matrix aggregation. Therefore, secondary matrix-
matched, serum-based materials have been developed for
apo A-1 and B. Value assignment of these materials,
SP1-01 for apo A-1 and SP3-07 for apo B-100, was by
the use of consensus methods, RIA for apo A-1 and INA
for apo B, and purified materials, BCR-393 for apo A-1
and LDL freshly prepared by ultracentrifugation (density
1.030-1.050 kg/L) for apo B [6,7]. An apo A-1 value of
1.50 g/L was assigned to SP1-01, and an apo B value of
1.22 g/L was assigned to SP3-07. These reference
materials have become the WHO-IFCC International
Reference Reagents for apo A-1 and apo B-100.
Current preferred methods are end-point and rate
immunonephelometric assays available on dedicated
protein analyzers or end-point immunoturbidimetric
assays available on automated chemistry analyzers.
Specimen
The optimum apolipoprotein specimen is a fresh serum
sample and is the preferred sample type in INA (Tables
1a and 1b, Methods 4 and 5). Plasma-heparin and
plasma-EDTA may be suitable in ITA methods,
depending on the specific assay to be used (Tables 1a
and 1b, Methods 1 through 3). It is reported that apo A-1
and B remain stable for approximately a week at 2C to
8C, about 6 months at 20C, and for over 5 years Similar to the serum lipids, apolipoproteins are
when stored at 50C to 80C [8]. In another study, influenced by biological variation. Whereas there are
apo A-1 was shown to rise slightly in some serum
specimens stored at 4C after a couple of weeks,
whereas apo B was stable for at least 4 weeks [9].
Commercial assays make specific recommendations for
sample type and specimen stability, which have been
tested for these assays.
Interferences
Similar to ITA and INA methods for other specific
proteins, apo A-1 and B measurement is affected in
particular by turbidity from large triglyceride particles in
grossly lipemic samples. These contribute to excess light
scattering that is not due to apo A-1 or apo B immune
complexes. Other particulate matter present in samples
(e.g., fibrin, cellular debris, etc.) can also cause excess
scatter unrelated to the analyte concentration.
Commercial assays make specific recommendations for
levels above which interference may occur. In general,
triglyceride interference is reported to occur at a
concentration of 500 to 1,000 mg/dL and above. Other
interferents include icterus (bilirubin > 30 to 60 mg/dL),
hemolysis (hemoglobin > 500 to 1,000 mg/dL), and
paraproteins, especially monoclonal IgM, may affect apo
A-1 and apo B results by both ITA and INA methods.
Reference Intervals and Interpretation
Until recently, the large-scale introduction of
apolipoprotein measurement was hampered by the lack
of reference materials and methods, which resulted in the
use of different calibration methods and a lack of
comparability of results. It was soon realized that the
preparation, selection, and calibration of appropriate
serum-based reference materials were key to obtaining
international standardization and harmonization of
immunoassays for measurements of apo A-1 and B-100
[10-13] and would enable the establishment of
standardized reference values and decision cutoff limits
in different ethnic populations (Tables 2a and 2b). Apo
A-1 material SP1-01 and apo B-100 material SP3-07
(more recently SP3-08) are used to transfer assigned
values to manufacturers calibrators [6,7].
Through the use of IFCC standardized methods traceable
to SP1-01 and SP3-07 and the availability of these
certified reference materials that can be used by
diagnostic manufacturers to value-assign their assay
calibrators, the value distributions of apo A-1 and apo B
in various populations have been described. These
include studies in the United States (NHANES III and
Framingham Offspring studies) and in Finland, Italy, and
Sweden [14-19]. From this work, clinically meaningful
cut-points for risk stratification have been established in
various populations (Tables 2a and 2b). The studies
showed the percentile distribution of apo A-1 levels was
nearly identical between populations, whereas apo B-100
levels were slightly higher in the Finnish and Swedish
populations, compared with the American and Italian
populations. The higher apo B-100 levels in the
Scandinavian groups were matched by higher cholesterol
levels.
marked differences in high-density lipoprotein
cholesterol (HDL-cholesterol) and low-density
lipoprotein cholesterol (LDL-cholesterol) between
fasting and nonfasting patients, apo A-1 and B-100
concentration in lipoproteins is more stable than the
lipids concentration, which may vary according to
presence of metabolic disease and diet. In healthy
subjects within-individual variation for apo A-1 and B is
approximately 6% to 7% [8]. Similar to lipids and
lipoproteins, in severe illness the apolipoproteins may
decrease substantially in concentration [20]. During
pregnancy, apo A-1 and B levels increase by 30% and
60%, respectively [21].
Apo A-1 is important in lipoprotein transport and has
other physiological functions, including (1) structural
protein for chylomicrons and HDL; (2) cofactor activity
in the enzyme lecithin:cholesterol acyl transferase
(LCAT), which esterifies free cholesterol with fatty
acids from phospholipids; (3) ability to remove excess
cholesterol from tissues; and (4) ligand for HDL-
receptor. These processes are important for the anti-
atherogenic reverse cholesterol transport pathway back
to the liver. In addition, apo A-1 seems to play a role in
prostacyclin stabilization, leading to improved
vasodilation and the inhibition of platelet aggregation,
183
Apolipoproteins A-1 and B
these functions playing an important role in the
protection against development of atherosclerosis.
Elevated apo A-1 concentration occurs in familial
hyperalphalipoproteinemia, familial cholesteryl ester
transfer protein deficiency, which results in a doubling
of apo A-1 concentration over normal, exercise, liver
disease, moderate alcohol intake, estrogens and
pregnancy, and is associated with decreased risk for
coronary heart disease (CHD) and atherosclerosis.
Measurement of apo A-1 enables identification of
patients with certain heritable abnormalities of
lipoprotein metabolism not detectable using other
methods. Decreased apo A-1 concentration occurs in
familial hypoalphalipoproteinemia, familial combined
hyperlipidemia (FCHL), fish-eye disease, which is due
to LCAT deficiency and apo A-1 concentrations 15% to
30% of normal, Tangier disease, cholestasis, acute
hepatitis, sepsis, and in insulin-treated diabetics.
Decreased apo A-1 is generally associated with
increased risk of CHD and atherosclerosis. Generally,
the apo A-1 variants are associated with low HDL-
cholesterol levels and are incomplete forms of the
protein. Tangier disease, for example, is a rare
autosomal-recessive disorder with very low apo A-1,
total cholesterol, and HDL-cholesterol levels. Plasma
apo A-1 levels are 1% to 3% of normal in homozygotes
and half that of normal in heterozygotes [22]. In contrast,
the mutation called A-1Milano with low levels of apo A-1
and HDL-cholesterol is associated with longevity, and
carriers rarely have coronary heart disease [23,24].
Apo B is the main major protein moiety component of
all lipoproteins other than HDL, that is, LDL,
intermediate-density lipoprotein (IDL), very-low-density
lipoprotein (VLDL) classes, and also chylomicrons. Apo
B accounts for approximately 95% of the total protein
content of LDL. It is important for the secretion of
triglyceride-rich lipoproteins both from the liver and the
intestine and is essential to the formation and release of
lipoproteins into the plasma. Apo B-100 acts as a
structural protein for chylomicrons, VLDL, IDL, and
LDL, with a single molecule of apo B present in each
particle. Lipoprotein (a) (Lp[a]) is also an apo B-
containing lipoprotein and will contribute to total apo B-
100 concentration, the amount depending on the Lp(a)
concentration (average apo B-100 is 5 mg/dL for normal
Lp[a] levels). Unlike the smaller apolipoproteins, such as
apo A-1 and apo C, very-high-molecular-weight
apolipoproteins such as apo B-100 do not migrate from
one particle to another but remain bound during
assembly, secretion, and catabolism of the lipoprotein
particles. Apo B-100 interacts with the B/E (LDL)
receptors of peripheral cells, thereby functioning in the
recognition of cellular receptors for the catabolism of
LDL. Synthesis of apo B-100 is regulated by steroid
hormone receptors and thyroid hormones and also by
lipid-lowering drugs such as HMG-CoA reductase
inhibitors.
Measurement of apo B also enables identification of
patients with certain heritable abnormalities of
lipoprotein metabolism not detectable using other
methods. Elevated apo B-100 concentration occurs in
familial hypercholesterolemia, FCHL, familial
hyperapobetalipoproteinemia in which LDL-cholesterol
levels are below the decision limit for risk of CHD, but
plasma apo B-100 and LDL-apo B-100 concentrations
are elevated [25], in individuals with elevated Lp(a),
nephrotic syndrome, hypothyroidism, and biliary
obstruction, and is associated with increased risk for
CHD and atherosclerosis. Decreased apo B-100
concentration is found in abetalipoproteinemia due to an
absence of apo B in the intestine and liver, familial
hypobetalipoproteinemia in which plasma apo B-100 is
low but not as low as in homozygous
abetalipoproteinemia and is 25% to 50% of normal
levels in heterozygotes, in chronic anemia, liver disease,
and neuromuscular degeneration.
Clinical Significance of Apolipoprotein A-1 and B
There is a considerable evidence base linking high levels
of certain plasma lipoproteins to accelerated
atherogenesis and CHD. Apo A-1, the major protein in
HDL, and apo B-100, the major protein in LDL, serve as
important predictors of cardiovascular disease risk. The
non-HDL lipoproteins, VLDL, IDL, and LDL that
contain apo B-100, have been identified as the vehicles
in which cholesterol is transported into the artery wall
and are regarded as atherogenic particles. Apo B-100
interacts with the mucopolysaccharide, chondroitin
sulfate B, at the artery wall and probably leads to in-situ
degradation of the lipoproteins. Concentrations of apo B-
100 and non-HDL cholesterol are closely correlated
[26].
Whereas plasma apo B-100 reflects LDL primarily, a
proportion of non-HDL cholesterol is found in VLDL
and IDL. Measurement of apo B-100 reflects the number
of particles presenting cholesterol to the arterial wall and
may give a better prediction of risk of vascular events
than measurement of total cholesterol alone. LDL (and
thus the usual lipid parameters) is not a uniform
lipoprotein but a very heterogeneous macromolecule.
Although LDL particles contain one molecule of apo B,
they can vary in their cholesterol content by almost 25%,
resulting in large, buoyant particles enriched in
cholesterol and small, dense LDLs that contain less
cholesterol [27]. Small LDL particles can enter the
arterial wall more easily than larger LDLs, they have a
greater affinity for the glycoproteins of the arterial
intima, and they are more susceptible to oxidation
leading to enhanced macrophage uptake and foam-cell
formation. Hence, by determining LDL-cholesterol, it is
not possible to make assumptions regarding the total
LDL mass or the total number of LDL particles.
By contrast, the measurement of apo B-100 provides
information regarding the number of LDL particles and
the relative density of this lipoprotein. Such information
is useful, since small and dense LDLs (higher apo B
concentration) are more atherogenic than buoyant ones
(lower apo B concentration). In hypertriglyceridemic
patients with impaired glucose tolerance and type II
diabetes, and where total cholesterol is similar to the
non-diabetic population, apo B-100 can differentiate
184
Apolipoproteins A-1 and B
small, dense LDL enriched with cholesterol ester from
less atherogenic particles [27].
Apo B/apo A-1 Ratio
Diagnostic Relevance of Apolipoprotein Measurements
The Adult Treatment Program recommends LDL-
cholesterol as the most clinically useful lipid parameter
to assess risk of vascular disease and to guide treatment
therapy. An alternate paradigm, based on atherogenic
lipoprotein particles, suggests that rather than equating
risk due to LDL and the plasma cholesterol
concentration of LDL-cholesterol, or to VLDL and
triglyceride concentration, the lipoprotein atherogenic
burden is best represented by total plasma apo B.
Current apo B assays measure the sum of apo B-100 and
apo B-48 lipoprotein particles, with approximately 90%
of plasma apo B derived from LDL and IDL particles
and the remainder largely from VLDL. Chylomicrons
and their remnants contribute minimally to total apo B.
The quantitative measurement of apo A-1 and apo B-100
is therefore an alternative to conventional lipid and
lipoprotein analysis. Increasing evidence has
accumulated linking apolipoproteins to atherosclerosis,
indicating that their measurement is as reliable as
measurements of lipoproteins for coronary artery disease
(CAD) risk assessment and response to lipid-lowering
therapy. Elevated concentrations of apo B-100 may be
found in normolipidemic patients with early CAD, even
when levels of total and LDL-cholesterol are normal.
Apo B-100 measurement may be more relevant to CAD
risk assessment, because the amount of apo B-100 per
LDL particle is relatively constant, whereas that of
cholesterol is variable [1,28]. Numerous prospective
epidemiological studies have shown apo B to be superior
to LDL-cholesterol in predicting the risk of
cardiovascular events and disease progression and in
determining the adequacy of LDL-cholesterol lowering
therapy [29-37].
Apo B seems to be useful for assessment of the
effectiveness of pharmacological treatment with lipid-
lowering drugs. Statins lower both LDL-cholesterol and
apo B. However, there is faster removal of LDL-
cholesterol from the circulation than number of LDL
particles, with triglyceride replacing some of the
cholesterol in LDL particles. Use of apo B as a marker
for LDL particle number in the assessment of persons at
a moderate risk of vascular events (i.e., at LDL-
cholesterol concentrations below the 50th percentile)
may be a better discriminator than LDL-cholesterol [38].
Whereas the relationship between risk and LDL-
cholesterol concentration flattens out at lower levels,
there is a linear relationship between risk and apo B
from the lowest to highest decile of apo-B concentration
according to the results of the AMORIS and
INTERHEART epidemiological health studies [34,39].
The ratio of apo B/apo A-1 allows those subjects with
and without CAD to be distinguished [25,40-42]. A high
ratio of apo B to apo A-1 can be used as a marker of
increased risk for CAD and a decreasing ratio as an
indicator of lowering of the risk following lipid-lowering
treatment. The AMORIS study, with a mean follow-up
period of 66.8 months for 98,722 males and 64.4 months
for 76,831 women, demonstrated that the apo B/apo A-1
ratio was significantly more informative of risk than the
conventional cholesterol parameters [34]. Adding further
weight to the predictive power of the apo B/apo A-1
ratio is the data from the INTERHEART study, which
involved 30,000 subjects from 52 countries and showed
that the ratio accounted for approximately 50% of
cardiovascular events [39]. The apo B/apo A-1 ratio, or
so-called Cholesterol Balance, indicates an increased
risk of vascular events at values > 0.9 for men and > 0.8
for women.
Apolipoprotein Performance Goals
Standardization Efforts Early standardization efforts
occurred in the 1980s. Initially the Centers for Disease
Control and Prevention (CDC), in collaboration with the
International Union of Immunological Societies,
undertook an apolipoprotein survey of 28 participating
laboratories and found that comparability of
measurements was 13% CV at 112.4 mg/dL apo A-1 and
27% CV at 58.9 mg/dL apo B-100, respectively. Sources
of variation were different calibrators, matrix interaction,
and lack of assay linearity and parallelism [43].
In a further international standardization study
implemented by the International Federation of Clinical
Chemistry and Laboratory Medicine (IFCC) and
coordinated by the Northwest Lipid Research
Laboratories (NWLRL) in collaboration with diagnostic
manufacturers and research institutes, candidate
reference materials and serum pools for apo A-1 and apo
B-100 were tested for analytical performance,
commutability of material, and comparability of method
comparison values before and after common calibration.
Whereas apo B-100 concentrations before the uniform
calibration showed a large among-assay variation
(>19%) due to different assay standardization, this was
significantly reduced to a mean of 6% after uniform
calibration. Of the 26 reference materials for apo A-1
and apo B proposed by the manufacturers, 15 for apo A-
1 and 11 for apo B, two lyophilized preparations for apo
A-1 and two liquid-stabilized preparations for apo B
were selected for further evaluation in Phase 2 of the
study [44]. Using these materials as the common
calibrator, results again showed that by uniform
calibration of 37 test systems, harmonization of results
could be accomplished. Among-systems variation in apo
A-l levels improved from 9% to 5.4% using the common
calibrator and from ~ 20% to 7% for apo B. The
candidate reference materials, SP1 and SP3, gave
analytical characteristics closest to that of the serum
pools, and there were no significant matrix effects in the
tested assays. Following recalibration, 50 fresh-frozen
serum samples covering a wide range of apo A-1 and
apo B-100 concentrations were analyzed to test for
comparability between various immunoassays. The
results obtained by the diagnostic companies were
excellent, with an interlaboratory variation for results of
2% to 6% for apo A-1 and 3% to 7% for apo B [6,7].
185
Apolipoproteins A-1 and B
Between-Method Result Comparability
The desirable quality specifications for precision, bias,
and total allowable error for apo A-1 and apo B
measurement are an imprecision (CV) of 3.3% and 3.5%
(based on 50% of the intraindividual biological
variation), a bias of 3.7% and 6.0%, and a total error of
9.1 % and 11.6% for apo A-1 and apo B, respectively
[8,45]. According to the 2007 American College of
Pathologists (CAP) survey of apolipoproteins A-1 and B,
the number of laboratories measuring these proteins is
small compared with the number measuring lipids and
lipoproteins. Method principles are ITA and INA in
approximately equal numbers. The measurement
variation for apo A-1, at a mean level of 128.0 mg/dL,
was 6.6% CV (range: 5.0% to 6.7% CV for all method
principles and 115 participating laboratories). For apo B,
at a mean level of 71.1 mg/dL, the variation was 7.7%
CV (range: 3.8% to 7.6% CV for 112 laboratories;
another 10 labs used Roche Cobas Integra, with a 10.6%
CV at a mean apo B concentration of 66.5 mg/dL).
According to the CAP survey, not all apo A-1 and apo B
methods showed acceptable precision. Although current
assays are traceable to the certified secondary reference
materials for apolipoproteins, bias may contribute to
concentration differences between methodsfor
example, apo A-1 for the same sample as above ranged
between 124.5 and 134.9 mg/dL (mean 128.0 mg/dL)
and apo B between 66.5 and 73.0 mg/dL (mean 71.1
mg/dL).
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43. Smith SJ, Cooper GR, Henderson LO, Hannon
WH and the international collaborative study on
standardization of apolipoproteins A-I and B.
Part 1. Evaluation of a lyophilized candidate
187
Apolipoproteins A-1 and B

reference and calibration material. Clin Chem. 45. Fraser CG. Biological variation from principles
1987;33:2240-2249. to practice. Washington, DC: AACC Press;
44. Albers JJ, Marcovina SM, Kennedy H. 2001:139-141.
International Federation of Clinical Chemistry
standardization project for measurement of Acknowledgement
apolipoproteins A-I and B. part 2. evaluation Data on current external quality assessment were kindly
and selection of candidate reference materials. provided by Sharon Burr from the American College of
Clin Chem. 1992;38:658-662. Pathologists.
Tables

Table 1a: Apo A-1 Methods Summary Table

Method 1: End-point ITA with sample blanking and using polyclonal goat anti-human apo A-1 antibody
Principle of analysis: Light scattering by antigen/antibody complexes increases turbidity, which is
measured over a defined time interval
Comments: Serum and plasma (Li heparin, Na heparin, EDTA); avoid interference due to grossly
lipemic samples and paraproteins.

Method 2: End-point ITA with sample blanking and using polyclonal goat anti-human apo A-1 antibody
Principle of analysis: Same as Method 1
Comments: Serum and plasma-heparin; avoid interference due to grossly lipemic samples.

Method 3: End-point ITA with sample blanking and using polyclonal sheep anti-human apo A-1 antibody
Principle of analysis: Light scattering by antigen/antibody complexes increases turbidity, which is
measured over a defined time interval
Comments: Serum and plasma (Li, Na, NH4+-heparin, EDTA); avoid interference due to grossly
lipemic samples and paraproteins.

Method 4: End-point INA with sample blanking and using polyclonal rabbit anti-human apo A-1 antibody
Principle of analysis: Antigen/antibody complexes cause light scattering, with the scattered-light
intensity measured at an angle to the incident beam of light over a defined time interval
Comments: Serum is preferred; avoid interference due to particulate matter in the sample and from
grossly lipemic samples.

Method 5: Rate INA with sample blanking and using polyclonal goat anti-human apo A-1 antibody
Principle of analysis: Measures the rate of increase in light scattered by antigen/antibody complexes
after sample blanking check. Scattered-light intensity is measured at an angle to the incident beam of
light.
Comments: Serum is preferred; avoid interference due to particulate matter in the sample, grossly
lipemic samples, and IgM paraproteins.
188
Apolipoproteins A-1 and B

Table 1b: Apo B-100 Methods Summary Table

Method 1: End-point ITA with sample blanking and using polyclonal goat anti-human apo B-100 antibody
Principle of analysis: Light scattering by antigen/antibody complexes increases turbidity, which is
measured over a defined time interval
Comments: Serum and plasma (Li heparin, Na heparin, EDTA); avoid interference due to grossly
lipemic samples and paraproteins.

Method 2: End-point ITA with sample blanking and using polyclonal goat anti-human apo B-100 antibody
Principle of analysis: Same as Method 1
Comments: Serum and plasma-heparin; EDTA plasma NOT RECOMMENDED; sample stable when
stored at 18C to 28C 1 day; 2C to 8C 3 days; 70C 1 year, but storage at 20C NOT
RECOMMENDED; avoid interference due to grossly lipemic samples.

Method 3: End-point ITA with sample blanking and using polyclonal sheep anti-human apo B-100 antibody
Principle of analysis: Light scattering by antigen/antibody complexes increases turbidity, which is
measured over a defined time interval
Comments: Serum and plasma (Li, Na, NH4+-heparin, EDTA); avoid interference due to grossly
lipemic samples and paraproteins.

Method 4: End-point INA with sample blanking and using polyclonal rabbit anti-human apo B-100 antibody
Principle of analysis: Antigen/antibody complexes cause light scattering with the scattered light
intensity measured at an angle to the incident beam of light over a defined time interval
Comments: Serum is preferred; avoid interference due to particulate matter in the sample and from
grossly lipemic samples.

Method 5: Rate INA with sample blanking and using polyclonal goat anti-human apo B-100 antibody
Principle of analysis: Measures the rate of increase in light scattered by antigen/antibody complexes
after sample blanking check. Scattered light intensity is measured at an angle to the incident beam of
light
Comments: Serum is preferred; avoid interference due to particulate matter in the sample, grossly
lipemic samples, and IgM paraproteins.
Avoid interference due to grossly lipemic samples and paraproteins.
189
Apolipoproteins A-1 and B

Table 2a:Apo A-1 Reference Intervals and Cut-points

Country Method Age (y) Apo A-1 (mean SD) in mg/dL

(10th percentile)

Male n Female n

Finland [17] ITA Orion kit 27-67 136 22 286 158 29 289
(Kone Olli CD
analyzer) (109) (125)

Northern Italy Rate INA 49.8 16.0 141 21 298 156 23 315
[18] (Beckman Array
360) (116) (127)

Sweden [19] ITA Orion kit M: 46.8 13.2 136 22 83,112 151 24 64,46
(Kone C/D 4
analyzer) F: 49.5 15.2 (110) (122)

US ITA Orion kit M: 51.9 10.2 134 23 1,879 154 28 1,939

Framingham (Abbott Spectrum
[15,16] CCX analyzer) F: 51.3 10.6 (107) (122)

US NHANES RID and later rate 20 136 22 6,000 151 23 6,000

III [14] INA (Beckman
Protein System) (111) (120)

Table 2b:Apo B Reference Intervals and Cut-points

Country Method Age (y) Apo B-100 (mean SD) in mg/dL

(75th percentile)

Male n Female n

Finland [17] ITA Orion kit 27-67 121 32 286 109 32 289
(Kone Olli CD
analyzer) (141) (129)

Northern Italy Rate INA 49.8 16.0 111 29 298 110 35 315
[18] (Beckman Array
360) (129) (131)

Sweden [19] ITA Orion kit M: 46.8 13.2 131 35 83,112 122 36 64,464
(Kone C/D
analyzer) F: 49.5 15.2 (153) (142)

US ITA Orion kit M: 51.9 10.2 103 24 1,880 96 26 1,944

Framingham (Abbott Spectrum
[15,16] CCX analyzer) F: 51.3 10.6 (118) (111)

US NHANES RID and later rate 20 107 25 6,000 103 28 6,000

III [14] INA (Beckman
Protein System) (122) (119)
 190
Aspartate Aminotransferase
Aspartate Aminotransferase
James J. Miller
Name: Aspartate aminotransferase, AST, L-aspartate:2-oxoglutarate
aminotransferase, formerly serum glutamate oxaloacetic transaminase (SGOT)
Clinical significance: Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Enzyme number: EC 2.6.1.1
Molecular weight: Cytoplasmic: 93,000 D; mitochondrial: 90,400 D
Chemical class: Enzyme, protein
Biochemical reaction: Amino transfer catalyzed by AST (see Figures at end of text)
Methods: CLSI RS2-A
Principles of Analysis and Current Usage i
Aspartate aminotransferase (AST) catalyzes the transfer of
an amino group from specific amino acids (L-glutamate or
L-aspartate) to specific ketoacids (-ketoglutarate or
oxaloacetate) (see biochemical reaction in Figures at end
of text). Although at physiological pH, the reaction is
energetically favored toward the formation of L-aspartate
and -ketoglutarate (to the left, as in the equation), in vivo
the reaction goes to the right to provide a source of
nitrogen for the urea cycle. The glutamate thus produced is
deaminated by glutamate dehydrogenase, resulting in
ammonia and regeneration of -ketoglutarate.
Two methods of AST analysis have enjoyed the widest
popularity. The coupling of oxaloacetate with 2,4-
dinitrophenylhydrazine (DNPH) to produce a blue
hydrazone (Method 1, AST Methods Summary Table) was
introduced in 1957 [1] and gained popularity because of its
simplicity and relative accuracy. In this method, as in all
others, it is necessary to force the AST reaction toward the
energetically less favored products, of which only
oxaloacetate is easily quantitated. Thus L-aspartate and -
ketoglutarate are added in excess. After incubation, the
reaction is stopped with the addition of DNPH, which
reacts with both -ketoglutarate and oxaloacetate. After
condensation, the pH is made alkaline, and the absorbance
at 505 nm is measured. Because the absorptivity of the
oxaloacetate hydrazone is much greater than the
corresponding hydrazone of -ketoglutarate (present in
iAST
Previous and current authors of this method:
First edition: Robert L. Murray
Methods edition: Robert L. Murray
Second edition: Robert L. Murray
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: James J. Miller
great excess when the patient specimen has low AST
activity), little error is introduced.
In the second approach, first described by Karmen [2],
oxaloacetate is again the product quantitated. Malate
dehydrogenase (MDH) is used to convert the oxaloacetate
to malate (Table 1, Method 2). The decrease in absorbance
at 340 nm, caused by NADH (reduced nicotinamide
adenine dinucleotide) consumption in the second reaction,
is used to follow the course of the AST reaction. More
than 80% of reporting survey laboratories use the Karmen
reaction.
A number of photometric techniques have been described,
all of which followed a similar principlethe coupling of
an aryldiazonium salt to the active methylene of
oxaloacetate (Table 1, Method 3) [3]. In all cases, the aryl
substitute (Ar) is a dye with absorbance characteristics that
change with the formation of the azo bridge to
oxaloacetate. The reaction is thus monitored by
measurement of the increase in absorbance of the
diazonium reaction product.
A colorimetric method for AST is also available. This
method is employed in one of the dry-film assays available
for AST measurement (Table 1, Method 4). In this
procedure, oxaloacetate formed in the deamination of L-
aspartate is converted to pyruvate and carbon dioxide by
oxaloacetate decarboxylase. The pyruvate is next oxidized
by pyruvate oxidase to acetyl phosphate and hydrogen
peroxide. The final indicator reaction step involves the
oxidation of a leuco dye by peroxidase to produce a
colored dye. Samples that contain increased concentrations
of pyruvate will result in substrate depletion and must be
diluted. The thin-film assays are also interfered with by the
presence of increased protein concentrations, especially
samples with increased immunoglobulin concentrations
such as those found in patients with multiple myeloma [4].
Identification and quantitation of AST isoenzymes has
been reported [5-7]. The catalytic and physicochemical
properties of mitochondrial and soluble (cytoplasmic) AST
 191
Aspartate Aminotransferase
are compared in Table 3 below. Measurement of AST
isoenzymes has not become routine. However, several
reports have suggested clinical utility in the differential
diagnosis of liver disease and for timing of acute
myocardial infarctions [8-11].
Reference and Preferred Methods
There have been many minor modifications in the
enzymatic technique since its introduction by Karmen
[2,12]. In addition, the specifications listed in the reference
method published by the International Federation of
Clinical Chemistry (IFCC) have been modified over the
years, the most recent modification in 2002 being to
optimize conditions for 37C [13-15]. The main use of the
IFCC reference method is to assay calibrators for use in
routine methods. This allows routine methods to be
traceable to the IFCC reference method, with the goal of
reducing the biases between methods.
In the most recent IFCC procedure (2002) [15], 0.20 mL of
serum is preincubated for 5 minutes in 2.00 mL of a
mixture that contains all reactants except -ketoglutarate.
During this preincubation period, the added lactate
dehydrogenase (LD) rapidly converts the endogenous
pyruvate in the serum to lactate, and the pyridoxal
phosphate cofactor joins with any inactive apoenzyme to
form an increased amount of active AST. With the
addition of 0.20 mL of -ketoglutarate, the primary
reaction is initiated, and the concentrations shown in Table
2 are reached, exclusive of the small increases caused by
the presence of endogenous material in the serum. After
steady state is reached, the rate of NADH oxidation is
monitored repeatedly at 339 nm. The rate of change in
absorbance is corrected for a reagent blank.
Most reference methods, including the current IFCC
procedure [15], have included pyridoxal phosphate. The
need for addition of this cofactor has been widely debated.
The essential nature of the cofactor has long been
recognized, but because it is usually present in human
serum in adequate amounts, many investigators do not add
this component to the reaction mixture. In the occasional
patient with severe vitamin B deficiency, this could lead to
a serious underestimation of AST activity. Addition of
pyridoxal phosphate results in an increased activity,
reportedly ranging up to 50% [16]. The higher values
might be expected in severely ill patients, especially if
elevated AST activity is present, and in patients suffering
from pyridoxal phosphate deficiency.
The presence of aminotransferases in the reagents is
possible if the LD is not carefully prepared. Good-quality
enzymes will not pose a problem; in any event, a blank
determination will identify the problem. The presence of
pyruvate is a potential source of error, since in the
presence of endogenous LD, pyruvate will be converted to
lactate with simultaneous consumption of NADH. This
problem is circumvented by the addition of a large excess
of LD, so that endogenous pyruvate is converted during
the preincubation period, eliminating interference during
the measurement period.
Some older reference methods based on the Karmen
coupled enzymatic method used phosphate buffer, which
retards the recombination of added pyridoxal phosphate
with the apoenzyme. If a large amount of the inactive
enzyme is present, a falsely low activity will be observed.
Activation of apoenzyme is more efficient in Tris buffer.
However, NADH is somewhat less stable in Tris buffer
than it is in phosphate buffer. For this reason, the Tris
concentration is kept relatively low at 80 mmol/L.
Most routine methods today use the Karmen coupled
enzymatic method and may be traceable to the current
IFCC reference method.
The American Association for Clinical Chemistry
proposed a method for the small clinical chemistry
laboratory that differs from the IFCC in that (1) a single
reagent is used to avoid a two-step addition procedure, (2)
the reaction is read after 150 seconds for the following 180
seconds, and (3) pyridoxal phosphate is not added [17].
The drawbacks of the dinitrophenylhydrazine method are
that (1) the oxaloacetate produced by the reaction results in
feedback inhibition of AST, and thus specimens exhibiting
high activity are spuriously lowered; and (2) any ketone in
serum can react, though most do not result in an
absorbance change in the region measured. However,
acetoacetic acid and hydroxybutyric acid, both components
of ketosis, do cause false elevations. The diazonium
method similarly is limited by nonspecificity in that any
activated methylene group will react. This is seen in
patients on certain drugs and those with renal failure.
The U.S. National Institute of Standards and Technology
(NIST) Standard Reference Material No. 909b is a
lyophilized human serum preparation intended for use in
evaluating the accuracy of reference methods. It is
available to manufacturers and laboratories for the
validation of AST methods.
Specimen
Serum or plasma may be used. Heparin, oxalate, EDTA,
and citrate do not cause enzyme inhibition [18].
Anticoagulants with ammonium as the cation should be
avoided to reduce the possibility of error. Because of the
high activity of AST in red blood cells, hemolyzed
samples are unacceptable. For the colorimetric thin-film
procedure, serum or heparinized plasma only are
acceptable specimens.
Interferences
Potential sources of error in the enzymatic method include
endogenous serum glutamate dehydrogenase,
aminotransferases (in the reagent preparation), very high
serum pyruvate concentration, or the use of phosphate
buffer. Glutamate dehydrogenase is present in some
pathological sera, particularly in serum from patients with
 192
Aspartate Aminotransferase
liver disease. This enzyme introduces error by catalyzing
the formation of glutamate with NH4+ to form -
ketoglutarate, with reduction of NADH to NAD+. Since
this source of error is possible only when ammonium ions
are present, the IFCC method eliminates all deliberate
addition of these. (Older methods sometimes included
ammonium sulfate in the formulation.)
Aspartate Aminotransferase Reference Interval
IFCC, with pyridoxal phosphate, 37C
Adults [19]: M: 14-45 U/L
F: 13-37 U/L
Infants: AST activity in neonates and infants is
approximately twice that in adults, but these decline to
adult levels by approximately 6 months of age [20,21].
Interpretation
There are two forms of ASTmitochondrial and soluble
forms [9,12]. The isoenzymes have significant similarities
in amino acid sequence (approximately 50%) and in
active-site positions. Each enzyme comprises two identical
subunits. The molecular weights of these dimers are
93,000 for the soluble and 90,400 for the mitochondrial
isoenzyme. A summary of the physical and catalytic
properties of these isoenzymes is presented in Table 3. The
mitochondrial form of the enzyme accounts for only about
12% of the total activity in normal serum.
The tissue distribution of total AST and the ratio of the
activity of AST in each tissue to the AST activity in serum
are presented in Table 4 [22,23]. Per gram of wet tissue,
heart, liver, skeletal muscle, and kidney have similar
amounts of AST. While enzyme release is not specific,
high enzyme activity does indicate tissue damage. The
mitochondrial form represents 81% of the total AST
present in human liver [24].
Alanine aminotransferase (ALT) activity is usually higher
than AST in most types of liver disease, in which the
activity of both enzymes is predominantly from the
hepatocyte cytosol. When liver necrosis is substantial, as
in individuals with alcoholic and viral hepatitis,
mitochondrial AST is also released into the blood, and
AST activity is usually higher than ALT. The ratio of AST
to ALT, sometimes called the De Ritis Ratio, is often used
to evaluate alcoholic liver disease [25] and severity of liver
disease in viral hepatitis [26,27]. This ratio is only
pertinent in isolated liver disease when comorbidities that
increase AST activity are not present.
Aspartate Aminotransferase Performance Goals
The current target for total error in AST measurements is
20% (CLIA). Biological variation data suggest that in
patients with stable AST activities, a total error of <15% is
required for optimum use [28]. The interlaboratory
coefficients of variation listed for various AST methods in
the 2007 College of American Pathologists Participant
Summary Report (C-B) are all <5%, indicating that this
target is easily met using current AST methods.
References
1 Reitman S, Frankel S. A colorimetric method for
the determination of serum glutamic oxalacetic
and glutamic pyruvic transaminase. Am J Clin
Pathol 1957; 28: 56-63.
2 Karmen A. A note on the spectrophotometric
assay of glutamic oxalacetic transaminase in
human blood serum, Appendix: p. 131-3 in
Karmen A, Wroblewski F, LaDue JS.
Transaminase activity in human blood. J Clin
Invest 1955; 34: 126-33.
3 Sax SM, Moore JJ. Determination of glutamic
oxalacetic transaminase activity by coupling of
oxalacetate with diazonium salts. Clin Chem
1967; 13: 175-85.
4 Tang M, Sullivan M, Gibson D, Truskolawski C.
Kinetic error on Kodak Ektachem: A clue in
diagnosis of myeloma. Clin Chem 1994; 40: 166.
5 Bodansky O, Schwartz MK, Nisselbaum JS.
Isozymes of aspartate aminotransferase in tissues
and blood of man. Adv Enzyme Regul 1966; 4:
299-315.
6 Sampson EJ, Hannon WH, McKneally SS,
McKenzie C, Miller SA, Whitner VS, Burtis CA.
Column chromatography and immunoassay
compared for measuring the isoenzymes of
aspartate aminotransferase in serum. Clin Chem
1979; 25: 1691-6.
7 Rej R, Bretaudiere J, Graffunder N. Measurement
of aspartate aminotransferase isoenzymes: six
procedures compared. Clin Chem 1981; 27: 535-
42.
8 Nalpas B, Vassault A, Le Guillou A, Lesgourgues
B, Ferry N, Lacour B, Berthelot P. Serum activity
of mitochondrial aspartate aminotransferase: a
sensitive marker of alcoholism with or without
alcoholic hepatitis. Hepatology 1984; 4: 893-6.
9 Panteghini M. Aspartate aminotransferase
isoenzymes. Clin Biochem 1990; 23: 311-9.
10 Panteghini M, Pagani F, Cuccia C. Effects of
therapeutic coronary reperfusion on aspartate
aminotransferase isoenzymes in sera of patients
with acute myocardial infarction. Clin Chem
1989; 35: 909-12.
11 Yoneda K, Katayama Y, Koike T, Tanimizu I. A
homogeneous assay system of aspartate
aminotransferase iso-enzymes using proteases
and application for clinical evaluation of
myocardial infarction. J Clin Lab Anal 1992; 6:
362-7.
12 Rej R. Measurement of aminotransferases: Part 1:
Aspartate aminotransferase. Crit Rev Clin Lab Sci
1984; 21: 99-186.
13 Bergmeyer HU, Bowers GN Jr., Horder M, Moss
DW. Provisional recommendations on IFCC
methods for all measurements of catalytic
 193
Aspartate Aminotransferase
concentrations of enzymes, part 2, IFCC method
for aspartate aminotransferase. Clin Chem 1977;
23: 887-99.
14 Bergmeyer HU, Horder M, Rej R. Approved
recomendation (1985) on IFCC methods for the
measurement of catalytic concentrations of 20
enzymes, Part 2. IFCC method for aspartate
aminotransferase (L-aspartate: 2-oxoglutarate
aminotransferase, EC 2.6.1.1). J Clin Chem Clin 21
Biochem 1986; 24: 497-510.
15 Schumann G, Bonora R, Ceriotti F, Ferard G, 22
Ferrero CA, Franck PF, et al. IFCC primary
reference procedures for the measurement of 23
catalytic activity concentrations of enzymes at
37C, part 5, reference procedure for the
measurement of catalytic concentration of 24
aspartate-aminotransferase. Clin Chem Lab Med
2002; 40: 725-33.
16 Bruns DE, Savory J, Titheradge AC, Cross RE, 25
Wills MR. Evaluation of the IFCC-recommended
procedure for serum aspartate aminotransferase as
modified for use with a centrifugal analyzer, Clin 26
Chem 1981; 27: 156-159.
17 Butler TJ, Civantos F, Steele BW, Airan RS,
Klotzsch SG. Aspartate aminotransferase, AST
(provisional). In: Faulkner WR, Meites S, editors.
Selected methods of clinical chemistry.
Washington, D.C.: American Association for
Clinical Chemistry; 1982. p. 101-7. 27
18 Demetriou JA, Drewes PA, Gin JN. Enzymes. In:
Henry RJ, Cannon DC, Winkelman JE, editors.
Clinical chemistry: principles and technics. 2nd ed.
Hagerstown, MD: Harper & Row; 1974. p. 815-
1001. 28
19 Stromme JH, Rustad P, Steenland H, Theodorsen
L, Urdal P. Reference intervals for eight enzymes
Table 1: Methods of Aspartate Aminotransferase (AST) Analysis
Method 1: Dinitrophenylhydrazine coupling (colorimetric) (Reitman and Frankel [1]); quantitative
Principle of analysis*: click here
Comments: Serum
Method 2: Enzymatic (ultraviolet monitoring) (Karmen [2]); quantitative
Principle of analysis*: click here
Comments: Serum
Method 3: Diazonium dye coupling (colorimetric); quantitative
Principle of analysis*: click here
Comments: Serum
Method 4: Enzymatic leuco dye (visible); quantitative
Principle of analysis:
_
Aspartate + -Ketoglutarate ASTOxaloacetate + Glutamate
Oxaloacetate Oxaloacetate decarboxylasePyruvate + CO2
_
Pyruvate + Phosphate + O2 Pyruvate oxidase H2O2 + Acetyl phosphate
_
H2O2 + Leuco dye Peroxidase Colored dye
Comments: Infrequently used *Asp, Aspartic acid; -KG, -ketoglutaric acid; Gl, glutamic acid; Oa, oxaloacetic acid
in blood of adult females and males measured in
accordance with the International Federation of
Clinical Chemistry reference system at 37C: part
of the Nordic Reference Interval Project. Scand J
Clin Lab Invest 2004; 64: 371-84.
Meites S, editor. Pediatric clinical chemistry.
Washington, D.C.: American Association for
Clinical Chemistry: 1981. p. 117-20.
Soldin SJ, Hicks JM, editors. Pediatric reference
ranges. Washington, D.C.: AACC Press: 1995.
Wroblewski F. Serum transaminase activity. Adv
Clin Chem 1958; 1: 313-51.
Whitby LG, Percy-Robb IW, Smith AF. Lecture
notes on clinical chemistry. 3rd ed. London:
Blackwell Scientific Publications; 1984. p. 141.
Rej R. Aspartate aminotransferase activity and
isoenzyme proportions in human liver tissues.
Clin Chem 1978; 24: 1971-9.
Majhi S, Baral N, Lamsal M, Mehta KD. De Ritis
ratio as diagnostic marker of alcoholic liver
disease. Nepal Med Coll J 2006; 8: 40-2.
Giannini E, Risso D, Botta F, Chiarbonello B,
Fasoli A, Malfatti F, et al. Validity and clinical
utility of the aspartate aminotransferase-alanine
aminotransferase ratio in assessing disease
severity and prognosis in patients with hepatitis C
virus-related chronic liver disease. Arch Intern
Med 2003; 163: 218-24.
Giannini EG, Zaman A, Ceppa P, Mastracci L,
Risso D, Testa R.. A simple approach to
noninvasively identifying significant fibrosis in
chronic hepatitis C patients in clinical practice. J
Clin Gastroenterol 2006; 40: 521-7.
Fraser CG. Biological variation: from principles
to practice. Washington, DC: AACC Press; 2001,
p. 140.
 194
Aspartate Aminotransferase

Table 2: Conditions of the 2002 IFCC AST Reference Method

Component/Condition Concentration/Value
L-aspartate (mmol/L) 240
2-Oxoglutarate (mmol/L) 12
Buffer & concentration (mmol/L) Tris 80
pH 7.65
Pyridoxal phosphate (mmol/L) 0.1
NADH (mmol/L) 0.18
MDH (U/L) 600
LDH (U/L) 900
Volume fraction (v/v) 0.0833
Temperature (C) 37.0
Wave length (nm) 339
Band width (nm) 2
Light path (mm) 10
Incubation time (s) 300
Delay time (s) 90
Measurement interval (s) 180
Readings (measurement points) 6

Table 3: Properties of AST Isoenzymes

Mitochondrial Soluble
Catalytic
Km of oxaloacetate 9.0 10-6 3.7 10-5
Km of glutamate 3.8 10-2 1.0 10-2
Km of oxoglutarate 5.6 10-3 1.3 10-4
Km of L-aspartate 4.0 10-4 2.0 10-3
pH optimum Approximately 7.0 Approximately 8.0

Physicochemical
Molecular weight 90,400 D 93,000 D
Isoelectric point >9.0 5.15 to 5.80
Modified from Rej R: Aspartate aminotransferase, Crit Rev Clin Lab Sci 21:99-186, 1984.

Table 4: Tissue Distribution of AST in Normal Human Adult Tissues

Ratio of activity
-3
U 10 /g in tissue
Tissue wet tissue homogenate to that in serum
Heart 156 7800
Liver 142 7100
Skeletal muscle 99 4950
Kidney 91 4550
Pancreas 28 1400
Spleen 14 700
Lung 10 500
Plasma 0.02 1
From Wrblewski F: Serum transaminase activity, Adv Clin Chem 1:313-351, 1958.

Figures
 195
Aspartate Aminotransferase

Figure 1: Amino transfer catalyzed by AST.

Figure 2: Reaction of oxaloacetate with dinitrophenylhydrazine as in Method 1.

Figure 3: Conversion of oxaloacetate to malate with malate dehydrogenase as in Method 2.

Figure 4: Diazonium dye coupling to oxaloacetate as in Method 3.

Procedure: IFCC Recommended Procedure[15] Principle

In the presence of aspartate aminotransferase, L-
 196
Aspartate Aminotransferase
aspartate and 2-oxoglutarate exchange an amino group to
form oxaloacetate and L-glutamate. The rate of this
reaction is monitored by use of an indicator reaction in
which the oxaloacetate formed is converted to malate by
an excess of malate dehydrogenase. The decrease in
absorbance at 339 nm is monitored as the NADH is
consumed. The IFCC reference method is primarily used
to assay calibrators to allow traceability of routine methods
to the reference method.
Reagents
1. Tris-aspartate buffer (96.92 mmol/L Tris,
302.4 mmol/L-aspartic acid, pH 7.65). In a 100 mL
volumetric flask, dissolve 1.17 g of
Tris(hydroxymethyl)aminomethane, 4.02 g of L-aspartic
acid, and 0.052 g sodium azide in 80 mL of distilled water.
Adjust to pH 7.65 with 2 mol/L NaOH. Make up to 100
mL with distilled water. Stable for 3 months at 2C to 8C.
This is Solution 1.
2. Tris-HCl buffer (96.92 mmol/L Tris, pH 7.65).
In a 100 mL volumetric flask, dissolve 1.17 g of
tris(hydroxymethyl)aminomethane and 0.052 g sodium
azide in 80 mL of water, adjust to pH 7.65 with 1 mol/L
HCl, and bring to 100 mL volume with water. Stable for 3
months at 2C to 8C. This is Solution 2.
3. Pyridoxal phosphate, 6.3 mmol/L. Dissolve
16.7 mg of pyridoxal-
5-phosphoric acid monohydrate in 10.0 mL of the Tris-
HCl buffer. Store in a dark bottle at 2C to 8C. Stable for
1 week. This is Solution 3.
4. NADH, 11.34 mmol/L. Dissolve 16 mg of -
nicotinamide adenine dinucleotide reduced from disodium
salt in 2.0 mL Tris-HCl buffer. Stable for 1 week at 2C to
8C. This is Solution 4.
Diluent for reagent enzymes. Dissolve 1.2 g bovine
serum albumin and 0.9 g NaCl in 100 mL of water. Stable
at least 1 month at 2C to 8C.
5. Malate dehydrogenase/lactate dehydrogenase
(1.26 mkat/L [75.6 kU/L] and 1.89 mkat/L [113.4 kU/L]
at 37C, respectively). Separately dilute stock MDH and
LDH to twice these activities with diluent for reagent
enzymes, and mix equal volumes of the two enzyme
solutions. Stable for 2 days at 2C to 8C. This is Solution
5.
6. Reaction solution. Mix 10 mL of Tris-aspartate
(Solution 1) with 0.2 mL of pyridoxal phosphate (solution
3), 0.2 mL of NADH (Solution 4), and 0.1 mL of the
enzymes (Solution 5). Mix thoroughly and in a dark bottle.
Stable for 1 day at 2C to 8C.
7. Start reagent solution. Dissolve 326 mg of 2-
oxoglutaric acid, disodium salt in 10.0 mL of distilled
water. Stable for 1 week at 2C to 8C.
Assay
Equipment: Spectrophotometer with 2 nm band
pass at 339 nm with a constant temperature cuvette capable
of maintaining a constant temperature with less than 0.1C
fluctuation. A recording spectrophotometer is preferable.
1. Add 2 mL of reaction solution and 0.2 mL of
serum to the cuvette.
2. Mix and allow to stand for 5 minutes.
3. Add 0.2 mL of start reagent solution
5. Mix, wait 90 s, and record the change in
absorbance for an additional 180 s. If no recorder
is available, record at least every 30 s.
6. A reagent blank is run by replacing the sample
with 9 g/L sodium chloride in step 1.
7. A sample blank should be checked by replacing
the start reagent in step 3 with 9 g/L sodium
chloride. The sample blank is not included in the
calculation of AST activity, but a rate >1% of the
AST activity indicates that the material is not
suitable as a calibrator. For the reagent blank of
the sample blank, replace both the start reagent
and the sample with 9 g/L sodium chloride.
Calculations
If the change in absorbance per second (A/t) is
greater than 0.0022 per second, dilute the sample 5- to 10-
fold with 9 g/L NaCl, and repeat the measurement.
Correct for the blank reaction using the following
schema:
(A/t)A = Measured reaction rate with serum in step 1
(A/t)B = Measured reaction rate with NaCl in place of
serum in step 1
A/tAST = (A/t)A - (A/t)B and
AST activity = 1905 x A/tAST kat/L
AST activity in kat/L can be converted to U/L by
multiplying by 60.
197

B-Type Natriuretic Peptide, NT-proBNP and ProBNP

B-Type Natriuretic Peptide, NT-proBNP and ProBNP

Alan H.B. Wu i

Name: B-type natriuretic peptide, NT-proBNP, and proBNP

Clinical significance: See Chapter 28, Physiology of Body Water and Electrolytes, and Chapter 36,
Cardiac and Muscle Disease, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass:
Chemical class: Peptide hormone, prohormone metabolite and prohormone
Structure: (See Figure below)
The enzymatic cleavage of proBNP into BNP (active hormone, 32 amino acids) and NT-proBNP
(inactive peptide, 76 amino acids) by corin. Used with permission from Omland T, Hall C: N-terminal
pro-B-type natriuretic peptide. In Wu AHB: Cardiac Markers, ed 2, Totowa, NJ, 2003, Humana Press.

Figure 1
The enzymatic cleavage of proBNP into BNP (active hormone, 32 amino acids) and NT-proBNP
(inactive peptide, 76 amino acids) by corin. Used with permission from Omland T, Hall C: N-
terminal pro-B-type natriuretic peptide. In: Wu AHB: Cardiac Markers, ed. 2, Totowa, NJ, 2003,
Humana Press.
Principles of Analysis and Current Usage natriuretic peptide, and urodilantin. Pro-BNP is a 108
B-type natriuretic peptide (BNP) is a member of a family amino acid precursor protein of BNP found in the
of natriuretic peptides that include atrial natriuretic ventricles and atria of the heart. The gene for proBNP is
peptide (ANP), C-type natriuretic peptide, D-type found on chromosome 1 [1]. In situations of volume

i
B-Type Natriuretic Peptide, NT-proBNP and ProBNP
New method
Fifth edition: Alan H.B. Wu
198
B-Type Natriuretic Peptide, NT-proBNP and ProBNP
overload and myocardial muscle stretching, proBNP is
upregulated at the genomic level. A small amount of
BNP is found in cytoplasmic granules. Upon stimulation
for production and release, ProBNP is cleaved by corin
to equimolar amounts of the biologically active BNP
hormone and the biologically inactive amino-terminal
proBNP (NT-proBNP) (Figure 1). ProBNP is also
released into the circulation as the unprocessed precursor
protein. When the effects of this peptide on endothelial
and vascular smooth muscle cells [2] and cardiac
fibroblasts and myocytes [3] were evaluated, this peptide
did not appear to have any biological activity.
BNP is a 32amino acid peptide hormone with a
molecular weight of 3472 daltons. The molecule consists
of an N-terminal tail of 9 amino acids, a C-terminal tail
of 6 amino acids, and a 17-member ring that is closed by
a disulfide bond between two cysteine residues. Eleven
of the amino acids found in the ring are conserved across
the family of natriuretic peptides. BNP binds to
natriuretic peptide receptors A and C, causing the loss of
water and electrolytes, vasodilation, and inhibition of the
renin/aldosterone axis. BNP is cleared from the
circulation by neutral endopeptidases. The first two
amino acids from the N-terminal sequence (serine and
proline) are removed by dipeptidyl peptidase IV very
soon after BNP is released into the circulation [4]. BNP
has an estimated half-life of about 20 minutes in humans
[5]. The amino-terminal proBNP (NT-proBNP) is a 76
amino acid, biologically inactive peptide with a
molecular weight of 8460 daltons (Figure 1). Although
not exactly known, NT-proBNP has a much longer half-
life than BNP, estimated to be between 1 and 2 hours
[5]. This peptide exists as a straight chain and does not
contain any disulfide linkages.
All assays for BNP, NT-proBNP, and proBNP are
measured by dual-site sandwich immunoassays. There is
no standardization between manufacturers for BNP with
regard to results or the antibodies used in the assays. The
Beckman BNP assay uses the same antibodies and is
standardized to the Inverness (Biosite) Medical Triage
point-of-care BNP assay, and the Siemens assay uses the
same antibodies as the Shionogi assay (Table 1). Most
manufacturers have assigned a cutoff value of 100 ng/L
as the cutoff for diagnosis of heart failure. Figure 1
shows the epitopes for the capture and detection
antibodies for some of the commercially-approved BNP
tests [6]. For NT-proBNP, both the assay and antibodies
are standardized to the Roche Diagnostics Elecsys
platforms. This assay uses one antibody directed towards
the N-terminus of the peptide and the other towards the
central portion (Table 1). The intellectual property rights
for use of NT-proBNP belong to Roche. As a condition
for acquiring a license for clinical use, all manufacturers
of NT-proBNP assays must use the Roche antibodies
and calibrators.
There have been many studies that have documented the
clinical equivalence of BNP and NT-proBNP for patients
with heart failure. Although there are minor differences
in the performances of these tests, there is no current
need to measure both assays on the same patient. For
more information, see the Interpretation section.
Currently there are no commercial assays approved by
the U.S. Food and Drug Administration (FDA) for
proBNP. However, one company (Bio-Rad Laboratories)
has produced an assay that is currently undergoing
clinical evaluations [7]. The specificity of this assay is
dependent on the use of a monoclonal antibody directed
towards the hinge-76 region of the peptide, an epitope
present in proBNP and absent in both BNP and NT-
proBNP (Table 1). The second antibody is unspecified.
At this point, it is unclear whether or not assays for
proBNP will be superior, equivalent, or provide
supplementary information to that currently provided by
BNP and/or NT-proBNP. There are likely differences in
the release of proBNP from patients with heart failure
for example, there are genetic polymorphisms in the
gene encoding corin [8]. These and other questions will
be addressed in upcoming clinical studies.
Reference and Preferred Methods
There are no reference materials or reference methods
currently available for BNP, NT-proBNP or proBNP.
For BNP, the International Federation of Clinical
Chemistry Committee on the Standardization of Markers
of Cardiac Damage has undertaken the responsibility of
proposing a secondary reference standard from candidate
materials for future standardization consideration by
manufacturers. From there, development of a primary
standard would be possible. There is no current effort for
developing a reference method for BNP or a reference
standard or method for NT-proBNP or proBNP. Due to
matrix effects, a reference method will likely require
mass spectrometry. Reference methods for NT-proBNP
and proBNP are less of an issue, owing to the ownership
of the intellectual property rights for clinical use of these
assays by Roche and Bio-Rad, respectively (see previous
section).
All clinical assays for BNP and NT-proBNP are two-site
sandwich immunoassays and are acceptable methods.
The use of two antibodies improves the specificity of the
assay and affords freedom from cross-reactivity from
other structurally related peptides. Radioimmunoassay
techniques (e.g., Shionoria BNP) are acceptable for
research purposes but not for routine clinical practice.
Interferences
As with other immunoassays, the specificity of the
antibodies used in the tests determine the specificity of
the assay and freedom from interferences. There are
three classes of compounds that have been studied with
reference to BNP and NT-proBNP: structurally similar
peptides, precursor proteins, and other neurohormones;
drugs used in heart failure therapy; and general
photometric interferents. The peptides and proteins that
have been demonstrated to have no interference in BNP
and NT-proBNP assays include renin, aldosterone,
angiotensin I, II, and III, endothelin, adrenomedullin,
and vasopressin. Other natriuretic peptides that have
significant homology in amino acid sequences found in
199
B-Type Natriuretic Peptide, NT-proBNP and ProBNP
the ring structure (ANP, preproANP, CNP, and
urodilantin) also do not exhibit cross-reactivity. Selected
antibodies towards the non-homologous amino acids in
the ring structure are essential in ensuring the required
assay specificity. It is also important to demonstrate that
antibodies used in the BNP assays do not exhibit any
cross-reactivity with NT-proBNP and vice versa.
Because there are no amino acid sequences that are in
common to these peptides (Figure 1), cross-reactivity is
not expected. Cardiac drugs that show no interference in
BNP/NT-proBNP assays include -blockers,
angiotensin-converting enzyme inhibitors, angiotensin
inhibitors, diuretics, digoxin and digitoxin,
antiarrhythmics, calcium channel blockers, statins and
other lipid lowering agents, platelet inhibitors, and other
noncardiac medications, including some analgesics and
antibiotics [9].
All immunoassays for BNP and NT-proBNP are
heterogeneous (i.e., use a separation step between bound
and free antibodies), so there are no problems with
photometric interferents (i.e., hemoglobin, bilirubin, or
lipemia). One study showed that a wide hematocrit range
(28% to 58%), which can affect blood glucose
measurements, did not affect whole blood NT-proBNP
results using a point-of-care device [10]. There have
been no documented reports of interference due to the
presence of human anti-mouse antibodies (HAMA) or
heterophile antibodies. The abnormal antibodies have
been shown to produce false positive results for other
sandwich-type immunoassays for other cardiac and
noncardiac markers such as troponin and human
chorionic gonadotrophin (hCG), where low
concentrations have considerable clinical significance
(acute coronary syndromes and trophoblastic tumors,
etc.). The absence of these reports does not infer
freedom from these interferences. However, the clinical
significance of the presence of HAMA and heterophile
antibodies is diminished for BNP and NT-proBNP
assays; mild false-positive results do not have significant
clinical implications.
The cross-reactivity of assays for BNP and NT-proBNP
towards proBNP is an important consideration, since it is
now known that proBNP also circulates in the blood of
patients with heart failure. Because proBNP is the
precursor protein to both BNP and NT-proBNP, the
epitopes used to raise antibodies for these latter two
peptides are also present in the proBNP peptide
structure. ProBNP also exists in the blood of heart
failure patients as an O-linked glycoprotein [11]. There
are 7 potential sites for glycosylation from the center of
the molecule (from the non-BNP amino acid sequences).
There have been three studies that have tested the cross-
reactivity of proBNP towards commercial BNP and NT-
proBNP assays [2,3,12]. For BNP, the cross-reactivity
ranged from 17% to 38% for glycosylated proBNP and
from 5% to 14% for non-glycosylated proBNP [12]. For
NT-proBNP, the cross-reactivity ranged from 29% to
249% for non-glycosylated proBNP but with no cross-
reactivity towards glycosylated proBNP [3,12]. The
relative composition of blood with regard to the various
BNP, NT-proBNP, proBNP, and glycosylated forms
present in the blood of patients with heart failure is not
known. Therefore, comparing results of different
markers and different assays of the same marker will be
difficult.
Specimen
The in vitro stability of BNP and NT-proBNP has been
extensively studied. BNP is labile and must be collected
into tubes containing EDTA and tested ideally within 4
hours of collection. Serum, citrated and heparinized
plasma cannot be used. BNP can be stored for up to 24
hours at 2C to 8C for some assays. BNP is degraded
by contact activation of the blood coagulation system
[13] and can be stabilized by storage under frozen
conditions or with the addition of kallikrein protease
inhibitors [14]. In contrast, NT-proBNP is much more
stable than BNP and can be kept at refrigerated
temperatures for 3 days. NT-proBNP can be tested in
serum and plasma collected in either heparin or EDTA
[15]. When EDTA plasma is used, NT-proBNP
concentration is about 10% lower than for serum or
lithium heparin.
BNP, NT-proBNP, and ProBNP Reference Intervals
Reference intervals for BNP and NT-proBNP were
established by the first manufacturers of FDA-approved
assays (Inverness and Roche, respectively). For BNP, a
single cutoff of 100 ng/L was determined and validated
[16]. Commercial BNP assays developed and released
after the FDA clearance of the Inverness BNP assay
have configured their assay to maintain the use of this
cutoff concentration. For NT-proBNP, the manufacturer
(Roche Diagnostics) established two cutoff
concentrations: <125 ng/L for patients under 75 years of
age, and <450 ng/L for patients 75 years and older. All
other NT-proBNP assays make use of these limits as
well. Values for BNP and NT-proBNP in healthy
subjects are considerably lower than these limits and
show significant variance by age and gender. These
subgroups and lower cutoff concentrations have not been
incorporated into routine clinical practice. Figure 2
shows the relationship between age and gender for BNP
[17] and NT-proBNP [18]. The results show that males
have lower BNP/NT-proBNP concentrations than
females, and younger subjects have lower BNP/NT-
proBNP concentrations than older subjects. The reason
for these differences are not fully known. For gender,
some have suggested that the use of hormone
replacement therapy (HRT) in postmenopausal women
may result in higher BNP concentrations in this
population. Maffei et al. showed that treatment with
estrogen therapy caused BNP to increase from 12.6 to
19.8 ng/L after 3 months [19].
Decreasing renal function with age may be partly
responsible for the increase in BNP/NT-proBNP. In the
absence of heart failure, the concentration of BNP/NT-
proBNP increases in patients with a decreasing
glomerular filtration rate [20]. It is also possible that
200
B-Type Natriuretic Peptide, NT-proBNP and ProBNP
apparently healthy elderly subjects may have declining
cardiac function not detectable by current noninvasive
techniques. There is controversy with regard to which
marker, BNP or NT-proBNP, is more influenced by
renal dysfunction. Vickery et al. suggested that NT-
proBNP is more influenced than BNP in patients with
declining renal function [21]. However, deFilippi et al.
suggested that while both of these markers are
equivalent predictors of decompensated heart failure in
patients with kidney disease, NT-proBNP was a better
predictor of mortality than BNP (odds ratio of 4.5 vs.
2.6, respectively) [22]. There is currently no data on the
influence of declining renal function on measurements of
proBNP. Given that this marker is the same size as the
combination of BNP and NT-proBNP, it is likely that
this peptide will be retained to a greater extent in
patients with reduced glomerular filtration rates.
The body mass index is another variable that influences
the reference intervals for BNP and NT-proBNP. Obese
patients have lower BNP and NT-proBNP
concentrations than lean patients [23,24]. There have
been several theories as to the mechanism for these
observations. Natriuretic peptide receptors are found in
high concentrations in adipose tissues, suggesting that
BNP is degraded. However, this does not account for the
obesity effect seen for NT-proBNP, which is not
degraded by these receptors [23]. Others have suggested
that BNP may be a causative factor in development of
obesity [24]. The reduced clinical utility of BNP and
NT-proBNP in obese patients has prompted a search for
new serum biomarkers for heart failure that can
complement the use of these peptides.
Interpretation
Results for BNP and NT-proBNP assays are used as an
aid in the diagnosis and management of patients with
heart failure. The most documented usage is for patients
who present with acute symptoms of heart failure such
as dyspnea and shortness of breath. Two widely quoted
studies are the Breathing Not Properly (BNP) Trial for
BNP [25] and the Pro-BNP Investigation of Dyspnea in
the Emergency Department (PRIDE) Study for NT-
proBNP [26]. These studies were conducted on patients
presenting to the emergency department with symptoms
suggestive of heart failure. BNP and NT-proBNP testing
was conducted on blood collected at admission. Results
were compared to a final assessment conducted by two
attending physicians, using results of all diagnostic
procedures (symptoms, electrocardiography,
echocardiography, chest x-ray, clinical notes, response to
therapy, etc.), excluding the results of BNP or NT-
proBNP. A third physician was asked to adjudicate
discordances in results between the first two attendants.
The major objective of these studies was to determine
the clinical performance of these assays. Using a cutoff
of 100 ng/L in the BNP Trial, BNP produced a clinical
sensitivity and clinical specificity for diagnosis of heart
failure of 90% and 76%, respectively, with an area under
the receiver-operating characteristic curve (AUC-ROC)
of 0.91. Improved specificity can be achieved by
increasing the cutoff to 400 ng/L, prompting some
groups to advocate the use of two BNP cutoffs: 100 ng/L
for ruling out and 400 ng/L for ruling in heart failure
[27]. In the PRIDE study, a NT-proBNP cutoff of 450
and 900 ng/L for patients <50 and 50 years,
respectively, produced a clinical sensitivity of 93% and
91% and clinical specificity of 95% and 80%,
respectively. The AUC-ROC was 0.94. These studies
have also shown that BNP/NT-proBNP correlates with
the severity of heart failure, as assessed subjectively by
criteria established by the New York Heart Association.
Increased concentrations of BNP and NT-proBNP have
been associated with risk for cardiovascular death and
risk stratification for patients with heart failure [28],
acute coronary syndromes [29], stable heart disease [30],
and apparently healthy subjects without a history of
heart disease [31]. For patients with acute coronary
syndromes, the combination of BNP/NT-proBNP with
other markers such as cardiac troponin and C-reactive
protein further improves the odds ratio for prediction of
adverse events [32]. The acute release of BNP and/or
NT-proBNP appears to arise from the small cytoplasmic
pool and not from upregulation at the genomic level. The
success of these studies has led some investigators to
suggest a role for BNP/NT-proBNP for screening
patients for heart failure who are asymptomatic [33].
However, screening programs with natriuretic peptide
markers have not been adopted by national and
international clinical guidelines and therefore are not
currently used for this purpose.
There have been a few studies directly comparing the
clinical utility of BNP to NT-proBNP for use in patients
with heart failure. While there are minor differences in
performance, most studies have shown that the two
markers can be used interchangeably [34-36]. There
would be no benefit in a laboratory offering both tests.
Given that the markers are different, results from one
assay cannot be directly converted to the other.
Therefore, both assays should not be used on the same
patient.
BNP, NT-proBNP, and ProBNP Performance Goals
The biological variability of BNP and NT-proBNP has
been extensively studied in healthy subjects and patients
with stable heart failure [37-39]. Results vary according
to the time interval between serial collections. For short-
term (day-to-day) variation, BNP and NT-proBNP have
intraindividual imprecision (CVI) of 20% to 25%. The
analytical imprecision of analytical assays for BNP and
NT-proBNP (CVA) range from 3% to 5% for automated
immunoassays and 10% to 15% for point-of-care testing
assays and manual assays. Using 1/2CVI as the criteria,
all manufacturers of BNP and NT-proBNP assays are
able to meet imprecision goals based on biological
variability. The CVI is also used to compute the relative
change value (RCV)the difference in test results from
serial collections that is considered statistically
significant. With 95% confidence, the RCVs for BNP
and NT-proBNP range from 30% to 40% [39]. For
patients admitted to a hospital with decompensated heart
201
B-Type Natriuretic Peptide, NT-proBNP and ProBNP
failure, most studies have shown that the BNP and/or
NT-proBNP decreases by more than the RCV when
patients are successfully treated over the period of
hospital admission (compensation of heart failure),
suggesting that these tests are useful for short-term
monitoring. For longer-term (week-to-week) variation,
BNP and NT-proBNP have CVIs of 35% to 50% and
RCVs of 50% to 70%. Studies on the use of serial BNP
and NT-proBNP measurements for monitoring the
success of drug therapy on an outpatient basis do not
usually exceed these RCV limits. This puts into question
the value of BNP and NT-proBNP testing for long-term
monitoring of patients treated with chronic heart failure.
The College of American Pathologists offers an external
QC program for BNP and NT-proBNP. As of 2007,
there were about 2300 participants in the BNP survey
and 700 for NT-proBNP.
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index on diagnostic and prognostic usefulness
of amino-terminal pro-brain natriuretic peptide
in patients with acute dyspnea. Arch Intern Med
2007;167:400-7.
25 Maisel AS, Krishnaswamy P, Nowak RM,
McCord J, Hollander JE, Duc P et al, for the
BNP Multinational Study Investigators. Rapid
measurement of B-type natriruetic peptide in
the emergency diagnosis of heart failure. N
Engl J Med 2002;347:161-7.
26 Januzzi JL Jr., Camargo CA, Anwaruddin S,
Baggish AL, Chen AA, Krauser DG et al. The
N-terminal pro-BNP investigation of dyspnea in
the emergency department (PRIDE) study. Am
J Cardiol 2005;95:948-54.
27 Silver MA, Maisel A, Yancy CW. BNP
Consensus Panel 2004: a clinical approach for
the diagnostic, prognostic, screening, treatment
monitoring, and therapeutic roles of natriuretic
peptides in cardiovascular disease. Cong Heart
Fail 2004;10 (suppl 3):1-30.
28 Doust JA, Pietrzak E, Dodson A, Glasziou P.
How well does B-type natriuretic peptide
predict death and cardiac events in patients with
heart failure: systematic review. BMJ
2005;330:625.
29 Morrow DA, de Lemos JA, Blazing MA,
Sabatine MS, Murphy SA, Jarolim P et al.
Prognostic value of serial B-type natriuretic
peptide testing during follow-up of patients
with unstable coronary artery disease. JAMA
2005;294:2866-71.
30 Bibbins-Domingo K, Gupta R, Na B, Wu AHB,
Schiller NB, Whooley MA. N-terminal pro-B-
type natriuretic peptide and incident heart
failure in ambulatory patients with coronary
disease: data from the Heart and Soul Study.
JAMA 2007; 297:169-176.
31 Wang TJ, Larson MG, Levy D, Benjamin E,
Leip E, Omland T et al. Plasma natriuretic
peptide levels and the risk of cardiovascular
events and death. N Engl J Med 2004;350:655-
63.
32 Sabatine MS, Morrow DA, de Lemos JA,
Gibson CM, Murphy SA, Rifai N et al.
Multimarker approach to risk stratification in
non-ST elevation acute coronary syndromes:
simultaneous assessment of troponin I, C-
reactive protein, and B-type natriuretic peptide.
Circulation 2002;105:1760-3.
33 Hobbs FD, Davis RC, Roalfe AK, Hare R,
Davies MK, Kenkre JE. Reliability of N-
terminal pro-brain natriuretic peptide assay in
diagnosis of heart failure: cohort study in
representative and high risk community
populations. BMJ 2002;324:1498.
34 Hammerer-Lercher A, Neubauer E, Muller S,
Pachinger O, Puschendorf B, Mair J. Head-to-
head comparison of N-terminal pro-brain
natriuretic peptide, brain natriuretic peptide and
N-terminal pro-atrial natriuretic peptide in
diagnosing left ventricular dysfunction. Clin
Chim Acta 2001;310:193-7.
35 Tsutamoto T, Sakai H, Ishikawa C, Fujii M,
Tanaka T, Yamamoto T et al. Direct
comparison of transcardiac difference between
brain natriuretic peptide (BNP) and N-terminal
pro-BNP in patients with chronic heart failure.
Eur J Heart Failure 2007;9:667-73.
36 Clerico A, Fontana M, Zyw L, Passino C,
Emdin M. Comparison of the diagnostic
accuracy of brain natriuretic peptide (BNP) and
the N-terminal part of the propeptide of BNP
immunoassays in chronic and acute heart
failure: a systematic review. Clin Chem
2007;53:813-22.
37 Wu AHB, Smith AC, Mather JF, Duncan B,
White CM, Ahlberg A et al. Biological
variation for NT-pro- and B-type natriuretic
peptides and implications for therapeutic
monitoring of patients with congestive heart
failure. Am J Cardiol 2003,92:628
38 Bruins S, Fokkema MR, Pomer JWP,
DeJongste MJL, van der Dijs FPL, van den
Ouweland JMW et al. High intraindividual
variation of B-type natriuretic peptide (BNP)
and B-type proBNP in patients with stable
chronic heart failure. Clin Chem 2004;50:2052-
8.
39 Wu AHB. Serial testing of B-type natriuretic
peptide and NTpro-BNP for monitoring therapy
of heart failure: the role of biologic variation in
the interpretation of results. Am Heart J
2006;152:828-34.
203

B-Type Natriuretic Peptide, NT-proBNP and ProBNP

Table 1: Commercial Natriuretic Peptide Assay Characteristicsa

Assay Antigen Capture Ab Detection Ab

Abbott AxSYM BNP BNP a.a. 1-32 Scios (ring) Shionogi

(C-terminus)

Siemens Centaur BNP BNP a.a. 1-32 Shionogi Shionogi (ring)

(C-terminus)

Inverness Triage BNP a.a. 1-32 Scios (ring) Inverness

(Beckman Access) BNP (not characterized)

Shionogi BNP BNP a.a. 1-32 Shionogi Shionogi (ring)

(C-terminus)

Roche Elecsys NT-proBNP 1-76 Roche Roche

(Dade Dimension, Response (N-terminus) (central)
Biomedical)
NT-proBNP

Bio-Rad ProBNP 1-108 Hinge 76 region BNP (unspecified

(not FDA approved) (between BNP region)
and NT-proBNP)

a
Adapted from Apple FS. Personal communication.

Table 2: Methods of BNP, NT-proBNP, and ProBNP Analysis

Method 1: BNP, automated
Principle of analysis: Immunoassay
Comments: No standardization among automated BNP testing platforms. A diagnostic cutoff of 100 ng/L has
been established for all commercial BNP assays.

Method 2: BNP, POC

Principle of analysis: Immunochromatography coupled with immunoassay
Comments: Inverness (Biosite) Triage assay standardized to the Beckman Access assay.

Method 3: NT-proBNP, automated

Principle of analysis: Immunoassay
Comments: All NT-proBNP assays standardized to the Roche Elecsys platforms.

Method 4: NT-proBNP, POC

Principle of analysis: Immunochromatography coupled with immunoassay
Comments: All NT-proBNP assays standardized to the Roche Elecsys platform.

Method 5: Pro-BNP
Principle of analysis: Immunoassay
Comments: No commercial assay available. One manufacturer has developed a prototype assay.
204

B-Type Natriuretic Peptide, NT-proBNP and ProBNP

BNP (Inverness Triage) NT-proBNP (Roche)

70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
45-54 55-64 65-74 75-83 18-29 30-39 40-49 50-59 60-69

Age, years

Fig. 2. Reference intervals for BNP (Inverness Medical, n=2042) and NT-proBNP (Roche Diagnostics, n=1980) by age
and gender. Results in ng/L for both tests. Data from Redfield et al. [17] and Hess et al. [18] Aqua is male, blue is female
205

Barbiturates

Barbiturates
i
Steven C. Kazmierczak

Name: Amobarbital Butabarbital sodium Hexobarbital

Clinical significance: Refer to Chapter 47, Nervous System, in the 5th Edition of Clinical Chemistry:
Theory, Analysis, Correlation.

Molecular formula: C11H18N2O3 C10H15N2NaCO3 C12H16N2O2

Molecular weight: 226.27 D 234.23 D 236.26 D
Merck Index: 592 1471 4598
Chemical class: Barbiturate, organic heterocyclic
Structures: See Figures

Name: Pentobarbital sodium Phenobarbital Secobarbital sodium

Clinical significance: click here for all
Molecular formula: C11H17N2NaO3 C12H12N2O3 C12H17N2NaO3
Molecular weight: 248.6 D 232.23 D 260.27 D
Merck Index: 6998 7109 8268
Chemical class: Barbiturate, organic heterocyclic
Structures: See Figures

Principles of Analysis and Current Usage i Qualitative

Barbiturates are a group of drugs classified as hypnotics
and are prescribed for a number of conditions. They act
as sedatives and are used to help control seizure
disorders. Most commonly, phenobarbital is taken for
the control of epilepsy, and its determination, as part of
therapeutic drug monitoring, is usually the most
frequently performed analysis for this group of
compounds. Recently, the use of high-dosage
pentobarbital has become important in the therapy of
head trauma. However, barbiturates are also abused
substances; thus barbiturate determination in the
overdose patient is one of the major components of the
toxicological drug screen. Both qualitative and
quantitative tests are performed by laboratories. The
qualitative tests are selected for their rapid performance
in drug screening and usually react with any drug of the
barbiturate class. Most qualitative analyses are
performed on urine or gastric contents, since these body
fluids are best for drug screening. In contrast,
quantitative analyses are performed on blood, since
blood concentrations correspond most closely with
pharmacological effect.
ii
Barbiturates
Previous and current authors of this method:
First edition: Amadeo J. Pesce
Methods edition: Amadeo J. Pesce
Second edition: Amadeo J. Pesce
Third edition: Mark W. Linder
Fourth edition: Steven C. Kazmierczak
Fifth edition: Not updated
Qualitative analytical procedures for barbiturates using a
mercury complex colorimetric test [1] or absorbance
spectroscopy [2] are available and provide rapid analysis
for barbiturates in most body fluids or from tissue
sources [3]. However, the specificity of the colorimetric
mercury complex test is poor and the spectrophotometric
tests require sophisticated spectrophotometric equipment
which may not be available in modern clinical
laboratories (Table 1, Methods 1, 2, and 3).
Ultraviolet absorbance spectroscopy may be used to both
qualitatively identify as well as quantify barbiturates.
Following extraction, an absorbance spectrum is
obtained from 230 to 340 nm (Figure 1). The
characteristic spectrum of each barbiturate (Barbiturates:
Figure 2) allows for the qualitative identification. A
variation of this method, using differential spectroscopy,
allows for quantitation of the barbiturates [4] (Table
1,method 3).
Chromatographic procedures are also useful for the
identification of barbiturates; these include thin-layer
chromatography and gas chromatography. Thin-layer
chromatography (Table 1, method 4) is usually
performed on urine that has been acidified and extracted
into an organic phase. One places the sample on a thin-
layer chromatographic plate and identifies the position of
the barbiturate after chromatography by developing a
color reaction with one of several sprays, such as
potassium permanganate, mercuric sulfate, or
diphenylcarbazone [5]. Alternatively, a commercial thin-
layer method is available for the identification of several
barbiturates in urine which requires minimum
206
Barbiturates
manipulation of the specimen. The Rf value and the
color are the specific criteria for identification.
Gas chromatography detection (Table 1, method 5a) is
performed by the extraction of the acidified urine
specimen with toluene or other selected organic solvents
and the separation of the extracted analyte. An OV-
series column with a flame ionization detector is the
detection system most commonly used as part of an
overall screening procedure for drugs including
barbiturates [6]. Derivatization (methylation) improves
chromatographic behavior of these relatively nonvolatile
drugs. Figure 3 demonstrates the difference in retention
time and peak shape between chromatography of a
mixture of derivatized versus underivatized barbiturates.
The relative retention time is specific for each barbital
derivative and is used for presumptive identification.
A dedicated HPLC system for drug detection has also
been developed (Table 1, method 5b). Both serum and
urine can be analyzed with minimal sample preparation.
Following the addition of internal standard to the
specimen, a sample is automatically injected into the
system. The effluent is scanned by ultraviolet light in
the region of 190 to 300 nm and the combination of
retention time and ultraviolet absorbance characteristics
allow the identification of approximately 400 different
compounds. Unfortunately, the sensitivity of the system
for detecting barbiturates in urine is unacceptable. Also,
the system is subject to interference and
misidentification of drugs caused by co-eluting
compounds [7, 8].
Alternatively, the extract, as described in Method 5a, can
be analyzed with a gas chromatograph-mass
spectrometer (GC/MS) and identified by the pattern of
mass fragmentation (Table 1, method 6). Solid-phase
extraction methods have advantages over the common
liquid/liquid extractions and coupled to capillary gas
chromatography provide for a rapid and convenient
method for the qualitative identification of barbiturates
in urine [9].
Competitive binding immunoassays (Table 1, method 7)
for the detection of drugs of abuse in urine have
advanced in popularity because of the ease of use and
the rapidity of the analysis. With respect to barbiturates,
these assays rely on the cross-reactivity of the
structurally similar barbiturates with an antibody
developed against a specific barbiturate, commonly
secobarbital. These assays are available for automation
on a variety of random access analyzers and provide for
a rapid screen for the presence of barbiturates. These
assays have found wide acceptance in both forensic and
emergency toxicology. Because of the nature of these
assays, it is highly recommended that positive results be
confirmed by a more specific assay such as gas
chromatography/mass spectrometry [10].
The enzyme-multiplied immunoassay technique (EMIT)
for barbiturate in serum and/or urine is a homogeneous
enzyme immunoassay (EIA). The barbiturate
secobarbital is conjugated to the enzyme glucose-6-
phosphate dehydrogenase (G6PDH). The presence of
anti-barbiturate antibodies in the reaction mixture
suppresses G6PDH activity. Barbiturate introduced to
the reaction in the patient specimen competes for the
limited number of antibodies resulting in a dose-
dependent increase in G6PDH activity monitored by the
production of NADH at 340 nm [11].
Additional qualitative tests for barbiturates in urine and
serum based on competitive binding immunoassays are
available in formats applicable to both automated
analysis and formats which do not require
instrumentation. Other common automated procedures in
addition to EMIT, include the FPIA procedure and a
procedure based on the kinetic interaction of
microparticles in solution (KIMS) [12]. In the latter
method, absence of drug in the patient specimen, drug-
microparticle conjugates are aggregated into a lattice
through the interaction of anti-barbiturate antibodies
leading to increased absorbance of incident light. Drug
(barbiturate) in the sample competes for drug-
microparticle conjugate for a limited number of
antibodies in the reaction mixture. Binding of antibody
to drug in the sample inhibits microparticle lattice
formation (nephelometric inhibition) and diminishes the
increasing absorbance in proportion to the concentration
of drug in the sample.
Instrument-independent methods have also become very
popular for point-of-care use [13]. These devices allow
for visual detection of barbiturates and other abused
drugs [14]. One device utilizes colloid gold particles
conjugated to the drug(s) of interest and monoclonal
antibodies directed toward these drugs. Addition of
specimen containing drug(s) results in competition of
antibody for free drug(s) in urine and drug bound to gold
colloid. If the specimen contains drug(s) above the
detection limit of the system, some of the drug(s) bound
to the gold colloid particles will not be bound by
antibody. The reaction mixture is next transferred to a
nylon membrane with monoclonal antibodies bound to
the membrane. Specimens that have no drug(s) present
will have no colloid-bound drugs not bound to antibody
and thus pass through the membrane. Specimens, which
have drug(s) present above the detection limit of the
system will have some colloid-bound drugs not linked to
antibody and thus be able to bind to antibody on the
nylon membrane. Binding of the gold colloid to the
nylon membrane results in the appearance of a colored
bar which appears in the area containing antibody
directed toward the drug of interest.
These assays may be employed outside of the typical
laboratory environment. Although these methods have
proven useful, the user must comprehend the limitations
of the analyses and results should be confirmed by a
method which does not rely on competitive binding
immunoassay.
Quantitative
Techniques of quantitation include gas chromatography,
high performance liquid chromatography (HPLC),
ultraviolet spectroscopy, and a variety of competitive
207
Barbiturates
binding immunoassays. The various immunoassays are
the same in the respect that specificity of the analysis is
based on the binding specificity of the antibody for the
drug. The unique aspect of the various methods
available is with respect to the measured signal
generated by the presence of drug in the patient sample
(Table 1, method 8).
The fluorescent polarization immunoassay (FPIA), in
which fluorescein-labeled phenobarbital competes with
phenobarbital in the patient specimen for a limited
amount of antibody, is commonly used. The amount of
bound labeled phenobarbital is inversely proportional to
the phenobarbital in the patient sample. The intensity of
the polarized fluorescence is proportional to the amount
of antibody-bound labeled phenobarbital. The polarized
fluorescence is inversely proportional to the
concentration of phenobarbital in the patient specimen.
In general, quantitative immunoassays are used only
when the specific drug is known, such as phenobarbital.
In these procedures drug in the specimen competes with
labeled drug (method dependent) for a limited number of
antibody binding sites. Increases in specimen
barbiturate concentration leads to measurement of either
a proportional increase or decrease in the assay signal. It
should be noted that the antibodies employed in these
assays have a high degree of specificity for the intended
barbiturate, most commonly phenobarbital.
HPLC has been adapted for the measurement of the
barbiturates, but the most common procedure quantifies
phenobarbital in the presence of other antiepileptic
drugs, such as primidone and phenytoin. The
chromatographic system uses a 30 0.4 cm Porasil
column. The drug is detected with an ultraviolet
spectrophotometer, which monitors the absorbance at
254 nm [15].
Reference and Preferred Methods
Qualitative
Qualitative analysis of barbiturates is of clinical value in
screening for overdose or abuse. The identification of
barbiturates requires two independent methods, a class-
specific screening assay followed by a more specific
method for identification and confirmation. In the case
of an overdose, urine is the fluid of choice for screening.
In this circumstance, a competitive binding
immunoassay with general specificity for the barbiturate
class and good sensitivity is the best choice for a rapid
screen. The immunoassay and thin-layer
chromatography methods are the most commonly
utilized procedures and offer comparable clinical utility.
Following are the typical lower limits of detection
employed for the barbiturate immunoassay screens in
urine:
Secobarbital 200 ng/mL
Pentobarbital 500 ng/mL
Butabarbital 1000 ng/mL
Phenobarbital 6000 ng/mL
The presence of barbiturates should be confirmed by
thin-layer chromatography or the preferred method, gas-
liquid chromatography with mass spectrometric
detection.
Quantitative
For the analysis of mixtures of barbiturates, only the
gas/liquid and high performance liquid chromatography
procedures are applicable because they provide for
separation of the barbiturates prior to quantitation.
Phenobarbital is the most commonly quantitated
barbiturate in serum. Several immunoassays are
available with applications compatible with most testing
formats. Quantitation of the other barbiturates by
immunoassay is limited by availability of
immunoassays. For pentobarbital, both gas
chromatography and HPLC are acceptable.
When individual barbiturates are known to be present,
the ultraviolet procedure yields accurate results.
A comparison of the gas liquid chromatography, EMIT,
and fluorescence polarization is given in Table 2.
Specimen
The specimen can be blood, plasma, serum, urine, or
tissue extract, depending on the calibration of the
method. The drug is stable in any of these specimens for
1 week. However, most laboratories refrigerate or freeze
samples until analysis.
Interferences
The presence of detergents and liquid soap have been
reported to increase the barbiturates when measured
using FPIA [16].
Barbiturates Therapeutic Concentrations
Therapeutic concentrations in serum
1. Amobarbital Up to 8g/mL (<35 mol/L)
2. Butabarbital Up to 8 g/mL (<34 mol/L)
3. Pentobarbital Up to 4g/mL (<16 mol/L)
4. Phenobarbital 15 to 49 g/mL (6472 mol/L)
5. Secobarbital Up to 6 g/mL (<23 mol/L)
Barbiturates Performance Goals
For qualitative analysis of barbiturates, the evaluation
limits (CLIA-88) requires 80% participant consensus.
Interpretation
The barbiturates belong to the sedative hypnotic class of
drugs, and they function as central nervous system
depressants. Barbiturates have been used as hypnotics
for more than 80 years. They reversibly depress the
activity of all excitable tissues, inhibiting synaptic
responses.
There are many chemical derivatives of barbituric acid.
Those with hypnotic action have amyl of alkyl groups
substituted at the C-5 position. The barbiturate
derivatives are termed long acting, intermediate acting,
short acting, and ultrashort acting; the designation is
related to lipid solubility and time required to induce
208

Barbiturates

sleep. A classification summary of some barbiturates is estimation, and identification of barbiturates in

presented follows: biological material. Biochemistry 1956; 63:207-
13
Action Drug 4 Schumann GB, Lauenstein K, LeFever D,
Long Phenobarbital Henry JB. Ultraviolet spectrophotometric
Intermediate Amobarbital, butabarbital analysis of barbiturates. Am J Clin Pathol 1976;
Short Hexobarbital, pentobarbital, secobarbital 66:823-30
Ultrashort Sodium pentothal 5 Ganshirt H. Pharmaceutical products. In Stahl,
E., editor: Thin-layer chromatography: a
laboratory handbook, New York, 1962
Academic Press, 318-319.
The short-acting barbiturates are highly lipid soluble and
6 Sunshine I. Methodology for analytical
rapidly pass through the bloodbrain barrier. The
toxicology. Cleveland, 1975, CRC Press, Inc.
molecule is a weak acid, and the carbonyl group at the
34-44.
C-2 position tautomerizes between the lactam and lactim
7 Adams AK, Essien H, Binder SR.
configurations. The pI for barbituric acid is 7.4.
Identification of drugs in physiological fluids
following on-line liquid chromatographic
The most commonly prescribed barbiturate is
purification and analysis. Ann Biol Clin
phenobarbital, which is used in the therapy of seizure
1991;49:291-7
disorders.
8 Binder SR, Adams A., Regalia M, Essien H,
Rosenblum R. Standardization of multi-
Pentobarbital is used in very high concentrations in
wavelength UV detector for liquid
severe trauma to reduce cerebral edema. The mode of
chromatography-based toxicological analysis. J
action is not known, but the barbiturates in high
Chromatogr 1991;551:449-59
concentrations reduce central nervous system blood flow
9 Pocci R, Dixit V, Dixit VM. Solid-phase
and metabolism and can reduce oxygen requirements by
extraction and GC/MS confirmation of
50%.
barbiturates from human urine. J Anal Toxicol
1992;16:45-7
The barbiturates can be habit forming, and patients can
10 Urine testing for drugs of abuse, National
develop tolerance to the drug. Withdrawal symptoms
institute on drug abuse (NIDA) Research
can occur in abusers of the drug, and seizure is a
Monograph 73, 1986.
common sign of withdrawal. The removal of barbiturate
11 Ollerich M: Enzyme immunoassays in clinical
from a patient can be enhanced when the urine is made
chemistry: Present status and trends. J Clin
alkaline. However, the principal route of elimination of
Chem Clin Biochem. 1980;18:197-206.
barbiturates is metabolism in the liver. Several
12 Adler FL, Liu CT. Detection of morphine by
mechanisms take place: oxidation of the substituent
hemagglutinationinhibition. J Immunol
groups at the C-5 position, N-dealkylation, and
1971;106:1684-5
destruction of the barbiturate ring. The barbiturates
13 Biosite Triage and Roche Abutrack.
modulate cytochrome P-450 dependent metabolism of
14 Wu AHB, Wong SS, Johnson KG, Callies J,
other drugs either by inhibiting the biotransformation
Shu DX, Dunn WE, Wong SH. Evaluation of
thus increasing the serum half-life of the drug, or by
the Triage System for emergency drugs-of-
enhancing metabolism of certain drugs through
abuse testing in urine J Anal Toxicol
induction of specific enzymes of the cytochrome P-450
1993;17:241-5
system.
15 Kabra PM. Simultaneous liquid-
chromatographic determinations of 12 common
References
sedatives and hypnotics in serum Clin Chem.
1 Curry AS. Rapid quantitative barbiturate
1978;24:657-62,
estimation, Br Med J 1964; 1:354-5
16 Schwarzhoff R, Cody JT. The effects of
2 Walker JT, Fisher RS, and McHugh JJ.
adultering agents on FPIA analysis of urine for
Qualitative estimation of barbiturates in blood
drugs of abuse. J Anal Toxicol 1993;17:14-7
by ultraviolet spectrophotometry, Am J Clin
17 Data taken from College of American
Pathol 1948; 18:451-61
Pathologists (CAP) Profiency testing survey,
3 Broughton PMG. A rapid ultraviolet
1993 specimens Z-11, Z-12 and Z-13.
spectrophotometric method for the detection,
209

Barbiturates

Tables

Table 1: Methods of Barbiturate Analysis

Qualitative
Method 1. Extraction and colorimetric reaction
Principle: Barbiturate extracted from slghtly acidic solution and complexed with Hg2+; reacts with
diphenylthiocarbazone to form an orange colored product
Usage: Blood, plasma, serum, urine, gastric contents
Comments: Nonspecific for barbiturates
Method 2. Extraction and ultraviolet spectroscopy
Principle: Barbiturate extracted free of other ultraviolet-absorbing compounds; ultraviolet spectrum is
characteristic and specific for each drug
Usage: Blood, plasma, serum, urine, gastric juice, tissue
Comments: Original specific method; some interferences; can be quantitative; not recommended for mixtures
Method 3. Ultraviolet difference spectroscopy
Principle: As in Method 2, extracted barbiturate is divided into aliquots and difference between spectra at pH
greater than 13 and at pH 10.5 recorded
Usage: Blood, serum
Comments: More specific than ultraviolet spectroscopy; requires recording dual beam spectrophotometer
Method 4. Thin-layer chromatography
Principle: Extracted barbiturate chromatographed, its Rf factor (relative mobility factor) and reaction with stain
recorded; Rf factor and color reactions presumptive for barbiturates
Usage: Blood, plasma, serum, urine, gastric juice, tissue
Comments: Used as either qualitative or confirmatory test; can separate mixtures of barbiturates
Method 5a. Gas chromatography
Principle: Extracted barbiturate chromatographed and its retention time recorded; parent drug and possible
degradation products have specific retention times
Usage: Blood, plasma, serum, urine, gastric juice, tissue
Comments: Not quantitative; degradation of some barbiturates; not a gaussian elution pattern; can separate
mixtures
Method 5b. High-performance liquid chromatography
Principle: Extracted barbiturate chromatographed and its retention time recorded; detection by ultraviolet
spectra; parent drug and possible degradation products have specific retention times
Usage: Blood, plasma, serum, urine, gastric juice, tissue
Comments: Not quantitative; can separate mixtures
Method 6. Gas chromatographymass spectrometry
Principle: Steps identical to those for method 5; mass spectrophotometric detector can identify specific
barbiturates and resolve mixtures
Usage: Blood, plasma, serum, urine, gastric juice, tissue
Comments: Improves identification of simple gas chromatographic method
Method 7. Competitive binding immunoassays
Principle: Competition between labeled drug (method dependent) and drug in the sample for a limited number of
antibodies in the reaction mixture
Usage: Urine; serum (limited use)
Comments: Positive results corresponds with an assay signal for the sample which exceeds the signal for a
known concentration calibrant (cutoff)

Quantitativeall barbiturates
Method 8. Ultraviolet spectroscopy (similar to method 2)
Principle: Extracted barbiturate has characteristic absorbance spectrum, with amount of absorbance proportional
to concentration; some identification depends on spectral characteristics
Usage: Blood, plasma, serum, urine, gastric juice, tissue
Comments: Original method, but subject to interference by endogenous compounds and other drugs; does not
work well on mixtures
Method 9. Enzyme Immunoassay
Principles: EMITRelief from inhibition of phenobarbital labeled G6PDH; G6PDH- dependent production of
NADH is the measured signal; the signal is proportional to sample phenobarbital concentration
Radial partition immunoassayPhenobarbital in the patient specimen and alkaline phosphatase (Alk
phos) labeled phenobarbital compete for binding to immobilized anti-phenobarbital antibodies; Alk phos
dependent production of a fluorescent product is the measured signal; the signal is inversely proportional to
sample phenobarbital concentration
Usage: EMITSerum
210

Barbiturates

RPIASerum
Comments: EMIT most commonly used method; applications available for a wide variety of automated
analyzers
RPIA common; limited instrument options
Method 10. Fluorescence Polarization
Principle: Competitive binding with fluorescence polarization; bound labeled drug has higher polarized
fluorescence than free; amount of polarized fluorescence inversely proportional to the concentration of the drug
Usage: Serum, plasma
Comments: Commonly used procedure; lowest coefficient of variation
Method 11. Turbidimetric inhibition (TI)
Principle: Inhibitions of insoluble particlephenobarbitalantibody aggregates; the signal (rate of particle
aggregation) is inversely proportional to the sample phenobarbital concentration
Usage: Serum, plasma
Comments: Available as liquid reagents
Method 12. Nephelometric inhibition
Principle: Competitive inhibition of precipitation; signal is inversely related to sample phenobarbital
Usage: Serum
Comments: Liquid reagents, limited instrumentation compatibility

Table 2: Accuracy and Precision Comparison of Competitive binding Immunoassays for Quantitative Analysis of
phenobarbital in serum [17].

Quantitative Analysis of phenobarbital in serum. 1

Assay Weighed in value Mean measured value S.D. C.V.
6.8 mg/L
FPIA 6.14 0.37 6.0%
EMITa 6.60 0.31 4.7%
RPIA 7.31 0.66 9.1%
TI 7.22 0.63 8.7%
EMITb 6.46 0.41 6.4%
26.4 mg/L
FPIA 23.75 1.00 4.2%
EMITa 24.87 1.06 4.3%
RPIA 28.53 2.62 9.2%
TI 24.43 1.39 5.7%
EMITb 24.54 1.70 6.9%

75.6 mg/L
FPIA 63.21 3.36 5.3%
EMITa 62.15 3.22 5.2%
RPIA 72.07 8.06 11.2%
TI 62.93 3.70 5.9%
EMITb 67.74 5.27 7.8%
aDuPont reagents. bSyva reagents.
1
Data taken from College of American Pathologists (CAP) Profiency testing survey, 1993 specimens Z-11, Z-12 and Z-13.
211

Barbiturates

Table 3: Comparison of Assay Conditions for Serum Phenobarbital

Enzyme-multiplied Fluorescence
Gas-liquid immunoassay polarization
chromatography technique immunoassay
Sample size (mL) 1 0.05 0.02
Sensitivity (mg/L) 2 5 0.34
Coefficient of 8.5 8.2 5.0
variation (%)
Interference Phenobarbital and Some barbitals Some barbit-
mephobarbital not urates
separated
Time 1h 15 to 30 min 20 min

Figures

Figure 1: Barbiturates

Absorption spectra of a barbitone solution, 13.5 mg/mL: in 0.45 M NaOH, solid tracing, and in borate buffer (pH 10.5),
dashed tracing. (From Broughton, P.M.G.: Biochem. J. 1956; 63: 207-213.)
212

Barbiturates

Figure 2: Barbiturates

Ultraviolet absorption spectra of barbiturates in methanol, solid; in 0.1 M NaOH, dashes; and in 0.1 M HCl, dots and
dashes. A, Phenobarbital; B, butabarbital; C, secobarbital.
213

Barbiturates

Figure 3: Barbiturates

Gas chromatographic tracing of barbiturates separated as underivatized (above) and derivatized (below) form. For
derivatization, procedure outlined in manual method was used. Mixed barbiturate standard was 10 mg of each barbiturate
dissolved in 10 mL of CH3OH. 1, Butabarbital; 2, amobarbital; 3, pentobarbital; 4, secobarbital; 5, phenobarbital; 6,
phenytoin. Comparative testing was performed with the use of Hewlett-Packard 583A with a flame ionization detector and
a 2 mm inner-diameter glass column 6 feet long packed with 3% OV-17 on Chromosorb W (HP) of 800/100 mesh.
Nitrogen was used as a carrier gas at a flow rate of 30 mL/min. Temperature programming was performed from 115 to
270 C at a 15 C/min increase after an initial hold time of 0.5 min. (Courtesy Hassan, F.M., and Fahey, C.: University of
Cincinnati.)
214

Barbiturates

Figure 4: Barbiturates

Elution pattern of barbiturate standards after HPLC. 1, Methyprylon; 2, phenobarbital; 3, butabarbital, 4, butalbital; 5,
pentobarbital; 6, amobarbital; 7, glutethimide; 8, secobarbital; 9, nitrazepam internal standard; 10, methaqualone.
the dissolved standard. This is mixed and frozen as 1-
Procedure: Gas Chromatography mL aliquots, which are stable for 1 year. The resultant
calibration mixture contains 10 g/mL (approximately
Principle
The barbiturates are extracted from acidified serum with 45 mol/L) of each of the barbiturates.
an organic solvent. They are then back-extracted into Assay
tetramethylammonium hydroxide. The extracts are Equipment: Gas chromatographic equipment,
introduced into a gas chromatograph, and the methylated including a flame ionization detector; a 6-foot glass
derivatives of the drugs are formed on the column. The column, 2 mm inner diameter, packed with 3% OV-1 on
gas chromatograph detects, separates, and quantitates the 80/100 mesh Chromasorb W (HP).
drugs of interest, using aprobarbital as an internal
standard.
Sample preparation
Specimen
a. To a 10 mL screw-capped tube, add 1.0 mL of
Reagents and Materials patient serum (or serum calibrator), 0.5 mL of
1. Toluene. Chromatoquality. This is stable for 2 1M H3PO4, 100 L of aprobarbital internal
years at room temperature. standard, and 5 mL of toluene.
2. Methanol. Chromatoquality. This is stable for b. Shake vigorously for 1 min and centrifuge.
2 years at room temperature.
c. Transfer upper toluene layer to a 5 mL conical
3. Tetramethylammonium hydroxide. This is
centrifuge tube.
stable for 2 years at room temperature.
4. Barbiturates. Obtained in pure form from d. Add 12 L of tetra methylammonium
manufacturer. These are stable for 2 years at room hydroxide, shake for 1 min, and centrifuge.
temperature. Discard the upper layer.
5. 1 M H3PO4. Add 6 mL of concentrated e. Inject 2 L of lower layer into gas
H3PO4 (phosphoric acid) to 80 mL of H2O. This is chromatograph. Start programmer as the
solvent peak is eluted.
stable for 1 year at room temperature.
6. Working aprobarbital, internal standard,
Chromatographic parameters
1000 g/mL (4.76 mol/mL). Add 50 mg of each of
aprobarbital, butabarbital, pentobarbital, and secobarbital a. Column program, 90 to 200 C at 8 C/min
in 50 mL of methanol. To 99 mL of serum add 1 mL of b. Injection port temperature, 275 C
215

Barbiturates

c. Detector temperature, 280 C 3. Phosphate buffer (pH 4.4, 0.15 mmol/L).

d. Nitrogen flow, 60 mL/min Prepare 1 mol/L KH2PO4 by dissolving 13.6 g
e. Attenuation, 16, range 10-12 of KH2PO4 in 100 mL of distilled water.
f. Recorder, 1 mV and 0.5 inches/min Prepare 0.9 mol/L phosphoric acid by adding
4. Sample run time is approximately 20 min. 6.125 mL of phosphoric acid to distilled water.
Bring the volume to 100 mL.
5. Run known quality-control specimens with
each run. 4. Add 300 L of 1 mol/L KH2PO4 to 1800 mL
of distilled water. Using a pH meter, adjust the
pH to 4.4 with 0.9 mol/L phosphoric acid
Calculations (approximately 50 L).
1. Measure retention times of each peak, and 5. Acetonitrile: phosphate buffer (mobile
compare to the serum calibrator standards. phase). Add 190 mL of acetonitrile to 810 mL
2. To calculate each corresponding drug: of the phosphate buffer; filter through a 0.2 m
pore filter and degas under vacuum for 5 min.
Peak height of drug = Ratio of drug to aprobarbital 6. Internal standard (nitrazepam). Stock
Peak height of internal standard solution 1 mg/mL. Dissolve 10 mg nitrazepam
in methanol, and bring to 10 mL with methanol.
Divide peak height ratios of unknowns by peak Store at 4 C. Stable for 1 year.
height ratios of the calibration standard. 7. Working internal standard solution, 0.01
Multiply resulting ratio by 10 g/mL: mg/mL. Dilute 1 mL of working standard to
100 mL with sodium acetate buffer. Store at 4
Ratio of unknown 10 g/mL = Unknown (g/mL) C. Stable for 6 months.
Ratio of calibrated material 8. Calibration standards. Dissolve the desired
barbiturate in methanol, and dilute to a
concentration of 5 g/mL in serum. For
Procedure: High-Performance Liquid methyprylon and phenobarbital, dilute to a final
Chromatography concentration of 20 g/mL. Stable for 3 months
Principle at 4 C.Assay
For the specific quantitative analysis of any of the Equipment: high performance liquid chromatography
following barbiturates and drugs that are extracted equipment such as Waters HPLC System including
together: injector column heater, pump, detector, recorder, and a
15 cm C18 Bondapak Column; Vac Elut extraction
Barbiturates Other drugs apparatus; and 1 mL C18 Bond Elut solid-phase
Amobarbital extraction columns.
Glutethimide 1. Sample preparation
Butabarbital Methaqualone
a. Set up a Bond Elut column for each
Butalbital Methyprylon
sample to be analyzed. With vacuum
Phenobarbital
on, prime columns twice with
Pentobarbital
methanol and twice with distilled
Secobarbital
water.
The above compounds are isolated by use of a b. Turn off vacuum.
C18 bonded-phase column and analyzed by HPLC with c. To the respective columns, add 0.5 mL
spectrophotometric detection at 214 nm, with of working internal standard followed
comparison of retention times and peak height ratios of by 0.5 mL of appropriate sample.
unknowns against known standards and controls. d. Turn the vacuum on to pull samples
through the columns.
Specimen e. Immediately wash columns twice with
Serum, plasma distilled water.
f. Turn off vacuum.
Reagents and Materials
1. Methanol (HPLC grade). This is stable for 2 g. Wipe needle tips inside Vac Elut, and
years at room temperature. position labeled collection tubes in
rack.
2. Sodium acetate buffer (pH 4.5, 0.1 mol/L).
Dissolve 8.2 g of sodium acetate in distilled h. Add 100 L of methanol; wait 20 sec.
water and bring to 1000 mL with distilled i. Turn on vacuum; then turn off after
water. Adjust the pH to 4.5 with acetic acid if methanol passes through.
necessary. Store at room temperature in brown j. Repeat with another 100 L of
bottle. Stable for 2 years; check monthly for methanol.
growth.
216

Barbiturates

k. Remove collection tubes, and with a d. A glutethimide metabolite has the

disposable glass pipet, transfer the same retention time as butalbital.
contents of a collection tube to a larger e. Hemolyzed, lipemic, and icteric
12 75 mm glass tube. samples do not interfere with the
l. Inject 20 L into the HPLC system. analysis.
2. Chromatographic parameters
a. Flow rate, 1.5 mL/min Pentobarbital, Quantitative by Gas Chromatography
b. Column temperature, 60 C
c. Detector AUFS, 0.1 (absorbance units I. Principle
at full scale)
d. Detector wavelength 214 nm Pentobarbital is isolated from acidified
e. Chart speed, 0.5 cm/min biological fluids by liquidliquid solvent
extraction, and, after concentration by
Calculations evaporation, can be separated from other
A sample chromatogram of a series of barbiturate barbiturates by a gas chromatographic capillary
standards is presented in Barbiturates: Figure 4. column, detected utilizing a nitrogen
phosphorus detector, and quantitated by its peak
Calculate the peak height ratio of drug of interest to height ratio to known internal standard and
internal standard. subsequent calculation from known calibrator
standards.
Peak height (mm) of drug
Peak height (mm) of internal standard = Peak height II. Specimens of Choice
ratio
Preferred specimen is serum 1 mL (minimum
Compare elution times of standards to the elution times volume of 0.5 mL), collected in a red-top tube
of all unknown peaks of interest. See Barbiturates: with no separator gel. Plasma, collected in
Figure 4 for elution order of the various drugs. either heparinized or EDTA tubes, is also
acceptable. Specimens may be icteric or
Concentration unknown (g/mL) = slightly hemolyzed, but should not be grossly
hemolyzed or lipemic. Specimen should be
Peak height ratio Concentration of
obtained during the acute phase of toxicity or,
in patient or control appropriate drug
for TDM purposes, as a trough level drawn
Peak height ratio in standard (g/mL)
of calibration standard immediately prior to the dose of drug, or during
IV infusion.
Notes
1. The following limits of sensitivity for each to III. Indications
the barbiturates have been observed:
Compound Limit (g/mL) To document overdose or monitor therapeutic
Amobarbital 1 dosing of pentobarbital.
Butabarbital 1
Butalbital 1 IV. Reagents and Materials
Phenobarbital 5
Pentobarbital 1 A. Aprobarbital internal standard (5
mg/100 mL). To a 100 ml volumetric
Secobarbital 1
flask add 5 mg of pure aprobarbital
Glutethimide 1 (Applied Science Laboratories).
Methaqualone 1 Dissolve in deionized water and dilute
Methyprylon 5 to mark with the same. Store in 100
mL reagent bottle at 4 C. Stable for 3
Specific negatives are reported either as none months.
detected,or less than an appropriate limit of B. Stock Pentobarbital Standard (1
quantitation. mg/mL). To a 10 mL volumetric flask
2. Specificity add 10 mg of accurately weighed
a. Phenytoin is known to have the same Pentobarbital (Applied Science
retention time as amobarbital. Laboratories). Dilute to mark with
b. Clonazepam is known to have the methanol. Transfer to two 5 mL drug
same retention time as methaqualone. standard storage vials and refrigerate
at 4 C. Stable for 1 year.
c. Carbamazepine has a similar retention
time to secobarbital. C. Working 20 mg/mL Pentobarbital
Calibration Standard. To a 100 mL
217
Barbiturates
volumetric flask, add approximately
50 mL of Utak Negative Serum Pool.
With a 2 mL volumetric pipet add 2
mL of stock pentobarbital standard.
Mix well while adding additional
Negative Pool to the mark. Aliquot 1
mL portions into 12 75 plastic tubes.
Cap and store frozen at -20 C. Stable
for 1 year.
D. Working Pentobarbital Calibration
Standards (for the establishment of a
full linear curve). To 10 mL 16 100
glass tubes add the following amounts
of stock pentobarbital standard and
Utak Negative Serum Pool, resulting
in the concentrations noted:
Standard Concentration
(mg/mL) L of Pool SerumL
of Stock Pentobarbital
0 500
5 497.5
10 495
20 490
50 475
80 460
100 450
Prepare fresh as needed and proceed to Section VI.A.2.
D. 16 100 mm glass culture tubes with
caps
E. Utak Negative Serum Pool (Utak
Associates)
F. Utak Pentobarbital Control Serum
(Utak Associates)
G. 300 L Eppendorf pipet
H. 0.5 mL Eppendorf Pipet
I. 1 10 mL serological pipets
J. Glass disposable pasteur pipets
K. Hamilton 10 and 50 L syringe
L. 0.5 N HCl - To a 1 L volumetric flask
add approximately 600 ml of
deionized water. Carefully add 42.8
mL of concentrated HCl. Slowly
dilute to mark with deionized water.
Transfer to a 1 L reagent bottle. Store
at room temperature. Stable for 1
year.
M. 5 mL glass Reacti-vials
N. Ethyl acetate (Chromatoquality)
O. Methanol (Chromatoquality)
V. Instruments and Parameters
A. Hewlett Packard 5970 Gas
Chromatograph Column: Ultra-1 (Hewlett Packard Co.)
Temperatures: Column temperature program 120 to 300
C at 15 C/min after a 0.5 min hold; detector temperature
250 C; injector temperature 260 C.
Attenuation: 4 (Note: May need to be
optimized/adjusted with each run)
Carrier gas: helium
Gas pressures at tank: air, 20; helium, 40; hydrogen, 30
mm Hg
Gas flows (mL/minute): air, 100; hydrogen, 3; helium,
25
B. Gas Chromatograph Method:
PENTOSR2
C. Heating block: 60 C with nitrogen gas
attachment
D. Extraction rocker
VI. 0 Procedure
Note: Each run will consist of a control, a 20
2.5mg/mL calibration standard, and patient
serum analyzed in singlecate.
5
A. Sample Preparation and Entering Run Identifiers
10
1. To a 10 mL 16 100 glass tube add 0.5
mL of control, frozen calibration standard, or patient
25 a 0.5 mL Eppendorf pipet.
serum with
2. With a 300 L Eppendorf pipet add
300 L40of aprobarbital internal standard to each tube.
Vortex mix 5 sec.
503. With a 1 mL serological pipet add 0.1
mL of 0.5 N HCl to each tube, then vortex mix.
4. With a 10 mL serological pipet add 5
mL of ethyl acetate. Cap tubes and extract on rocker for
5 min.
5. Centrifuge samples for 3 min (Note:
Break any emulsions, which occur with a glass stirring
rod, and recentrifuge).
6. With a glass Pasteur pipet transfer
upper solvent layers to premarked and preheated 16
100 glass tubes. Evaporate to dryness at 60 C under
nitrogen.
7. Reconstitute dried vials in 100 L of
methanol, then vortex for 10 sec.
8. Inject 1 L of reconstituted 20 g/mL
standard solution into the "B" detector (NPD) of the gas
chromatograph, then puch the GC START button.
VII. Calculations and Quality Control
A. Integrated Results
The 5890, after performing a calibration, will print
control and patient data directly, in g/ml concentration
units. When integrated, test results are questioned, or
when calibration cannot adequately be performed,
218

Barbiturates

proceed with manual calculation. 1. Optimal serum concentrations are specific and
relative to the drug's use:
B. Manual Calculation 3040 g/mL optimal during treatment for
intracranial pressure
1. Observe the peak height (PH) amounts noted 13 g/mL for conventional anesthetic uses
for pentobarbital and internal standard on the hardcopy
report. Compute the peak height ratio (PHR) for the 20 Toxic effects may be noted at concentrations
g/mL standard, the Utak Control, and patient greater than 5 g/mL, and life-threatening
chromatograms: levels are generally greater than 10 g/mL.

PHR = Peak height of pentobarbital 2. Run a complete pentobarbital curve to establish

Peak Height of Internal Std.
Note: If printed peak heights are not available, manual
peak heights should be measured in millimeters.
2. Compute the concentration of Pentobarbital in
the control and patient:
Concentration (g/ml) =PHR of Control or Patient 20
PHR of 20 g/ml Standard
C. An acceptable run includes control values
within acceptable limits (i.e., refer to "Internal QC"
Program).
linearity monthly, when changing GC columns,
or when other changes dictate (i.e., detector
repair).
3. For concentration above 100 g/mL, dilute the
patient specimen 1:1 (250 L plus 250 L) with
Utak Negative Pool, add internal standard, and
reanalyze. Multiply calculated g/mL result by
2.
4. The current method sensitivity for pentobarbital
is 1 g/mL. Negative results should be reported
as "less than 1 g/mL."
5. No known interfering compounds.

XI. references
Heinemeyer, G., Ther. Drug Monit. 8:145-150,
1986.

Notes and Interpretation

219
Bence Jones Protein
Bence Jones Protein
Stanley S. Levinson
Name: Bence Jones protein
Clinical significance: Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation
Molecular mass: 20,000 D
Merck Index: 1031
Chemical class: Protein
i trichloroacetic or sulfosalicylic acid or a protein-binding
Principles of Analysis and Current Usage
More than a century after Dr. Henry Bence Jones received
a peculiar urine specimen from Dr. Thomas Watson,
Edelman and Gally showed that the Bence Jones protein
(BJP) was identical to the free light chain (FLC) of IgG
[1,2]. The name Bence Jones protein is now commonly
used to describe monoclonal FLC in urine and will be used
here [3,4]. The urinary substance in the peculiar urine
specimen had the property of precipitating when heated to
a temperature between 45C and 60C and redissolving
when boiled (Table 1, Method 1). This property, which did
not show good sensitivity for BJP, was used as the basis
for the Bence Jones heat test, which served as the test for
this urinary protein in clinical laboratories until the early
1970s.
Although some continue to use high-resolution protein
electrophoresis (HRPE) (Table 1, Method 3) as a screen
for BJP, it is now recognized that immunofixation
electrophoresis (IFE) (Table 1, Method 5) is the most
sensitive and specific method for screening and
identification of BJP. This is the method recommended by
the College of American Pathologists (CAP) Diagnostic
Resource Committee [5] and the International Federation
of Clinical Chemistry (IFCC) Committee on Plasma
Proteins [5,6] (see Table 1, Method 5). A new, enhanced
nephelometric/turbidimetric method for identifying free
light chains in serum has recently been developed and is in
a continued process of being tested (Table 1, Method 6)
[7].
Reference and Preferred Methods
Reference Methods
There are no reference methods for any of the techniques
or procedures discussed in this paper.
Preferred Methods
Measurement of Total Protein
Most laboratories screen urine for total protein with a dip
strip impregnated with a pH indicator dye, followed by a
more specific confirmatory test such as precipitation with
i helpful for identifying severe prozoning of very large BJP
Bence Jones Protein
Previous and current authors of this method:
First edition: Gayle Birkbeck
Methods edition: Gayle Jackson
Second edition: Not updated
Third edition: Not updated
Fourth edition: Gayle Jackson
Fifth edition: Stanley Levinson
dye such as Coomassie blue or Ponceau S (Table 1,
Method 2). The degree to which these confirmatory
methods measure FLC is unclear. The strips mainly
measure albumin, and it is widely known that a mildly
positive or negative result by dip strip analysis, followed
by a grossly positive confirmatory result, may indicate the
presence of an unsuspected BJP. In actual fact, this type of
discrepancy cannot be relied on, because most patients
with renal disease are spilling albumin along with BJP into
the urine and test positive by both approaches. It is
important to follow up all cases of proteinuria with
electrophoretic analysis [5,8]. Except for the biuret method
that reacts with the peptide bonds in all proteins, most
methods detect albumin more effectively than globulins,
and as a result, these methods are poorly accurate. Also,
the calibrators may contain only albumin or an array of
proteins different from those found in samples producing
more variability.
Since some samples may contain large amounts of protein
with very little BJP, whereas others may contain large
amounts of BJP with little other protein, measurement of
urinary protein is not necessary prior to specifically
assaying for BJP and may be misleading. What is
important is to maximize the sensitivity of detecting very
low concentrations of BJP on IFE by concentrating all
samples > 100-fold for the initial screen [5].
Identification of BJP by Urine HRPE (UPE) and IFE (See
Immunoelectrophoresis Method)
UPE and IFE should be performed on all samples
suspected of Bence Jones proteinuria (BJPuria) (see Table
1, Methods 3 and 5). These initially should be performed
on samples concentrated approximately 100- to 150-fold
and directly applied to the gel without any dilution,
regardless of the amount of protein. If the gel is
overloaded and the pattern is confusing, it may be
necessary to rerun the assay after making dilutions with
saline. The sensitivity of UPE for BJP on concentrated
samples is in the order of 10 mg/L [6], but IFE is about
fivefold more sensitive [9]. Nevertheless, UPE is very
that may occur with IFE, so the two techniques
supplement one another and should be run together, as will
be discussed below [10]. Unless there is very strong
reason to suspect a BJP, for initial screening only, kappa
and lambda antisera against FLC and light chain bound to
heavy chain (intact) (F & B) should be run (antisera
against IgG, IgA, IgM, and free-light-chain-only antisera
220
Bence Jones Protein
is not needed).The older immunoelectrophoresis technique
is far less sensitive and should not be used for identifying
BJP in urine [5,6] (Table 1, Method 4) and is no longer
used.
If the initial screen with F & B kappa and lambda indicates
that there is a BJP, the assay should be repeated using
antisera against IgG, IgA, and IgM to ensure that the band
is not an intact immunoglobulin. As a cost cutting measure
and because of its low avidity compared to antibody
against F & B light chain, the IFCC Committee
recommends fixation only with F & B antisera and not
concomitantly with antisera against free light chain only,
unless it is suspected that a BJP is co-migrating with or
near an intact monoclonal protein [6].
Identification of Light Chains by Immunonephelometry or
Turbidimetry
Some laboratories measure kappa and lambda in
unconcentrated urine by routine nephelometric and
turbidimetric methods, using antibody against F & B
kappa and lambda (Table 1, Method 7) [11]. This
approach can be used as a screen. If the kappa-to-lambda
(ratio is increased or decreased, the urine can then be Importance of Identification
assayed by IFE after 100-fold concentration to see if there
is a BJP present. If the kappa and lambda are both
undetectable or the ratio is within the reference interval, it
is very unlikely that there is a BJP present [11]. The major
problem with this approach is that because of poor
methodological sensitivity, a majority of urines show only
a kappa or lambda that is undetectable while the other
light chain type is detectable, so a useful ratio cannot
be calculated (i.e., kappa < 1.5 mg/dL but lambda = 5
mg/dL or lambda < 5 mg/dL but kappa = 10 mg/dL).
Generally, this approach can eliminate about 30% of the
specimens from the need for IFE.
Recently an enhanced nephelometric assay using
polyclonal antibodies against FLCs attached to latex
particles was developed. It is commercially available and
has been and continues to be widely tested (Freelite from
The Binding Site Inc, San Diego, CA 92121) (Table 1,
Method 6). Studies suggest this approach is not very
useful for assaying FLC in urine [12], but because of its
great sensitivity (down to 1mg/L) [13], this method is
useful for identifying FLC in serum, where it shows
approximately 150-fold greater sensitivity than IFE
[7,14,15]. Practical problems associated with this method
include inaccuracy in recovery with dilution, inaccuracy
due to polymeric aggregates, concerns of incomplete
reaction between antibodies and structurally peculiar BJP,
and imprecision between different instruments [16]. This
method appears to be particularly useful for following
patients with nonsecretory myeloma and replacing UPE
for periodic monitoring of most patients with BJP to
follow the progress of their tumor load. It may also be
useful to help rule out amyloidosis AL when urine IFE is
negative before proceeding with testing for other types of
amyloidosis (Figure 1).
Specimen
The CAP committee recommends a 24-hour urine
collection [5,17], whereas the IFCC Committee
recommends a second morning-voided specimen, because
24-hour collections are cumbersome [6]. Either collection
is probably adequate for an initial screen, but if the
analysis is negative for a BJP and disease is still suspected
or to better rule out amyloid AL, a repeat 24-hour
collection should be performed. Random collections are
not sufficient to rule out BJPuria. Urine specimens stored
at 4C for 5 to 7 days remain stable for testing;
nevertheless, the IFCC Committee recommends that if a
24-hour collection is used, 1g sodium azide be added to
the container [6]. The urine specimen should be
concentrated > 100-fold before electrophoresis for reasons
that will be discuss below.
Interferences
Free hemoglobin, lysozyme and some beta-migrating
proteins may be identified as a paraprotein on UPE but
will not react on IFE.
Characteristics
Pathophysiological Characteristics of Free Light
Chains
Identification of and quantification of monoclonal FLC is
important for the following reasons:
1. In overt hematological malignancy, such as
multiple myeloma and Waldenstrms
macroglobulinemia, they portend a greater threat
of kidney disease and poorer prognosis,
especially lambda free light chains.
2. In about 10% of myeloma cases, only an FLC is
secreted, and its concentration can be used to help
gauge the effect of therapy and disease
progression.
3. In cases of amyloidosis not associated with an
overt hematological malignancy, where there is
not a clear overproduction of plasma cells or B-
lymphocytes, identification of a monoclonal FLC
is presumptive evidence for amyloidosis AL, and
failure to identify a monoclonal FLC suggests a
different form of amyloid.
Normal Physiology and Characteristics of Free Light
Chains
In some cases, BJP may exhibit deletions or other peculiar
modifications, owing to the malignant nature of the cells
producing them. However, the characteristics of BJP are
usually very similar to normal polyclonal FLC, with the
major difference being that BJP are produced by a single
clone that produces either kappa or lambda free light
chains but not both, whereas normal FLCs are produced
by multiple clones, including both kappa and lambda.
Polyclonal FLCs are synthesized in excess of heavy
chains, and normally very small amounts of polyclonal
FLCs are found in serum and spill over into the urine
[8,18]. The free light chains are peptides of about 22 kDa.
They may exist as monomers or as dimers with a
molecular weight of about 44 kDa [18]. The dimers may
be covalently disulfide-linked or noncovalently linked
[18]. Normally, more kappa than lambda chains are
synthesized, and since light chains measured in serum are
221
Bence Jones Protein
most commonly those in complete (intact)
immunoglobulin molecules (bound light chainsB) and Other Overt Hematological Malignant Proliferations
(mostly IgG), normal serum / ratios are about 2 to 1 [8].
Studies that measure only FLC and not intact light chains
suggest the normal polyclonal free light chain / ratio in
serum is lower than the intact ratio (between 0.3 and 1.6),
either because of the synthesis and secretion of more
lambda than kappa chains or because of preferential
filtration of -monomers and dissociable dimers through
the glomerulus, or both [7,19,20]. The / ratio in urine is
higher, about 3 to 1 [7,19,20].
The structure of light chains characteristically shows a 107
N-terminal half and a kappa C-terminal half, with 107
residues or a lambda C-terminal with 105 residues [3,4].
Amino acid sequence and immunochemical studies
indicate that the variable region consists of three regions
of extensive sequence variability (hypervariable region),
and regions of lesser variability that provide structural
framework (framework regions, FR). Homologies in
amino acid sequence, especially those in the first 22
lambda residues and first 23 kappa residues of the FR1
region, chemically define four subgroups of kappa and six
of lambda chains [4].
Clearance
The primary means for elimination of proteins with
molecular weights below 60 kDa, including FLCs, is by
filtration through the glomerulus, followed by reabsorption
and catabolism by the cells of the proximal tubules. Since
2 to 3 g of protein is filtered per day and only about 75 to
150 mg/L is excreted, most of the protein is reabsorbed.
Normally, FLCs account for about 40% of the
immunoglobulin in urine [8]. Persons with impaired
tubular reabsorption show an increased concentration of
polyclonal free light chains in urine [4,8].
Characteristics and Excretion of BJP in Disease
As would be expected on the basis of normal FLC
structure, most commonly -BJP appear as disulfide-
linked covalent dimers, and -BJP as noncovalent dimers,
stable monomers, or mixtures of the two [4,21]. BJP may
also be found as smaller fragments with molecular weights
< 22 kDa and as anomalously larger forms > 45 kDa.
Those with molecular weights of approximately 55 kDa
may contain covalently linked carbohydrates such as sialic
acid which are not normally associated with light chains
[9]. Less commonly, BJP have been identified as
trimolecular complexes, tetramers, and even higher
polymer forms [8,14].
BJP usually appears in the urine because of overflow
conditions in which the concentration of FLCs in the renal
tubules is greater than the amount that can be reabsorbed,
either because of high concentrations in plasma or because
of damage to the kidney. BJP may cause tubular disease in
the form of cast nephropathy or Fanconis syndrome [8],
or they may cause amyloidosis AL, usually leading to
glomerular failure. In early cases of tubular disease, serum
BUN and creatinine may not be elevated, but proteinuria
may appear.
Multiple Myeloma, Waldenstrms Macroglobulinemia,
[22]
Most frequently, BJP is seen in urine from patients with
multiple myeloma, which is a malignant proliferation of
plasma cells in bone derived from a single abnormal clone
(plasma cell dyscrasia). Table 2 illustrates criteria that
define myeloma. Besides multiple myeloma, BJP may be
associated with Waldenstrms macroglobulinemia (in
which small lymphocytes and plasmacytoid lymphocytes
in the bone marrow secrete IgM), with B cell lymphomas,
and with leukemia, especially advanced chronic
lymphocytic leukemia, where identification probably adds
little to the diagnosis or predicted outcome.
In most cases of myeloma and in Waldenstrms, an intact
monoclonal protein is found in sera, and it can be used as
the main noninvasive marker for following tumor burden.
In about 10% of myeloma cases, a BJP is found in the
absence of an intact monoclonal protein. In these cases,
estimation of the monoclonal FLC level is the best
noninvasive marker for disease progression.
Monoclonal Gammopathy of Undetermined Significance
(MGUS) (See Method 4, Immunoelectrophoresis)
In some cases, low concentrations of BJP are seen in the
urine of patients without apparent disease [23,24].
However, most persons with BJP develop overt disease
within 8 to 20 years [25]. It is therefore recommended that
such patients be kept under observation indefinitely. It is
far more unusual to find MGUS in the form of BJP in
urine than as intact immunoglobulins in serum. This may
be due to the toxicity of the BJP, as compared to intact
monoclonal proteins that may persist in serum for a
considerable period of time after its secretion without
causing harm [25].
Amyloidosis
Many proteins have a tendency to aggregate. This
tendency seems to be especially great when proteins are
denatured or in an abnormal conformation. In such cases,
aggregation of the abnormal proteins may take on an
amorphous form that deposits in tissue. When this
proteinaceous material is composed of organized fibrils
that are arranged in an antiparallel conformation and -
pleated sheet structure when examined by x-ray
diffraction, the deposits are called amyloid [26]. In
amyloidosis AL, the fibers are composed of FLC [27,28].
As compared to myeloma, where -type chains are seen
more frequently, chains occur more often in
amyloidosis. Systemic amyloidosis comprises a
heterogeneous group of diseases that have in common the
extracellular deposition in tissue of a hyaline-like material
which when stained with Congo red, exhibits apple-green
birefringence under the polarizing light microscope. This
appearance under polarized light is the most common
method used to identify amyloid. Although the electron
microscope is more sensitive and is employed for
suspected cases when light microscopy is nondiagnostic
[26], it usually adds little to a carefully performed Congo
red stain [8].
222
Bence Jones Protein
The cerebral amyloidosis of Alzheimers disease is a case
where amyloidosis is primarily found in the brain due to
the accumulation of beta-amyloid from a normal
transmembrane protein-amyloid beta-precursor protein.
Prion disease appears to be the case where abnormal
proteins (proteinaceous infectious particles) that cause
encephalopathy can actually induce the unfolding of other
proteins [29], although the amorphous form associated
with prion disease is different from that of amyloidosis.
Types of Systemic Amyloidosis
Table 3 shows the most common types of amyloidosis, the
causative protein, and clinical features [30]. Amyloid AL
is the most common. It affects the kidney, heart,
gastrointestinal tract, liver, tongue, nerves, skin, lungs,
adrenals, and thyroid, with involvement of the latter three
organs often going unrecognized [30].
Diagnosis of Systemic Amyloidosis
Because of the variety and widespread organ involvement
with diverse symptoms, amyloidosis may imitate other
syndromes, so it is often undiagnosed or misdiagnosed;
when diagnosed, it is usually well advanced. About 10%
of patients with myeloma develop amyloidosis [30]. In the
absence of myeloma or Waldenstrms, the disease may
be suspected in presentations of peripheral nervous system
disease (especially carpal tunnel syndrome), macroglossia,
or an echocardiogram that shows restrictive
cardiomyopathy with a granular sparkling appearance
[31]. It may be identified on a biopsy done to investigate
kidney or liver involvement. In the case of amyloid AL,
the first clue may be a urine electrophoresis that shows a
BJP in a patient with chronic renal disease. A rectal or fat-
pad biopsy with Congo red staining will be positive about
85% of the time for all types of amyloidosis [32,33]. If
suspicion is high and a fat-pad biopsy is negative, the
affected organ should be biopsied [30].
Misdiagnosis of Systemic Amyloidosis
Identification of amyloid deposits does not define the type
of disease. One study showed that 34 of 350 patients
(9.7%) were misdiagnosed as AL, when they actually
manifested other types [34]. Subsequent correct
identification showed 18 patients were AFib, 13 patients
were ATTR, 2 patients were AApoA-I, and 1 patient was
ALys. Some misdiagnosis occurred because a small serum
MGUS (often < 10 g/L) was seen in about a third of the
misdiagnosed patients, erroneously leading clinicians to
conclude the condition was amyloidosis AL.
Another reason for misdiagnosis is that although
immunohistochemical staining of effected tissue almost
always identifies amyloidosis AA, monoclonal light chains
indicative of AL are identified only about 30% of the time
[34], and amyloid deposits are only found in bone marrow
about 30% of the time. Moreover, in amyloidosis that is
unassociated with overt B-cell dysplasia, plasma cells in
the bone marrow are usually within the normal range or
only slightly elevated (< 5% to 10%). As a result, bone
marrow biopsy is usually nondiagnostic.
Importance of BJP Identification in Correct Amyloid
Diagnosis
Eighty-six percent of patients with AL show a BJP in
urine [34]. Thus the importance of identifying a BJP
associated with amyloidosis cannot be overemphasized. In
BJP-negative cases of AL amyloidosis, although the bone
marrow may show a normal or only slightly elevated
number of plasma cells, a clonal predominance of either -
or -type plasma cells can often be identified by
immunohistochemical staining of a biopsy [30]. Together,
these two techniques can allow for presumptive
identification of amyloidosis AL in most cases. Figure 1
shows a diagnostic algorithm for identifying the type of
amyloidosis [30].
Interpretation
HRPE and IFE Identification of BJP
Figure 2 shows HRPE UPE, illustrating two gel strips with
high-concentration BJPs. Usually BJP migrate in the beta
to gamma region. However, as illustrated in Figure 2,
where a BJP can be seen in the beta-1 region on track 1,
because of their small size, they may migrate anywhere
between the alpha and cathodal end of the gamma region.
These paraproteins should be definitively identified by IFE
to be certain they are monoclonal BJP and not intact
immunoglobulins or some other protein such as lysozyme
(see Figure 4).
Figure 3 shows a typical IFE from the sample seen on
track 4 from Figure 2, where a lambda BJP is confirmed,
since there is no band seen with IgG, IgA, IgM, or kappa
IFE. Figure 4 shows a lysozyme band on urine UPE that
could be confused with a cathodally migrating BJP. IFE
for immunoglobulins (not shown) was negative, and
lysozyme was confirmed by IFE, using antibody against
lysozyme as seen in Figure 4.
Problems of Overloaded Gels Secondary to 100-fold
Concentration of Urine
Figure 5 shows an overloaded gel. One difficulty with
performing IFE when the concentration of an
immunoglobulin is very high is that it may be in the zone
of antigen excess (see Method 4, Immunoelectrophoresis),
causing prozoning when its specific antisera is applied to
the gel. In the mild case, this may cause the band to look
like a donut, as seen on the strip under F & B kappa 0
(urine as collected, not concentrated or diluted). In Figure
5, as the sample is diluted, the antigen and antibody
concentrations become more equivalent, and the band
appears more and more discrete, as seen with the 1:10
dilution, with the faded area disappearing. In the figure,
the band on the initial UPE appears so large that it could
be mistaken for being polyclonal, but it is clearly
monoclonal. Thus a BJP is clearly apparent from analysis
of the 100-fold concentrate by IFE, since the dense area on
UPE combined with extensive prozoning when fixed with
F & B kappa, but not lambda, implicate a -BJP. For
demonstration, the IFE was repeated with dilutions. Notice
that antibody against free kappa does not show a reaction
with the 100-fold concentrate. This occurs because of its
lower avidity and is the reason the IFCC Committee on
Plasma Proteins does not recommend using free antibody
for fixation except when F & B antibody is used
concomitantly [6].
223
Bence Jones Protein
Very Low Concentration BJP
The reason it is necessary to perform IFE on very
concentrated urine is to maximize the sensitivity for
detecting low concentrations of BJP that are so important
for identifying amyloidosis AL. Figure 6 illustrates four
cases in which the BJP concentration is too low to be seen
with UPE, where only albumin is seen, but a BJP is
identified by the more sensitive IFE. If lesser concentrated
samples were used, some BJP may not have been
identified by either technique. Before these low
concentration BJPs were identified, these patients were
thought to have nephropathy due to hypertension and
diabetes. The correct diagnosis of amyloidosis was made
only after Congo red staining of a biopsy, based on the
finding of a BJP in urine.
Use of Antibody Against FLC Only
Antibody against free light chain only is useful for
identifying a BJP when it is migrating nearly in the same
place as an intact monoclonal protein, as illustrated in
Figure 7A. It is also effective in ruling out a BJP when an
intact monoclonal immunoglobulin without an apparent
BJP is present in urine, as illustrated in Figure 7B. Figure
7A shows a kappa BJP migrating very close to an intact
monoclonal IgG-kappa that would react with the F & B
kappa antibody and with the free-kappa-only antibody. On
the other hand, in Figure 7B, where there seems to be an
intact IgG-kappa and no BJP, no reaction with the
antibody against free light chain only is seen. The
antibody against free light chain only was diluted over a
range of concentrations to make sure a band was not
masked (no reaction) by prozoning due to the lower
avidity of the free-only antibody.
Problem of Polyclonal Multiband Patterns
Polyclonal FLCs may show diffuse patterns or multiple
banding patterns (ladder pattern) overlying diffuse patterns
upon IFE and isoelectric focusing. The ladder pattern,
observed as two to seven equally spaced bands, is more
often associated with kappa but may also be found on
analysis of lambda light chains and can be seen in most
urine specimens if the sample is highly concentrated [35-
37]. Figure 8 illustrates this type of pattern in samples
fixed for kappa and lambda in comparison with a sample
containing a true BJP. Multiple banding patterns usually
appear as two to five bands, with one being denser than
the others, largely depending on the resolution of the
agarose gel system. It was demonstrated that this pattern is
a property of normal free light chains by inducing the
transient appearance of multiple kappa bands with arginine
infusion in normal healthy volunteers [35]. The multiple
banding pattern appears to be largely a product of charge
differences [36], possibly due to the four kappa and six
lambda structural framework regions in the Fab regions
[3,9].
Owing to poorer resolving power, IFE devices from some
manufacturers do not appear to have the ability to
routinely distinguish this pattern. With some systems,
electrophoresis of polyclonal free light chains in high
concentration will often show ladder band patterns with
five to seven bands. Patterns on other systems will more
often show diffuse staining zones without ladder patterns,
although sometimes one to three bands will be seen.
Although a ladder pattern per se does not reflect an
abnormal condition, it may obscure a low concentration
BJP migrating coincidental with the pattern. In such a
case, the presence of a tiny amount of BJP may be
overlooked. Polyclonal multiband patterns may also
interfere with the interpretation of IFE if the denser band
is mistakenly classified as a BJP. These ladder patterns are
most commonly seen in samples from patients with
proteinuria that is unrelated to BJPuria. Sometimes they
are seen in very concentrated samples from normal
persons. Since IFE is not a quantitative technique with a
well-defined lower reference limit for detection, systems
with sufficient separation power to discern these ladder
patterns may offer little advantage over otherwise sensitive
HRPE IFE systems that do not.
Quantification of FLCs from Urine by UPE and
Densitometry
Quantification of BJP by electrophoresis is fraught with
problems for the following reasons:
1. Except for biuret, most urine total protein dye
assays react 20% to 40% less with
immunoglobulins than with albumin, and it is
unclear to what degree FLCs are measured.
2. BJP react poorly with most immunometric
quantification assays [38], so the common
approach for measuring them has been by
densitometry, and the densitometer relies on the
total protein concentration, so it must be semi-
quantitative at best.
3. BJP often migrate very close to or coincidental
with an intact immunoglobulin (Figure 7A) or
superimposed upon normal proteins that migrate
in the beta region of the gel (e.g., on transferrin
that has leaked into the urine due to renal
disease).
These features may make quantification difficult and in
some cases impossible. Nevertheless, sufficient
assessment of the amount can usually be made either
quantitatively or qualitatively, to be useful if needed for
prognosis or to follow therapy. Although the exact amount
determined by testing may vary from the actual amount of
BJP, it will generally be clear from visual evaluation of the
density of the band by UPE and IFE, whether the amount
is very large or not [38].
Analysis of FLC in Serum by Nephelometry or
Turbidimetry (Table 1, Method 6)
Studies have shown that the greater sensitivity of these
methods allows more complete identification of FLC in
the serum of persons with nonsecretory myeloma [13] and
amyloidosis with negative IFE results than do
electrophoretic analysis of serum and urine. It also appears
to be a more accurate marker of complete remission [15].
It has been suggested that when a monoclonal protein is
identified in serum by HRPE, it is not necessary to
perform a urine IFE study but to assay the same serum
using the FLC immunoassay [39]. If the serum shows a
normal FLC ratio, it is unlikely there is a monoclonal
FLC. If the FLC / ratio is abnormal, an FLC is likely,
224
Bence Jones Protein
and the urine would then be tested by IFE for
confirmation. This would eliminate the need to
concentrate and assay many urines by IFE, thereby
simplifying the process. It is important to remember that
the serum FLC assay measures polyclonal FLCs and is not
specific for monoclonal FLCs. An abnormal ratio is
considered presumptive identification of monoclonal
FLCs. Thus the method is apt to produce inaccurate results
when a monoclonal gammopathy is concomitant with a
polyclonal increase that is not uncommon, since the
ratio is likely to be within the normal range.
Also, greater sensitivity is a two-edged sword when used
for general screening; it identifies more true positives but
usually more false positives as well. Moreover,
identification of low-level monoclonal proteins is
problematic, since the great majority are found in older
people who will not develop disease from it in their
lifetime (MGUS) [40]. Even in patients with higher levels
of monoclonal proteins that are inadvertently identified in
serum, therapy is often withheld until symptoms develop
[22]. Therefore, more sensitive screening methods, apt to
identify more patients for questionable follow-up, may not
be desirable.
This method is most useful for following therapy in cases
of nonsecretory myeloma and most cases of amyloidosis
AL once the diagnosis is established. Even if accuracy is
not exact and varies between instruments and between
people [16], changes with therapy should have clinical
value. The change in magnitude should be related to the
initial value in the same person, and results should be
superior to the present densitometric scans or visual
evaluation from electrophoretic techniques discussed
above. That is to say, once a BJP has been identified in
urine, a baseline for free light chain can be established by
this test in serum. It is then much simpler to follow the
progress of the disease by monitoring the FLC in serum
than to continue to attempt to follow the BJP in
concentrated urine by densitometry. In some cases of
proven amyloidosis AL, IFE on urine is not sensitive
enough to identify a BJP in urine or a free light chain in
serum, but the enhanced assay shows an elevated FLC
[39,41]. In such cases, progress of the disease can continue
to be followed by the serum FLC assay without the need
for IFE on urine. It may also be useful for following (1)
early remission after therapy, since the FLC reflects the
status earlier than intact monoclonal proteins because of
more rapid turnover [41], and (2) in cases of documented
amyloidosis but a negative BJP in urine, where a normal
/ ratio would strongly suggest testing for types of
amyloidosis other than AL (Figure 1).
Quality Control and Performance Goals
Quality Control (QC)
Since the results of IFE (Table 1, Method 5) are
interpretive, exact control limits are not defined. Most
electrophoretic plates contain wells at the bottom of the
plates for control sera that qualitatively indicate whether
the reaction has occurred: a precipitant ring indicates a
positive reaction (see Figure 3). Most commercial controls
contain intact, bound light chain but not unbound free light
chain, so that F & B antisera will show a precipitant ring,
but free-light-chain-only antisera will not (see Figure 7).
For this reason, it is advisable to check each lot of new
free-light-chain antisera carefully against urines with
known BJP and against normal serum that does not
contain significant amounts of free light chain before use.
Control sera with exact cutoff limits are available for
methods that quantify kappa and lambda concentrations
(Table 1, Methods 6 and 7). Controls are supplied by the
same company that makes the kit.
Performance Goals
As with most testing, performance goals are best assessed
by proficiency testing. The CAP provides testing
assessments for monoclonal free light chains in urine
(Bence Jones Protein specimens). These require qualitative
identification by IFE as positive or negative (Table 1,
Method 5) and quantitative testing by immune-
onephelometry or turbidimetry (Table 1, Method 7).
Performance is judged acceptable on the basis of the
following:
1. Bands on IFE should be sharp (Table 1, Method
5) and meet all the QC requirements listed above.
2. The IFE findings from the CAP surveys should
be in accord with the majority of laboratories in
the CAP survey, as listed on the CAP critique.
These results are listed qualitatively as kappa or
lambda free light chain present or BJP absent.
3. Results of urine immunonephelometry or
turbidimetry (Table 1, Method 7) should be in
agreement with the quantitative mean obtained by
the laboratories participating in the survey and
within the limits of acceptability provided in the
survey critique.
There is as yet no proficiency testing for the ultrasensitive
serum FLC method (Table 1, Method 6). Laboratories
using this test should install their own proficiency testing.
One way to accomplish this is to obtain the serum of a
patient with a BJP in urine and assay the serum using the
serum FLC method.
Since QC sera is available from the manufacturer of the
kit, a positive result from a patient with a known BJP in
urine, along with daily QC results that are within the
manufacturers control range, should ensure proper
performance.
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algorithm for monoclonal gammopathies by using
Tables
Table 1: Methods of Bence Jones Protein Analysis
Method 1: Heat precipitation of urine; qualitative.
Principle of analysis: Bence Jones proteins precipitate from acidic solutions heated to 45C to 60C and
redissolve on boiling.
Comments: Of historical interest only.
Method 2: Total urinary protein analysis; quantitative
Principle of analysis: Measurement of total urinary protein excretion by dip stick, dye binding, or precipitation.
Very insensitive and nonspecific.
Comments: Usually a measure of albumin, since renal disease usually accompanies BJPuria so that most of the
protein is albumin. The dip stick only measures albumin. Not helpful for identifying BJP.
Method 3: Protein electrophoresis by HRPE after 100 concentration of urine; qualitative
Principle of analysis: Bence Jones proteins separate from other proteins and may be identified as a band in the
gamma region.
Comments: Common; not sufficiently sensitive and cannot identify BJP migrating in the beta region of the gel,
since nonspecific proteinuria often produces other proteins that migrate in this region. Nevertheless, this
procedure is useful when run concomitantly with IFE, because it helps in identifying prozoning that may occur
when very large BJP are present.
Normal reference range: no paraproteins
Method 4: Immunoelectrophoresis of urine; qualitative
Principle of analysis: Bence Jones proteins separated from other proteins by electrophoresis; react with
antibodies to light chains to form visible precipitate.
Comments: No longer recommended and rarely used.
Method 5: Immunofixation after 100 concentration of urine; qualitative
Principle of analysis: Similar to Method 4.
Comments: This is the most common and most sensitive of the electrophoretic procedures and the recommended
approach.
Normal reference range: No kappa or lambda FLC.
Method 6. Analysis of serum / ratio by an enhanced nephelometric or turbidimetric assay: quantitative for any single
patient but not between patients
Principle of analysis: Immunoassay using highly specific polyclonal antibodies for FLC only.
Comments: Proven to be useful as a marker for following nonsecretory myeloma and patients with proven BJP,
because it is much more exact and simple to run than attempting to quantify BJP by HRPE or IFE. Not yet
recommended for screening.
Normal reference range: Free kappa 3.3 to 19.4 mg/L; free lambda 5.7 to 26.3 mg/L, / ratio 0.26 to 1.65.
Method 7. Analysis of urine / ratios using standard immunonephelometric/turbidimetric methods for F & B FLC on
unconcentrated urine.
Comments: Very insensitive but can reduce the need for IFE on concentrated specimens by about 30%.
Normal reference range: / ratio normal for serum or kappa and lambda undetectable.
227
Bence Jones Protein

Figure Legends
Figure 1. Algorithm for identifying the type of amyloidosis (see Table 3 for definition of types).
228
Bence Jones Protein

Figure 2. Example of high-concentration Bence Jones proteins

(BJP) on agarose HRPE UPE after 100 concentration of urine.
1, 2, 3, 4 indicate tracks with different urine samples. Arrows
indicate the positions of the BJP. The region of migration for
common serum protein fractions is indicated. The direction of
migration is towards the anode.

Figure 3. Typical IFE showing definitive

identification of BJP from sample from Figure 2,
track 4. The wells at the bottom of the gel are
control wells showing a circular precipitate. F &
B antisera was used for fixation of light chains.
(See Chapter 6, Electrophoresis for
methodological details.)

Figure 4. Identification of a paraprotein as lysozyme.

Compare this with Figure 3, showing a BJP.
229
Bence Jones Protein

Figure 5. Overloaded gel. 100 concentrated urine was diluted as indicated for IFE. F & B indicates
antisera against free and bound light chain, and F indicates antisera against free light chain
only. For other details, see Figure 3.

Figure 6. Identification of very low concentration BJP by IFE. Arrows point to the BJP bands. Atisera
against F & B light chains used. For more details, see Figure 3.
230
Bence Jones Protein

Figure 7. Usefulness of antisera against free light chain only. A, Above, a kappa BJP migrating close to an intact
monoclonal protein (IgG-kappa). B, below, BJP is apparently ruled out by no reaction with free-light-chain-only
antisera over a range of dilutions. This suggests the reaction by F & B antisera is due to the intact IgG-kappa. F &
B indicates antisera against free and bound light chain and Free or F indicates antisera against free light chain
only.
231
Bence Jones Protein

Figure 8. Normal
multiple banding
(ladder) patterns.
Samples 1 and 3
show ladder
patterns. Sample
2 shows a true
lambda BJP.
Sample 4 shows
the most common
pattern seen with
normal samples
(no staining).
Arrows to the left
indicate the
positions of the
multiple bands.
Arrow to the
right, BJP.
Antisera against
free and bound
light chain used.
Other details
same as in Figure
3.

Table 2: Characteristics for Multiple Myeloma [42,43]

> 30% plasma cells in bone marrow (normal <5%).
Monoclonal protein in serum> 35 g/L IgG or > 20 g/L IgA.
>10% plasma cells in with other evidence: significant monoclonal protein in serum (usually IgG > 20
g/L or IgA >10 g/L or urine BJP, lytic bone lesions, or hypogammaglobulinemia (uninvolved
immunoglobulins).
> 1 g of monoclonal free light chain (BJP) in urine per 24 hours.

Table 3: More Common Systemic Amyloidosis

Type Fibril Composition Clinical Features
AL Immunoglobulin FLC Cardiomyopathy, hepatomegaly, proteinuria, renal
insufficiency, macroglossia, orthostasis,
autonomic and peripheral neuropathy, ecchymoses.

AA Amyloid A protein Underlying inflammatory disorder,

proteinuria, renal insufficiency,
hepatosplenomegaly, macroglossia, orthostasis.

ATTR Transthyretin Midlife onset of (prealbumin)

(prealbumin) autonomic and peripheral,
neuropathy, cardiomyopathy, vitreous opacities.

Other familial types:

AFib Fibrinogen A Neuropathy, hypertension
(Most common)

ALys Lysozyme Neuropathy, hepatomegaly

AApo-A-I Apolipoprotein A-I Polyneuropathy, neuropathy
232
Benzodiazepines
Benzodiazepines
Harold W. Peel
Name: Benzodiazepines
Clinical significance: Refer to Chapter 42, Nervous System, in the 4th Edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: see Benzodiazepine Table: Drugs
Molecular mass: seeBenzodiazepine Table: Drugs
Merck Index: seeBenzodiazepine Table: Drugs
Chemical class: Benzodiazepines
Principles of Analysis and Current Usage
The benzod1azepines are a group of nitrogen-containing
compounds that are related by a common 1,4-
benzodiazepine structure (Benzodiazepine Table: Drugs
). Early analytical methods for the benzodiazepines
required only the measurement of a single drug or, at
most, two or three additional compounds. However,
because of the large number of drugs and metabolites in
the benzodiazepine class, the number of reported
methods is now quite large, involving many procedural
approaches (Benzodiazepines Methods Summary Table)
such as gas-liquid chromatography (GLC), high-
performance liquid chromatography (HPLC),
spectrofluorescence, ultraviolet spectrophotometry (UV),
polarography, thin-layer chromatography (TLC), and the
immunoassay procedure of EMIT. The procedures
developed for whole blood are usually appropriate for
plasma or serum analysis.
Gas-Liquid Chromatography (GLC) Procedures
(see methods 1ac, Benzodiazepines Methods Summary
Table)
The early GLC methods for chlordiazepoxide and
diazepam required the hydrolysis of the blood extracts
with 6 M hydrochloric acid to form benzophenone
products, which are more easily chromatographed than
other products. In 1964, De Silva[1] described a GLC
method that used a tritium foil electron-capture detector
and a 2% Carbowax 20M column for the analysis of
diazepam. After an ether extraction of blood, diazepam
and desmethyldiazepam were hydrolyzed to form 2-
methylamino-5-chlorobenzophenone (MACB) and 2-
aminochlorobenzophenone (ACB). The method was
sensitive to 0.02 g/mL. The method was modified by
De Silva et al.[2] in 1966, correcting the problem of
rapid column deterioration by using a more stable
column consisting of 2% Carbowax 20M and
terephthalic acid. These two methods were also
applicable to chlordiazepoxide analysis, by analysis of
its benzophenone, ACB. Foster and Frings utilized the
flame ionization detector with a 3% SE-30 column, but
1
This chapter was originally prepared by:
Harold W. Peel
this method was applicable only to blood concentrations
above 0.3 g/mL.[3] De Silva and Puglisi in 1970 used
the more stable nickel electron-capture detector (ECD)
and the polysiloxane polymer stationary phases, OV-1
and OV-17, to analyze diazepam, medazepam, and their
metabolites in blood and urine and improved the
detection limits to about 0.04 g/mL.[4]
A 1971 study by Zingales described a GLC method for
chlordiazepoxide that resolved the compound from its
metabolites desmethylchlordiazepoxide and
demoxepam.[5] Whereas most earlier reports described
the use of primarily diethyl ether, benzene, or
chloroform as the extraction solvent, Zingales
recommended a solvent of n-heptane containing 1.5%
isoamyl alcohol. The method could detect plasma
concentrations of chlordiazepoxide of 0.015 g/mL.
A 1976 report by De Silva et al. described a
comprehensive extraction scheme for many
benzodiazepine drugs that included diazepam,
chlordiazepoxide, flurazepam, nitrazepam, clonazepam,
bromazepam, oxazepam, lorazepam, and some of their
metabolites.[6] Blood was extracted by different
procedures depending on the extractability of the
specific benzodiazepines from blood, at pH 9 or 12, by
use of diethyl ether or the solvent mixture
benzene:methyl chloride (9:1). Derivatization by
methylation or silylation was employed to reduce
adsorption or decomposition effects, with the
compounds detected as intact 1,4-benzodiazepine or
benzodiazepine-2-one. The method was reported as
simple and readily automated for large-scale clinical
procedures. Blood concentrations, after a single oral
therapeutic dose of diazepam, bromazepam, clonazepam,
or flunitrazepam, were detected from 1.0 to 10.0 ng/mL.
A rapid microprocedure for diazepam and
desmethyldiazepam that used a plasma volume of 10 to
100 L was reported by Rutherford[7] in 1977. Plasma
was extracted directly with n-butyl acetate that contained
prazepam as the internal standard. After thorough
mixing and centrifugation, a 2 L injection was made
onto a 3% OV-7 column, equipped with a 63Ni ECD
detector.
233
Benzodiazepines
In a procedure using flame ionization, Baselt et al[8]
quantified the thermolabile drugs chlordiazepoxide and
oxazepam by chromatographing their decomposition
products on a 2% OV-17 column, after direct extraction
of 4 mL of blood with n-butyl chloride. The procedure
was limited to overdose cases involving diazepam,
desmethyldiazepam, flurazepam, and
desalkylflurazepam at concentrations of 0.2 g/mL or
greater.
A combined GLC-ECD and HPLC procedure for
common benzodiazepines in blood or plasma was
described by Peat and Kopjak.[9] They reported the
chromatographic properties of six parent
benzodiazepines and some metabolites, including GLC
retention data on a 3% OV-17 column and HPLC data
on a C18 column. HPLC was used to confirm the
presence of the benzodiazepines and to quantify
chlordiazepoxide and desmethylchlordiazepoxide. More
recently, Levine et al. used this approach in their studies
on the stability of benzodiazepines in stored
samples.[10] They found that chlordiazepoxide,
desmethyldiazepam, and desmethylchlordiazepoxide
were not stable beyond a few days, unless refrigerated or
frozen. Wallace et al., who also used HPLC in similar
combination with GLC-ECD, were able to resolve
diazepam and nordiazepam more successfully using an
SP-2250 DB stationary liquid phase.[11] In 1979, Kelly
et al. reported a sensitive method (from 0.05 g/mL),
using a 1 mL blood sample, for measuring diazepam,
chlordiazepoxide, oxazepam, flurazepam, and
chlorazepate.[12] The procedure used an extraction
solvent consisting of toluene, hexane, and isoamyl
alcohol (78:20:2) and a 3% OV-17 column. Oxazepam
chromatographed reproducibly as its quinazoline
rearrangement product. Chlordiazepoxide was the only
drug examined that was not well quantified by this
procedure because the thermal degradation products for
the parent drug, desmethylchlordiazepoxide and
demoxepam, both had retention times similar to that of
nordiazepam.
In 1980 Peel and Perrigo,[13] in reviewing the previous
methodology, reported a broad screening procedure
requiring 1 mL of blood or serum for 22 compounds
consisting of parent benzodiazepines, some of their
metabolites, and benzophenone hydrolysis products. A
choice of two columns (10% OV-1, 3% OV-17) with
detection by a 63Ni detector was offered for general
screening or quantification. This system overcame many
of the problems of close retention times reported in
previous reports. An optional step that formed the
benzophenone product provided confirmation using gas
chromatography or thin-layer chromatography.
Concentrations of flurazepam and its blood metabolites
were reported in an earlier method that used a 3% QF-1
column and electron-capture detector and had sufficient
sensitivity to detect levels from 0.005 to 0.01
g/mL.[14] Other investigators used a column of 1%
SP-1000, developed by a temperature program and
employing a nitrogen selective detector to analyze
therapeutic concentrations of flurazepam and its
metabolites.[15] Methods for directly determining
lorazepam in plasma were reported by de Groot et al[16]
and later by Greenblatt et al[17] Although both methods
used a similar 3% OV-1 column, Greenblatt et al[17]
were able to improve the sensitivity to a range of 1 to 3
ng/mL, probably because of the selection of an
extraction solution consisting of benzene containing
1.5% isoamyl alcohol. Following an analytical approach
similar to that used for lorazepam, Greenblatt et al[18]
analyzed plasma volumes of 0.5 to 2.0 mL for the potent
benzodiazepines triazolam and alprazolam. The
sensitivity was reported to be 0.25 ng/mL. Jochemsen
and Breimer[19] reported extracting 2 mL plasma with
the solvent pentane:dichloromethane (4:3) and injecting
the residue with a solid injection system onto a SCOT
column consisting of a 0.5% PE-21 and 3% OV-17. The
sensitivity was 0.5 ng/mL.
The use of fused silica capillary columns offers excellent
potential for improved GLC analysis of benzodiazepines.
In particular, the nonpolar DB-1 columns have excellent
characteristics for broad screening with good baseline
stability. Coupled with the 63Ni electron-capture
detector, this system gives highly resolved peaks for
very low amounts of drug. Anderson and Stafford[20]
have recently shown the advantages of using a capillary
column coated with a nonpolar phase and a flame
ionization detector for routine drug screening.
A summary of some of the gas chromatographic
procedures used for benzodiazepine analysis is presented
in Benzodiazepines Table: Summary of GLC and HPLC
methods.
High-Performance Liquid Chromatography
Procedures
(method 2, Benzodiazepines Methods Summary Table)
The HPLC procedures, which involve direct extraction
and chromatography of the intact drug, may be
preferable for thermolabile drugs such as
chlordiazepoxide. Both normal- and reversed-phase
chromatography systems have been reported.
One technique[21] employed a silica column, Partisil 10,
with a mobile phase of n-heptane:isopropanol:methanol
for the quantitative analysis of diazepam and
desmethyldiazepam. The sensitivity was reported to be
0.25 g/mL, using a 2 mL sample of blood.
Greizerstein and Wojtowicz[22] reported a micromethod
for chlordiazepoxide and desmethylchlordiazepoxide
that required 50 L of blood to be extracted with
subsequent analysis on a Bondapak C18 column with a
solvent-gradient program of aqueous potassium
phosphate and methanol. It was reported that as little as
0.1 g/mL of the compounds in a 50 L sample was
routinely detected.
234
Benzodiazepines
Oxazepam, diazepam, and nordiazepam were analyzed
using[23] a 2 mL blood sample that was extracted,
purified by back-extraction, and subsequently separated
by a reversed-phase column, Partisil-10, using a mobile
phase of acetonitrile and 0.1 M aqueous sodium acetate
(pH 4.6). The sensitivity of this method was 0.3 g/mL
for oxazepam and desmethyldiazepam and 0.4 g/mL
for diazepam. Brodie et al[24] proposed a less
complicated extraction procedure for benzodiazepine
analysis in 2 mL plasma samples. The method analyzed
diazepam and desmethyldiazepam with a sensitivity of
10 and 2 ng/mL, respectively, using a reversed-phase
C18 Bondapak column with an elution solvent of
methanol and water.
Ascalone[25] recommended reversed-phase HPLC as the
most suitable method for determining chlordiazepoxide
and its metabolites in body fluids because of its
specificity, sensitivity, simplicity, and speed. The
method used a LiChrosorb RP-18 column with a
precolumn and solvent of acetonitrile and 0.1%
ammonium carbonate to separate chlordiazepoxide,
desmethylchlordiazepoxide, demoxepam, and
desmethyldiazepam.
In developing a method for determining therapeutic
levels of diazepam and its metabolites in serum, urine,
and saliva, Tjaden et al[26] achieved a separation of nine
benzodiazepines within 12 min using a high-
performance column of methyl-silica and a 50% aqueous
methanol solvent.
By employing a solvent extraction of plasma with
benzene:isoamyl alcohol as in previous GLC procedures,
Divoll et al.[27] separated chlordiazepoxide and its two
metabolites using a C18 Bondapak column with a
mobile phase of methanol, acetonitrile, and aqueous
sodium acetate. A sensitivity of 0.05 g/mL was
achieved. Chlordesmethyldiazepam was used as the
internal standard.
Sutheimer and Sunshine[28] described a broad method
for the analysis of chlordiazepoxide,
desmethylchlordiazepoxide, flurazepam,
desalkylflurazepam, diazepam, and desmethyldiazepam
in a 1 mL sample of blood, plasma, urine, bile, or gastric
content. Clonazepam was used as an internal standard
for quantification and was contained in the extraction
solvent of n-butyl chloride. The evaporated extract was
reconstituted in the elution solvent, acetonitrile, and
0.015 M, pH 3.3 phosphate buffer and separated on an
Altex Ultrasphere-ODS (octadecyl sulfate) column. The
reported sensitivity was generally 0.01 g/mL.
In an attempt to develop a procedure that could be used
for clonazepam, as well as for a wide variety of other
drugs requested in therapeutic monitoring, Shaw et
al[29] methylated extracts from serum. The derivatized
products were chromatographed on a C18 reversed-
phase column using a complex solvent mixture of
acetonitrile:methanol:diethylamine:phosphoric
acid:pentanesulfonic acid. The methylation step was
necessary because carbamazepine had the same retention
time as clonazepam. The retention times of 45 other
common basic drugs, along with four benzodiazepines
and their metabolites, were included.
Wong[30] described a single-step extraction with
toluene and a choice of either a Bondapak C18 or
Bondapak phenyl column to separate 15
benzodiazepine types of compounds and metabolites.
The mobile phase found to be particularly good for
benzodiazepines was methanol:water (65:35), as
originally used by Brodie.[24] The method normally
requiring 0.5 mL of blood was sensitive from 0.05 to 0.1
g/mL and was reportedly applicable for triazolam
detection with a 2 mL sample.
A summary of some of the high-performance liquid
chromatographic procedures used for benzodiazepine
analysis is presented in Benzodiazepines Table:
Summary of GLC and HPLC methods.
UV and Fluorometric Procedures
Ultraviolet spectrophotometric (UV) and fluorometric
methods (see method 3, Benzodiazepines Methods
Summary Table) have limited use for the analysis of
benzodiazepines because of their lack of specificity in
discriminating between various parent compounds and
metabolites. In addition, UV is not sufficiently sensitive
for analysis of many of the newer, more potent drugs.
The UV methods described by Walberg[31] for
chlordiazepoxide and diazepam both followed similar
extraction procedures on 2 mL of serum. Selective
reextraction with 0.5 N sulfuric acid separated
chlordiazepoxide. The concentration for
chlordiazepoxide was determined by the following
difference: A245 nm A 290 nm and A 240 nm A 290
nm for diazepam. The limit of detection was reported to
be about 3 g/mL. In a procedure designed for screening
and semiquantitative analyses of total
benzodiazepines, Valentour et al.[32] hydrolyzed
extracts from blood to produce the benzophenones,
which were then converted to the fluorescent product 9-
acridanone. With an excitation wavelength of 270 nm,
the test samples were scanned from 400 to 480 nm,
giving the procedure a reported sensitivity of 0.025
g/mL for diazepam, desmethyldiazepam, oxazepam,
chlordiazepoxide, demoxepam, and desalkylflurazepam.
However, the sensitivity for flurazepam and
desmethylchlordiazepoxide was only 0.25 g/mL.
Thin-layer chromatography (TLC) (see method 4,
Benzodiazepines Methods Summary Table) has been
used to separate and detect benzodiazepines, either as the
intact compound or as the benzophenone products.
Reviews by Hailey[33] and Clifford and Franklin
Smyth[34] describe various solvent systems and special
detection methods for TLC.
Chlordiazepoxide and its metabolites were assayed by
Straughn et al[35] after separation by thin-layer
235
Benzodiazepines
chromatography, following extraction from 2 mL of
serum. The Quanta-Gram LQD plates were exposed to
red fuming nitric acid and then heated to 100 C to
produce intensely fluorescing benzodiazepine spots that
were measured using a spectrodensitometer equipped
with a fluorescent attachment. The sensitivity was about
0.1 g. In a similar approach, van der Merwe and
Steyn[36] used silica gel 60 plates and exposed them to
hydrogen chloride gas followed by radiation at 245 nm
for 45 min. This procedure converted diazepam to
fluorescent compounds that were measured
quantitatively on a spectrofluorometer equipped with a
scanning TLC attachment (exc, 360 nm; em, 460 nm).
Palermo and Poklis[37] included TLC data for
flurazepam, hydroxyethylflurazepam, oxazepam,
diazepam, and chlordiazepoxide in their evaluation of
TLC method for flurazepam in urine. The systems,
which were all developed on silica gel GF plates, were
as follows:
System A: ethylacetate:ethanol:n-butanol:ammonia (70:35:5:1).
System B: benzene:chloroform:acetone (40:40:20).
System C: benzene:methanol:acetic acid (90:10:10).
System D: ethanol:acetic acid:water (50:30:20).
System E: benzene:dioxane:ethanol:ammonia (50:40:5:5).
System F: ethyl acetate:methanol:ammonia (85:10:15).
Peel and Perrigo[13] described the TLC characteristics
(Rf, visualization) of nine parent benzodiazepines and
their metabolites and benzophenone products in three
TLC systems, as a means to confirm GLC identification.
The limit of sensitivity for most of the TLC methods
reported was about 1 to 2 g.
Using differential pulse polarography (see DPP, method
5, Benzodiazepines Methods Summary Table) as an
approach for selective screening of total benzodiazepine
concentration in blood, Brooks et al[38] assayed blood
for chlordiazepoxide, diazepam, chlorazepate,
exazepam, and their common metabolites before
quantification by more specific methods. This technique
is possible because the output signal in differential pulse
polarography is much stronger than the signal of
conventional direct current polarography. The
benzodiazepine analysis is based on the reduction of the
4,5-azomethine group. Reduction peak potentials (volts)
for chlordiazepoxide, flurazepam, and diazepam are
0.655, 0.635, and 0.715, respectively.
Chlordiazepoxide and its desmethyl metabolite are also
characterized by the N4-oxide reduction peak at 0.335
V. The DPP method was adapted to direct analysis of
diluted urine or gastric content.
Immunoassay Procedures
A wide variety of immunoassay procedures including
EIA, ELISA, FPIA, and agglutination or kinetic
interaction of microparticles are the most common
methods in use today. These methods demonstrate
variable degrees of cross reactivity to benzodiazepines
so that the different methods will not all recognize all
benzodiazepines and their metabolites.[39] Hydrolysis
of urine to convert glucuronide metabolites to more
readily detectable immunoreactive compounds improves
the sensitivity of some methods.
The homogeneous enzyme-multiplied immunotechnique
(EMIT) (see method 6, Benzodiazepines Methods
Summary Table) for benzodiazepine urine screens was
reported by Haden et al.[40] This direct method of
analysis detected several benzodiazepines and their
metabolites with an assay response equivalent to 0.5 g
of oxazepam per milliliter. Poklis[41] evaluated the
EMIT urine assay and found no false-positive results,
but about 10% inconclusive or false-negative results.
Using reconstituted organic extracts of blood, bile, or
tissue, Slightom[42] adapted this commercially available
kit to toxicological screening applications.
Reference and Preferred Methods
The number of benzodiazepines and their metabolites,
their variable chemical characteristics, and the wide
range of concentrations encountered in therapeutic and
toxicological analyses require that the clinical laboratory
use a flexible method that is useful for qualitative
screening as well as for quantification purposes.
Methods involving ultraviolet spectrophotometry are
generally limited by insufficient sensitivity and
specificity. For example some drugs that may be
extracted in a similar fashion to and exhibit a maximum
absorbance wavelength in nanometers in the same region
as diazepam (240), or desmethyldiazepam (245),
include methapyrilene (238), flurazepam (239),
desmethyldiazepam (239), amitriptyline (240),
lorazepam (240), prazepam (240), and imipramine
(247). The conventional ultraviolet methods for
benzodiazepines are limited to the microgram-per-
milliliter level because the extinction coefficients are of
the order of 104 L mol-1 cm-1.
The pulse polarographic procedure, as reported by
Brooks et al,[38] was more sensitive (0.25 to 0.5 g/mL)
than ultraviolet spectrophotometry but was limited in
specificity. Resolution of oxazepam from diazepam or
desmethyldiazepam was difficult because of the poor
separation of the azomethine peaks. Although useful for
single-drug monitoring, this dedicated instrumentation is
primarily useful for research applications.
For large qualitative drug screening programs of urine,
TLC and homogeneous assay (EMIT) procedures are
most useful. The degree of sensitivity of the TLC
procedure is quite dependent on the visualization
technique, with some benzodiazepines fluorescing when
as little as 0.2 g of drug is present. Combining TLC
techniques with fluorodensitometry for quantification of
drugs and their metabolites is possible but not efficient
for routine or clinical stat analyses since the procedures
for the chromophore development usually require 45 to
60 min.
236
Benzodiazepines
The EMIT procedure is sensitive (0.5 g/mL) and very
useful for efficiently screening large numbers of urine
samples for the benzodiazepine class of drugs. It has the
distinct advantage of being very fast, since there are no
extraction procedures involved. However, EMIT is
limited as a quantitative method because it gives only a
total measurement of combined parent drug and
metabolites. There are no reports of EMIT being used
for detecting the more potent alprazolam and triazolam
drugs.
HPLC is often preferred to GLC techniques for
benzodiazepine analysis because of the milder column
conditions. However, chlordiazepoxide is probably the
only benzodiazepine drug that may be adversely affected
by packed column gas chromatography. Although some
specific HPLC methods using ultraviolet detectors have
reported a sensitivity as low as 2 ng/mL, sensitivity in
the range of 10 ng/mL is more common. The modified
method of Sutheimer and Sunshine[28] can be
recommended because it includes sufficient range and
sensitivity, which are very useful in the toxicology
laboratory.
A distinct advantage of GLC procedures is that the
conditions for a broad qualitative screen are also suitable
for virtually all quantitative analyses. The technique
does not require individual adjustments to accommodate
the various benzodiazepines. For those situations where
a project will require several quantitative analyses for a
later-eluting benzodiazepine, the temperature can be
increased over a minimum equilibration time, and so the
total analysis time is shorter. This type of flexibility for
broad screens and adaptability for quantitative
procedures is more easily accomplished by use of gas
chromatography.
The GLC procedures using flame ionization detectors
have sufficient sensitivity for many of the
benzodiazepines that are present at higher concentrations
in the blood (that is, above 0.2 g/mL) but would not be
applicable to the newer, more potent drugs even at toxic
doses.
Gas-liquid chromatography using a 63Ni electron-
capture detector is the most versatile technique, with
sufficient sensitivity to be of use for broad
benzodiazepine analysis. The GLC methods that
hydrolyze the extracted benzodiazepines to their
benzophenones are usually sufficiently sensitive for
therapeutic concentrations (0.02 g/mL), but one can
presume only a general identification because one
benzophenone can result from several parent
compounds. The easily prepared benzophenone may be a
useful corroborative procedure to use in some cases after
initial identification. Careful selection of the extraction
solvent, buffer pH, and injection solvent can overcome
most of the problems of the various drugs, including the
poor recovery of less than 45% (in the case of
oxazepam) and the thermal decomposition, that were
encountered in many of the earlier methods.
The extremely thermolabile nature of chlordiazepoxide
and the decline of detector response to this drug limit the
use of GLC as a method of choice for this particular
drug. However, by using an internal standard and
developing the calibration graph during each
chromatographic session, one can obtain very good
precision. The flexibility of being able to increase the
temperature to quantify later-eluting drugs, or to apply
temperature programming for selective isolation of
specific peaks, makes GLC the single method of choice
for this large class of drugs. Changing operating
parameters is not as easily accomplished with HPLC.
A comparison of the EMIT, GLC, and HPLC procedures
is found in Benzodiazepine Table: Comparison of
methods.
Specimen
Serum or plasma are acceptable samples for analysis.
Whole blood (ante mortem or post mortem) is also
acceptable for most of the procedures. Storing samples at
4 C or frozen is generally sufficient to ensure stability
for benzodiazepine analysis, though Levine et al[10]
recommend that analysis for chlordiazepoxide be
performed as soon as possible. There is no indication of
interferences incurred by commonly used anticoagulants
or preservatives.
Urine specimens can be analyzed, but quantification is
not usually useful in judging toxic situations. Since the
benzodiazepines are mostly present as glucuronide
conjugates in the urine, conventional glucuronidase
hydrolysis at pH 5 is used to free the bound compounds.
Interferences
Concentrations in patients taking benzodiazepines tend
to be lower in smokers than in nonsmokers. The
presence or addition of beta-glucuronidase to urine
specimens can result in increased sensitivity of
immunoassay methods.
Benzodiazepine Therapeutic Concentration Ranges
All the benzodiazepines have therapeutic concentrations
in serum ranging from 0.1 to 1500 ng/mL, depending on
the particular drug. The concentration values presented
in Benzodiazepines Table: Pharmacological and
therapeutic concentration data can be used only as a
general guide because the effects of these drugs vary
considerably because of the occurrence of active
metabolites related to the therapeutic regime (single or
chronic dose schedule). Unlike many other drug
overdoses, plasma concentrations of the benzodiazepines
are often not related to or predictive of clinical
outcome.[43]
Interpretation
The benzodiazepine group of drugs comprises a
relatively large number of compounds that are in
prominent use as anticonvulsants, muscle relaxants,
hypnotics, and anxiolytics because of their clinical
effectiveness and relatively low toxicity
(Benzodiazepines Table: Pharmacological and reference
237
Benzodiazepines
interval data). All the benzodiazepines undergo hepatic
biodegradation by dealkylation, hydroxylation, and other
pathways to produce numerous metabolites that are
significant because of their biological activity
(Benzodiazepines Table: Benzodiazepines with active
metabolites) or their extended excretion rate. Some
metabolites are common to more than one parent drug.
For example, desmethyldiazepam and oxazepam are
metabolites that occur in the blood after administration
of chlordiazepoxide, diazepam, or chlorazepate.
Extensively bound to serum albumin, benzodiazepines
are excreted in the urine primarily as glucuronide
conjugates of hydroxylated metabolites and related
compounds produced in the liver. Only negligible
amounts of unchanged drug are found in urine.
The benzodiazepines have emerged as drugs
with a useful range of clinical applications and with
relatively few undesired side effects. However, because
of their widespread use, a significant number of cases
involving intoxication or undesirable episodes do occur.
The most common adverse effect that does occur is an
excessive degree of sedation. Toxic concentrations may
be manifested as drowsiness, or coma. Acute overdoses
of benzodiazepines taken alone are seldom fatal, but,
because of their depressant effects, they can be very
dangerous when ingested with other central nervous
system depressants, such as alcohol and barbiturates.
Patients with plasma benzodiazepine concentrations 2 to
10 times above the therapeutic concentrations will
generally exhibit toxic symptoms. Long-term usage has
resulted in cases of benzodiazepine withdrawal
syndrome, as reported by Greenblatt and Shader.[44]
Abrupt cessation of these drugs can result in symptoms
similar to those associated with the withdrawal of other
central nervous system depressants. Chlordiazepoxide
has been used as a substitute during the withdrawal
process. In updating earlier reports on benzodiazepine
overdose by Greenblatt et al[45] and Finkle et al,[46]
again affirms that serious toxicities from these drugs
alone are rare and deaths almost negligible.
References
1 De Silva JA, Schwartz MA, Stefanovic V, et al.
Determination of diazepam (Valium) in blood
by gas chromatography. Anal Chem
1964;36:2099-105.
2 De Silva JA, Koechlin BA., Bader G. Blood
level distribution patterns of diazepam and its
major metabolite in man. J Pharmacol Sci
1966;55:692-6,.
3 Foster LB, Frings CS. Determination of
diazepam (Valium) concentrations in serum by
gas-liquid chromatography, Clin Chem
1970;16:177-9.
4 De Silva JAF, Puglisi, CV. Determination of
medazepam (Nobrium), diazepam (Valium) and
their major biotransformation products in blood
and urine by electron capture gas-liquid
chromatography. Anal Chem 1970;42:1725-36.
5 Zingales IA. Determination of chlordiazepoxide
plasma concentrations by electron capture gas-
liquid chromatography, J Chromatogr
1971;61:237-52.
6 De Silva JA., Berkersky I, Puglisi CV. et al.
Determination of 1,4-benzodiazepines and -
diazepin-2-ones in blood by electron-capture
gas-liquid chromatography. Anal Chem
1976;48:10-19.
7 Rutherford DM. Rapid micro-method for the
measurement of diazepam and
desmethyldiazepam in blood by gas-liquid
chromatography. J Chromatogr 1977;137:439-
48.
8 Baselt RC, Stewart CB, Franch SJ.
Toxicological determination of benzodiazepines
in biological fluids and tissues by flame-
ionization gas chromatography. J Anal Toxicol
1977;1:10-13.
9 Peat MA., Kopjak L. The screening and
quantitation of diazepam, flurazepam,
chlordiazepoxide and their metabolites by
electron-capture gas chromatography and high
pressure liquid chromatography. J Forensic Sci
1979;24:46-54.
10 Levine B, Blanke RV, Valentour JC. Post-
mortem stability of benzodiazepines in blood
and tissues. J Forensic Sci 1983;28:102-15.
11 Wallace JE, Schwertner HA, Schimek EL.
Analysis for diazepam and nordiazepam by
electron-capture gas chromatography and by
liquid chromatography. Clin Chem
1979;25:1296-1300.
12 Kelly RC, Anthony RM, Krent L, Thompson
WL, Sunshine O. Toxicological determination
of benzodiazepines in serum: methods and
concentrations associated with high-dose
intravenous therapy with diazepam. Clin
Toxicol 1979;14:445-57.
13 Peel HW, Perrigo BJ. Toxicological analysis of
benzodiazepine-type compounds in postmortem
blood by gas chromatography. J Anal Toxicol
1980;4:105-113.
14 De Silva JA, Puglisi CV, Brooks MA, Hackman
MR. Determination of flurazepam (Dalmane)
and its major metabolites in blood by electron-
capture gas-liquid chromatography and in urine
by differential pulse polarography. J
Chromatogr 1974;99:461-83.
15 Ferrara SD, Tedeschi L, Marigo M, Castagna F.
Concentrations of phenobarbital, flurazepam
metabolites in autopsy cases. J Forensic Sci
1979;24:61-9.
16 De Groot G, Maes RA, Lemmens HH.
Determination of lorazepam in plasma by
electron capture G.L.C. Arch Toxicol
1976;35:229-34,.
17 Greenblatt DJ, Franke K, Shader RI. Analysis
of lorazepam and its glucuronide metabolite by
electron capture gas-liquid chromatography. J
Chromatogr 1978;146:311-20
18 Greenblatt DJ, Divol M, Moschitto LJ, Shader
RI. Electron capture gas chromatographic
analysis of the triazolo-benzodiazepines
238
Benzodiazepines
alprazalom and triazolam. J Chromatogr
1981;225:202-7.
19 Jochemsen R, Breimer DD. Assay of triazolam
in plasma by capillary gas chromatography. J
Chromatogr 1981;223:438-44,.
20 Anderson WH, Stafford DT. Applications of
capillary gas chromatography in routine
toxicological analysis. J High Resolut
Chromatogr Commun 1983;6:247-54.
21 Bugge A. Quantitative high-performance liquid
chromatography of diazepam and n-
desmethyldiazepam in blood. J Chromatogr
1976;128:111-16.
22 Greizerstein HB, Wojtowicz C. Simultaneous
determination of chlordiazepoxide and its n-
desmethyl metabolite in 50-microL blood
samples by high pressure liquid
chromatography. Anal Chem 1977;49:2235-6.
23 Kabra PM, Stevens GL, Marton LJ. High
pressure liquid chromatographic analysis of
diazepam, oxazepam, and n-desmethyldiazepam
in human blood. J Chromatogr 1978;150:355-
60,.
24 Brodie RR, Chasseaud LF, Taylor T. High-
performance liquid chromat-ographic
determination of benzodiazepines in human
plasma. J Chromatogr 1978;150:361-6.
25 Ascalone V. Determination of chlordiazepoxide
and its metabolites in human plasma by
reversed-phase high-performance liquid
chromato-graphy.J Chromatogr 1980;181:141-
6.
26 Tjaden VR, Meeles MT, Thys CP, Van der
Kaay M. Determination of some
benzodiazepines and metabolites in serum,
urine and saliva by high-performance liquid
chromato-graphy. J Chromatogr 1980;181:227-
41.
27 Divoll M, Greenblatt DJ, Shader RI. Liquid
chromatographic determine-ation of
chlordiazepoxide and metabolites in plasma.
Pharmacology 1982;24:261-6.
28 Sutheimer C, Sunshine I. Benzodiazepines by
reverse-phase HPLC. In Sunshine I, Jatlow PI.
editors: Methodology for analytical toxicology.
Boca Raton, FL: CRC Press; 1982; p 25-30.
(vol. 2)
29 Shaw W, Long C, McHan J. An HPLC method
for analysis of clonazepam in serum, J Anal
Toxicol 1983;7:119-22,.
30 Wong AS. An evaluation of HPLC for the
screening and quantitation of benzodiazepines
and acetaminophen in post mortem blood. J
Anal Toxicol 1983;7:33-9.
31 Walberg CB. Diazepam (Valium). In Sunshine
I, Jatlow PI. editors: Methodology for analytical
toxicology. Boca Raton, FL: CRC Press; 1976.
p. 76-9, p. 119-21.
32 Valentour JC, Montforte JR, Lorenzo B,
Sunshine I. Fluorometric screening method for
detecting benzodiazepines in blood and urine.
Clin Chem 1975;21:1976-9.
33 Hailey DM. Chromatography of the 1,4-
benzodiazepines. J Chromatogr 1974;98:527-
69.
34 Clifford JM, Franklin Smyth W. The
determination of some 1,4-benzodiazepines and
their metabolites in body fluids, a review.
Analyst 1974;99:241-72.
35 Straughn JL, Cathcart-Rake WF, Shoeman DW,
Azarnoff DL. Quantitation of chlordiazepoxide
and its major metabolites in biological fluids by
thin-layer chromatography. J Chromatogr
1978;146:473-80
36 Van der Merwe PJ, Steyn JM. Thin-layer
chromatographic method for determination of
diazepam and its major metabolite, n-
desmethyldiazepam, in human serum. J
Chromatogr 1978;148:549-52.
37 Palermo SF, Poklis A. Evaluation of thin-layer
chromatographic methods for the determination
of flurazepam and n-1-hydroxymethyl
flurazepam. Can Soc Forensic Sci J 1977;10:77-
82.
38 Brooks MA, DArconte L, Hackman MR, De
Silva J.A. Toxicologic analysis of 1,4-
benzodiazepines in differential pulse
polarography, J. Anal. Toxicol. 1977;1:179-83.
39 Drummer OH. Methods for measurement of
benzodiazepines in biological samples. J
Chromatogr B Biomed Sci App 1998;713:201-
25.
40 Haden BH, McNeil KG, Huber NA. An EMIT
assay for benzodiazepines in urine. Clin Chem
1976;22:1200.
41 Poklis A. An evaluation of EMIT-d.a.u.
benzodiazepine metabolite assay for urine drug
screening J Anal Toxicol 1981;5:174-6.
42 Slightom EL. The analysis of drugs in blood,
bile and tissue with an indirect homogeneous
enzyme immunoassay. J Forensic Sci
1978;23:292-303.
43 Divoll M, Greenblatt DJ, Lacasse Y, Shader RI.
Benzodiazepine overdosage: plasma
concentrations and clinical outcome.
Psychopharmacology 1981;73:381-3.
44 Greenblatt DJ, Shader RI. Dependence,
tolerance and addiction to benzodiazepines:
clinical and pharmacokinetic considerations.
Drug Metab. Rev 1978;8:12-8
45 Greenblatt DJ, Allen MD, Noel BJ, Shader RI.
Acute overdosage with benzodiazepine
derivatives. Clin Pharmacol Ther 1977;21:497-
514.
46 Finkle BS, McCloskey KL, Goodman L.S.
Diazepam and drug-associated deaths. JAMA
1979;242:429-34
239

Benzodiazepines

Tables
Benzodiazepine drugs
Merck Molecular Molecular Structure
Drug Index formula weight R1 R2 R3 R4
Bromazepam 1357 C14H10BrN3O 316.2 H H 1
2 (N)* Br
General Structure of 1,4-Benzodiazepine
Clonazepam 2352 C15H10C1N3O3 315.2 H H C1 NO2
General Structure of 1,4-Benzodiazepine
Diazepam 2967 C16H13ClN2O 284.7 CH3 H H C1
General Structure of 1,4-Benzodiazepine
Flurazepam 4100 C21H23ClFN3O 387.9 C2H4N(C2H5)2 H F C1
General Structure of 1,4-Benzodiazepine
Halzepam 4472 C17H15ClN2O16 298.8 C2H5 H H C1
General Structure of 1,4-Benzodiazepine
Lorazepam 5400 C15H10ClN2O2 312.2 H OH C1 C1
General Structure of 1,4-Benzodiazepine
Oxazepam 6799 C15H11ClN2O2 286.7 H OH H C1
General Structure of 1,4-Benzodiazepine
Temazepam 8976 C16H13ClN2O2 300.7 CH3 OH H C1
General Structure of 1,4-Benzodiazepine
Chlordiazaepoxide
2049 C16H14ClN3O3 299.8 click here
Clobazam 2325 C16H13ClN2O2 300.7 click here
Medazepam 5609 C16H15ClN2 270.8 click here
Alprazolam 303 C17H13ClN4 308.8 click here
Triazolam 9418 C17H12Cl2N4 343.2 click here
Chemical structures go here for: 1,4-Benzodiazepine; Clobazam; Alprazolam; Chlordiazepoxide; Medazepam; and
Triazolam
*C-5 pyridyl group.
240

Benzodiazepines

Methods of benzodiazepine analysis

Method 1: Gas-liquid chromatography (GLC); chromatographic separation, quantitative
a. Derivative formation, electron-capture detector
b. Direct GLC flame ionization detector
c. Direct GLC electron-capture detector
Principle of analysis:
a. Extraction of alkalinized blood, formation of benzophenone by acid hydrolysis, and analysis by GLC
with electron-capture detector
b. Extraction of alkalinized sample, evaporation, reconstitution, and GLC analysis
c. Extraction of alkalinized sample, evaporation, reconstitution, and GLC analysis on a 10% OV-1
column with a 63Ni electron-capture detector
Comments:
a. Single drug determination; not specific, but can be corroborative if linked to other methods
b. Screens and quantification in toxicological cases; limited sensitivity, 0.2 g/mL
c. Method of choice; most flexible for screening and quantification; sensitivity of 0.01 to 0.5 g/mL
Method 2: High-performance liquid chromatography; chromatographic separation, quantitative
Principle of analysis: Extraction of alkalinized sample, evaporation, reconstitution, separation, usually on a
reversed-phase column, and detection at 254 nm
Comments: In common use for general quantification; particularly useful in chlordiazepoxide analysis;
sensitivity about 0.01 g/mL
Method 3: Spectrophotometric; ultraviolet, fluorescent, qualitative
Principle of analysis: Extraction into solvent, separate appropriate fractions, measure ultraviolet absorbance
Comments: Single drug determinations; both have limited specificity; ultraviolet methods not sensitive
Method 4: Thin-layer chromatography; chromatographic separation, qualitative
Principle of analysis: Extracted drugs separated on silica gel and detected fluorometrically
Comments: Drug screens; sensitivity of 0.2 g/mL; not useful for stat work
Method 5: Polarographic; differential pulse polarography (DPP), quantitative
Principle of analysis: Extraction of alkalinized blood and DPP analysis for identification
Comments: Drug screens; sensitivity of 0.25 to 0.50 g/mL
Method 6: Immunoassay; EMIT (enzyme-multiplied immunoassay technique), qualitative
Principle of analysis: Competitive binding assay; drug attached to enzyme glucose-6-phosphate dehydrogenase
and unknown compete for antibody; cross-reactivity for antibody allows for reaction with many benzodiazepines
Comments: Drug screens, normally using urine; variable sensitivity to 0.5 g/mL equivalent oxazepam
241

Benzodiazepines

Summary of GLC and HPLC methods for benzodiazepines

Gas-liquid chromatographySource: De Silva et al., flurazepam, lorazepam, nitrazepam,
1964 [1] oxazepam, and metabolites
Column: 2% Carbowax 20M Sensitivity: 1 to 10 ng/mLSource: De Groot
Temperature (F): 190 et al., 1976 [16]
Column: 3% OV-1
Detector: 3H, electron-capture detector
Temperature (F): 240
(ECD)
Internal standard: Detector: 3H, ECD
Compounds measured: MACB (diazepam); Internal standard: Griseofulvin
ACB (desmethyldiazepam) Compounds measured: Lorazepam
Sensitivity: 20 ng/mLSource: De Silva et al., Sensitivity: 10 ng/mL
1966 [2] Source: Rutherford, 1977 [7]
Column: 2% Carbowax 20M terephthalic Column: 3% OV-7
acid Temperature (F): 250
Temperature (F): 215 Detector: 63Ni, ECD
Detector: 3H, ECD Internal standard: Prazepam
Internal standard: Compounds measured: Diazepam,
Compounds measured: MACB (diazepam); desmethyldiazepam
ACB (desmethyldiazepam) Sensitivity: 20 ng/mLSource: Greenblatt et
Sensitivity: 20 ng/mLSource: Foster and al., 1978 [17]
Fringes, 1970 [3] Column: 3% OV-17
Column: 3% SE-30 Temperature (F): 280
Temperature (F): 205 Detector: 63Ni, ECD
Detector: Flame ionization detector Internal standard: Oxazepam
Internal standard: Compounds measured: Lorazepam
Compounds measured: Diazepam Sensitivity: 1 to 3 ng/mLSource: Kelly et al.,
Sensitivity: 330 ng/mL 1979 [12]
Source: De Silva and Puglisi, 1970 [4] Column: 3% OV-17
Column: 3% OV-17 Temperature (F): 265
Temperature (F): 230
Detector: 63Ni, ECD
Detector: 63Ni, ECD Internal standard: Medazepam
Internal standard: Compounds measured: Chlorazepate,
Compounds measured: Diazepam, chlordiazepoxide, diazepam, flurazepam,
medazepam, and metabolites and metabolites
Sensitivity: 4 ng/mL Sensitivity: 5 ng/mL
Source: Zingales, 1971 [5] Source: Peat and Kopjak, 1979 [9]
Column: 2% OV-17 Column: 3% OV-17
Temperature (F): 275 Temperature (F): 240
Detector: ECD
Detector: 63Ni, ECD
Internal standard:
Internal standard: Flunitrazepam
Compounds measured: Chlordiazepoxide
Compounds measured: Diazepam,
and metabolites
flurazepam, and metabolites
Sensitivity: 15 ng/mLSource: De Silva et al.,
Sensitivity: 100 ng/mLSource: Ferrara et al.,
1974 [14]
1979 [15]
Column: 3% QF-1
Column: 1% SP-1000
Temperature (F): 220
Temperature (F): 260
Detector: 63Ni, ECD Detector: Nitrogen
Internal standard: Diazepam Internal standard: Diazepam
Compounds measured: Flurazepam and Compounds measured: Flurazepam and
metabolites metabolites
Sensitivity: 5 ng/mLSource: De Silva et al., Sensitivity: 10 ng/mL
1976 [6] Source: Wallace et al., 1979 [11]
Column: 3% OV-17 Column: 3% SP-2250-DB
Temperature (F): Various isothermal Temperature (F): 235
Detector: 63Ni, ECD Detector: 63Ni, ECD
Internal standard: Internal standard: Prazepam
Compounds measured: Bromazepam, Compounds measured: Diazepam,
clonazepam, chlordiazepoxide, diazepam,
242
Benzodiazepines
desmethyldiazepam
Sensitivity: 10 ng/mL
Source: Peel and Perrigo, 1980 [13]
Column:10% OV-1 or 3% OV-17
Temperature (F): 230
Detector: 63Ni, ECD
Internal standard: Prazepam or chloroquin
Compounds measured: Chlordiazepoxide,
clonazepam, diazepam, flurazepam, lorazepam,
nitrazepam, oxazepam, prazepam, 5
metabolites, 9 benzophenones
Sensitivity: 10 to 100 ng/mL
Source: Greenblatt et al., 1981 [18]
Column: 1% OV-17
Temperature (F): 290
Detector: ECD
Internal standard: U-31485
(triazalobenzodiazepine)
Compounds measured: Triazolam, alprazolam
Sensitivity: 0.25 ng/mL
Source: Jochemsen and Breimer, 1981 [19]
Column: SCOT, 0.5% PPE-21 and 3% OV-17
Temperature (F): 250
Detector: 63Ni, ECD
Internal standard: Clonazepam
Compounds measured: Triazolam
Sensitivity: 0.05 ng/mL
High-performance liquid chromatography
Source: Bugge, 1976 [21]
Column: Partisil 10
Solvent: n-Heptane, isopropanol, methanol
(40:10:1)
Detector: 232 nm (variable)
Internal standard:
Compounds measured: Diazepam,
desmethyldiazepam
Sensitivity: 25 ng/mL
Source: Greizerstein and Wojtowicz, 1977 [22]
Column: Bondapak C18
Solvent: 1 mM KH2PO4 (pH 8), methanol
(60:40, programmed)
Detector: 254 nm (fixed)
Internal standard: Chlorpromazine
Compounds measured: Chlordiazepoxide and
metabolites
Sensitivity: 100 ng/mL
Source: Kabra et al., 1978 [23]
Column: Partisil 10
Solvent: 0.01 M acetonitrile, sodium acetate
(pH 4.6) (35:65)
Detector: 240 nm (variable)
Internal standard: Prazepam
Compounds measured: Oxazepam, diazepam,
desmethyldiazepam
Sensitivity: 300 ng/mL
Source: Brodie et al., 1978 [24]
Column: Bondapak C18
Solvent: Methanol, water (65:35)
Detector: 254 nm (fixed)
Internal standard: Prazepam
Compounds measured: Diazepam,
desmethyldiazepam
Sensitivity: 10 ng/mL
Source: Ascalone, 1980 [25]
Column: LiChrosorb RP-18
Solvent: Acetonitrile, 0.1% ammonium
carbonate (31:69)
Detector: 260 nm (variable)
Internal standard: Nitrazepam
Compounds measured: Chlordiazepoxide
Sensitivity: 30 ng/mL
Source: Tjaden et al., 1980 [26]
Column: Methyl silica
Solvent: 50% methanol
Detector: 254 nm (fixed)
Internal standard:
Compounds measured: Bromazepam,
clonazepam, chlordiazepoxide, diazepam,
flurazepam, lorazepam, nitrazepam, oxazepam,
prazepam, and metabolites
Sensitivity: 1 ng/mL
Source: Divoll et al., 1982 [27]
Column: Bondapak C18
Solvent: 450 mL of methanol and acetonitrile
(50:50), 550 mL of water, 1 mL of sodium
acetate
Detector: 254 nm (fixed)
Internal standard: Chlordesmethyldiazepam
Compounds measured: Chlordiazepoxide and
metabolites
Sensitivity: 50 ng/mL
Source: Sutheimer and Sunshine, 1982 [28]
Column: Ultrasphere-ODS
Solvent: 0.015 M, pH 3.3 phosphate buffer,
acetonitrile
Detector: 254 nm (fixed)
Internal standard: Clonazepam
Compounds measured: Chlordiazepoxide,
diazepam, and metabolites
Sensitivity: 10 ng/mL
Source: Shaw et al., 1983 [29]
Column: C18, reversed phase
Solvent: 800 mL of methanol, 1200 mL of
water, 4 mL of diethylamine, 1 mL of
phosphoric acid, 1.5 g of pentanesulfonic acid,
pH to 6.5
Detector: 308 nm (variable)
Internal standard: Desalkylflurazepam
Compounds measured: Clonazepam
Sensitivity: 20 ng/mL
Source: Wong, 1983 [30]
Column: Bondapak C18
Solvent: Methanol, water (65:35)
Detector: 254 nm (variable)
Internal standard: Prazepam
Compounds measured: Chlordiazepoxide,
clonazepam, diazepam, demoxepam,
desalkylflurazepam,
desmethylchlordiazepoxide,
desmethyldiazepam, lorazepam, oxazepam,
nitrazepam, triazolam, and other metabolites
Sensitivity: 50 ng/mL
243

Benzodiazepines

Comparison of methods for benzodiazepine analysis

Parameter: Sample volume EMIT*: Tris HCl: 0.55 mol/L with
Urine: surfactant
TLC: 5 mL Serum:
EMIT*: 50 L GC:
Serum: HPLC: Mobile phase: 30%;
GC: 1 mL acetonitrile: 70%; 0.5 mol/L phosphate buffer
HPLC: 0.5 mL EMIT*: Tris HCl: 0.55 mol/L with
EMIT*: 50 L surfactant
Parameter: Fraction of sample volume Parameter: Sensitivity
Urine: Urine:
TLC: 1.0 TLC: 1 g/mL
EMIT*: 0.0016 EMIT*: 0.3 g of oxazepam/mL,
Serum: varies from 2 to 3 g/mL for other
GC: 1.0 benzodiazepines
HPLC: 1.0 Serum:
EMIT*: 0.0061 GC: 50 ng/mL for diazepam and
Parameter: Temperature clonazepam
Urine: HPLC: 0.5 g/mL
TLC: Ambient EMIT*: 0.3 g of diazepam/mL,
EMIT*: 30 C varies from 2 to 5 g/mL for other
Serum: benzodiazepines and metabolites
GC: 230 C Parameter: Linearity
HPLC: 60 C Urine:
EMIT*: 30 C TLC:
Parameter: pH EMIT*: 0.3 to 10 g/mL for
Urine: oxazepam varies for other benzodiazepines
TLC: Serum:
EMIT*: 8.0 GC: 100 to 1000 ng/mL
Serum: HPLC: 0.5 to 10 g/mL
GC:
EMIT*: 0.3 to 2.0 g of
HPLC: 4.4
diazepam/mL; varies for other benzodiazepines
EMIT*: 8.0
*Enzyme-multiplied immunoassay technique
Parameter: Final concentration of reagents
Method presented in text.
Urine:
TLC:

Retention data on 3% OV-17

Compound Reaction index Retention time (min)
Oxazepam 2805 3.40
Lorazepam 2925 4.26
Diazepam 2940 4.57
Desalkylflurazepam 3030 5.72
Desalkyldiazepam 3085 6.47
Chlordiazepoxide I 3125 6.85
Prazepam 3160 7.46
Flurazepam 3245 9.16
Hydroxyethylflurazepam 3255 10.08
Nitrazepam 3490 15.75
Clonazepam 3540 19.24
Chlordiazepoxide II 3575 19.31
Desmethylchlordiazepoxide 3770

Retention data on 10% OV-1

244

Benzodiazepines

Compound Retention index Retention time (min)

Medazepam 2215*
Halazepam 2270*
Oxazepam 2305 3.65
Lorazepam 2380 4.45
Diazepam 2405 4.73
Desalkylflurazepam 2430 4.96
Desmethyldiazepam 2490 5.62
Chlordiazepoxide I 2500 5.70
Clobazam 2535*
Temazepam 2575*
Bromazepam 2575*
Prazepam 2630 8.43
Hydroxyethylflurazepam 2655 8.90
Flurazepam 2775 12.08
Clonazepam 2860*
Alprazolam 2920*
Triazolam 3075*
Desmethylchlordiazepoxide 3100*
*Calculated retention index.

Pharmacological and reference interval data for commonly used benzodiazepines

Therapeutic Toxic
Daily Half- concentration concentration
Compound dose (mg) life (hours) (ng/mL) (g/mL)
Alprazolam 0.53 1220 222
Chlordiazepoxide 15100 627 5001600 >3000
Chlorazepam 810 1850 730 >70
Diazepam 240 2137 301500 >3000
Desalkylflurazepam 1530 47100 30110 >500
(flurazepam)
Lorazepam 26 1015 20240 >300
Oxazepam 3060 411 1001500 >500
Triazolam 0.1250.5 2.55 0.18

Benzodiazepines with active metabolites

Parent Metabolite
Chlorazepate Desmethylchlorazepate
Chlordiazepoxide Desmethylchlordiazepoxide
Demoxepam
Clobazam Desmethylclobazam
Diazepam Desmethyldiazepam
Hydroxydiazepam
Oxazepam
Flurazepam Desalkylflurazepam
Medazepam Desmethylmedazepam
Diazepam
Desmethyldiazepam
Prazepam Desmethyldiazepam
245

Benzodiazepines

Figures
Benzodiazepines: Figure 1

Standard (calibration) curves for some

common benzodiazepines using the GLC
(gas-liquid chromatography) method
described in text: response ratio (peak area
analyte divided by peak area of internal
standard) versus concentration of analyte.

Benzodiazepines: Figure 2

Typical GLC chromatograms of

common benzodiazepines on either
10% OV-1 (left) or 3% OV-17
(right).
246

Benzodiazepines

Benzodiazepines: Figure 3

A, Typical HPLC chromatogram of benzodiazepine standards: 1,demoxepam; 2, nitrazepam (internal standard); 3,

clonazepam; 4, norchlordiazepoxide; 5, nordiazepam; 6, chlordiazepoxide; 7, diazepam. B, Patient sample analyzed for
benzodiazepines by HPLC: 1, demoxepam; 2, unknown; 3, internal standard; 4, norchlordiazepoxide; 5, chlordiazepoxide.

Benzodiazepines: Figure 4

Extraction scheme for

isolation of amphoteric,
basic, and neutral drugs for
TLC (thin-layer
chromatography) analysis.
247
Benzodiazepines
Procedure: Quantitative Benzodiazepine Analysis in
Blood by Gas Chromatography
Principle
A general gas chromatographic procedure that employs a
10% OV-column and 63Ni detector is used. The
benzodiazepine and metabolites are extracted from 1 mL
of buffered blood or serum into n-butyl chloride,
concentrated, and analyzed on a gas chromatograph.[13]
Reagents
All reagents are stored in glass containers.
1. Glass distilled solvents. n-Butyl chloride;
injection solventbenzene/acetone/methanol (80:15:5).
2. Internal standard solution (200 mg/L).
Twenty mg of prazepam is dissolved in 100 mL of
absolute ethanol and diluted with water (1:100) just
before use. If prazepam is not available as a standard or
is interfered with by another peak in the chromatogram,
a solution of chloroquine diphosphate in water, 0.02
mg(base)/mL, is acceptable. When stored in glass and
refrigerated, the standard is stable for about a year.
3. Buffer solution pH 10, 0.05 M. (Potassium
carbonatepotassium boratepotassium hydroxide.
4. Stock solution of appropriate benzodiazepine
standard (250 mg/L, as base). Bromazepam,
clonazepam, chlordiazepoxide, diazepam, flurazepam,
lorazepam, and oxazepam are dissolved in absolute
ethanol. The stock solutions are stable for about 6
months or longer if stored in amber bottles under
refrigeration. The chlordiazepoxide solution is the least
stable and should be checked after 3 to 4 weeks for
decomposition.
5. Benzodiazepine standard curve. To aliquots
of blood or serum known to be free of the drug, add 10,
20, 30, and 40 L of stock solution, and bring to 10 mL
volume with blood or urine. If lower concentrations are
necessary for the more potent drugs (such as triazolam),
further dilution can be made. The controls should be
stored in a frozen condition.
Assay
Equipment: Gas chromatograph equipped with a 63Ni
electron-capture detector, molecular sieve trap on carrier
gas line, injection port equipped with septum swinger,
and electronic integrator. The column is a 10% OV-1 on
Chromosorb G/HP (80/100), glass (3 feet, 4 mm inner
diameter).
1. Sample preparation:
a. Pipet 0.5 mL of buffer solution and 0.5
mL of internal standard into a 10 mL
centrifuge tube.
b. Add dropwise 1 mL of blood or serum,
and mix.
c. Add 4 mL of n-butyl chloride, and
shake for 5 min.
d. Centrifuge the mixture for 5 min at
2000 rpm (approximately 500 g), and
remove the n-butyl chloride layer
carefully into a 5 mL evaporation vial.
e. Evaporate the extract to dryness under
air or nitrogen, and take up the residue
by vortex mixing the vial with 0.1 mL
of injection solvent.
2. Chromatographic parameters:
a. Temperature: oven 230 C; injection
port 250 C; detector 300 C
b. Carrier gas: argon/methane (95:5) at
50 mL/min
3. Inject 2 L of sample or standard to begin
analysis.
Calculations
A calibration graph is prepared for each benzodiazepine
or its metabolite using triplicate analysis for each
concentration. A plot of the ratio of area of
benzodiazepine to area of internal standard times 100
versus blood concentration is prepared. Linear regression
analysis is carried out as follows:
(Area benzodiazepine/Area internal standard) 100 =
m (conc.) + b or y = mx + b
Graphical representation of standard curves for some
common benzodiazepines is shown in Benzodiazepines:
Figure 1. A carefully prepared calibration graph is
accurate for many months but should be replaced if a
new column is used or if quality control tests indicate
changes.
Notes
1. The retention data for the benzodiazepines and
some metabolites are presented in
Benzodiazepines Tables: Retention data on 3%
OV-17 and Retention data on 10% OV-1. The
retention time of the chlordiazepoxide
decomposition products are described as
chlordiazepoxide I and chlordiazepoxide II. In
the conditions shown, the chlordiazepoxide
peak (II-RI 2800) is predominant over its
degradation products (99:1).
2. The procedure is applicable to most
benzodiazepines and their metabolites.
However, since chlordiazepoxide is
thermolabile and its analysis is greatly affected
by a variable detector response (that is, not
similarly exhibited by the internal standard), the
procedure is somewhat limited for this drug. If
a calibration graph for quantification is
prepared at the time the chlordiazepoxide
sample is analyzed, the precision is similar to
that of other benzodiazepines. One can increase
the column temperature to 260 C for ideal
quantification of chlorodiazepoxide and other
compounds that are eluted after prazepam in
248
Benzodiazepines
order to reduce analysis time and improve peak
shape.
3. The limit of sensitivity is 10 to 50 ng/mL for
diazepam, clonazepam, and
desalkylflurazepam; 50 to 100 ng/mL for
flurazepam, lorazepam, and
desmethyldiazepam; and 100 to 150 ng/mL for
oxazepam. The limit for chlordiazepoxide is
generally sufficient for concentrations
encountered in therapy.
4. If the blood sample or reagents are stored in
plastic tubes before analysis, a phthalate ester
(retention index, 2500) can be extracted into the
sample and interfere with desmethyldiazepam
(retention index, 2490). One can overcome the
problem of concomitant drug therapy and drugs
with similar retention indices by reviewing lists
of drug-retention indices published for the
similar SE-30 column.
5. To use the procedure for quantification of the
very potent drug triazolam, one should extract 3
mL of serum or blood twice with 5 mL of n-
butyl chloride and then clarify it by passing
through Na2SO4 before evaporation. The GLC
conditions are changed to the following: initial
temperature 270, increase by 8/min; final
temperature 290 for 16 min.
Benzodiazepines Therapeutic Concentration Range
click here
Procedure: Qualitative Benzodiazepine Analysis
Amadeo J. Pesce
Gas chromatographic screen. A qualitative screen for
the numerous benzodiazepines and their metabolites can
be efficiently carried out by gas chromatography using a
63Ni detector. Although the 10% OV-1 column is
suitable for quantification and general qualitative
analysis, the commonly used 3% OV-17 column is also
very useful for screening. The instrument conditions are
as follows: 183 0.2 cm inner diameter glass column
containing 3% OV-17 on Chromosorb W-HP, 80 to 100
mesh, at 260 C. The retention data for some of the
benzodiazepines are included in Benzodiazepines
Tables: Retention data on 3% OV-17 and Retention data
on 10% OV-1. Following the extraction procedure
previously described and using either of these two
columns, one can attain a sensitive screen (10 to 100
ng/mL). A typical gas chromatogram for each column is
shown in Benzodiazepines: Figure 2.
Benzodiazepines Reference Interval
click here
Procedure: Serum Benzodiazepines by HPLC
Principle
After the addition of buffer and internal standard
(nitrazepam) to the sample, the benzodiazepines
(diazepam, nordiazepam, chlordiazepoxide,
norchlordiazepoxide, and demoxepam) are separated
from the serum by adsorption onto a Bond Elut silica gel
column. The analytes are then eluted from the Bond Elut
column with methanol and injected into the HPLC
system where separation of the benzodiazepines occurs.
The concentration of each drug is then quantitated
through the use of a proportional calculation.
Reagents
1. Stock calibration standard. Place 10 mg of
each of the following in a volumetric flask: demoxepam,
nordiazepam, diazepam, norchlordiazepoxide,
chlordiazepoxide. Dissolve the drugs in methanol and
bring the solution to a final volume of 10 mL. Stable for
2 months at 4 C stored in the dark.
2. Stock internal standard. Dissolve 10 mg of
nitrazepam in methanol. Bring to a final volume of 10
mL with methanol. Stable for 2 months at 4 C stored in
the dark.
3. Plasma working standards. Obtain plasma
from the blood bank and filter through Whatman no. 50
paper. Add 50, 100, 200, 500, and 100 L of the stock
calibration standard to 100 mL aliquots of the filtered
plasma. This yields standards of 0.5, 1.0, 2.0, 5.0, and
10.0 g/mL. Aliquot the pooled plasma standards into 2
mL portions and store frozen at -20 C. Stable for 6
months.
4. Working sodium carbonate buffer, 0.01
mol/L. Dissolve 1.06 g of Na2CO3 in 80 mL of distilled
water and bring to a final volume of 100 mL with
distilled water. Stable for 6 months at room temperature.
5. Acetonitrile. HPLC grade.
6. Working phosphate buffer, 192 mol/L (pH
4.4). Dilute 300 L of 1 M potassium phosphate and 50
L of 0.9 M phosphoric acid to 1800 mL with distilled
water. The pH must be measured and adjusted with
small amounts of phosphoric acid. Proper pH value is
critical. Stable for 3 months at 4 to 8 C.
7. Phosphoric acid (0.9 mol/L). Dilute 6.125 mL
of 14.7 M phosphoric acid to 100 mL with distilled
water. Stable for 1 year at room temperature.
8. Potassium phosphate (1 mol/L). Dissolve
13.6 g of KH2PO4 in 80 mL of distilled water. Bring to
a final volume of 100 mL with distilled water. Stable for
1 year at room temperature.
Assay
Equipment: The HPLC equipment includes a solvent
delivery system, absorbance detector, and a recorder.
Other equipment include a water bath (60 C), Bond Elut
extraction columns, and a Vac Elut processing station.
1. Insert the needed number of Bond Elut columns
into the Vac Elut holder, and after turning the
vacuum on, prime each column twice with
methanol and then twice with distilled water.
Turn the vacuum off.
2. Into separate 12 75 mm disposable test tube
add the following:
a. 500 L of sample or control.
b. 500 L of working internal standard.
249
Benzodiazepines
c. 500 L of sodium carbonate buffer.
Vortex mix for 10 sec and pipet onto the Bond
Elut column.
3. Turn vacuum on, and draw sample through the
column. Then rinse the column twice with
distilled water.
4. Remove lid of the Vac Elut processing chamber
and wipe off tips inside. Label the appropriate
number of 10 75 mm test tubes, and place in
metal rack. Put rack inside, and replace lid.
5. Add 100 L of methanol to each column, wait
20 sec, and turn vacuum on. Repeat one more
time.
6. Take eluate and inject into HPLC.
7. The HPLC parameters are as follows:
a. Column: Bondapak C18
b. Mobile phase:
(1) 300 mL of acetonitrile
(2) 700 mL of phosphate buffer
Filter each using an organic filter, mix, and
degas the solution for 15 min.
c. Pump:
(1) Flow rate = 1.0 mL/min
(2) Pressure limit = 4000 psi
d. Detector:
(1) Model 441 absorbance detector set at
254 nm
(2) Sensitivity = 0.02 AUFS (absorbance
units at full scale)
e. Recorder: chart speed = 0.5 inch/min
8. Elution of all the peaks requires approximately
25 min. An example of chromatograms for
standards and for a patient with an overdose of
benzodiazepines is seen in Benzodiazepines:
Figure 3.
Calculations
1. Determine all relative retention times of
standards, controls, and patients.
2. Calculate the peak height of each
benzodiazepine (benzo) and for the internal
standard (IS), nitrazepam. Determine the peak
height ratio:
Peak height ratio (PHR) = Peak height of benzo
Peak height of 1S
Concentration of unknown =
Peak height ratio of unknown benzo Concentr of benzo in standard
PHR of that benzo standard
Benzodiazepines Therapeutic Concentration Range
Procedure: TLC Drug Screen Including
Benzodiazepines
Principle
Some of the benzodiazepines can be detected by a
general drug screen using thin-layer chromatography if
they are present in high therapeutic or toxic
concentrations in blood, urine, or stomach aspirate. The
limiting factor for benzodiazepine detection is the
sensitivity of the conventional TLC sprays used during
the drug screen. The following procedure has been found
useful for screening basic and neutral drugs, including
benzodiazepines. The schematic procedure is shown in
Benzodiazepines: Figure 4.
Reagents
1. Concentrated ammonium hydroxide (15.1
mol/L).
2. n-Butyl chloride (reagent grade).
3. 0.2 N H2SO4 (0.1 mol/L). Slowly add 5.5 mL
of concentrated sulfuric acid to approximately 800 mL
of distilled water in 1 L volumetric flask. Mix, and allow
to cool to room temperature. Bring to mark with distilled
water, and mix well. Stable at room temperature for 1
year.
4. Sodium carbonate (solid). Reagent grade.
5. Chloroform and isopropanol. Reagent grade.
6. TLC sprays.
Assay
Equipment: Silica TLC plates, spraying equipment, TLC
chromatographic tanks, ultraviolet lamp, and oven (80
C).
1. Mix blood, urine, filtered stomach aspirate, or
tissue homogenate (5 mL) with ammonium
hydroxide (0.1 mL) in a screw-capped test tube
and extract twice with n-butyl chloride (5 mL
portions) for 3 min. Separate the n-butyl
chloride extracts from the aqueous layer using a
disposable pipet, combine, and centrifuge.
2. Bring the aqueous layer to pH 8.5 with excess
sodium carbonate and extract 5 min with 60 L
of a 1:1 mixture of chloroform/isopropanol.
(This step is optional if hydrolyzed urine is to
be screened for morphine or other amphoteric
drugs.)
3. Extract the combined organic extracts for 3 min
with 0.2 N sulfuric acid (5 mL) in a screw-
capped test tube. Remove the n-butyl chloride
layer (containing neutral drugs) using a
disposable pipet, and evaporate to dryness
under a stream of nitrogen.
4. Bubble air or nitrogen through the sulfuric acid
extract for about 1 min to remove the residual
n-butyl chloride. This acid extract may be
screened by ultraviolet spectrophotometry
before you proceed.
5. Add 0.8 mL of ammonium hydroxide to 4.5 mL
of the sulfuric acid extract in a 5 mL centrifuge
tube. Shake the basic solution for 2 min and
centrifuge. The chloroform extract contains
basic drugs.
6. Screen the three extracts containing basic drugs,
neutral drugs, and amphoteric drugs by thin-
layer chromatography. The TLC system is a
common system for use in broad drug screens.
250

Benzodiazepines

TLC system: The practice of sequential spraying and viewing is very

Plates: Silica gel G or silica gel GF, 250 m useful in differentiating compounds with similar
Developing solvent: retention values that may belong to different classes of
Ethylacetate/methanol/ammonia (17:2:1). A drugs. The Rf values of a series of benzodiazepines are
small beaker of fresh ammonia is placed in the tank. listed in Benzodiazepines Table: Relative migration
Visualization sequence: values in TLC system tested in text.
1. Observe under ultraviolet lamp.
2. Spray plate with 50 g of Flu-ram Benzodiazepines Therapeutic Concentration Range
(fluorescamine) per liter of acetone; observe under click here
ultraviolet lamp.
3. Spray plate with 0.1% ninhydrin in
acetone; heat at 80 C for 5 min and observe.
4. Observe under ultraviolet lamp.
5. Spray with potassium iodoplatinate.
 251
Beta-hCG (Beta-human Chorionic Gonadotropin)
Beta-hCG (Beta-human Chorionic Gonadotropin)
James J. Miller
Name: Human chorionic gonadotropin, hCG, HCG, -hCG
Clinical significance: Refer to Chapter 44, Pregnancy, in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Molecular mass: 47,000 D
Chemical class: glycoprotein, hormone
Principles of Analysis and Current Usage i
Human chorionic gonadotropin (hCG) is a glycoprotein
composed of two noncovalently linked polypeptides: the
alpha and beta subunits. The individual subunits lack
biological activity but become active when associated to
form the intact complex. For clarity and for historical
reasons, we will use hCG as the abbreviation for this
hormone rather than HCG or -hCG. The designation
-hCG is used to represent specifically the beta subunit of
hCG, not the entire hormone. The term human, though
usually superfluous in a clinical setting, is in common use
with chorionic gonadotropin and with growth hormone and
is indicated with a lowercase h. All other hormone
references are in uppercase letters and do not include a
species designation.
The polypeptide chains contain various amounts of
carbohydrate moieties, including D-galactose, D-mannose,
and N-acetylneuraminic acid, the last of which is essential
for the biological activity of the hormone [1]. The -hCG
subunit is essentially identical to the alpha chain of several
other pituitary polypeptide hormones, such as thyroid-
stimulating hormone (TSH), follicle-stimulating hormone
(FSH), and luteinizing hormone (LH). It is the individual
beta subunit that gives each of these hormones their
specific biological characteristics.
There is also a great degree of similarity between the beta
subunit of hCG and the beta subunit of LH. In fact, LH and
hCG bind to the same receptor, the LHCG receptor [2]. In
the past, using polyclonal antibodies in competitive
immunoassays, this similarity of structure led to cross-
reactivity between hCG and LH. The carboxyterminal 24
amino acid peptide (CTP) of the -hCG chain is absent in
LH [3], and many current -hCG assays use monoclonal
i Beta-hCG (Beta-human Chorionic Gonadotrophin)
Previous and current authors of this method:
First edition: Lawrence A. Kaplan
Methods edition: David C. Hohnadel, Lawrence A. Kaplan
Second edition: David C. Hohnadel, Lawrence A. Kaplan
Third edition: Steven C. Kazmerczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: James J. Miller
antibodies directed toward this amino acid region [4,5].
Current two-site immunometric sandwich assays using
monoclonal antibodies have decreased the cross-reactivity
to about 0.1% [6]. Besides the heterogeneity described
above, hCG and subunits may dissociate and the
subunit may be cleaved (nicked) in various positions,
including removal of the CTP [4]. A hyperglycosylated
form of hCG has been described [7]. In addition, during
the lifetime of hCG molecules in the circulation [4], they
lose N-acetylneuraminic acid and possibly other sugars
from the carbohydrate side chains [8]. Thus although the
major form of hCG in early pregnancy is the intact ,
dimer, a variety of forms are present, and the assortment of
these forms changes throughout pregnancy.
Since it is the beta chain that specifies the biological
activity of hCG, many current assays for hCG are designed
to specifically detect this portion of the hCG molecule.
Thus many commercial assays are known as -hCG assays
even though they may be specific for the intact hCG
molecule, the -hCG subunit, or both. Most hCG assays
detect the various altered forms of hCG to some extent.
Because of differences in antibody specificity, it is
possible to obtain different results from the same samples
with different hCG kits, even if they are standardized with
the same standard material.
The hormone hCG is synthesized by the placenta, and it
appears in urine and serum relatively soon after
implantation of the developing embryo; the presence of
this hormone serves as the basis for pregnancy testing. In
an acute care setting, knowledge of pregnancy is used in
deciding whether to submit or exclude a patient for
abdominal roentgenograms, drug treatment, or surgery and
to rule out an ectopic or molar pregnancy. Qualitative
pregnancy tests have thus been developed for use in acute
care situations to provide this information accurately
within 5 to 15 minutes of sample receipt.
In the urine, a significant portion of hCG is present as a
metabolic fragment commonly referred to as the -core
fragment (hCGcf) [9,10]. The hCGcf consists of two
polypeptide chains derived from the subunit of hCG,
which are joined by disulfide bridges. This fragment still
retains the unique immunological determinants found in
both intact hCG and the subunit. Urine of pregnant
women contains large quantities of hCGcf, while it is
 252
Beta-hCG (Beta-human Chorionic Gonadotropin)
essentially undetectable in serum. Some urine pregnancy
tests detect hCGcf and may cross-react with the
corresponding metabolite of LH, the LH -core fragment
(LHcf) [11]. This is rarely a problem, however, since the
urinary concentration of hCGcf would typically be much
greater than the urinary concentration of LHcf in early
pregnancy.
Quantitative hCG measurements have many other
applications besides detection of routine pregnancy and
estimation of gestational age [12]. In threatened or
spontaneous abortions, lower than expected or declining
hCG concentrations may be found, whereas hCG is often
elevated in non-uterine pregnancies (ectopic, molar), in
trophoblastic growths (choriocarcinomas), and in the
presence of certain cancers, such as seminomas, teratomas,
hepatoblastomas, and bronchiogenic carcinomas. In these
patients with ectopic hCG-secreting tumors or
trophoblastic diseases, serum hCG concentrations are
followed to assess the success of surgical or drug therapy.
Measurements of hCG are also useful in monitoring
hormone-treated or subfertile patients when ovulation is
induced.
The need for hCG analysis has produced a wide variety of
methods. These procedures differ in their sensitivity and
specificity, speed and ease of analysis, and cost. The most
important considerations for use of any stat assay for hCG
are the sensitivity of the assay and the time necessary to
report a result.
Qualitative Assays
Qualitative tests for serum and urine hCG are a widely
divergent group of assays with many different physical
forms. Most are variants of the sandwich immunometric
assay (Table 1, Method 2). A capture antibody to intact
hCG or the -hCG subunit is bound to a solid surface. The
surface can be a membrane, a plastic bead, a plastic
dipstick or paddle, or a coated tube. After incubation with
the sample (serum or urine), any hCG in the sample will be
bound to the surface antibody. A second enzyme- or color-
tagged detection antibody to intact hCG or the -hCG
subunit is added. For enzyme-tagged detection, a substrate
for the enzyme reaction is added, and development of a
color is determined visually. Color-tagged detection is
directly visible. Only samples with hCG present can form
the sandwich (first antibodyhCG enzyme linked or
colored second antibody) and ultimately any color. The
antibodies may be monoclonal or polyclonal.
In most cases, these assays have been designed to detect -
hCG-intact or free subunits and may also detect the
hCGcf. They react within a few minutes at sensitivities of
10 to 25 mIU/mL [13,14]. This is an area of testing
currently undergoing constant improvement and change.
Although these assays are qualitative in nature, it has been
shown that with serum as specimen, an approximation of
the hCG concentration can be obtained (up to 1000
mIU/mL) by measuring the time (in seconds) from sample
or reagent addition to the appearance of a positive test
result [15].
Quantitative Assays
Quantitative hCG assays are usually used only for serum
(or plasma) samples. In the past, radioimmunoassays
(RIAs) had been used for measurement of hCG levels
(Table 1, Method 1). RIAs were typical competitive
binding assays in which the hCG in the sample competed
with hCG labeled with radioactive iodine for binding sites
on an hCG antibody. Both solid-phase and double-
antibody procedures were used. Some quantitative RIA
procedures were modified to shorten the reaction time and
allow the procedure to be used as a qualitative, stat
pregnancy test. However, with the appearance of good
qualitative and quantitative immunometric sandwich
assays, the use of RIA for hCG testing has virtually
disappeared.
Virtually all current quantitative hCG assays utilize the
two-site sandwich immunometric assay technique (Table
1, Method 2). The capture antibody (monoclonal or
polyclonal) may be bound to a microparticle of glass,
plastic, or magnetic material. This reacts with the hCG in
the sample. Then a labeled detection antibody (monoclonal
or polyclonal) binds to the hCG to form a sandwich.
After the reaction is complete, the unbound labeled anti-
hCG molecules are washed away. The amount of label
captured in the sandwich is measured and is directly
proportional to the hCG concentration. This is illustrated in
Figure 1-A. The label may be an enzyme, such as
horseradish peroxidase or alkaline phosphatase, or (more
commonly today) a chemiluminescent molecule. These
assays are sensitive to approximately 0.5 mIU/mL.
Reference and Preferred Methods
At this time there, is no reference method for hCG.
Theoretically, a reference method should measure not only
intact hCG but also all of its partially degraded forms on
an equimolar basis. This is unlikely to be possible with an
immunoassay but could be possible with liquid
chromatographicmass spectrometric techniques.
The preferred method depends on whether a qualitative or
quantitative assay is needed and whether the assay needs to
be used for urine, serum, or both. In addition, the method
of choice may depend on a balance between the need for a
rapid result and the need for an accurate result. A false
negative can result in delayed recognition of an ectopic or
molar pregnancy or a fetus being subjected to x-irradiation
or threatened by a surgical procedure. On the other hand, a
false-positive pregnancy test in cases of acute lower
abdominal pain in women may cause helpful diagnostic
procedures and pharmacological therapies not to be used.
Based on 2008 College of American Pathologists (CAP)
 253
Beta-hCG (Beta-human Chorionic Gonadotropin)
proficiency testing survey participant summaries, over 30
professional (not over-the-counter), qualitative urine hCG
devices are used by laboratories in the United States. Many
of these are also approved for qualitative serum hCG
analysis. Most of these have analytical sensitivities
between 20 and 25 mIU/mL. For pregnancy tests in
physician-office laboratories, emergency rooms, or other
clinical areas such as radiology, any of these kits should be
acceptable. For hospital laboratories serving emergency
rooms, it may be preferable to use one of the devices that
can also be used with serum samples.
Standardization
The first and second World Health Organization (WHO)
International Standards (1st IS and 2nd IS) were prepared
in 1938 and 1964, respectively. Both were crude hCG
preparations. They were replaced by the 1st International
Reference Preparation (1st IRP; hCG preparation 75/735)
in 1978. The 1st IRP was prepared from highly purified
hCG and in 1986 was renamed the 3rd International
Standard (3rd IS; hCG preparation 75/537). In 1999, a
second batch of the 1st IRP was prepared and released as
the 4th IS (hCG preparation 75/589). All of these hCG
standards were purified from crude extracts of urine from
pregnant women [16,17] and contain approximately 9%
nicked hCG [16]. All current commercial hCG assays use
calibrators traceable to either the 3rd or 4th IS.
On a recent (2008) CAP proficiency testing challenge,
there was a difference of 40% to 50% in hCG measured on
the same sample between the methods detecting the lowest
and highest concentrations (e.g., 724 versus 1092
mIU/mL). Even after removing the lowest and highest
methods, there was 25% to 30% difference between the
second lowest and the second highest methods (e.g., 729
versus 915 mIU/mL). Clearly, additional efforts to
standardize hCG methods are needed. Toward that end, a
new preparation, the 1st Reference Reagent (RR; hCG
preparation 99/688), was recently prepared from a fresh
urine extract and is in the process of being released. This
new preparation was further purified to remove nicked
hCG [18]. It remains to be seen whether this new standard
will improve the agreement between assays. Even when a
pure hCG-IS is available, it may not be possible to
improve the harmonization between assays. The various
methods do not actually use the IS for calibrating the assay
but have their working calibrator assayed against the IS
that is, the working calibrator nominal concentration is
traceable to the IS. The working calibrator may not be and
is probably not as pure as the IS. Given this fact, the
variability of hCG forms in different samples, and
differences in specificity of the antibodies used in different
assays, it may not be possible for the various assays to
agree [19].
Specimen
Urine
Urine samples should be collected into a clean container.
Any random urine sample is adequate, but first morning
voids have slightly higher hCG concentrations. Samples
containing particulate matter should be centrifuged before
analysis. Urine samples can be stored for up to 8 hours at
room temperature or refrigerated for up to 72 hours. Urine
samples should not be frozen.
Serum/Plasma
For most methods, serum or plasma may be used, although
some methods may be affected by chelating anticoagulants
such as EDTA. Remove the serum from the clot as soon as
possible. Use of serum or plasma separator tubes is
acceptable. Most methods recommend analysis as soon as
possible, probably because of gradual nicking of hCG, but
some methods allow samples to be at room temperature for
up to 8 hours. If analysis cannot be performed within a
short time, refrigeration for 48 to 72 hours is acceptable, or
the sample can be frozen.
Interferences
The most common interference in hCG assays is due to
interfering antibodies. The reagent excess conditions of
two-site immunometric assays, which makes them rapid,
also introduces interference by antibodies present in some
patients serum or plasma. These interfering antibodies are
of various types, including human anti-mouse antibodies
(HAMA), other anti-animal antibodies, heterophile
antibodies, and rheumatoid factor [20-23]. When present,
such interfering antibodies may bind together the capture
antibody and detection antibody in the absence of the
analyte. This is illustrated in Figure 1-B. All
immunometric assays include blocking agents such as
preimmune serum, gamma globulin, or other proprietary
products to minimize the effects of interfering antibodies.
Rarely, patients with high concentrations of interfering
antibodies may have false-positive or falsely increased
results in immunometric assays, including hCG assays.
This type of interference, though rare, has caused
unnecessary chemotherapy and surgery [24-26].
Because of the similarity in structure, LH could cross-react
in some hCG assays. However, because these are two-site
immunometric assays, cross-reactivity is quite low (about
0.1 %), and LH would have to be 10,000 mIU/mL to give
an apparent hCG of 10 mIU/mL. LH could only be that
elevated in a patient with an LH-secreting tumor. The
highest reported LH in a patient with an LH-secreting
tumor is 707 mIU/mL [27]. Thus it is unlikely that LH
could cause a false elevation of hCG due to cross-
reactivity.
Depending on the assay design, some immunometric
assays are prone to interference due to antigen excess,
whereby high concentrations of the analyte may give
falsely low results. This type of interference is called the
hook effect. In some immunometric assays, the capture
antibody, sample, and detection antibody are added
simultaneously. These assays are called one-step assays. In
 254
Beta-hCG (Beta-human Chorionic Gonadotropin)
other assays, only the capture antibody and sample are
incubated first, followed by a wash step. Then the
detection antibody is added, followed by a second
incubation and wash. These assays are called two-step
assays. One-step assays are more rapid but more prone to
the hook effect. The possibility of a hook effect is
eliminated in two-step assays. In practice, most hCG
assays use the more rapid one-step approach and avoid the
hook effect by setting an appropriate upper limit of the
analytical range.
In general, hemolysis, icterus and lipemia (turbidity) do
not interfere with hCG assays, but product labeling should
be checked, since these may interfere with some detection
systems.
Beta-hCG Reference Interval
The absolute amount of hCG present in the serum during
pregnancy varies greatly with gestational age and between
patients. Following is a table of approximate values for the
first trimester (ranges will vary by method).
Serum hCG Levels With Gestational Age
Gestational Age hCG (mIU/mL)
(Weeks)
0.2 1 5 50
12 50 500
23 100 5000
34 500 10,000
45 1,000 50,000
56 10,000 100,000
68 15,000 200,000
9 13 10,000 100,000
Interpretation
During the first 8 weeks of pregnancy, hCG doubles
(100% increase) approximately every 2 days.
Concentrations begin to decline after the eighth week,
reaching a plateau 15% to 20% of peak concentration
around the 18th week until term [28]. In twin pregnancies
at same stage of gestation, hCG concentrations are
approximately twice those seen in singleton pregnancies
during weeks 15 to 22 [29]. Healthy, nonpregnant,
premenopausal females have hCG concentrations of < 5
mIU/mL [30]. Some postmenopausal females have hCG
concentrations as high as 13 mIU/mL [30,31]. Healthy
males have low circulating concentrations of hCG of < 1
mIU/mL [32].
Higher concentrations of hCG than normal may be
associated with twin pregnancies, molar pregnancies, and
choriocarcinoma. Elevated values of hCG in males have
been associated with testicular tumors (seminomas,
teratomas) and ectopic hCG-producing tumors [33].
A qualitative serum hCG assay that becomes positive at 15
to 25 mIU/mL can be used to diagnose pregnancy or the
presence of hCG-producing tissue. A negative result rules
out all cases of pregnancy except those seen immediately
(1 to 2 days) after conception. If the result is positive and
abdominal pain or vaginal bleeding is present, there is the
possibility of ectopic (non-uterine) pregnancy.
In ectopic pregnancies, hCG values are lower than in
uterine pregnancies, but ectopic pregnancies are difficult to
distinguish on the basis of hCG values alone because of
the large overlap of hCG values with hCG values seen in
normal pregnancies. The rate of increase of hCG is more
helpful. The rise in hCG in 2 days varies somewhat from
53% to 228%. A rise slower than 53% in 2 days is
associated with a higher risk of ectopic pregnancy or
spontaneous abortion [34].
The diagnostic sensitivity of an hCG that is apparently low
for gestational age can be increased when it is combined
with ultrasonography. If the hCG is greater than 6000 to
6500 mIU/mL, ultrasonography will usually demonstrate
an intrauterine pregnancy; if no sac is present, an ectopic
pregnancy is 94% probable [35]. For hCG values less than
6000 mIU/mL, in nonemergency cases, sequential
sampling over several days or weeks can be used to predict
when ultrasonography will prove diagnostically useful.
When serum hCG is used as a tumor marker, the results
are used to monitor or stage the tumor [33,36]. In these
cases, progression or regression of the cancer will be
monitored by changes in serum hCG levels. With the
current analytical sensitivity of quantitative hCG methods,
a serum value of less than 2 mIU/mL would indicate that
no hCG-secreting tumor was present.
hCG Performance Goals
In laboratory practice, hCG is an analyte regulated for
proficiency testing. Acceptable performance criteria
require reporting laboratories to be within 3 SD of the
mean of the peer group laboratories. On a recent CAP
proficiency-testing participant summary, the median
coefficient of variation (CV) was 5.6%. This represents a
total allowable error of approximately 17%, Suggested
medical decision limits are 25 mIU/mL and 10,000
mIU/mL.
References
1. Ross GT. Clinical relevance of research on the
structure of human chorionic gonadotropin. Am J
Obstet Gynecol 1977;129:795-805.
2. Ji I, Zeng H, Ji TH. Receptor activation of and
signal generation by the
lutropin/choriogonadotropin receptor.
Cooperation of Asp397 of the receptor and alpha
Lys91 of the hormone. J Biol Chem
1993;268:22971-4.
3. Maston GA, Ruvolo M. Chorionic gonadotropin
has a recent origin within primates and an
evolutionary history of selection. Mol Biol Evol
 255
Beta-hCG (Beta-human Chorionic Gonadotropin)
2002;19:320-35.
4. Cole LA. Immunoassay of human chorionic
gonadotropin, its free subunits, and metabolites.
Clin Chem 1997;43:2233-43.
5. Cole LA, Abbott JT, Armstrong EG. Comment on
the nature and specificity of Bayer Corporation
and Chiron Diagnostics hCG immunoassays. Clin
Chem 1998;44:2001-2.
6. Cole LA, Sutton JM, Higgins TN, Cembrowski
GS. Between-method variation in human
chorionic gonadotropin test results. Clin Chem
2004;50:874-82.
7. OConnor JF, Ellish N, Kakuma T, Schlatterer J,
Kovalevskaya G. Accuracy of home pregnancy
tests at the time of missed menses. Prenat Diagn
1998;18:1232-40.
8. Sutton JM. Charge variants in serum and urine
hCG. Clin Chim Acta 2004;341:199-203.
9. Kato Y, Braunstein GD. Beta-core fragment is a
major form of immunoreactive urinary chorionic
gonadotropin in human pregnancy. J Clin
Endocrinol Metab 1988;66:1197-201.
10. Wehmann RE, Blithe DL, Flack MR, Nisula BC.
Metabolic clearance rate and urinary clearance of
purified beta-core. J Clin Endocrinol Metab
1989;69:510-7.
11. Iles RK, Javid MK, Gunn LK, Chard T. Cross-
reaction with luteinizing hormone beta-core is
responsible for the age-dependent increase of
immunoreactive beta-core fragment of human
chorionic gonadotropin in women with
nonmalignant conditions. Clin Chem
1999;45:532-8.
12. Tyrey L. Human chorionic gonadotropin assays
and their uses. Obstet Gynecol Clin North Am
1988;15:457-75.
13. O'Connor RE, Bibro CM, Pegg PJ, Bouzoukis JK.
The comparative sensitivity and specificity of
serum and urine hCG determinations in the ED.
Am J Emerg Med 1993;11:434-6.
14. Cole LA, Sutton-Riley JM, Khanlian SA,
Borkovskaya M, Rayburn BB, Rayburn WF.
Sensitivity of over-the-counter pregnancy tests:
comparison of utility and marketing messages. J
Am Pharm Assoc 2005;45:608-15.
15. Mishalani SH, Seliktar J, Braunstein GD. Four
rapid serum-urine combination assays of
choriogonadotropin (hCG) compared and
assessed for their utility in qualitative
determination of hCG. Clin Chem 1994;40:1944-
9.
16. Birken S, Gawinowicz MA, Kardana A, Cole LA.
The heterogeneity of human chorionic
gonadotropin (hCG). II. Characteristics and
origins of nicks in hCG reference standards.
Endocrinology 1991;129:1551-8.
17. Canfield RE, Ross GT. A new reference
preparation of human chorionic gonadotrophin
and its subunits. Bull World Health Organ
1976;54:463-72.
18. Birken S, Berger P, Bidart J-M, Weber M,
Bristow A, Norman R et al. Preparation and
characterization of new WHO reference reagents
for human chorionic gonadotropin and
metabolites. Clin Chem 2003;49:144-54.
19. Ekins R. Immunoassay standardization. Scand J
Clin Lab Invest Suppl. 1991;205:33-46.
20. Kricka LJ, Schmerfeld-Pruss D, Senior M,
Goodman DB, Kaladas P. Interference by human
anti-mouse antibody in two-site immunoassays.
Clin Chem 1990;36:892-4.
21. Ward G, McKinnon L, Badrick T, Hickman PE,
Heterophilic antibodies remain a problem for the
immunoassay laboratory. Am J Clin Pathol
1997;108:417-21.
22. Kricka LJ. Human anti-animal antibody
interferences in immunological assays. Clin
Chem 1999;45:942-56.
23. Levinson SS, Miller JJ. Towards a better
understanding of heterophile (and the like)
antibody interference with modern
immunoassays. Clin Chim Acta 2002;325:1-15.
24. Cole LA. Phantom hCG and phantom
choriocarcinoma. Gynecol Oncol 1998;71:325-9.
25. Cole LA, Rinne KM, Shahabi S, Omrani A.
False-positive hCG assay results leading to
unnecessary surgery and chemotherapy and
needless occurrences of diabetes and coma. Clin
Chem 1999;45:313-4.
26. Rotmensch S, Cole LA. False diagnosis and
needless therapy of presumed malignant disease
in women with false-positive human chorionic
gonadotropin concentrations. Lancet
2000;355:712-5.
27. Hirshberg B, Conn PM, Uwaifo GI, Blauer KL,
Clark BD, Nieman LK. Ectopic luteinizing
hormone secretion and anovulation. N Engl J
Med 2003;348:312-7.
28. Braunstein GD, Rasor J, Danzer H, Adler D,
Wade ME. Serum human chorionic gonadotropin
levels throughout normal pregnancy. Am J Obstet
Gynecol 1976;126:678-81.
29. Wald N, Cuckle H, Wu TS, George L. Maternal
serum unconjugated oestriol and human chorionic
gonadotrophin levels in twin pregnancies:
implications for screening for Downs syndrome.
Br J Obstet Gynaecol 1991;98:905-8.
30. Snyder JA, Haymond S, Parvin CA, Gronowski
AM, Grenache DG. Diagnostic considerations in
the measurement of human chorionic
gonadotropin in aging women. Clin Chem
2005;51:1830-5.
31. Gronowski AM, Fantz CR, Parvin CA, Sokoll LJ,
Wiley CL, Wener MH, Grenache DG. Use of
serum FSH to identify perimenopausal women
with pituitary hCG. Clin Chem 2008;54:652-6.
 256
Beta-hCG (Beta-human Chorionic Gonadotropin)
32. Lee CL, Hes R, Shephert JH, Hudson CN, Chard
T. The purification and development of a
radioimmunoassay for beta-core fragment of
human chorionic gonadotrophin in urine:
applications as a marker of gynecological cancer
in premenopausal women. J Endocrinol
1991;130:481-9.
33. Stenman UH, Alfthan H, Hotakainen K. Human
chorionic gonadotropin in cancer. Clin Biochem
2004;37:549-61.
34. Barnhart KT, Sammel MD, Rinaudo PF, Zhou L,
Hummel AC, Guo W. Symptomatic patients with
an early viable intrauterine pregnancy: hCG
curves redefined. Obstet Gynecol 2004;104:50-5.
35. Kadar N, Caldwell BV, Romero R. A method of
screening for ectopic pregnancy and its
implications. Obstet Gynecol 1981;58:162-6.
36. Rule AH, DeLellis RA, Papish SW. Alpha-
fetoprotein and beta-chorionic gonadotropin in
the diagnosis and management of testicular germ
cell tumors. J Clin Immunoassay 1983;6:213-20.
Tables
Table 1: Methods for hCG Measurements
Method 1: Radioimmunoassay (RIA)
Type of analysis: Competitive
Principle: Radiolabeled (radioactive iodine,
125I) hCG competes with sample analyte for
binding to anti-hCG. Increased hCG in sample,
decreased bound radioactivity.
Usage: Rarely used today
Comments: Of historical interest
Method 2: Immunometric assay
Type of analysis: Two-site sandwich assay
Principle: Labeled anti-hCG reacts with sample
hCG bound to solid-phase anti-hCG. Amount of
bound label directly proportional to amount of
hCG in sample.
Usage: Most frequently used assay
Comments: One or both antibodies are usually
monoclonal. Reagent excess conditions make this
method rapid but subject to rare false-positive
results due to heterophile antibodies in patients
samples. Sensitivity of 0.1 to 1 mIU/mL. Assay
time 15 to 20 minutes.
 257
Beta-hCG (Beta-human Chorionic Gonadotropin)

Figures

Beta-hCG: Figure 1

Immunometric Assay. A, Incubation of immobilized capture antibody, sample containing hCG, and labeled antibody results in
a complex (sandwich). Label captured is proportional to hCG concentration. B, Interfering antibody (heterophile, HAMA,
etc.) forms the sandwich in the absence of hCG, causing a false-positive or falsely elevated result.

Beta-hCG: Figure 2

Mean serum hCG levels throughout normal pregnancy. Arithmetical scale used on ordinate. Bars represent 1 standard error of
the mean [28].
258
Beta-2-Microglobulin
Beta-2-Microglobulin
James J Miller
Name: Beta-2-microglobulin, 2M, B2M, thymotaxin
Clinical significance: see Interpretation,
Molecular mass: 11,800 D
Chemical class: Protein, light-chain, of the MHC class I antigens
i Blood:
Principles of Analysis and Current Usage
Beta-2-microglobulin (B2M) is the light chain of the
major histocompatibility complex (MHC) class I antigen
and is found on the surface of all human cells expressing
this antigen. It is noncovalently bound to the MHC class
I heavy chain and is shed into the circulation [1,2]. B2M
has a molecular mass of 11.8 kDa, with 100 amino acids
in a single polypeptide chain with one disulfide bond. It
was first isolated from urine by Berggard and Bearn in
1968 [3]. B2M was found to be identical to a T-cell
precursor chemotactic factor in the thymus called
thymotaxin [4], and in 1999, it was found to be an
apoptosis-inducing factor [5].
Early assays for B2M used radial immunodiffusion [6]
(Table 1, Method 1) and immunoprecipitation methods
[7]. These methods lacked sufficient sensitivity for
measurements in serum and required concentration of
urine before measurement. Sensitive and specific
radioimmunoassays were developed in the early 1980s
[8]. In the late 1980s and 1990s, latex agglutination
(Table 1, Method 2) [9], turbidimetric (Table 1, Method
3) [10], nephelometric (Table 1, Method 4) [11], and
nonisotopic immunometric [12,13] assays (Table 1,
Method 5) were developed.
Reference and Preferred Methods
There is no reference method for B2M.
Radial immunodiffusion and immunoprecipitation
methods are inexpensive and relatively simple to
perform, but they are labor intensive and not suitable for
rapid analysis. Latex agglutination methods are rapid but
imprecise. Radioimmunoassay methods are sensitive,
specific, and relatively inexpensive to perform, but they
are rarely used today because of the short reagent shelf
life, the hazards of radioactivity, and the expense of
radioactive waste disposal. Currently, most laboratories
use nephelometric or turbidimetric assays because they
are faster and readily automated procedures.
Specimen
i
Beta-2 microglobulin
Previous and current authors of this method:
First edition: Not done
Methods edition: Donald T. Forman
Second edition: Not updated
Third edition: Donald T. Forman
Fourth edition: Not updated
Fifth edition: James J. Miller
Serum is the preferred specimen. If possible, lipemic
specimens should be avoided. Samples should be
analyzed fresh or refrigerated for up to 3 days. Samples
may be frozen until assayed.
Urine:
B2M is unstable in acid urine [14]. Random urine
samples should be adjusted to a pH of 7 immediately
after collection; 24-hour urine collections should have a
neutral buffer added before collection, and the collection
should be refrigerated during collection. Aliquots of
random or 24-hour urines should be centrifuged before
analysis.
CSF should be centrifuged and assayed immediately or
stored refrigerated for <3 days. Samples may be frozen
until assayed.
Interferences
Lipemia may interfere with B2M measurements in some
nephelometric and turbidimetric methods [15].
Beta-2-Microglobulin Reference Interval
Serum: 0.11-0.23 mg/dL (1.1-2.3 mg/L) [8]
Urine: 0.04-0.36 mg/d (0.4-3.6 mg/d) [8]
Clearance: 8-130 L/min [8] =
(B2MUr V 1000 L/mL)/(B2MS t),
where B2MUr = urine B2M in mg/dL;
B2MS = serum B2M in mg/dL;
V = urine volume in mL;
and t = duration of collection in min.
CSF: 0.06-0.22 mg/dL (0.6-2.2 mg/L) [16]
Interpretation
Because of its small size, B2M is most used for
assessing renal function, especially for differentiating
glomerular and tubular proteinuria. High serum B2M
concentrations are associated with low glomerular
filtration rate, inflammation, and various neoplastic
conditions, especially those involving B lymphocytes
(e.g., multiple myeloma). With normal glomerular
function, even high serum B2M concentrations do not
usually lead to elevated urine B2M levels because of its
catabolism in the proximal tubules. On the other hand,
elevated urinary B2M concentration is found in acute
tubular necrosis and failing renal transplants [8].
B2M has not proven to be a useful tumor marker, but it
does have prognostic value for patients with multiple
myeloma [17]. A role for B2M as a prognostic marker in
259
Beta-2-Microglobulin
human immunodeficiency virus (HIV) infection has
been explored [2]. More recently, B2M has been found
to be the cause of dialysis-related amyloidosis, first
discovered by Assenat et al. [18] in 1980.
In acute leukemia and lymphoma with central nervous
system involvement, the concentration of B2M is
increased in cerebrospinal fluid [19].
Beta-2-Microglobulin Performance Goals
B2M survey data from the 2007 College of American
Pathologists Participant Summary Report shows median
interlaboratory imprecision values (% coefficient of
variation) of 6% to 8%. Based on biological variability,
desirable imprecision should be <3.0%; desirable bias
should be <4.1%; and desirable total error should be
<9.0% [20].
References
1 Harris HW, Gill TJ III. Expression of class I
transplantation antigens. Transplantation 1986;
42: 109-17.
2 Miyata T, Jadoul M, Kurokawa K, Van
Ypersele de Strihou C. Beta-2 microglobulin in
renal disease. J Am Soc Nephrol 1998; 9: 1723-
35.
3 Berggard I, Bearn AG. Isolation and properties
of a low molecular weight beta2-globulin
occurring in human biological fluids. J Biol
Chem 1968; 243: 4095-4103.
4 Dargemont C, Dunon D, Deugnier MA,
Denoyelle M, Girault JM, Lederer F, et al.
Thymotaxin, a chemotactic protein, is identical
to beta 2-microglobulin. Science. 1989; 246:
803-6.
5 Mori M, Terui Y, Ikeda M, Mishima Y,
Tomizuka H, Ikeda M, Itoh T, et al. Beta(2)-
microglobulin identified as an apoptosis-
inducing factor and its characterization. Blood
1999; 94: 2744-53.
6 Petherson PA, Evrin PE, Berggaard I.
Differentiation of glomerular, tubular and
normal proteinuria: Differentiations of urinary
excretion of 2-microglobulin, albumin, and
total protein. J Clin Invest 1969; 48: 1189-98.
7 Hemmingsen L, Skaarup P. The 24 hour
excretion of plasma proteins in the urine from
apparently health subjects. Scand J Clin Lab
Invest 1975; 35: 347-53.
8 Woo J, Floyd M, Longley MA, Cannon DC. 2-
Microglobulin clearance as measured by radio-
immunoassay Clin Chem 1980; 26: 1193-7.
9 Itoh Y, Enomoto H, Kawai T. A latex
agglutination test for urinary beta-2-micro-
globulin. Contrib Nephrol 1988; 68: 172-8.
10 Rifai N, Morales A. Immunoturbidimetry of 2-
microglobulin in serum. Clin Chem 1989; 35:
1996-7.
11 Viedma JA, Pacheco S, Albaladejo MD.
Determination of 2-microglobulin in serum by
a microparticle-enhanced nephelometric
immunoassay. Clin Chem 1992; 38: 2464-8.
12 Tienhaara A, Eskola JU, Manto V. Double
monoclonal time-resolved immunofluorometric
assay of 2-microglobulin in serum. Clin Chem
1990 ; 36: 1961-4.
13 Kandoussi A, Cachera C, Pagniez D, Saile R,
Tacquet A. Quantification of 2-microglobulin
and albuminin plasma and peritoneal dialysis
fluid by a nonbompetitive immunoenzymo-
metric assay. Clin Chem 1993; 39: 93-6.
14 Donaldson M D, Chambers RE, Woolridge
MW, Whicher JT. Stability of alpha-1-
microglobulin, beta-2-microglobulin, and
retinol binding protein in urine. Clin Chim Acta
1989; 179: 73-7.
15 IMMAGE Immunochemistry Systems Beta-2-
Microglobulin product labelling. Beckman
Coulter, Inc. 2001.
16 Lutz CT, Cornell SH, Goeken JA.
Establishment of a reference interval for beta-2-
microglobulin in cerebrospinal fluid with use of
two commercial assays. Clin Chem 1991; 37:
104-7.
17 Greipp PR, San Miguel J, Durie BG, Crowley
JJ, Barlogie B, Blade J, et al. International
staging system for multiple myeloma. J Clin
Oncol 2005; 23: 3412-20.
18 Assenat H, Calemard E, Charra B, Laurent G,
Terrat JC, Vanel T. Hemodialysis: carpal tunnel
syndrome and amyloid substance. Nouv Presse
Med 1980; 9: 1715.
19 Clausen N, Ibsen KK. Central nervous system
relapse surveillance by serial beta-2-
microglobulin measurements in childhood acute
lymphoblastic leukemia. Acta Paediatr Scand
1984; 73: 848-54.
20 Fraser CG. Biological variation: from principles
to practice. Washington, DC: AACC Press;
2001, p. 140.
260
Beta-2-Microglobulin

Table 1: Beta-2-Microglobulin: Methods Summary Table

Method 1: Radial immunodiffusion (RID); quantitative

Principle of analysis: Diffusion of B2M through medium containing anti-B2M antibody
Usage: Seldom used today
Comments: Serum, plasma, manual; requires very long incubation time

Method 2: Latex agglutination

Principle of analysis: Agglutination of latex particles coated with anti-B2M antibodies due to presence of B2M
results in formation of visible aggregates; size of aggregates related to B2M concentrations
Usage: Seldom used today
Comments: Semiquantitative; may be useful as rapid screen

Method 3: Immunoturbidimetry
Principle of analysis: Reaction of B2M with anti-B2M antibody results in formation of antigen-antibody
complexes which absorb light
Usage: Common quantitative method for B2M
Comments: Sensitive, specific; automated; can be implemented on most current random access chemistry
analyzers

Method 4: Immunonephelometry
Principle of analysis: Reaction of B2M with anti-B2M antibody results in formation of antigen-antibody
complexes which scatter light
Usage: Most common quantitative method for B2M in use
Comments: Sensitive, specific; automated; requires specialized instrumentation (nephelometer).

Method 5: Immunometric assay

Principle of analysis: Two anti-B2M antibodies are used. One is a capture antibody immobilized on a
microparticle; the other detection antibody is labeled with an enzyme or a fluorescent or chemiluminescent
compound. After incubation and removal of unbound reagents by washing, B2M in the sample sandwiches the
capture and detection antibody, and the captured label is proportional to the B2M concentration.
Usage: Not commonly used for B2M
Comments: Sensitive, specific; automated; requires specialized instrumentation; more expensive than
nephelometry and turbidimetry.
261
Bilirubin
Bilirubin
R Swaminathan
Name: Bilirubin
Clinical significance: Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Structure: See figure 1
Principles of Analysis and Current Usage i
Bilirubin was first demonstrated to be present in normal
serum by van den Bergh and Snapper [1]. They found
that bilirubin in normal serum reacted with Ehrlichs
diazo reagent (diazotized sulfanilic acid) only when
alcohol was added. It was later observed that pigment in
human bile reacted with the diazo reagent without the
addition of alcohol [2]. The pigment that reacted in the
absence of alcohol was termed direct. The pigment
that required the presence of alcohol was termed the
indirect bilirubin fraction. The response of serum to
van den Berghs test, with or without the presence of
alcohol, has been the basis for several classifications of
jaundice.
It is now clear that indirect bilirubin is unconjugated
bilirubin bound to albumin en route to the liver from the
reticuloendothelial system, where it is formed. The
unconjugated bilirubin is a nonpolar molecule and is not
soluble in water. Consequently it will react with diazo
reagent only in the presence of an agent (historically
called an accelerator) such as alcohol, in which both
bilirubin and the diazo reagent are soluble. (The alcohol
also enhances the intensity of the color formed.) Because
of the nonpolar nature of the unconjugated bilirubin and
its strong affinity to albumin, unconjugated bilirubin is
not filtered at the glomerulus and is not normally found
in urine.
In contrast, bilirubin conjugated with glucuronate is a
polar and water-soluble compound that exists in plasma
loosely bound to albumin. When blood levels of
conjugated bilirubin are high, it is filtered at the
glomerulus and excreted in the urine.
Conjugated and unconjugated bilirubin fractions have
historically been differentiated by the time of the
reaction and solubility of the fractions. Bilirubin that
reacts quickly in the absence of solvent is called direct,
i
Bilirubin
Previous and current authors of this method:
First edition: John E. Sherwin, Ronald Overholte
Methods edition: John E. Sherwin, Ronald Overholte
Second edition: John E. Sherwin, Ronald Overholte
Third edition: Steven C. Kazmierczak
Fourth edition: John E. Sherwin, Charles Thompson
Fifth edition: R. Swaminathan
or conjugated, bilirubin, whereas in the presence of a
solvent, both forms of bilirubin readily react. The
bilirubin that requires solvent to react is termed indirect
or unconjugated bilirubin. However, it has been known
for some time that a portion of the unconjugated
bilirubin is available for reaction in the absence of
solvent and that the extent of the reaction is dependent
on the time and temperature of the reaction and on the
final concentration of reagents [3]. Decreasing the time
of reaction to minimize reactivity of the unconjugated
bilirubin in the absence of solvent can increase the
specificity of the conjugated (direct) bilirubin assay. A
third bilirubin fraction has also been described [4-7].
This fraction, called delta () bilirubin, is a covalent
conjugate between albumin and bilirubin. The -
bilirubin fraction was identified by high-performance
liquid chromatography (HPLC) and made available to
the routine clinical laboratory as a result of the dry-film
technology used for measuring bilirubin. The -bilirubin
fraction reacts like conjugated bilirubin in the diazo
reaction and is usually present in very small amounts in
the blood. However, when the levels of conjugated
bilirubin increase, so does the concentration of -
bilirubin. The -bilirubin fraction does not react
quantitatively in most current diazo reactions [8].
Modifications in diazo concentration and time of
reaction are necessary to fully react the -fraction.
Qualitative analysis of serum bilirubin as indirect or
direct, according to the type of van den Bergh reaction,
has long been replaced by quantitative determinations of
the amount of conjugated bilirubin and total bilirubin, in
which the difference between the total and direct
reaction is assumed to represent the indirect or
unconjugated bilirubin.
Methods for Total Bilirubin
Diazo Reagent Methods
Commonly used procedures for measurement of
bilirubin and its fractions are based on the diazo reaction
[9,10]. In this reaction, bilirubin reacts with diazotized
sulfanilic acid (diazo reagent) to form a colored
chromophore (Figure 1). The diazotized sulfanilic acid
reacts at the central methylene carbon of bilirubin to split
the molecule, forming a mixture of two isomers of
azodipyrroles (azobilirubin). The reaction can be carried
out at various pH; in the original method of Malloy-
Evelyn (Table 1, Method 1), the procedure was
performed at pH 1.2. At this pH, the azobilirubin is red
262
Bilirubin
purple in color with an absorption maximum of
approximately 560 nm (Figure 2) . The rate of reaction
of the diazo reagent with the bilirubin varies for the
different forms of bilirubin. Conjugated bilirubin reacts
very rapidly [11] and -bilirubin reacts more slowly
[12]. The rate of reaction of unconjugated bilirubin is pH
dependent [13]. To accelerate the reaction at pH 6, a
compound usually called the accelerator is added. In the
original Malloy-Evelyn method, methanol was used as a
solubilizer of the unconjugated fraction, but many other
chemicals have been employed, such as dimethyl
sulfoxide and nonionic detergents. The Malloy-Evelyn
method is seldom used in practice now because of
several problems. These include matrix effects, protein
precipitation by methanol (causing turbidity) [14],
negative interference from hemoglobin, and relatively
long reaction time [15]. Other compounds used as
accelerator include caffeine, sodium benzoate,
dyphylline, sodium dodecyl sulphate, and surfactants
[16].
The Jendrassik-Grof modification (Table 1, Method 2) is
carried out at a pH near 6.5, but the absorbance of the
reaction is measured after alkalinization of the reaction
solution to pH 13 [17,18]. At this pH, the absorption
spectrum of the azobilirubin is shifted to a more intense
blue color measured at 600 nm. The original Jendrassik-
Grof method employed sodium benzoatecaffeine to
solubilize unconjugated bilirubin for the measurement of
total bilirubin.
Diazo methods have been successfully automated.
However, the modified Jendrassik and Grof method
requires the use of four separate reagents, whereas most
commonly used analyzers are designed to use two
reagents only. Furthermore, the azosulfanilic acid and
ascorbic acid reagents are only stable for a short period
of timeusually a day. In an attempt to improve the
assay, hydroxylamine salt was substituted for ascorbic
acid (U.S. Patent No. 2569,721). Others have used
different diazo reagents such as 2,4 dichlorophenyl or
2,5 dichlorophenyl diazonium salts to help with
automation [14]. In the Ektachem dry chemistry system
for total bilirubin, a very stable diazonium salt 4N-
carboxymethylsulphanyl-benzene-dazonium
hexafluorophosphate is used [19]. Another common
diazonium salt used in many automated analyzers is 2,5-
dichlorophenyldiazonium tetrafluoroborate (DPD),
which reacts very rapidly with bilirubin under acidic
conditions (Table 1, Method 3). In the Roche Modular
analyzer, DPD in strong acidic medium (pH 1.0 to 2.0) is
used together with a solubilizing agent to accelerate the
reaction and avoid protein precipitation. In the Abbott
Aeroset/Architect, 2,4-dichloroaniline is used as the
diazo reagent.
The dry-film technology method uses analytical reagents
layered onto a clear polyester film. In the total bilirubin
method, which is a modification of the Jendrassik-Grof
diazo reaction, dyphylline and a surfactant are used to
dissociate the unconjugated bilirubin from albumin
(Table 1, Method 4). Conjugated, unconjugated and -
bilirubin react with diazo reagent to produce
azobilirubin, which is then measured by reflectance at
540 nm and 460 nm. The measurement at 460 nm is used
for any spectral interference [19].
Spectrophotometric Methods
Bilirubin can also be measured by direct
spectrophotometry (Table 1, Method 5) [14]. This can be
done with or without prior dilution of the sample. This
method is applicable to samples from neonates up to 3
months of age, because carotenoids which are present in
older infants and adults also absorb in the 454 nm
region. It is, however, necessary to correct for the
presence of oxyhemoglobin (oxyHb), because oxyHb
also absorbs at 454 nm. This is accomplished by
subtraction of the absorbance at 540 nm, since oxyHb
absorbs equally at each of these wavelengths [20]. This
method requires either a spectrophotometer or a
bichromatic photometer (bilirubinometer) capable of
measurements at 454 nm and 540 nm. Direct
spectrophotometry can also be performed with or
without dilution of serum with a suitable buffer [21].
When using this method, it is assumed that only
unconjugated bilirubin is present in the sample and that
the only interfering substance is hemoglobin.
Instruments dedicated for this purpose
bilirubinometersare used in neonatal units, because
they are convenient and relatively easy to use. However,
blood samples need to be centrifuged and serum/plasma
introduced into the instrument. This is an additional step
for clinical staff to perform. With the availability of
whole-blood bilirubin measurements and transcutaneous
bilirubinometers, this type of meter is less often used at
present. Although results from a bilirubinometer
(UniStat) showed good correlation with routine methods
[22], in samples with a significant amount of -bilirubin,
the results are unreliable because this species of bilirubin
has much higher absorbance than unconjugated bilirubin
[12]. Methods in which predilution is used are affected
by protein concentration of the sample and the strength
of the pH of the buffer/diluent. Use of diluents
containing caffeine reduces this matrix-related
interference [23]. Predilution of samples with Tris buffer
before spectrophotometric measurements is used in some
automated instruments for the measurement of neonatal
bilirubin (e.g., Abbott Aerostat/Architect).
The direct spectrophotometric method has now been
incorporated into blood-gas analyzers to measure
bilirubin in whole blood. In the Radiometer
(Copenhagen, Denmark) ABL735 blood-gas analyzer,
multiple absorbance measurements are made on whole
blood between 478 and 672 nm over 128 wavelengths.
Red cells are hemolyzed by ultrasonic vibration and the
absorbance of the hemolysate measured accurately.
Because erythrocytes do not contain any bilirubin,
plasma bilirubin concentration is calculated using a
dilution factor based on the hematocrit to correct for
dilution of plasma by intracellular fluid. This bilirubin
value is then corrected for hemoglobin pigments. This
analyzer has the facility to correct for either adult or fetal
hemoglobin, which have different absorption spectra
[24]. A recent multicenter evaluation of this instrument
263
Bilirubin
found good agreement with laboratory-based
measurements [25]. This method has also been found to
give acceptable results for specimens from adult patients
as well [24]. Direct spectrophotometry methods are rapid
and convenient to use for point-of-care testing.
One of the methods employed in the dry-chemistry
system (Ektachem, Johnson & Johnson) is reflectance
spectrophotometry (Table 1, Method 6). In this method,
serum is exposed to a binding agent at pH 8.0, and the
light reflected is measured at two wavelengths to allow
for the different absorbance maxima of the unconjugated
(420 to 425 nm) and conjugated bilirubin (460 to 465
nm). This allows the simultaneous measurement of
conjugated and unconjugated species [26].
Direct spectrophotometry is the principle behind the
transcutaneous bilirubinometers (Table 1, Method 7).
Several noninvasive transcutaneous bilirubinometers are
now available for the measurement of bilirubin in the
neonate. In the original devices, the yellowness of the
skin was assessed by measuring the optical density of
blue and green light reflected from the skin. These
devices were influenced by skin pigmentation [27].
BiliCheck (SpectRx Inc., Norcross, GA, USA) and
JM103 (Minolta AirShields) are two commercially
available bilirubinometers. In the BiliCheck meter, white
light is transmitted onto the skin, and reflected light is
analyzed. Absorption from interfering substances
(hemoglobin, melanin, and dermal thickness) is isolated
mathematically from light absorption by bilirubin in the
capillary bed. Several studies have shown that these
bilirubinometers give results which are in agreement
with laboratory results, especially when the bilirubin
concentration is less than 240 mol/L(14 mg/dL) [28].
These devices, however, are no substitute for laboratory
measurement of serum bilirubin in deciding treatment
options, but they are useful in identifying infants who
require serum bilirubin determination [29].
Enzymatic Methods
An enzymatic method for determination of bilirubin has
been developed using bilirubin oxidase (EC 1.3.35)
isolated from Myrothecium verrucaria MT-1 (Table 1,
Method 8) [30]. The enzyme catalyzes the oxidation of
bilirubin to biliverdin. The reaction is monitored by
measurement of the decrease in absorbance at 405 to 460
nm. Sodium dodecyl sulfate (SDS) and/or sodium
cholate are included in the total bilirubin assay to
dissociate bilirubin from albumin and accelerate the
reaction.
Enzymatic methods has been automated [31], and the
results of the enzymatic methods are in good agreement
with those by the Jendrassik-Grof method. However,
differences were observed in samples with high
direct/conjugated bilirubin concentration, and this can be
reduced by measuring the absorbance at 425 nm instead
of 465 nm [32].
Hemoglobin was initially thought to cause negative
interference, but the inclusion of EDTA in the regent
reduced this effect [33].
The 2007 College of American Pathologists (CAP)
comprehensive chemistry quality assurance surveys [34]
showed that 60% of the participants used a Jendrassik-
Grof procedure (53% of these employed a sample
blank), 13% used a diazonium saltbased method, 16%
used a dry-film assay, 3.8% used an oxidation method,
and less than 1% used the bilirubin oxidase method [35].
Other Methods
A method based on the oxidation of bilirubin to
biliverdin by potassium ferricyanide was described by
OLeary et al, [36], and this has been adapted for
automated analyzers (Table 1, Method 9). The decrease
in absorbance at 480 nm is measured with blanking at
600 nm. This method is not affected by hemolysis, and
the reagents are stable. Nearly 25% of the laboratories in
the United Kingdom are using this method routinely.
Results by this method compare well with those by
Jendrassik-Grof methods [37].
Several other methods for measuring total bilirubin have
been described. A fluorimetric method based on the
fluorescence of bilirubin bound to protein or detergent
[38], an electrochemical technique [39], and a
chemiluminescence method based on the reaction of
bilirubin and peroxynitrite [40] have all been described.
However, none of these have been found to be useful in
routine clinical laboratory practice. An amperometric
method using bilirubin-imprinted poly(methacrylic acid-
to-ethyl glycol dimethylacrylate) film as a sensing
electrode has been recently described [41].
Measurement of Bilirubin Fractions
Liquid Chromatography
Several high-performance liquid chromatography
(HPLC) methods have been developed to separate and
quantitate the four bilirubin fractions, namely,
unconjugated (-bilirubin), monoconjugated (-
bilirubin), diconjugated (-bilirubin), and -bilirubin (a
fraction that is strongly bound to proteins). These
methods are also capable of detecting photoisomers
which are produced during phototherapy [42]. It has
been shown that in sera from patients with cholestatic
jaundice that the major fraction is -bilirubin (37%) and
di- and monoconjugated forms account for 24% and
13%, respectively. The remaining 27% is unconjugated
bilirubin [43]. Initially, normal HPLC was used to
separate bilirubin fractions after protein precipitation
followed by extraction [44]. Later, reverse-phase HPLC
was used after precipitation of globulins by sodium
sulphate [45], and this separated bilirubin into the four
fractions. Adachio et al. [42] have described a reverse-
phase HPLC method using a MicroMax RP-30 column
without protein precipitation. A more recent direct
injection method using a Shodex Asshipak GS-320 HQ
column was described with good results [46]. A method
for measuring -bilirubin using anion exchange
chromatography has also been reported [47]. The
precision and accuracy of the HPLC methods are not
adequate enough to consider this to be a reference
method. Calibration of HPLC methods is a problem. The
commonly used calibrant with unconjugated bilirubin is
264
Bilirubin
unsatisfactory because the molar absorptivity of the four
fractions may not be identical [45].
Spectrophotometric Methods
The conjugated and unconjugated bilirubin fractions can
also be estimated by a dry-film spectrophotometric
technique [26]. In the method used in the dry-chemistry
system, conjugated and unconjugated bilirubin are
measured simultaneously. Samples are treated with
caffeine, sodium benzoate, and surfactant to dissociate
unconjugated bilirubin from albumin. Unconjugated and
conjugated bilirubin then bind to a cationic polymer
called a mordant. This interaction of the bilirubin with
the mordant causes a spectral shift such that both species
of bilirubin have similar molar absorptivity at 400 nm,
but at 460 nm, unconjugated bilirubin has a higher molar
absorptivity. The concentration of conjugated and
unconjugated bilirubin are determined by measuring the
reflectance at these two wavelengths. The concentration
of -bilirubin is calculated as the difference between
total bilirubin (by the diazo dry-film method) and the
sum of conjugated and unconjugated bilirubin by the
spectral shift method [48].
Enzymatic Methods
Enzymatic methods using bilirubin oxidase have also
been used for the measurement of bilirubin fractions.
Direct bilirubin reacts at pH 3.7, and total bilirubin at pH
7.2 [33]. In the absence of detergents, conjugated
bilirubin and -bilirubin are oxidized by bilirubin
oxidase at acidic pH, and unconjugated bilirubin is
oxidized at alkaline pH. By altering the pH and by
addition of detergent, all three fractions can be
measured. Total bilirubin is measured at pH 10.0 in the
presence of SDS, direct bilirubin (conjugated and delta)
is measured at pH 3.7, and conjugated bilirubin at pH
10.0 in the absence of SDS. This method compares well
with results from an HPLC method [49,50]. In another
approach to measure conjugated bilirubin, the reaction of
bilirubin oxidase with -bilirubin and unconjugated
bilirubin is reduced by the addition of sodium fluoride
and N-acetylcysteine [51]. Morimoto et al. [52]
measured the decrease in absorbance at 450 nm caused
by the oxidation of conjugated bilirubin by bilirubin
oxidase in the presence of methylene phosphonic acids,
EDTA, manganese, and 4-hydroxy-2,2,6,6,-
tetramethylpiperidine 1-oxyl. It has been reported that up
to 5% of unconjugated bilirubin may be oxidized in
these enzymatic methods [53], and others have reported
differences between diazo methods and enzymatic
methods in the measurement of conjugated bilirubin
[54].
Diazo Methods
In the absence of an accelerator (such as caffeine
reagent), bilirubin fractions other than the unconjugated
fraction react with diazo reagents. This fraction is called
direct bilirubin. However, it is now known that some
unconjugated bilirubin and -bilirubin react under these
conditions [12,48]. Even at low pH, a small proportion
of unconjugated bilirubin will react with the diazo
reagent, especially if the sodium nitrite concentration is
high [55]. Preincubation of serum with HCl has been
recommended to reduce the reaction of unconjugated
bilirubin [11]. However, this will also reduce the
reactivity of conjugated and delta fractions. By carefully
selecting the reaction conditions, it is possible to
measure direct bilirubin, and this correlates well with the
HPLC method.
Other Methods
A thin-layer chromatography [56] and a capillary
electrophoresis method [57] to separate and quantitate
bilirubin fractions have also been described. None of
these are clinically useful, because the identification of
all bilirubin fractions do not add any diagnostic value.
Free (Unbound) Unconjugated Bilirubin
The fraction of unconjugated bilirubin which is not
bound to albumin is negligible in adults, but may be high
in neonates. This fraction is implicated in the
pathogenesis of kernicterus [58]. Based on the principle
that free bilirubin is rapidly oxidized by horseradish
peroxidase, whereas the albumin-bound fraction is
protected from this reaction [59], a method for
measuring free bilirubin has been described. Two
commercial analyzers are available to measure this
fraction. These are the UB analyzer (Arrows Co Ltd.,
Osaka, Japan)[60] and the FloPro analyzer (Global FIA,
Fox Island, WA) [61]. In the UB analyzer, the sample is
diluted 42-fold, whereas in the FloPro analyzer, the
sample is diluted only 2-fold. A recent study showed that
the use of assay buffer without chloride gave lower
values in the UB analyzer [62].
Other methods of measuring free bilirubin include
Sephadex column chromatography, where the unbound
bilirubin is retained, while the bound bilirubin is not
[63]. Assays based on the fluorescence of albumin-
bound bilirubin [64] and on the quenching of albumin
fluorescence by bilirubin binding [65] have also been
described to measure bilirubin binding capacity.
Urine bilirubin measurements are most often made on
fresh specimens using dipsticks (Chemstrip or Multistix)
impregnated with direct-reacting diazo reagent, such as
2,4-dichloroaniline diazonium salts. The Ictotest uses a
similar chemical reaction as the bilirubin pad on the
multi-reagent dipstick, but is presented in a tablet
format. The active reagent is p-nitrobenzene diazonium
p-toluenesulfonate . With a sensitivity of about 10 mg/L,
it is two to four times more sensitive than the dipstick
procedure. Dipstick methods are specific but subject to
interference. Medications such as phenazopyridine,
which color the urine, can cause false-positive results.
Other compounds reported to cause interference are
aminosalicylic cid, ascorbic acid, chlorhexidine, and
chlorpromazine and its metabolites [66].
Reference and Preferred Methods
A modified Jendrassik-Grof procedure that employs
caffeine-benzoate reagent to solubilize all the various
bilirubin fractions with excellent recovery has been
recommended as a reference method [11].
265
Bilirubin
Because the direct and total bilirubin methods have
limited specificity, the true concentrations and
proportions of conjugated and unconjugated bilirubin in
serum have not been known with any great certitude.
The development of HPLC procedures [44] that can
physically separate and quantitate each fraction has led
to a better understanding of the composition of bilirubin
in normal blood. The HPLC results show that in healthy
subjects, conjugated bilirubin is less than 5% of the total
and there is very little -bilirubin. Comparison with
HPLC methods show that most diazo methods greatly
overestimate the concentration of conjugated fraction. In
addition, the HPLC analysis indicates the presence in
serum of non-bilirubin compounds that are also diazo
reacting. Certain compounds present in urine, such as
mesobilifuscin and uroerythrin, are known to interfere
with urinary bilirubin analysis, and it is presumed that
these or similar compounds exist in plasma.
These studies indicate that chemical assays that give a
low proportion (15% or less) of direct-reacting bilirubin
in healthy persons probably give the most accurate
results. For a particular diazo method, the amount of
direct-reacting bilirubin apparent in normal serum
depends on the time and temperature of the reaction.
The primary advantages of the Jendrassik-Grof
procedures include the greater sensitivity and precision
(especially at low bilirubin concentrations) as a result of
the higher molar absorptivity of the azobilirubin under
alkaline conditions (approximately 7 104 L mol1
cm 1), relative insensitivity to hemoglobin interference,
rapid color development, and a large linear range (up to
1.7 absorbance units). Jendrassik-Grof procedures often
use ascorbic acid to minimize hemoglobin interference,
though a sample blank is also used to minimize the
effect of endogenous color.
The use of bilirubinometers to measure total bilirubin is
useful only in the neonatal population because of the
presence of carotenoid compounds in adult serum that
can cause strong positive interference. These instruments
are simple to use, however, require a small sample
volume, and have good accuracy and precision if
maintained properly. The recent introduction of blood-
gas analyzers with bilirubin measurement by direct
spectrophotometry makes the measurement of bilirubin
even easier, and some studies suggest that these
instruments can be used in older children and adults
[24].
There is no definitive method for bilirubin. At this time,
there seems to be no clear-cut reason to choose between
the diazo, enzymatic, spectrophotometry, or OLeary
methods. Since all have acceptable precision and are
adapted to many automated instruments, the choice will
depend on the available instrument or individual
laboratory preference.
The dry-film procedure for total, conjugated, and
unconjugated bilirubin analysis has compared well with
diazo procedures. The bias between the dry-film and
Jendrassik-Grof methods for direct bilirubin has been
ascribed to the ability of the dry-film methods to
differentiate between -bilirubin and bilirubin
glucuronide, both of which react in diazo procedures but
to different extents. There is very little interference from
hemoglobin [35]. Carotene does not interfere in the dry-
film assay. It continues to be the only method used in the
routine clinical laboratory that measures the bilirubin
fractions (conjugated, unconjugated, and -bilirubin)
rather than classifying them as direct or indirect reacting
groups.
Transcutaneous methods for measurement of bilirubin
have been developed that are not affected by skin color
and show good agreement with bilirubin measured in
serum [67]. However, one must be aware that
transcutaneous and blood bilirubin measurements do not
measure the same parameter. Transcutaneous methods
measure the amount of bilirubin that has moved from the
serum into tissue. Whether or not the transcutaneous
bilirubin measurements are indicative of the bilirubin
that is available to move into brain tissue is still
unanswered.
Perhaps a more significant problem than choosing
between the available methods is the problem of
choosing an appropriate calibrator. Many apparent
inaccuracies and differences between methods are a
result of differences in standardization.
Recommendations for a uniform bilirubin standard are
available, as are serum-based calibrated bilirubin
standards. However, concerned laboratories should
check the accuracy of serum-based calibrators by using
pure bilirubin standards available from the National
Institute for Standards and Technology (Standard
Reference Material No. 916a). Several methods are
available for preparing pure bilirubin standards, and
these are discussed below. Bilirubin conjugates have
been synthesized and are available commercially
(Ultimate D, Beckman, Brea, CA). There is evidence
that these conjugates react similarly to glucuronide
conjugates in diazo reactions and thus could be used for
more accurate standardization of direct bilirubin
analyses [4].
Specimen
Total bilirubin determinations using a diazo method
require either serum or plasma. Bilirubin is very
sensitive to and destroyed by light and heat, and
specimens should be protected from ambient light prior
to and during analysis. Concentrations may decrease by
30% to 50% per hour if exposed to direct sunlight. If
separated and stored in the dark, serum or plasma is
stable for 2 days at room temperature, 4 days at 4C and
at 20C for an indefinite period. Repeated freezing and
thawing has no significant effect.
Conjugated bilirubin may be determined in either serum
or plasma, but serum is the usual sample. Spinal fluid
samples can be analyzed for both total and direct
bilirubin. Urine samples can also be analyzed by direct
diazo methods, since the protein-bound unconjugated
bilirubin does not cross the glomerular barrier [68].
266
Bilirubin
Interferences
Diazo methods are prone to interference from turbidity,
hemoglobin, indican, metal ions, and some drugs such as
L-dopa [14]. Although metal ions, especially zinc, can
cause significant interference in clinical practice, it is
thought to be of little importance [11]. Interference from
turbidity and lipemia is reduced when caffeine or other
surfactants are used [23]. Interference from high protein
concentration (due to paraproteinemia) has been reported
to cause spurious elevation with the Roche automated
total bilirubin method and is thought to be due to
precipitate formation [69].
Indicans, which are present in high concentration in
uremia, can react with diazo reagents and give
spuriously high results. Methods using dichlorophenyl
diazonium salts are most affected, and diazotized
sulfanilic acid methods are least effected [70].
Interference from indicans can be reduced by shortening
the reaction time [71,72].
Hemolysis can cause interference in the diazo methods
by several possible mechanisms: interfering with the
diazo reaction, spectral interference [73], and destruction
of bilirubin prior to its reaction [74]. The degree of
interference depends on the formulation of the bilirubin
reagent [75], and the interference can be negative or
positive [76]. In neonates, the commonest method of
blood collection for bilirubin is by capillary heel-prick,
and this method is associated with some degree of
hemolysis [77]. The mean serum free hemoglobin
concentration in capillary heel-prick samples was found
to be 1.62 g/L (range < 0.5 to 6.00 g/L), and this can
cause significant negative interference when bilirubin is
measured by methods using 2,5-dichlorophenyl
diazonium tetrafluoroborate (DPD) [78].
The presence of lipemia has been shown to increase
measured bilirubin concentrations. Turbid serum creates
artifacts in the performance of spectrophotometers and
should not be directly analyzed. The use of gel-barrier
tubes for collection of specimen showed no significant
change in bilirubin concentrations after 1 week [79].
Bilirubin Reference Interval
Reference interval for bilirubin varies with the method.
For most diazo-based methods, the reference interval for
total bilirubin concentration in healthy adults is up to 26
mol/L (15 mg/L), with a median of 12 mol/L (7.0
mg/L). In both sexes, the median concentration rises
after puberty. After puberty, males have both higher
median values and a more pronounced skew to high
levels than females do [8]. The observation that men
have a higher serum value than women has been
confirmed, but the difference is probably not clinically
significant. In addition, significant racial differences
have been reported [80]. The racial differences were
more pronounced between the female groups than the
male. Concentrations decrease slightly during the sixth
through ninth decade of life [81].
Conjugated bilirubin levels up to 3.5 mol/L (2 mg/L)
are found in infants by 1 month of age, and conjugated
bilirubin remains at this level thereafter. Figure 3
summarizes serum bilirubin concentrations as a function
of age and sex [80].
Serum bilirubin shows diurnal variation, with evening
values being approximately 30% higher than in the
morning [82]. During pregnancy, serum unconjugated
bilirubin concentrations are about 55% lower compared
to nonpregnant values. Some of this change is due to
decreased albumin concentration during pregnancy [83].
A small seasonal variation (lower in summer) has also
been described [84]. In breastfed infants, the serum
bilirubin concentration is higher (up to 253 mol/L)
compared to bottle fed infants (up to 212 mol/L) [85].
Fasting tends to increase serum bilirubin; after 48 hours
of fasting, a 240% rise in bilirubin is observed.
Interpretation
The most common cause of an elevated bilirubin
concentration in the population is Gilberts syndrome, a
benign condition affecting up to 5% of the population
and due to reduced hepatic glucuronyl transferase
activity as a result of a mutation in the bilirubin-UDP-
glucuronosyltransferase (UGT1A1) gene. Total bilirubin
concentration is usually < 50 mol/L in this condition
and is predominantly unconjugated. Fasting tends to
increase the bilirubin concentration in Gilberts
syndrome [86].
Jaundice is common in the newborn and is most
commonly due to elevation in unconjugated bilirubin as
a result of immaturity of the liver (physiological
jaundice) or due to hemolytic disease of the newborn.
Serum unconjugated bilirubin may reach more than 400
mol/L in hemolytic disease.
In the adult, increased bilirubin and jaundice can arise
due to increased production (prehepatic), diseases of the
liver (hepatic), or obstruction to bile flow (posthepatic).
In prehepatic jaundice, the bilirubin is predominantly
unconjugated, and the concentration rarely exceeds 100
mol/L. In obstructive jaundice, the bilirubin is
predominantly conjugated bilirubin, and concentrations
in excess of 700 mol/L can be seen. In hepatic jaundice,
there is a mixture of conjugated and unconjugated
bilirubin.
Although it has been possible to measure -bilirubin, the
methodology is limited to HPLC, dry-chemistry, or
enzymatic methods and therefore not available in all
clinical laboratories. There has been a suggestion that
measurement of -bilirubin may be helpful in monitoring
biliary drainage. An increase in the percentage of -
bilirubin is an indication of good drainage [43].
Measurement of -bilirubin and conjugated bilirubin has
been reported to be useful in the early detection of
rejection after liver transplantation [87].
Apart from its use in the diagnosis of liver and biliary
diseases, serum bilirubin has also been suggested to be a
267
Bilirubin
marker of coronary heart disease. Bilirubin via its
antioxidant potential is inversely associated with risk of
coronary artery disease [88].
Bilirubin Performance Goals
The current target for acceptable performance for total
bilirubin measurements as mandated by The Clinical
Laboratory Improvement Amendments [89] is 0.7
mol/L (4.0 mg/L) or 20% of the peer-group mean
value, whichever is greater. The recommended target,
however, is 10% [90]. The interlaboratory coefficient of
variation (CV) is generally 6% to 11% at bilirubin levels
near normal ranges (approximately 10 mg/L) and 3% to
7% CV at bilirubin concentrations of 45 to 50 mg/L [91].
In healthy adults, bilirubin demonstrated a within-subject
variation of approximately 17% when measured over a
20-week period [92].
References
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Table 1: Methods for Total Bilirubin Analysis

Method Principle Usage Comments
Diazo Methods
Method 1 Mallory-Evelyn Reaction of diazotized sulfanilic Of historical Risk of protein
acid at pH 1-2 interest precipitation
Method 2 Jendrassik-Grof Reaction as above, but readings Most common
taken at alkaline pH method
Other Diazo Methods
Method 3 Dichlorophenyl- Rapid reaction with bilirubin Used in Roche
diazonium- under acidic conditions modular
tetrafluoroborate analyzer
(DPD)
Method 4 Dyphylline Similar to Method 2, Used in dry-
absorbance measurement at chemistry
540 nm system
Direct
Spectrophotometric
Methods
Method 5 Bilirubinometer Direct absorbance reading of Useful POCT Interference from
plasma at 454 nm device oxyHb
Method 6 Dry chemistry Reflectance spectrophotometry
Method 7 Transcutaneous Reflectance spectrophotometry Useful POCT May not be
bilirubinometer device applicable in
subjects with
dark skin
Oxidation
Method 8 Enzymatic Decrease in absorbance Used in
following oxidation of automated
bilirubin by the enzyme analyzers
bilirubin oxidase
Method 9 Chemical oxidation As above but oxidation using Used in
potassium ferricyanide automated
analyzers
271
Bilirubin

Figures

Figure 1

Formation of diazotized sulfanilic acid and its reaction with esterified and nonesterified forms of bilirubin to form
azobilirubin derivatives. Me, CH3 group; R, CH=CH2 group.

Figure 2

Spectra of alkaline azobilirubin chromophores. Dashed

curve, reagent versus water; dashed-dotted curve, sample
(serum containing 65 mg/L total bilirubin) reaction versus
water; solid curve, sample reaction versus reagent blank.
272
Bilirubin

Figure 3

Figure 3
Serum total bilirubin concentrations in percentiles. In both sexes, median concentration rises after puberty, falls during
third decade, and thereafter remains stable. Notice that similar shifts are more pronounced at 90th and 95th percentile
levels and that 5th and 10th percentile levels hardly change. After puberty, males have both higher median values and a
more pronounced skew at high values than females do. (From [80] Werner M et al. Z Klin Chem 1970;8:105-115.)

Preparation of Bilirubin Standard
Pure bilirubin can be obtained from several commercial
sources. However, the best source is the National
Institute for Standards and Technology (Standard
Reference Material No. 916a). It should be used as
described below to calibrate serum that is used as the
reference or standard in the total bilirubin procedure.
Thus the commercially available reference pools or
laboratory-prepared pools can be checked for the
accuracy of the stated bilirubin values. If the procedure
is carefully carried out, one should use the value
obtained by the validation method rather than the
manufacturers stated value if there is a substantial
difference between the two.
The following procedure must be carried out in a well-
darkened room:
Stock Bilirubin Standard (200 mg/L) Weigh about 20
mg of bilirubin SRM916a to the nearest 0.01 mg in a
plastic weighing boat, and transfer to the bottom of a
100-mL volumetric flask using 1 mL of dimethyl
sulfoxide (DMSO). Disperse the bilirubin by gently
swirling the contents of the flask. Wash the plastic dish
and the neck of the flask with 2.0 mL of 0.1 M sodium
carbonate solution (solution 10). Swirl the flask gently
until a crystal-clear red-orange solution is obtained.
Make up to 100 mL with the BSA diluent. Wrap the
flask in aluminium foil to protect from light. This
standard is stable for at least 6 months at 70C, but
deteriorates at 20C.
273
Bilirubin
Inspect an aliquot of the stock standard after
centrifugation at 1500 g for 10 minutes. If there is any
orange sediment visible, discard the stock and prepare it
again.
Analyze the stock standard solution in triplicate using
the method described below. Omit the addition of
ascorbic acid before the tartrate, since there is no
hemoglobin. Analyze the BSA diluent in duplicate as a
test, and subtract its absorbance from that of the stock
standard. Calculate the molar absorptivity of the
azopigments at 598 nm. Acceptable limits for the molar
absorptivity are from 74,750 to 78,380. If the molar
absorptivity falls outside these limits, prepare a new
stock standard.
Working Standards: Dilute the stock standard with BSA
diluent to give bilirubin concentrations of 5, 10, 15, 20,
50, and 100 mg/L. Dispense these and the stock
standards into polypropylene tubes in suitable aliquots
and store at 70C. These standards are stable for at
least 6 months.
This standard is suitable for methods using caffeine or
dyphylline as accelerator but is not suitable for direct
spectrophotometric methods or diazo methods that use
other accelerators. BSA (Cohen fraction V, 40g/L)
solution is an acceptable diluent for the stock calibrant of
unconjugated bilirubin.
An alternative method for preparation of stock standard
in protein matrix is to dissolve pure bilirubin using
sodium hydroxide. Weigh out approximately 22 to 24
mg of bilirubin in a tared 50-mL beaker. Add 4 mL of
buffered NaOH, pH 11.3. {To prepare, dilute 1 mL of 5
N NaOH to 100 mL with 0.067 M pH 7.4 Srensens
phosphate buffer. [This buffer is made by mixing 41.3
mL of 0.067 M monopotassium phosphate (9.073 g
KH2PO4/L) with 51.7 mL of 0.067 M disodium
phosphate (11.87 g Na2HPO4 2H2O/L)]}. Stir gently
with a glass rod, but do not form foam or bubbles. The
bilirubin will dissolve completely in 2 to 4 minutes to
form a clear, red-orange solution. Add 40 mL of 40 g/L
bovine serum albumin solution dissolved in 0.067 M pH
7.4 Srensens phosphate buffer and stir. An alternative
to the 40 g/L albumin solution is Plasmonate (Cutter
Laboratories, Berkeley, CA). Completely transfer the
solution to a 100-mL volumetric flask with the aid of
additional albuminpH 7.4 Srensens phosphate buffer,
and bring to volume with this solution.
Procedure: Jendrassik-Grof Method for Total
Bilirubin
Principle
In this method, the specimen is added to a caffeine
reagent and then diazotized sulfanilic acid solution.
After a period of incubation, ascorbic acid and alkaline
tartrate are added. During the incubation period,
bilirubin reacts with the diazo reagent to form
azobilirubin. Alkalinization of the mixture produces a
blue-green solution which is measured at 598 nm.
Reagents
1. Sulfanilic acid (26.2 mmol/L in 0.18 mol/L
HCl). Add 5.0 g of sulfanilic acid to about 500 mL of
distilled water, followed by 15 mL of concentrated
hydrochloric acid (approximately 12 mol/L), and make
up to 1 L with water. Store at room temperature.
2. Sodium nitrite stock (0.725 mol/L). Dissolve
0.5g sodium nitrite (NaNO2) in distilled water, and
dilute to 100 mL. Prepare fresh every 2 weeks.
3. Caffeine-benzoate reagent. Dissolve 56 g of
anhydrous sodium acetate, 56 g of sodium benzoate, 1 g
of disodium ethylenediamine tetra acetic acid (EDTA)
and 37 g of caffeine in about 700 mL of distilled water.
Make up to 1 L. This reagent is stable at room
temperature for at least 6 months.
4. Hydrochloric acid 0.05 mol/L.
5. Diazo reagent. Mix 20 mL of the sulfanilic
acid with 0.5 mL of sodium nitrite solution. Prepare
fresh daily and store at 4C.
6. Ascorbic acid solution (40 g/L). Dissolve 200
mg of ascorbic acid in 5 mL of water. Prepare fresh daily
and store at 4C.
7. Alkaline tartrate solution. Dissolve 75 g of
sodium hydroxide and 320 g of sodium potassium
tartrate tetrahydrate in about 700 mL of water. Allow the
solution to cool, and make up to 1 L. If the solution is
turbid, filter. This reagent is stable for 6 months at room
temperature.
8. Serum-based calibrant and controls.
9. Bovine serum albumin (BSA) diluent (40
g/L). Dissolve 10.0 g of BSA in 200 mL of Tris buffer,
adjust the pH to 7.35 0.05 if necessary, and make up to
250 mL with Tris buffer. Store at 4C for 2 to 3 days or
at 20C (in aliquots) for long-term storage. This diluent
is required for the preparation of standards.
10. Sodium carbonate (0.1mol/L). Dissolve 1.06 g
of anhydrous sodium carbonate, and make up to 100 mL.
Assay
Equipment: spectrophotometer, with band pass 8 nm,
capable of reading at 598 nm. For accurate results, the
analysis should be performed in subdued light and away
from windows.
1. Pipette 4.0 mL of caffeine reagent into a series
of glass tubes.
2. Pipette 0.5 mL of sample (standard, serum, or
QC), mix, and allow to stand for 10 min.
3. Add 1.0 mL of freshly prepared diazo reagent,
mix immediately, and let stand for 10 min (add
the diazo reagent at timed intervals, and
observe the same interval for the addition of
alkaline tartrate and for the absorbance
measurement).
4. Add 0.1 mL of ascorbic acid solution and mix.
5. Add 3.0 mL of alkaline tartrate, mix
thoroughly, and let stand for 10 min before
measuring the absorbance.
6. Sample blanks. Follow the procedure as above
from 1 to 5, but substitute sulfanilic acid
solution instead of diazo reagent.
7. Reagent blank. Prepare a regent blank by
mixing 20 mL of caffeine reagent, 2.5 mL of
274
Bilirubin
water, 5 mL of diazo reagent, and 15 mL of
alkaline tartrate. A reagent blank is required to
check for photometric drift during the assay.
8. Absorbance. With the reagent blank, set the
instrument to zero absorbance at 598 nm.
Measure the absorbance of all the sample
blanks and of the BSA diluent (analyzed as test)
and then the absorbance of all the tests. For
each test sample, subtract the absorbance of the
corresponding blank for the test absorbance.
Correct the absorbance of the standards by
subtracting that of the BSA diluent.
9. Using the standard curve or the regression
equation of the standards, calculate the
concentration of total bilirubin in each of the
unknowns.
Calculation
Notes
1. Volume of samples and reagents can be reduced
to half of what is described without
compromising the precision of the assay.
2. The large excess of sulfanilic acid in relation to
sodium nitrite is necessary for obtaining a fast
reaction rate in the second stage of the reaction.
Procedure for Direct Bilirubin
Principle
In the direct bilirubin method, the sample is acidified
with hydrochloric acid and then mixed with diazo
reagent to produce azobilirubin. Only conjugated
bilirubin reacts with diazo reagent in the absence of
accelerator caffeine-benzoate. Addition of ascorbic acid
stops the reaction, and after addition of alkaline tartrate
followed by caffeine reagent, the absorbance is
measured at 598 nm. The tartrate reagent provides an
alkaline pH to produce the intense blue color of
azobilirubin.
Reagents
See Total Bilirubin Procedure: Reagents.
Assay
Equipment: See the total bilirubin assay.
1. Add 0.25 mL of sample to 1.0 mL of 0.05
mol/L HCL, and incubate for 5 min. Add 0.5
mL of diazo reagent an incubate for 10 min.
2. Add 0.1 mL of ascorbic acid, followed by 1.5
mL of alkaline tartrate and 2.0 mL of caffeine
reagent.
3. Set up a sample blank exactly as the test, except
substitute sulfanilic acid for diazo reagent.
4. Measure the absorbance of test and blank
against water at 598 nm, and subtract
absorbance of the blank from the test.
5. Calculate the concentration of conjugated
bilirubin in the sample against a standard of
unconjugated bilirubin analyzed by the total
bilirubin method (dilute the sample with
caffeine regent instead of HCl, and add the HCl
after the alkaline tartrate).
Notes
1. A commercially available conjugate of bilirubin
(Ultimate D; SmithKline-Beckman, Brea, CA) can
be used to standardize this reaction.
2. The dilution of the sample with HCl for 5
minutes keeps the unconjugated bilirubin from
reacting.
3. Ascorbic acid destroys the excess diazo reagent.
Otherwise, the diazo reagent will react with
unconjugated bilirubin when the alkaline tartrate is
added.
4. Hemoglobin may interfere with this assay, more
than in the total bilirubin method. The negative bias
due to hemoglobin is caused by (1) oxidation of
conjugated bilirubin to biliverdin, which does not
react with diazo reagent, and (2) destruction of the
azo pigment by hydrogen peroxide produced when
hemoglobin is converted to methemoglobin.
5. Under the conditions of the assay described
here, there is minimal reaction of the unconjugated
bilirubin with the diazo reagent.
Calculation
The blank-corrected absorbance of cuvette C, the
absorbance of the standard, and the concentration of the
standard are used to determine the concentration of
conjugated bilirubin in the sample (see total bilirubin
procedure).
275
Blood-Gas Analysis and Oxygen Saturation
Blood-Gas Analysis and Oxygen Saturation
Goce Dimeski
Name: Partial pressure of oxygen (PO2), carbon dioxide (PCO2), and the pH of whole
blood
Clinical significance: Refer to Chapter 29, Acid-Base Control and Acid-Base Disorders, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
i chloride. The calomel electrode consists of a glass tube
Blood-gas analyses are usually performed to assess the
acid-base status and respiratory status of the patient. It
includes measurement of blood pH, PCO2, and PO2 and
may include any or all of the calculated parameters of
oxygen content, oxygen saturation, total CO2,
bicarbonate, and base excess. These calculated
parameters are discussed in Chapter 25 of Clinical
Chemistry: Theory, Correlation, Analysis and are not
considered further in this chapter.
Analysis of pH
Although pH is not a gas, it is commonly analyzed
concomitantly with PCO2 and PO2 in blood specimens
so that patient acid-base status can be assessed.
Maintenance of the blood pH is partly dependent on the
respiratory processes that eliminate CO2 from the blood
and take up O2. In addition, the calculation of
bicarbonate, total CO2, and base excess requires
knowledge of the blood pH.
The components of the blood pH-measuring system are
the glass electrode, reference electrode, and the liquid
junction between the two electrodes [1]. The hydrogen
ions in the blood sample exchange with metallic ions in
the glass membrane of the glass electrode, creating a
potential difference across the membrane that is
proportional to the hydrogen-ion concentration in the
blood specimen. A solution of constant pH inside the
electrode is in contact with the glass membrane. A
silversilver chloride wire that is connected to a
voltmeter lies inside the solution of constant pH and
measures the potential difference created by the
hydrogen-ion concentration in the blood specimen, as
shown in Figure 1.
The potential difference measured by the glass electrode
is assessed against the reference electrode (Figure 1).
The reference electrode, which is also connected to the
voltmeter, is usually a calomel electrode or silversilver
i solution inside the electrode, and the following reaction
Blood-Gas Analysis and Oxygen Saturation
Previous and current authors of this method:
First edition: John E. Sherwin, Berndt B. Bruegger
Methods edition: John E. Sherwin, Berndt B. Bruegger
Second edition: John E. Sherwin, Berndt B. Bruegger
Third edition: John E. Sherwin
Fourth edition: John E. Sherwin, Christopher Tennant
Fifth edition: Goce Dimeski
containing a platinum wire that extends into a calomel
paste of mercurous chloride (Hg2Cl2) and mercury (Hg)
and a solution of potassium chloride (KCl) at a constant
concentration. One end of the glass tube usually contains
a cotton wick to keep the calomel paste in contact with
the platinum wire and allow the system to be permeated
with the KCl solution that is inside the glass tube and
surrounding it. The KCl solution is usually a saturated
solution, although some pH electrode systems use lower
concentrations of KClfor example, the Radiometer
ABL uses a 20% KCl solution. The silversilver chloride
electrode contains a silver wire coated with silver
chloride and an ion-permeable polymer saturated with
KCl. The KCl block in the reference chamber allows for
the concentration of Cl- to be maintained constant in the
solution, ensuring the reference sensor maintains a
constant electrical potential.
A liquid junction is present between the reference
electrode and blood specimen being analyzed for its pH.
Generally the reference electrode has a permeable
membrane through which the KCl solution inside the
electrode escapes, coming into contact with the
specimen and thus forming the junction. The KCl
solution is at a high concentration so that the difference
in ionic composition of the blood specimen does not
change the constant potential on the reference electrode.
A representation of the entire pH-measuring system is
shown in Figure 2. The only variation in potential in the
system is at the interface of the blood specimen being
analyzed and the glass electrode, so that any voltage
change measured by the voltmeter is attributable to the
pH of that blood specimen.
Analysis of PCO2
PCO2 is measured by isolating a glass electrode in a
weak bicarbonate buffer and separating this buffer from
the blood sample by a gas-permeable membrane. The
membrane is permeable to the carbon dioxide (CO2) in
the specimen. The CO2 diffuses into a bicarbonate buffer
occurs:
CO2 + H2O H2CO3 H+ + HCO3
The change in hydrogen concentration in the buffer is
then measured inside the PCO2 electrode by the same
type of components found in the pH electrode. A glass
276
Blood-Gas Analysis and Oxygen Saturation
electrode inside the PCO2 electrode develops a potential
difference because of a change in concentration of H+
ions, while a reference electrode also inside the PCO2
electrode has a constant potential that is the standard to
which the change in potential at the glass electrode is
compared. The glass electrode and reference electrode
are connected to a voltmeter, as shown in Figure 3. The
gas-permeable membrane of the PCO2 electrode
excludes hydrogen ions in solution, so the pH of the
specimen will not affect the observed pH change inside
the PCO2 electrode induced by the diffusion of the CO2
from the specimen into the electrode.
Analysis of PO2
The measurement of PO2 relies on the electrochemical
measurement of the oxygen that diffuses across a gas-
permeable membrane into an electrolyte buffer that is
within a Clark electrode [2]. The gas-permeable
membrane allows oxygen but not ions to cross into the
electrode. Inside the electrode is an electrolyte solution
consisting of potassium chloride and phosphate buffer,
or buffered potassium hydroxide, a silversilver chloride
anode, and a platinum cathode. The cathode is held at a
constant potential with respect to the anode so that when
oxygen diffuses into the electrode, the oxygen is reduced
at the cathode as follows:
O2 + 2H+ + 4e H2O2 + 2e 2OH There is a small amount of diffusion of gas from the
The reduction of oxygen causes a current to flow
between the cathode and anode. The current is measured
and is proportional to the amount of oxygen diffusing
into the electrode from the analyzed specimen. Figure 4
illustrates the components of the PO2 electrode. The
electrolyte solution buffers against the accumulation of
peroxide (H O ) and hydroxide (OH), as well as
2 2
serving as the conduction medium for the diffusing
oxygen and the current. In contrast to the pH and PCO2
electrodes, the oxygen electrode is a destructive,
nonequilibrium electrode. This has led to controversy
concerning the most accurate way to calibrate a PO2
electrode [3,4]. Calibration can be achieved either with a
flowing gas or a single equilibrated liquid sample.
The majority of the older models of blood-gas analyzers
use certified gases saturated with water for calibration.
This technique has the advantage of providing a constant
and highly reproducible calibration mixture.
Nevertheless, it suffers from the fact that it is a gas
rather than a liquid and therefore is not equivalent to the
patient samples being analyzed. It is essential that the
gas-flow rate through the measuring chamber be strictly
controlled. Variations will result in electrode cooling and
incomplete humidification, both of which result in
significant calibration errors. The new-model analyzers
use the second calibration technique, tonometered (gas-
buffered) liquid for calibration. The advantages of this
technique are that it is similar to the unknown samples
being analyzed, the calibration is independent of the
flow rate of the gas, and humidification is not a
consideration. This technique results in a positive bias of
about 5% to 8% for both PCO2 and PO2 as compared to
the gas calibration technique, and it is important that the
results from instruments using different calibration
techniques be identified. If multiple instruments are
used, the calibrations can be cross-correlated to
minimize this difference.
Noninvasive In-Vivo Blood-Gas Analysis
In the past few years, noninvasive means of measuring
PO2 and PCO2 through the skin have been developed,
evaluated, and found to be useful [5]. These
transcutaneous monitors allow continual assessment of
the blood-gas status of a person. This is especially
important for monitoring neonates suffering from
respiratory distress. Too low an O2 content can lead to
brain damage in a neonate, whereas too high a level can
lead to blindness through damage of the retinal
capillaries by oxygen.
The transcutaneous measurement of PCO2 and PO2
currently relies on miniaturized electrodes of the same
basic design as the electrodes of blood-gas analyzers.
These transcutaneous, noninvasive measurements are
complicated by the presence of the skin, which acts as a
second gas-permeable membrane.
capillary bed in the skin toward the surface of the skin. If
the skin is heated so that local blood flow is maximally
increased, the PO2 at the surface of the skin correlates
well with the arterial PO2. Temperatures of 42C to 43C
have been found to be best for maximal blood flow
while minimizing the risk of burns, so a major feature of
the transcutaneous PO2 (tcPO2) electrode is a heating
element maintaining the 42C to 43C temperature. The
tcPO2 electrode uses the same concept for measuring
PO2 as the Clark polarographic PO2 electrode described
earlier. A membrane, through which PO2 diffuses, lies
between the skin and the electrode, and a heating device
is present inside the electrode (Figure 5). A sealing ring
around the outer edge of the electrode needs to be
present to prevent nonregulated diffusion of atmospheric
oxygen to the electrode. Because the PO2 electrode
consumes oxygen during measurement, the area of the
cathode is kept as small as possible, minimizing O2
consumption by the electrode so that measurements will
correlate with arterial PO2 values.
The transcutaneous PCO2 (tcPCO2) electrode is based on
the same principles as the in-vitro PCO2 electrode
discussed previously, except that a thermostatted internal
heater has been added. The tcPO2 electrode is sealed
from the atmosphere so that only CO2 from the skin is
detected. The skin is heated by the electrode for maximal
capillary flow. However, increased temperature also
increases skin metabolism, which increases the PCO2.
277
Blood-Gas Analysis and Oxygen Saturation
Additionally, the tcPCO2 electrode does not consume the
CO2 as the tcPCO2 electrode consumed O2, so the PCO2
is higher at the electrode than at the area of the capillary
closest to the surface of the skin. The tcPCO2 electrode
system uses a correction factor to compensate for this
increase in PCO2 resulting from the electrode
arrangement. The tcPCO2 electrode uses two gas
mixtures to calibrate CO2, in contrast to the tcPO2
electrode which uses surrounding atmospheric oxygen.
In-vivo pH monitoring is being developed but at this
time is still invasive. A pH electrode probe can be
inserted into muscle tissue to measure the interstitial
fluid pH. Some assumptions in using the data derived are
that interstitial fluid pH reflects the status of the blood
pH and that the insertion of the pH electrode probe does
not cause local tissue changes that alter the pH.
Analysis of Oxygen Saturation (sO2)
The oxygen saturation is a ratio of the amount of
hemoglobin (Hb) bound to oxygen to the total amount of
hemoglobin able to bind oxygen, expressed as
percentage. Oxygen saturation can be accurately
calculated by use of equations 1 to 3 below, directly
determined from the measured oxyhemoglobin (O2Hb)
and reduced hemoglobin (HHb) (equation 4) fractions or
determined by pulse oximetry.
Estimated oxygen saturation does not account for
variations in 2,3 DPG levels, or the presence of
dysfunctional hemoglobins (COHb, MetHb). These
equations assume no presence of dysfunctional
hemoglobins. If any of these conditions change, the
estimated sO2 will be inaccurate. Oxygen saturation can
be calculated from the measured parameters PO2 and pH
on the basis of standard oxygen-dissociation curves [6].
Equation 1: sO2 = {[(PO2 + 150 PO2)-1 23 400] +1}-1
The Severinghaus [7] equation assumes the following
parameters are normal: pH = 7.40, PCO2 = 40 mm Hg
and 2,3-diphosphoglycerate = 5 mmol/L.
Equation 2: The Radiometer ABL uses the following
Hill equation to determine oxygen saturation from the
measured pH and O2:
in which,
Found in the equation is a Bohr factor of 0.48 and a
standardized P50 of 26.6 mm Hg.
Equation 3: The Siemens (formerly Bayer) analyzers use
the relationship described by Kelman [8] and Thomas
[9]:
N = pO2 10[0.48(pH(37) -7.4) 0.0013BE(B)]
Equation 4: O2 saturation = ((O2Hb/(O2Hb + HHb))
100
The O2 saturation is often referred to as the fractional
sO2 (SaO2), since it is determined from the two
hemoglobin fractions. This is the most accurate method
for sO2 estimation because it is a direct measure of the
parameters that affect sO2. CO-oximetry is considered
the gold standard for oxygen saturation measurement.
The benchtop blood-gas instruments with oximeters
calculate oxygen saturation using this formula.
Pulse oximetry is a third method for sO2 estimation. The
saturation calculated from pulse oximetry is a functional
saturation, SpO2. It is the amount of HbO2 compared
with the sum of O2Hb and HHb only. These systems do
not measure COHb and metHb, so the SpO2 is falsely
elevated in the presence of dysfunctional hemoglobins.
In the presence of normal SpO2 with cyanosis, CO-
oximetry must be used to determine the saturation status
and presence of any dysfunctional hemoglobin.
Calibration of Gas Electrodes
Gases used to calibrate blood-gas analyzers are bubbled
through humidifiers at 37C to saturate the gases with
water vapor. The reason for saturating the gases with
water is to eliminate the problem of maintaining a totally
dry gas for calibrating the blood-gas instruments.
Some residual moisture will remain in the measuring
chamber and contaminate a dry gas. The pressure of
water vapor will reduce the partial pressures of the other
gases in the mixture and introduce a variable error. For
instance, at 37C, a water-saturated gas will have a
partial pressure of 47 mm Hg, contributed by the water
vapor. So to produce a reproducible gas mixture of
known partial pressure of O2 and CO2, calibration
requires a fixed partial pressure of water vapor. Since the
specimen to be analyzed is aqueous, this is best achieved
with a water-saturated gas. The cartridge-based
analyzers contain all the calibrants, including gases, in
the single cartridge. This eliminates the need to have
separate gas bottles, and the gases are water saturated.
Modern Instruments
In the past, instrument design led to a variety of required
sample volumes for blood-gas analysis. All the modern,
278
Blood-Gas Analysis and Oxygen Saturation
commonly used blood-gas analyzers have the capability
of analyzing microsamples of < 100 L to measure pH,
PO2, and PCO2 and usually the same volume to measure
all of the on-board analytes. The other range of analytes
available on many blood-gas instruments include
sodium, potassium, chloride, ionized calcium, ionized
magnesium, lithium, lactic acid, glucose, urea, and
creatinine (all by analyte-specific electrodes) and
oximeters to measure hemoglobin fractions. The actual
analyses that are available depend on the electrode
configuration adapted for each instrument. Most
instruments currently available automatically aspirate the
sample, thus reducing errors resulting from manual
introduction of the sample. Therefore in total, < 1.0 mL
of whole-blood sample is required in a syringe to
complete all the analytes and allow for dead volume.
Most blood-gas instruments are now microprocessor
controlled so that calibration occurs automatically at
preset intervals, and both the measured parameters and
the calculated parameters are displayed when analysis is
complete. Maintenance has been greatly simplified by
the use of microprocessors to monitor all system
parameters and indicate potential malfunctions. The
basic principles of electrode operation are unchanged
from the original equipment, and all available
instruments use equivalent electrodes. Nonetheless, the
electrode configuration within the sample compartment
can significantly affect both the sample volume and the
analysis time. Thus there can be statistical differences in
measured parameters between instruments produced by
different manufacturers, as well as between different
models produced by the same manufacturer. A review of
the 2007 blood-gas proficiency surveys of the College of
American Pathologists shows similar precisions for the
analyzers included in the survey. The pH coefficients of
variation (CVs) across the different concentration levels
for about 95% of the analyzers are 0.1%. The remaining
5% of the analyzers have CVs between 0.1 and 0.5%.
The PCO2 CVs are slightly higher at the low end (PCO2
< 20 mm Hg), ~ 5%, and decrease down to ~ 3% with
values at the high end (PCO2 ~ 60 mm Hg). The greatest
variation in both medians and CVs is seen with the PO2.
For samples with PO2 values < 100 mm Hg, the CV for
PO2 with some analyzers is as high as 11.2%. Samples
with PO2 values > 100 mm Hg had much lower CVs,
averaging < 5%the predominant reason being the
quality of samples used in the survey (aqueous and
poorly buffered). Thus even small exposure to air during
the sample aspiration/presentation process to the
analyzers has the potential to expose the sample to air
and significantly change the PO2 concentration.
Therefore, the differences seen can be ascribed to sample
aspiration/presentation technique, the different methods
of gas calibration, or the juxtaposition of the electrodes
to each other.
Blood-gas analyzers are also available for use in point-
of-care sites (e.g., surgery sites, intensive care,
emergency departments, etc.), as bench tops, the
traditional blood-gas analyzer, or as portable or hand-
held systems with disposable single-use or multiple-use
cartridges. These instruments use the same fundamental
electrode technology as conventional instruments and
are designed so that non-laboratory staff can use them.
Most of these have been designated as moderately
complex under the Clinical Laboratory Improvement
Amendments of 1988 (CLIA 88) rules. These new
instruments utilize microelectrode technology. This
permits individual electrode packets to contain not only
the analytic electrode but also the reference electrodes
and calibration solutions. The calibration and quality
control of these electrode assemblies is similar to that
used to measure electrolytes using dry-film technology.
A new technique for measurement of blood gases at the
bedside has recently been described. It is based on dry
chemistry and optical technology [10]. The measurement
principles of this technology include three-wavelength
infrared spectroscopy of dissolved CO for
2
determination of PCO , of O induced changes in the
2 2
decay time of phosphorescence, and of the absorbance
change spectral change of an azo-dye color indicator for
pH.
Reference and Preferred Methods
Although there are no available reference methods for
pH, PO2, or PCO2 estimation, currently available blood-
gas analyzers provide acceptable methods for blood-gas
estimation.
However, attention has now been focused on techniques
that eliminate the need to collect a blood specimen, that
is, the use of noninvasive instruments that measure PO2
and PCO2 [11]. No doubt if noninvasive methods can
provide pH measurement and improved correlation with
conventional blood-gas analyzer methods and cause no
patient harm (e.g., skin burns) [12], they would become
the preferred methods.
Specimen
The skill with which arterial specimens need to be drawn
and the potential harm to the patient limits the personnel
who can collect such specimens. Thus the capillary
specimen is frequently utilized as a compromise,
providing an arterialized specimen that is easily
collected. A capillary specimen can be drawn by a
competent phlebotomist under conditions that produce
pH, PCO2, and PO2 values closely paralleling arterial
blood. Conditions to arterialize capillary blood involve
warming the limb for several minutes at about 45C
immediately before the specimen is collected. The
warming dilates the capillaries, and the increased blood
flow in that area decreases the accumulation of
metabolic products of tissue respiration and ensures
more blood of an arterial than a venous composition.
Alternatively, a histamine cream can be used to produce
local hyperemia so that an arterialized specimen can be
collected.
Blood-gas measurements must be performed on whole
blood that is unclotted and well mixed. An anticoagulant
279
Blood-Gas Analysis and Oxygen Saturation
is used to inactivate the clotting mechanisms so that the
sample remains unclotted in the syringe. Whole blood is
collected in heparinized capillary tubes which may
contain a small metal mixing wire (flea), which is added
to facilitate mixing with the magnet provided. These are
then sealed at one or both ends. The capillaries are
placed into a labeled test tube or plastic bag and
delivered to the laboratory in crushed ice or wrapped in a
cold pack to prevent deterioration of the sample. Since
excess heparin can alter the pH of the specimen, sample
tubes containing heparin should be properly filled to
avoid heparin effect.
The time necessary for completion of the test should not
exceed 15 minutes at room temperature (~21C). This
includes drawing of sample, delivery to the laboratory,
log-in, analysis, and reporting of results. Samples for
blood-gas analysis are stable for a maximum of 45
minutes if properly stored in crushed ice [13]. If this
maximum time limit is adhered to, no difference is
observed between specimens collected in plastic and
glass syringes. Samples placed in crushed ice or ice
slush (ice/water) must be properly sealed and clean of
any exterior blood, particularly around the base of the
cap, to prevent leakage and contamination of the ice
slush. Ideally, samples should be placed in a biohazard
bag before placing in crushed ice. Alternatively, samples
placed in biohazard bags can be transported wrapped in
cooled (~4C) gel packs.
Syringes have been developed that allow air from the
sample to be expelled through the cap. The cap contains
absorbent material which is the same as that used in
autoventing syringes. Once the blood wets the material
in the cap, it is air sealed. Such caps prevent the need to
expel air, minimizing exposure to blood by collectors
and preventing soiling/contaminating of the bench or
other surfaces. Other syringes contain a metal ball
(Radiometer Pico Syringes for the ABL 800FLEX
system) in the barrel. The syringe is placed on the
system platform, which contains a magnet to spin the
ball to create a uniformly mixed sample.
Glass Versus Plastic Syringes
Early studies indicated that plastic substances may
absorb so much oxygen that the use of plastic syringes
for blood-gas samples would be ill advised. More recent
studies have not substantiated these observations [14]. In
essence, pH and PCO2 values are not affected, whereas
PO2 values in excess of 400 mm Hg drop more rapidly
for samples in plastic than in glass syringes. The rate of
O2 diffusion is also dependent on the temperature of the
sample and the time required before analysis is
completed, with diffusion increasing as both variables
increase. Smeenk et al. [15] studied patients receiving
100% oxygen with initial PO2 of ~ 500 mm Hg and
found PO2 decreased after 30 min in both an ice bath
(~11.2 mm Hg) and at room temperature (~40.5 mm Hg)
[15]. Arterialized samples with PO2 of ~ 96 mm Hg
stored in plastic syringes for 30 minutes at 0C to 4C or
at room temperature both increased the PO2 by 11.9 to
13.7 mm Hg [16]. Glass syringes have been preferred in
the past for the following reasons:
1. The barrel has minimal friction with the syringe
wall, and the pulsating arterial pressure is
clearly visible as the blood fills the syringe, and
thus the nature of the sample is clearly
identified to the clinician.
2. There is seldom a need to pull back on the
barrel, a maneuver that can cause air bubbles to
enter the sample around the barrel.
3. Small air bubbles adhere tenaciously to the side
of a plastic syringe, making it difficult to expel
the air from the samples.
Newer plastic syringes overcome many of these
concerns and when properly used are as good as glass
syringes.
Temperature During Transport and Storage
Blood is a living tissue in which oxygen continues to be
consumed and carbon dioxide continues to be produced,
even after the blood is drawn into a syringe. Table 1
shows the changes in blood-gas parameters at different
temperatures. A sample at 37C shows changes in all
three parameters at a faster rate than a sample held at
4C. The Clinical and Laboratory Standards Institute
(CLSI, formerly NCCLS)-approved guideline # C27-A
1993 cautions against placing plastic blood-gas syringes
in ice/water as this will tend to raise the PO2 by drawing
O2 through the plastic wall of the syringe. Therefore, the
time between placing the specimen in ice/water and the
analysis should be as short as possible in order to
maintain the O2 content of the sample. If the sample will
be brought to the work station within a few minutes of
draw, no ice is needed.
Pneumatic Tube Transport
The transport of samples for blood-gas analysis by
pneumatic tube systems (PTS) does not alter PCO2 and
pH values but does have significant effects on PO2
values. The PO2 changes are due to the presence of
microbubbles in the sample combined with pressure
effects from the delivery system. Samples for blood-gas
analysis should be free of air bubbles, and if transported
via PTS it is best to use a pressure-sealed container to
avoid artifacts in the PO2 [17,18].
Interferences
Oxalates, EDTA, and citrates are not acceptable for
blood-gas samples because they significantly alter the
blood sample [19]. Heparin is the anticoagulant used in
syringes. When blood-gas analyzers were only used to
measure blood-gas parameters, the heparin
concentration was high, > 100 IU/mL. With the
introduction of electrolytes, namely ionized calcium,
heparin caused ionized calcium binding and false
decreases. Syringe manufacturers
responded by either decreasing the heparin concentration
down to ~ 7 IU/mL, which increased the frequency of
clot formation, or titrated or electrolyte-balanced the
heparin to minimize ionized calcium binding while
keeping the heparin concentration at high levels, ~ 35
IU/mL, to prevent clot formation. However, too much
280
Blood-Gas Analysis and Oxygen Saturation
heparin may affect several parameters. The pH of
sodium heparin solutions is approximately 7.0, and the
PCO2 and PO2 of heparin approach room-air values. It
has been shown that 0.05 mL of sodium heparin (1000
units/mL or 10 mg/mL) will adequately anticoagulate 1
mL of blood, whereas up to 0.1 mL per 1 mL of blood
will not affect pH, PCO2, or PO2 values. If more than 50
L of solution remains in a 1-mL syringe and a minimal
specimen (0.2 mL) is collected, a negative error of about
5 mm Hg of PCO2 is observed. When a 5-mL syringe is
washed with sodium heparin and then ejected, the dead
space of the syringe will contain approximately 0.15 to
0.25 mL of sodium heparin. Thus 2 to 4 mL of blood
will theoretically contain at least 0.05 mL of heparin per
1 mL of blood.
Room air contains a PCO2 of essentially zero and a PO2
of approximately 150 mm Hg. Air bubbles that mix with
a blood sample will result in gas equilibration between
the air and the blood. Air bubbles may thus lower the
PCO2 values of the blood sample and cause the PO2 to
approach 150 mm Hg. The greater the amount of air
mixed with a blood sample, the greater the error. The
syringe must be immediately sealed with a cap after the
sample is obtained. It is recommended that any sample
obtained with more than minor air bubbles should have
the PO2 results carefully interpreted and either withheld
or discarded. As a general guide, if the PO2 value is >
110 mm Hg and the patient is not oxygen supplemented,
air contamination should be considered.
Blood-Gas Reference Interval (at 37C)
The unit commonly used to express partial pressure of
blood gases is millimeters of mercury (mm Hg) or its
equivalent, the torr. However, in the International
System of Units, the kilopascal (kPa) is the
recommended unit of expression, though it is not yet
widely used. One millimeter of mercury, or 1 torr, is
equal to 0.133 kPa, or divide mm Hg by 7.5 to convert to
kPa.
The three types of blood specimens assayed for blood-
gas content are arterial, venous, and capillary. The above
outline compares the normal ranges for arterial and
venous blood-gas parameters. Arterial blood is not so
affected by the metabolic activity of the area being
sampled as venous blood is. The slightly lower pH and
PO2 values and the slightly higher PCO2 in the venous
blood reflect the changes induced by tissue metabolic
activity, which releases acids and uses oxygen. Arterial
blood specimens reflect the state of pulmonary activity,
which introduces oxygen to the blood and releases
carbon dioxide from the blood to the air spaces of the
lungs. Although the usually small differences in pH
between arterial and venous blood specimens allow the
venous specimens to be of value in interpreting the acid-
base status of the patient, arterial blood is the specimen
of choice to understand the dynamics of the bodys
respiratory system.
Interpretation
See Chapter 29, Acid-Base Control and Acid-Base
Disorders, in Clinical Chemistry: Theory, Analysis,
Correlation, 5th edition.
Blood-Gas Performance Goals
Under CLIA 88 regulations, blood-gas analysis must
include at least one sample of control material each
eight hours and one sample of calibration or control
material each time patients [samples] are tested unless
automated calibration internally verifies calibration at
least every 30 minutes (Federal Register, Vol. 57[40],
February 28, 1992, p.7168). The same publication states
(p. 7158) that CLIA rules have also established
minimum criteria for acceptable performance for blood-
gas analyses on proficiency sample testing. These are as
follows:
PCO2Target value + 5 mm Hg or + 8% (whichever is
greater)
PO2 Target value + 3 S.D.
pH Target value + 0.04
References
1 Clark LC. Monitor and control of blood and
tissue oxygen tensions. Trans Am Soc Artif
Intern Organs 1956;2:41-48.
2 Evans NTS, Naylor PFD. The systemic oxygen
supply to the surface of human skin. Respir
Physiol 1967;2:21-27.
3 Fleischer WR, Hartmann AE. Blood Gases and
Their Measurement. Parts I and II. Chicago:
American Society of Clinical Pathologists; 1976.
4 Thimnig R. A Practical Course in the Techniques,
Applications, and Maintenance of the Radiometer
Acid-Base Laboratory. Cleveland: The London
Co; 1977.
5 Huch R, Huch A. Continuous Transcutaneous
Blood Gas Monitoring. New York: Marcel
Dekker; 1983.
6 Rem J, Siggaard-Andersen O, Norgaard-Pedersen
B, Storensen S. Hemoglobin pigments:
photometer for oxygen saturation,
carboxyhemoglobin, and methemoglobin in
capillary blood. Clin Chim Acta 1972;42:101-8.
7 Severinghaus JW. Simple, accurate equations for
human blood O2 dissociation computation. J Appl
Physiol 1979;46:599-602.
8 Kelman GR. Digital computer subroutine for the
conversion of oxygen tension into saturation. J
Appl Physiol 1966;21:1375-6.
9 Thomas LJ. Algorithm for selected blood acid-
base and blood gas calculations. J Appl Physiol
1972;33:154-8.
10 Boalth N, Wandrup J, Larsson L, Frischauf PA,
Lundsgaard FC, Andersen WL et al. Blood gases
and oximetry: calibration-free new dry-chemistry
and optical technology for near-patient testing.
Clin Chim Acta 2001;307:225-233.
11 Huch R, Lubbers DW, Huch A. Reliability of
arterial PO2 in newborn infants. Arch Dis Child
1974;49:213-218.
12 Parker SM, Gibson G J. Evaluation of a
transcutaneous carbon dioxide monitor
281

Blood-Gas Analysis and Oxygen Saturation

(TOSCA) in adult patients in routine
respiratory practice. Respir Med 2007;101:261-
264.
13 Mller-Plathe O, Heyduck S. Stability of blood
gases, electrolytes and haemoglobin in
heparinized whole blood samples: influence of
the type of syringe. Eur J Clin Chem Clin
Biochem 1992;30:349-55.
14 Evers W, Racz GB. A comparative study of
plastic (polypropylene) and glass syringes in
blood gas analysis. Anesth Analg 1972;50:92-7.
15 Smeenk F, Janssen J, Arends B, Harff G, van den
Bosch J, Schonberger J, Postmus PE. Effects of
four different methods of sampling arterial blood
and storage time on gas tensions and shunt
calculation in the 100% oxygen test. Eur Respir J
1997;10:910913.
16 Knowles TP, Mullin RA, Hunter JA, Douce FH.
Effects of syringe material, sample storage time,
and temperature on blood gas and oxygen
saturation in arterialized human blood samples.
Respir Care 2006;51:732-6.
17 Zaman Z, Demedts M. Blood gas analysis: POCT
versus central laboratory on samples sent by a
pneumatic tube system. Clin Chim Acta
2001;307:101-106.
18 Collinson PO, John CM, Gaze DC, Ferrigan LF,
Cramp DG. Changes in blood gas samples
produced by a pneumatic tube system. J Clin Path
2002;55:105-107.
19 Shapiro BA, Harrison RA, Walton JR. In:
Clinical Application of Blood Gases. 2nd ed.
Chicago: Year Book Medical Publishers; 1977.
20 Fleisher WR, Gambino SR. Blood pH, pCO2, and
Oxygen Saturation. Chicago: American Society
of Clinical Pathologists; 1976.
21 Lehmann H, Huntsman RG. Mans
Haemoglobins. Philadelphia: Lippincott; 1974.
22 Bunn HF, Forget BG, Ranney HM. Human
Hemoglobins. Philadelphia: Saunders; 1977.
23 Siggaard-Andersen O, Wimberley PD, Gothgen I,
Siggaard-Andersen M. A mathematical model of
the hemoglobin-oxygen dissociation curve of
human blood and of the oxygen partial pressure
as a function of temperature. Clin Chem
1984;30:1646-51.
24 Van Holde KE. Physical Biochemistry.
Englewood Cliffs, NJ: Prentice Hall; 1971.
25 Bellingham AJ. The red cell in adaptation to
anaemic hypoxia. Clin Haematol 1974;3:577.
26 Nelson MG, Savage GA, Cooke PJ, Lappin TR.
Determination of the oxygen dissociation curve
and P50 of whole blood. Am J Clin Pathol
1981;75:395-9.

Table
Table 1: Changes in Blood-Gas Parameters at Different Temperatures*
Parameter 37C 4C
pH 0.01/10 min 0.001/10 min
PCO2 1 mm Hg/10 min 0.1 mm Hg/10 min
PO2 33 mm Hg/10 min 3 mm Hg/10 min
*Approximate changes with time and temperature after sample is drawn into syringe.
A temperature of 37C implies that the blood remains at body temperature in the syringe.

Figures
Figure 1
282

Blood-Gas Analysis and Oxygen Saturation

Schema of a pH electrode and calomel reference electrode. A potential develops across pH-sensitive
glass membrane of pH electrode because of difference in pH of outer sample and inner electrode
buffer solution. Calomel electrode is in electrical contact with sample and pH electrode through a KCl
salt bridge and acts as a reference against the potential developed at pH electrode.

Figure 2

Schema of contact sequence of specimen and glass electrode and reference electrode. One area of
variable potential is at area of contact between specimen and pH-sensitive glass and is dependent on
pH of specimen.
283

Blood-Gas Analysis and Oxygen Saturation

Figure 3

Schema of PCO2 electrode. CO2 diffuses across the outer membrane, which is not permeable to

HCO3 or H+, into a layer of bicarbonate solution. Reaction of diffused CO2 and H2O to form
carbonic acid changes the pH of bicarbonate solution, which is detected by inner pH electrode.
284

Blood-Gas Analysis and Oxygen Saturation

Figure 4

Schema of Clark polarographic electrode. A constant voltage is generated between platinum cathode
and silver anode. Oxygen molecules that diffuse past outer membrane into a layer of electrolyte
solution in contact with platinum cathode are reduced at cathode, producing a current that can be
measured and related to amount of diffusing oxygen.
285

Blood-Gas Analysis and Oxygen Saturation

Figure 5

Schema of a transcutaneous PO2 electrode. Oxygen diffuses from a heated area of skin across oxygen-
permeable membrane into electrolyte solution, which is in contact with platinum cathode and silver
anode that are generating a constant voltage. Oxygen molecules are reduced at cathode, producing a
current which can be measured and related to amount of diffusing oxygen.
286

Blood-Gas Analysis and Oxygen Saturation

Figure 6

Oxygen-dissociation curve in middle is developed under following conditions: pH is 7.40, PCO2 is 40

mm Hg, and temperature is 37C. P50 is derived from that curve. Also indicated are factors that shift
curve to right and left.

P50 Oxygen-Dissociation Curve torr for adults and about 21 torr for newborns, with
standard conditions of pH 7.4, PCO2 40 torr, and 37C.
The term P50 is defined as the partial pressure of
oxygen (PO2) at which hemoglobin is 50% saturated The relationship between PO2 and O2 saturation follows
[3,19,20]. A graph of the percent oxygen saturation a sigmoidal curve (Figure 6). The curve can be
versus PO2 is referred to as the Hill plot or as the expressed in mathematical terms, and this expression is
the Hill equation [23,24]:
oxygen-dissociation curve (Figure 6). A shift of the
curve to the right represents a decrease in the affinity of
hemoglobin for oxygen and an increase in P50. The P50
value increases because a greater O2 partial pressure is
required to saturate 50% of the hemoglobin. This shift to
the right can be caused by an increase in PCO2,
temperature, or 2,3-diphosphoglycerate (DPG)
concentration or by a decrease in pH. The effect of pH
and PCO2 on the oxygen affinity of hemoglobin is in which K is the equilibrium constant and n equals 2.6.
known as the Bohr effect. A shift of the curve to the left The n factor, sometimes termed the interaction
represents an increase in the affinity of hemoglobin for coefficient, derives from the cooperative oxygen-binding
oxygen and a decrease in P50; that is, a smaller O2 partial process of the four sites on the hemoglobin molecules.
Because one or more molecules of O2 are bound to a
pressure is required to saturate 50% of the hemoglobin.
The shift to the left can be caused by an increase in pH hemoglobin molecule, a greater affinity for oxygen
or a decrease in PCO2, temperature, or DPG develops at the other sites on the hemoglobin molecule.
If there were no cooperative binding, n would be equal
concentration. Varying the DPG from 1 to 24 mmol/L to 1, and the oxygen-dissociation curve would not be
changes the P50 from 17 to 44 torr. The change in P50 is sigmoidal. This is the case for myoglobin. The value of n
affected more by acidotic conditions than by alkalotic has been derived empirically.
conditions. When the temperature is increased from
37C to 41C, the P50 is raised from 27 to 34 torr. The The Hill equation can be rearranged as follows:
influence of PCO2 is relatively small. Some abnormal
hemoglobins have been found to have an increased
affinity for oxygen, with decreased P50 values ranging
from 12 to 18 torr. Polycythemia generally develops
with this condition to compensate for the reduced
amount of oxygen reaching the tissues. Other abnormal
hemoglobins have increased P50 values, some reaching
40 torr, and a mild anemia is often associated with this
condition [21,22]. The normal P50 is approximately 27
287

Blood-Gas Analysis and Oxygen Saturation

dissociation curve is constructed. The P50 value is

determined directly from the curve. The different
instruments use different procedures to handle the
control of temperature, pH, and PCO2 of the blood
sample and the measurement of pH, PCO2, and
carboxyhemoglobin. The measurement of P50 is not
ordinarily performed in routine clinical laboratories
because of its limited clinical utility. The predominant
use of P50 is in (1) indicating the presence of abnormal
hemoglobin that affects the oxygen transport mechanism
and (2) as an indirect measure of the 2,3 DPG
concentration. It is also useful to indicate changes in pH,
PCO2, and temperature [23].

Analyzing a specimen for O2 saturation and PO2 and

substituting those parameters into equation 6, one can
solve for K. Once K is known, the PO2 at which O2
saturation is 50% can also be determined from equation
6, by substituting in the K factor and 50% for oxygen
saturation. In using equation 6, one assumption that is
made is that n is independent of other variables.
However, n is not always constant, and this difference is
particularly evident with hemoglobin mutants [25].

A number of methods are available that do not depend

on making the assumption of the constancy of n [26].
These are important for analyzing blood from patients
with multiple hemoglobin types, such as newborns with
hemoglobins F and A. One method involves
equilibrating aliquots of a blood sample with gases of
different oxygen composition, as can be done with
tonometry. The aliquots of blood are analyzed for PO2,
pH, PCO2, and oxygen saturation. After correcting the
pH values to a pH of 7.4 and the PCO2 values to 40 torr,
one plots the oxygen-saturation values and
corresponding corrected PO2 values as in Figure 6 to
form an oxygen-dissociation curve. The corrections
allow comparison with normal oxygen-dissociation
curves. From the curve, a P50 value can be determined.

Another method, which has been automated on a number

of instruments, involves continuous monitoring of blood-
gas parameters while O2 content of the blood sample is
changed. Although the process and measured parameters
will differ on the different instruments available, a
general description can be presented. An aliquot of blood
is first deoxygenated and is then subjected to increasing
partial pressures of oxygen. During the diffusion
process, the PO2 and oxygen saturation can be measured.
As more oxygen diffuses into the blood sample, the
parameters are measured continually, and an oxygen-
288
C-Reactive Protein (CRP)
C-Reactive Protein (CRP)
Odette Youdell
Name: C-reactive protein, CRP, acute-phase reactant
Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, and to Chapter 37, Coronary artery
disease: lipid metabolism, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation
Molecular mass: 118,000 D
Chemical class: Protein
Principles of Analysis and Current Usagei
In 1930, Tillet and Francis [1] described a substance in
the sera of patients with pneumococcal pneumonia
which closely paralleled the clinical course of the
infection. It was noted that in the presence of calcium
ion, this protein could form a complex with
pneumococcal Cpolysaccharide in a flocculation
reaction [2]. It was from this reaction that the term C-
reactive protein (CRP) was derived. In the decades to
follow, CRP has become one of the most popular clinical
laboratory tests for the evaluation of patients with
various inflammatory disorders [3].
CRP is synthesized in the liver and is under the direct
transcriptional control of interleukin 6 (IL-6) and
indirect control of IL-1, tumor necrosis factor and
other cytokines [4]. It is a 1.18 kDa protein which
consists of five identical, noncovalently bonded
subunits, each approximately 23 kDa and consisting of
206 amino acids. It has been shown to bind to bacteria
and tissue membranes and enhance phagocytosis. In
addition, CRP can activate the classical complement
pathway [5].
Today, circulating CRP concentrations previously
considered normal have been found to provide
valuable information with regard to cardiovascular risk
stratification for future coronary events [6-9]. As a result
of this new application, high-sensitivity CRP (hsCRP)
assays have been developed to quantify concentrations
below the limit of conventional assays.
Qualitative/Semiquantitative Assays
Until the late 1970s, CRP was measured using
qualitative or semiquantitative methods. These methods
required precipitation or agglutination of latex particles
by CRP to form large, visible aggregates which were
then graded on a scale of +1 to +4. This limited the use
of CRP as a differential diagnostic test in that any degree
i
C-Reactive Protein
Previous and current authors of this method:
First edition: Not done
Methods edition: Not done
Second edition: Not done
Third edition: Not done
Fourth edition: Steven C. Kazmierczak
Fifth edition: Odette Youdell
of inflammation produced a positive result. As a
consequence, the use of CRP for monitoring disease
activity declined significantly, being replaced by tests
for other acute-phase proteins and measurement of the
erythrocyte sedimentation rate (ESR).
Early methods for CRP analysis included radial
immunodiffusion and latex agglutination (Table 1,
Method 1) and have been described as insensitive,
semiquantitative, and subjective in assessment [10].
Further advances in assay and antibody development
have allowed for more sensitive and quantitative
methods.
Quantitative Assays
Since the late 1970s, purification of CRP by affinity
chromatography enabled the development of antibodies
that could be used for quantitative methods [11].
The major methods reported in a 2007 College of
American Pathologists (CAP) Participation Survey
(1490 participants across 19 different platforms) were
immunoturbidimetry, (77%), nephelometry (13%),
enzyme immunoassay (9%), and chemiluminescence
(<1%).
Immunoturbidimetric procedures for CRP are currently
the most popular method for CRP analysis (Table 1,
Method 2). Along with immunonephelometric methods
(Table 1, Method 3) they allow for CRP analysis which
is sensitive (0.4 mg/L), automated, and rapid [4]. Results
for the nephelometric and turbidimetric methods
correlate fairly well with other techniques, with intra-
assay biases generally being less than 20% [12].
Enzyme immunoassay (Table 1, Method 4) is another
quantitative method for CRP. Like the other
immunochemical methods, enzyme immunoassay assays
for CRP are automated, rapid, and show good sensitivity
and specificity.
Other assay techniques for quantitative CRP
measurement which may still be used by some
laboratories include immunofluorescence assays, latex
agglutination (Table 1, Method 5), radial
immunodiffusion (RID), and radioimmunoassay.
289
C-Reactive Protein (CRP)
High-Sensitivity/Ultrasensitive Quantitative CRP
Assays
The concentration of CRP measured to assess
cardiovascular risk is much lower than that measured in
acute inflammation. Manufacturers have responded to
this challenge by developing high-sensitivity or ultra-
sensitive CRP methods. The most common methods
utilize particle-enhanced turbidimetry or nephelometry.
With these techniques, monoclonal or polyclonal
antibodies are adsorbed onto latex particles in order to
enhance the measurement signal at low concentrations.
In a comparison study of nine such assays, the limit of
detection ranged from 0.02 to 0.32 mg/L [13].
Given that CRP results are interpreted in the context of
internationally established decision limits, ideally all
commercial assays should give equivalent results.
Quality-control surveys in Western Europe and the
United States during the late 1980s and early 1990s
showed that reported concentration for some proteins
varied by as much as 100%, depending on the calibrator
used [14]. The use of a single international reference
material by all manufacturers should reduce the
variability to a large degree.
There are two widely recognized standards for CRP. The
first is the World Health Organization (WHO) 1st
International Reference Preparation for C-Reactive
Protein (85/506) introduced in 1986, with a CRP
concentration of 98 mg/L, and the second is the Certified
Reference Material 470 (CRM 470) introduced in 1993-
94, with a CRP concentration of 39.2 mg/L. The second
reference material is sometimes also referred to as the
Reference Preparation for Proteins in Human Serum
(RPPHS). CRP values in CRM 470 were assigned from
WHO IRP 85/506 and so are directly traceable to the
WHO material [14,15].
A number of comparison studies [13,16-18]
demonstrated significant differences between hsCRP
methods. Since the target value 39.2 is much higher than
nominated decision limits used for cardiovascular risk
stratification, the reliability of using a diluted form of the
CRM 470 to further minimize these differences is
currently being investigated [15].
Reference and Preferred Method
There is no reference method for the measurement of
CRP. Laboratories choosing a method must first
determine the clinical application and consider the
advantages and disadvantages of high sensitivity versus
a wide measuring range in the context of laboratory
consolidation and automation.
Radioimmunoassay (RIA) methods are capable of
detecting as little as 1 to 5 ng/mL of CRP. Although RIA
is sensitive and also relatively inexpensive, it is not
commonly employed because of the availability of other
faster automated procedures.
To accommodate requirements for cardiovascular risk
prediction and acute-phase concentrations,
manufacturers have developed assays to provide both
high sensitivity and a wide measuring range. A recent
study evaluating concentrations spanning 0.15 and 290
mg/L between two particle-enhanced immuno-
turbidimetry based assays concluded that both needed
improvements to meet high-sensitivity criteria [19].
Specimen
CRP is a robust analyte. Common laboratory variables
such as specimen type, delays in specimen processing,
and storage temperature have little effect on
concentration. Many studies report little or no difference
between serum and plasma values, with a negative bias
up to 16% with EDTA blood [10,17,20,21]. Specimen-
processing delays of up to 6 hours and varying storage
conditions ranging from 70C to 21C for up to 3
weeks had little effect on CRP concentration [22-24]. It
is also stable for up to 7 freeze/thaw cycles [24,25].
No apparent difference has been described between
fasting and nonfasting specimens [3]. In a study of 13
healthy subjects with hourly samples over a period of 24
hours, no diurnal variation was reported [26]. However,
in a study looking at diurnal, seasonal, and blood-
processing patterns, Rudnicka et al. [27] reported both a
diurnal pattern and borderline evidence of seasonality
(higher in winter months). They concluded, however,
that the combined effects of variation on CRP, was less
than 1%.
Serum and CSF are the matrices primarily used in
diagnosis. Studies suggest that consumption of CRP in
CSF may result in falsely decreased values [28]. CRP
has also been detected in synovial, amniotic, pleural,
ascitic, and blister fluids [10]. As CRP does not cross the
placental membrane, a raised CRP in a newborn whose
mother has presented with prolonged rupture of amniotic
membranes and/or amnionitis should be evaluated for
neonatal bacterial infection [29].
Interferences
An interference study undertaken on three automated
hsCRP immunoassays highlighted the need to confirm
manufactures claims regarding hemolysis, icterus, and/or
lipemia. Based on 10% of the target value, moderate
hemolysis (above 4g/L) was found to interfere with two
of the assays tested, and one of the assays was affected
by triglyceride concentrations above 2.5 g/L, which was
lower than the manufacturers claims of 7.4 g/L. None of
the assays were affected by bilirubin (at concentrations
up to 60 mg/dL) [18].
Specimens with high titers of rheumatoid factor have the
potential to interfere with immunoassays for CRP, but
most reagent manufacturers incorporate dithiothreitol to
destroy rheumatoid factor by reducing disulfide bonds.
In inflammatory states, CRP can increase dramatically to
very high concentrations from baseline values, resulting
in falsely low results due to antigen excess. This prozone
effect may be seen for single-antibody nephelometric
and turbidimetric methods [13]. A number of sites have
reported evidence of prozone effects at varying CRP
290
C-Reactive Protein (CRP)
concentrations ranging from as little as < 50 mg/L to 510
mg/L [13,17,28]. In one study, four of the nine assays
tested were affected [28].
Reference Interval
Baseline CRP concentrations in newborns are very low.
Increases due to infection or other inflammatory
processes may only be detectable with hsCRP assays
[30].
In the absence of acute inflammation, healthy adults
usually have CRP concentrations less than 10 mg/L,
although studies have found mean concentrations tend to
increase slightly in women during late pregnancy [14]
and with increasing age [31-33].
Rifai and Ridker [34] proposed an algorithm for risk
assessment in both genders established by retrospective
epidemiological studies of coronary heart disease in
conjunction with LDL-cholesterol decision limits
recommended by the National Cholesterol Education
Program (NCEP) (Figure 1).
Interpretation
A variety of stimuli, including infection, major trauma,
surgery, and chronic inflammatory conditions, can result
in a marked increase in CRP production (Table 2) [35].
Following stimuli, concentrations of CRP rise within
hours, doubling 5 to 8 hours thereafter [25]. It is
generally accepted that mild inflammation and viral
infections cause elevation of CRP concentration within
the 10 to 40 mg/L range, whereas active inflammation
and bacterial infection result in concentrations of 40 to
200 mg/L. Concentrations over 200 mg/L are found in
severe bacterial infections and burns [36]. CRP has a
half-life of 19 hours [38].
Population studies indicate no major racial or ethnic
differences exist [31,32,34]. Higher CRP concentrations
have been found among obese individuals [38,39,40].
Obesity is associated with low-grade inflammation. It is
thought IL-6 production by adipose tissue induces
hepatic CRP synthesis and hence promotes the onset of
cardiovascular complications [39]. Smokers have higher
CRP concentrations than those who have never smoked
[13,31].
For both sexes, the 50th percentile of CRP measured in
various study populations was about 1.5 mg/L (Table 3),
hence gender-specific cutoff values are not required for
risk stratification [33,34].
There is consensus that baseline CRP concentrations
significantly increase following hormone replacement
treatment [41,42]. One study suggests that this increase
may be related to metabolic hepatic activation and not to
an acute-phase response [43].
In the context of cardiovascular risk prediction, the
American Heart Association (AHA) and the Centers for
Disease Control and Prevention (CDC) recommend three
decision limits be used: < 1.0 mg/L (low risk), 1.0 to 3
mg/L (average risk), and > 3.0 mg/L (high risk). An
individuals CRP should be measured twice, either
fasting or not fasting, with the average expressed in
mg/L. If the hsCRP concentration is 10.0 mg/L, then
the test should be repeated and the person examined for
sources of infection or inflammation [44].
Studies carried out to determine the usefulness of CRP
as an independent predictor of cerebrovascular events
and to evaluate prognosis after stroke conclude that at
present, there is insufficient evidence to support testing
[45,46].
CRP Performance Goals
The CAP performance criteria set for CRP is 3 standard
deviations of the target mean, which is determined by
each analyzers peer-group mean. For the 2007
Immunology Survey (CRP-01, CRP-02), 90% of the
assays are either turbidimetric (77% across 13 platforms)
or nephelometric (13% across 3 platforms). Overall the
turbidimetric assays perform marginally better, with
imprecision values (% coefficient of variation [CV])
ranging from 4.3% to 8.2% at a value around 20 mg/L
versus nephelometric, 4.9% to 10%. At a value around
40 mg/L, CVs are 3.4% to 8.6% versus 4.5% to 8.5%,
respectively.
The performance criteria set by CAP for hsCRP testing
is 30% of the target value as determined by each
analyzers peer-group mean. For the 2007 Cardiac Risk
Survey (hsCRP-01, hsCRP-02), at a mean of 1.28 mg/L,
CVs ranged from 4.5% to 29.5%, compared with a mean
of 3.02 mg/L CVs ranging from 2.2% to 13.8%. The
exact analytical performance criteria required for
accurate classification into risk categories have not yet
been established. However, it has been suggested that
the within-run precision of hsCRP assays should not
exceed 10% at a concentration of 0.2 mg/L [14].
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19 iman AR, Kme T, Ta G, Akan P, Tuncel P.
Comparison and evaluation of two C-reactive
protein assays based on particle-enhanced
immunoturbidimetry. J Clin Lab Analysis
2007;21:71-76.
20 Nordin G, Samuelsson I, Andersson B. C-
reactive protein: the difference between
quantitation in serum and EDTA plasma. Scand
J Clin Lab Invest 1996;56:123-127.
21 Dupuy AM, Michon AL, Badiou S, Cristol JP.
Adaptation and evaluation of the Randox Full-
Range CRP Assay on the Olympus AU2700.
J Clin Lab Analysis 2007;21:34-39.
22 Hartweg J, Gunter M, Perera R, Farmer A, Cull
C, Schalkwijk C et al. Stability of soluble
adhesion molecules, selectins, and C-reactive
protein at various temperatures: implications for
epidemiological and large-scale clinical studies.
Clin Chem 2007;53:1858-1860.
23 Eijsden M, van der Wal MF, Hornstra G,
Bonsel GJ. Can whole-blood samples be stored
over 24 hours without compromising stability
of C-reactive protein, retinol, ferritin, folic acid,
and fatty acids in epidemiologic research? Clin
Chem 2005;51:230-232.
24 Macy EM, Hayes TE, Tracy RP. Variability in
the measurement of C-reactive protein in
healthy subjects: implications for reference
intervals and epidemiological applications. Clin
Chem 1997;43:1:52-58.
25 Aziz N, Fahey JL, Detels R, Butch AW.
Analytical performance of a highly sensitive C-
reactive protein-based immunoassay and the
effects of laboratory variables on levels of
protein in blood. Clin Diagn Lab Immunol
2003;652-657.
26 Meier-Ewert HK, Ridker PM, Rifai N, Price N,
Dinges DF, Mullington JM. Absence of diurnal
variation of C-reactive protein concentrations in
healthy human subjects. Clin Chem
2001;47:3:426-430.
27 Rudnicka AR, Rumley A, Lowe GDO, Strachan
DP. Diurnal, seasonal and blood-processing
patterns in levels of circulating fibrinogen,
fibrin D-dimer, C-reactive protein, tissue
plasminogen activator, and von Willebrand
factor in a 45-year-old population. Circulation
2007;115:996-1003.
28 Sindic CJM, Collet-Casart D, Depre A, Laterre
ED, Masson PL. C-reactive protein in serum
and cerebrospinal fluid in various neurological
disorders. J Neurol Sci 1984;63:339-344.
29 Salzer HR, Genger H, Muhar U, Lischka A,
Schatten C, Pollak A. C-reactive protein: an
early marker for the neonatal bacterial infection
due to prolonged rupture of amniotic
membranes and/or amnionitis. Acta Obstet
Gynecol Scand 1987;66:365-367.
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C-Reactive Protein (CRP)

30 Wasunna A, Whitelaw A, Gallimore R, 43 Silvestri A, Gebara O, Vitale C, Wajngarten M,

Hawkins PN, Pepys MB. C-reactive protein and Leonardo F, Ramires JAF et al. Increased levels
bacterial infection in preterm infants. Eur J of C-reactive protein after oral hormone
Pediatr 1990;149:424-427. replacement therapy may not be related to an
31 Ford ES, Giles WH, Myers GL, Mannino DM. increased inflammatory response. Circulation
Population distribution of high-sensitivity C- 2003;107:3165-3169.
reactive protein among U.S. men: findings from 44 Pearson TA, Mensah GA, Alexander RW,
the National Health and Nutrition Examination Anderson JL, Cannon RO, Criqui M et al.
Survey 1999-2000. Clin Chem 2003;49:4:686- Markers of inflammation and cardiovascular
690. disease: application to clinical and public health
32 Imhof A, Frohlich M, Loewel H, Helbecque N, practice. A statement for healthcare
Woodward M, Amouyel P et al. Distribution of professionals from the Centers for Disease
C-reactive protein measured by high-sensitivity Control and Prevention and the American Heart
assays in apparently healthy men and women Association. Circulation 2003;107:499-511.
from different populations in Europe. Clin 45 Curb JD, Abbott RD, Rodriguez BL, Sakkinen
Chem 2003;49:4:669-672. P, Popper JS, Yano K, Tracy RP. C-reactive
33 Hung J, Knuiman MW, Divitini ML, Davis T, protein and the future risk of thromboembolic
Beilby JP. Prevalence and risk factor correlates stroke in healthy men. Circulation
of elevated C-reactive protein in an adult 2003;107:2016-2020.
Australian population. Am J Cardiol 46 Di Napoli M, Schwaninger M, Cappelli R,
2008;101:193-198. Ceccarelli E, Di Gianfilippo G, Donati C et al.
34 Rifai N, Ridker PM. Population distributions of Evaluation of C-reactive protein measurement
C-reactive protein in apparently healthy men for assessing the risk and prognosis in ischemic
and women in the United States: implication for stroke: a statement for health care professionals
clinical interpretation. Clin Chem from the CRP Pooling Project members. Stroke
2003;49:4:666-672. 2005;36:1316-1329.
35 Pepys MB, Hirschfield GM. C-reactive protein:
a critical update. J Clin Invest 2003;111:1805-
1812.
36 Clyne B, Olshaker JS. The C-reactive protein. J
Emerg Med 1999;17:6:1019-1025.
37 Vigushin DM, Pepys MB, Hawkins PN.
Metabolic and scintigraphic studies of
radioiodinated human C-reactive protein in
health and disease. J Clin Invest 1993;91:1351-
1357.
38 Fogarty AW, Glancy C, Jones S, Lewis SA,
McKeever TM, Britton JR. A prospective study
of weight change and systemic inflammation
over 9 years. Am J Clin Nutr 2008;87:30-35.
39 Bastard JP, Maaachi M, Lagathu C, Kim MJ,
Carson M, Vidal H et al. Recent advances in the
relationship between obesity, inflammation, and
insulin resistance. Eur Cytokine Netw
2006;17:4-12.
40 Bahceci M, Gokalp D, Bahceci S, Tuzcu A,
Atmaca S, Arikan S. The correlation between
adiposity and adiponectin, tumor necrosis factor
, interlukin-6 and high-sensitivity C-reactive
protein levels. Is adipocyte size associated with
inflammation in adults? J Endocrinol Invest
2007;30:210-214.
41 Ridker PM, Hennekens CH, Rifai N, Buring JE,
Manson JE. Hormone replacement therapy and
increased plasma concentration of C-reactive
protein. Circulation 1999;100:713-716.
42 Cushman M, Legault C, Barrett-Connor E,
Stefanick ML, Kessler C, Judd HL et al. Effect
of postmenopausal hormones on inflammation-
sensitive proteins: the Postmenopausal
Estrogen/Progestin Interventions (PEPI) study.
Circulation 1999;100:717-722.
293

C-Reactive Protein (CRP)

Table 1: Methods of Analysis for C-Reactive Protein

Method 1: Single radial immunodiffusion (RID); quantitative

Principle of analysis: Diffusion of protein through medium containing specific antibody
Usage: Seldom used
Comments: Serum, plasma, manual; requires very long incubation time

Method 2: Immunoturbidimetry
Principle of analysis: Reaction of CRP with anti-CRP antibody results in formation of antigen-antibody
complexes which absorb light
Usage: Most common quantitative method for CRP
Comments: Sensitive, specific; automated; requires specialized instrumentation

Method 3: Immunonephelometry
Principle of analysis: Reaction of CRP with anti-CRP antibody results in formation of antigen-antibody
complexes which scatter light
Usage: Very common quantitative method for CRP in use
Comments: Sensitive, specific; automated; requires specialized instrumentation.

Method 4: Florescence-labeled ELISA

Principle of analysis: CRP binds to enzyme-labeled antibodies and to antibodies bound to microparticles;
enzyme in complex releases fluorescent 4-methylumbelliferone, which is measured.
MEIA monoclonal Abs coat microparticle
MicroparticleAb + 2M + AbALP
Microparticle Ab2MAbALP + 2M +
AbALP + 2MAbALP
1. Wash step
2. 4-MU product substrate
3. Quench
Measure 4-MU product fluorescence at 448 nm
Usage: Very common quantitative method for CRP
Comments: Serum, plasma, urine; semiautomated technique; sensitive, specific; automated; requires specialized
instrumentation

Method 5: Latex agglutination

Principle of analysis: Agglutination of latex particles coated with anti-CRP antibodies due to presence of CRP
results in formation of visible aggregates; size of aggregates related to CRP concentrations
Usage: Commonly used as semiquantitative procedure; also can be used to report quantitative results
Comments: Semiquantitative; point-of-care test kit has been developed; useful as rapid screen
294

C-Reactive Protein (CRP)

Table 2: CRP Responses in Disease.

Major CRP Acute-Phase Response

Infections Bacterial
Systemic/severe fungal,
mycobacterial, viral

Allergic complications of infection Rheumatic fever

Erythema nodosum

Inflammatory disease Rheumatoid arthritis

Juvenile chronic arthritis
Ankylosing spondylitis
Psoriatic arthritis
Systemic vasculitis
Polymyalgia rheumatica
Reiters disease
Crohns Disease
Familial Mediterranean fever

Necrosis Myocardial infarction

Tumor embolization
Acute pancreatitis

Trauma Surgery
Burns
Fractures

Malignancy Lymphoma
Carcinoma
Sarcoma

Modest or Absent CRP Acute-Phase Response Systemic lupus erythematosus

Scleroderma
Dermatomyositis
Ulcerative colitis
Leukemia
Graft-versus-host disease
From Clyne B, Olshaker JS. The C-reactive protein. J Emerg Med 1999;17(6):1019-1025 [36].

Table 3: Population Distribution of C-Reactive Protein in Men and Women by Percentile.

5th 10th 25th 50th 75th 90th 95th

Women* 0.19 0.29 0.61 1.52 3.48 6.61 9.14

Men 0.28 0.40 0.80 1.50 3.20 6.05 8.55

*
Women were not taking hormone replacement therapy.
From Rifai N, Ridker PM. Population distributions of C-reactive protein in apparently healthy men and
women in the United States: implications for clinical interpretation. Clin Chem 2003;49(4):666-672 [34].
295

C-Reactive Protein (CRP)

Figure 1: Proposed algorithm for risk assessment of coronary heart disease in men and
women. (From Rifai N, Ridker PM. Population distributions of C-reactive protein in
apparently healthy men and women in the United States: implication for clinical
interpretation. Clin Chem 2003;49[4]:666-672 [34].)

4
>3

(mg/L)
2 1-3

CRP
<1.0
0
>1600 1300-1600 <1300
LDL-Cholesterol (mg/L)
296
Calcium

Calcium
Randal J. Schneider

Name: Calcium
Clinical significance: Refer to Chapter 33, Bone Disease, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Atomic symbol: Ca
Atomic mass: 40.08 D
Merck Index: 1613
Chemical class: Alkaline earth element
Number of forms: Free Ca2+, protein bound, or in inorganic complexes
NRSCL Definitive and
Reference Methods: NCCLS RS9-P
i
Principles of Analysis and Current Usage
Within the circulation, calcium is found in several A sensitive fluorescent method for the microanalysis of
different forms, with 46% free, 32% bound to albumin, serum or urine is available [3]. In one method, the
8% bound to globulins, and 14% associated in freely calcium is added to an alkaline solution containing
diffusible calcium complexes. All calcium is ionized no calcein (Table 1, Method 3). The calcium combines with
matter what it is bound to, but it may not be dialyzable the calcein, which at high pH fluoresces at 520 nm
or completely reactive with a chosen chromogen when (excitation at 490 nm). The fluorescent complex is
complexed to another compound. Ion-specific electrode titrated with ethylene glycol-bis(-aminoethylether)-
techniques for measuring the activity of the unbound N,N-tetraacetic acid (EGTA), which binds the calcium
fraction are available and are discussed elsewhere. from the complex with a resulting decrease in
fluorescence. When the baseline fluorescence is
The oldest procedures used for the clinical determination achieved, the volume of EGTA required to titrate the
of total calcium involved the quantitative precipitation of complex is directly proportional to the concentration of
calcium with an excess of anions from oxalic, calcium. It has been reported that magnesium and
chloranilic, or naphthylhydroxamic acids (Table 1, phosphate do not interfere with this method.
Method 1) [1,2].
Direct spectrophotometric measurements of calcium in
Methods using chloranilate or naphthylhydroxamate ions serum or urine are also based upon formation of color
to precipitate calcium were based upon quantification of complexes between calcium and organic molecules.
the color of the precipitating ion. An excess of Examples of compounds that give colored reaction
chloranilate was added to react with the serum calcium. products with calcium are (1) glyoxal-bis(2-
The calcium chloranilate complex was precipitated and hydroxyanil), (2) alizarin, (3) chlorophosphonazo III, (4)
redissolved in alkaline solutions of methylthymol blue, (5) o-cresolphthalein complexone,
ethylenediaminetetraacetic acid (EDTA). The and (6) arsenazo III. Of these calcium-complexing,
absorbance of the intense red-purple color of the colorimetric reagents, the o-cresolphthalein and arsenazo
liberated chloranilate acid (Amax,520 nm) was III dye are the most commonly used for routine calcium
proportional to the concentration of calcium in the analysis. Approximately 45% of the participants in the
specimen (Table 1, Method 2). Similarly, 2007 College of American Pathologists (CAP)
naphthylhydroxamate has been used to precipitate Participant Summary Reports indicated that they used
calcium. The precipitate was dissolved in alkaline the o-cresolphthalein method, while approximately 30%
EDTA, and when acid ferric nitrate was added, a red- used the arsenazo III dye. The remaining laboratories
orange color was produced. These precipitation methods reported using an ion-selective electrode.
are rarely used today, because they are time consuming
and insensitive. The reaction of calcium with o-cresolphthalein produces
a red complex (quantitated at 570 to 575 nm) at pH 10 to
12 (Table 1, Method 4). The spectrum of the complex
i formed with calcium is given in Figure 1. The reaction
Calcium
product is stabilized by the addition of KCN (potassium
Previous and current authors of this method:
cyanide), which also acts to eliminate interference from
First edition: E. Christis Farrell
heavy metals, and interference by magnesium ions is
Methods edition: E. Christis Farrell
eliminated by the addition of 8-hydroxyquinoline.
Second edition: E. Christis Farrell
Similarly, assays using the arsenazo III dye involve a
Third edition: Steven C. Kazmierczak
reaction with dye and calcium to produce a color
Fourth edition: Steven C. Kazmierczak
complex (blue-purple) that can be measured
Fifth edition: Randal J. Schneider
spectrophotometrically. In this reaction under acidic
297
Calcium

conditions, the color complex is measured at 650 nm, include atomic absorption spectroscopy, induction
and like the o-cresolphthalein reaction is proportional to coupled plasma atomic emission spectroscopy, ion
the calcium concentration in the sample. chromatography, and isotope-dilution mass
spectroscopy.
Dry-slide technology uses the arsenazo III dye in a dry-
reagent layered coating system. When patient fluid is The preferred clinical method for total calcium
applied to the slide, the arsenazo dye migrates from the determinations is atomic absorption spectroscopy,
bottom layer to a mordant layer and is trapped. The although this method is usually limited to large reference
calcium in the sample migrates to the mordant layer, laboratories for reasons mentioned below.
complexing with the dye and changing its color. The Acceptable methods include the dye-complex methods
absorption is recorded by reflectance photometry at 680 based on cresolphthalein and arsenazo III or indirect
nm [4]. potentiometric methods. Proficiency results for 2007
indicate all three methods to be satisfactory for reporting
Calcium present in protein or inorganic complexes is clinical calcium results.
detectable by flame atomic absorption only when steps
are taken to dissociate the calcium from these Additional methods listed in Table 1 are either not
complexes. Acid is used for the dissociation of protein- practical for most clinical laboratories or do not meet
Clinical Laboratory Improvement Amendments (CLIA)
bound calcium, and lanthanum (La2+) or strontium
requirements for total allowable error.
(Sr2+) ions are added to displace Ca2+ from phosphate, The older oxalate precipitation and titration methods are
oxalate, citrate, and other complexes. The La2+ or Sr2+ no longer in use for routine calcium analysis because
is added to the sample diluent. Serum is diluted 1:50 they were quite laborious and imprecise.
with 1% La2+, whereas urine is diluted 1:50 with 5%
The most commonly used reference method for accurate
La2+ in order to overcome the higher phosphate
calcium measurement is atomic absorption. Although it
concentration found in urine. Protein is precipitated by
is not the definitive method, atomic absorption is more
treatment of the sample with acid. Precipitation results in
commonly available and is thus often used as a reference
a 2% to 3% reduction in sample volume when the
method for comparison studies of new methods. The
precipitate is removed. One can mathematically correct
precision of atomic absorption over the range of
for the reduced volume. Acid treatment without protein
physiological concentrations is of the order of 2% to 3%.
precipitation requires increased maintenance of the
Atomic absorption methods are very sensitive, requiring
burner head to prevent accumulation of debris.
10 to 20 L of sample for analysis. The method has been
semiautomated with automatic sample aspiration after
A relatively recent technique for measurement of total
dilution with semiautomatic diluters.
calcium in serum and urine is with the use of indirect
potentiometry (Table 1, Method 7). This method utilizes
The o-cresolphthalein methods have been adapted to a
a calcium ionselective electrode in conjunction with a
wide variety of automated analyzers, with a resulting
sodium reference electrode. Sample is mixed with a
high precision. A significant problem with this technique
buffered solution before its interaction with the calcium
is the requirement for good temperature control during
electrode. Interaction of calcium with the selective
the analysis, since the chromogens molar absorptivity is
membrane results in changes in electrical potential that
temperature dependent.
are compared with the sodium reference electrode.
On the basis of precision and accuracy, the preferred
In atomic absorption, interference by magnesium and
clinical method is certainly atomic absorption. The
other elements is reduced by the use of narrow band-
major drawbacks of atomic absorption are the high level
pass, diffraction-grating spectrophotometers for specific
of maintenance and care the equipment requires and the
isolation of the atomic absorption line of calcium (422.7
limited throughput of specimens for high-volume
nm). Interference by sodium is eliminated by the
laboratories.
addition of physiological concentrations of sodium ions
to calcium standards. Atomic absorption is not
Specimen
frequently used for routine analysis, probably because it
Serum or heparinized plasma is separated from cells as
has rarely been automated for high sample throughput.
rapidly as possible to avoid the uptake of calcium by
This method has been approved as a reference method
erythrocytes. Stasis changes concentration of protein-
for measurement of serum calcium [5].
bound Ca2+, and concentrations of free Ca2+ will be
changed by pH shifts.
The definitive method for calcium measurements is
isotope-dilution mass spectroscopy (Table 1, Method 8)
Serum or heparinized plasma samples may be stored at
[6]. This method, available in only a few institutions, is
room temperature for up to 8 hours at 4C for 1 day or
the accuracy standard against which all methods should
frozen for up to 1 year. Calcium in specimens collected
be compared.
and stored in gel-separator tubes did not show any
difference compared with specimens collected and
Reference and Preferred Methods
stored in tubes not containing gel [8].
Currently there are 5 reported candidate reference
methods for measuring total calcium. These methods
 298
Calcium

Urine calcium can be kept dissolved by addition of 10
mL of 6 M HCl to the collection container before a 24-
hour specimen is collected. Urine should be well mixed
during the collection period.
Interferences
Most o-cresolphthalein methods using direct analysis
after dilution appear to be sensitive to interferences from
lipemia, hemoglobin, and myeloma protein [9, 10-12].
Atomic absorption is not affected by important
interferences from common compounds such as
hemoglobin, bilirubin, or lipemia. However, calcium-
chelating compounds interfere with this method. The
potentiometric method for total calcium is affected by
bromide, which causes a positive bias.
bound fraction. Although the concentration of ionized
calcium may vary during alkalosis and acidosis, the total
calcium concentration is relatively unaffected.
A major cause of hypercalcemia is malignancy, with
increases observed in bone diseases such as multiple
myeloma and with metastatic tumors with secondary
bony deposits. Only 10% to 15% of such metastatic
cancers result in hypercalcemia.
Critical values indicating the possibility of morbidity are
less than 6.0 mg/dL (1.5 mmol/L) and greater than 14.0
mg/dL (3.5 mmol/L).
A list of diseases causing hypercalcemia and
hypocalcemia is presented in the following boxes:

Calcium Reference Intervals Causes of Hypercalcemia

Many sources report serum calcium levels of 90 to 110 Hyperparathyroidism
mg/L for disease-free persons (normals). Newer Thyrotoxicosis
methods show lower intervals of about 80 to 105 mg/L Addisons disease
There is no clinically significant difference in serum Withdrawal of steroids
calcium concentrations between men and women. Urine Tumors
calcium concentration varies considerably and is only Vitamin D and vitamin A intoxication
meaningful if the patient is kept on a low-calcium, Sarcoidosis
neutral-ash diet for 3 days before collection. A low Idiopathic hypercalcemia of infancy
output is of the order of 50 to 100 mg/day, with the Immobilization
average being 100 to 300 mg/day. Values are lower in Subcutaneous fat necrosis in infants
persons older than 70 years. Diurnal variation in serum Thiazide diuretics
calcium has been described, with lowest concentrations Milk-alkali syndrome
seen at approximately 8:00 am and highest values Benign familial hypercalcemia
occurring in early afternoon [13].
Population Serum Ca UrineCa
mg/L (mmol/L) mg/24h(mmol/24hr) Causes of Hypocalcemia
Adults Vitamin D
Atomic absorption 80105 (2.02.6) Men<275 (<6.87)
Women <250 (<6.25) Decreased solar exposure and endogenous synthesis
Hypercalcemic >300 (>7.50) Decreased intestinal intake (malabsorption, dietary
Cresolphthalein deficiency)
complexone 80105 (2.02.6) Average diet 100250 Altered hepatic metabolism of vitamin D (hepatic
(2.56.25)
Pediatric (by atomic absorption)
disease, anticonvulsants)
Premature infants 60100 (1.5-2.5) Decreased renal synthesis of calcitriol (vitamin D
Full-term infants 73120 (1.8-3.0) dependency, renal failure)
1 to 2 years 100120 (2.5-3.0) Parathyroid
Hypoparathyroidism (primary and secondary)
Interpretation Pseudohypoparathyroidism
Total serum calcium concentration is influenced by the Calcitonin
serum albumin concentration, position of the body when Calcitonin or mithramycin infusion
the calcium is drawn, and tourniquet stasis. The ionized Calcium
calcium is not influenced by these factors and is the Intestinal malabsorption
better indicator of the hypercalcemic or hypocalcemic Acute pancreatitis
state. It may be calculated from total protein albumin Infusion of agents complexing calcium
and calcium values, but direct measurement is Alkalosis decreasing ionized calcium
preferable. Magnesium
Magnesium deficiency
The amount of albumin-bound calcium present is Phosphorus
dependent on the albumin concentration. A decrease of Renal failure
serum albumin of 1 g/L will decrease the total serum Phosphate infusion
calcium by about 0.08 mg/L. Cows milk formulas
Posture changes the serum calcium concentration; going
from an upright to a recumbent posture causes a decrease
of approximately 4% (range 2% to 7%). Calcium Performance Goals
Survey data from the 2007 CAP Participant Summary
The fraction of calcium bound to protein in serum is pH- Report show imprecision values (% coefficient of
dependent. Acidosis increases the ionized fraction variation [CV]) for measurement of calcium with a
(decreases binding), whereas alkalosis increases the
299
Calcium

concentration of 10.57 mg/dL to range from
approximately 1.4% to 2.9% for the arsenazo III dye and
cresolphthalein complexone methods. The indirect
potentiometric technique shows imprecision values that
range from 1.1% to 2.4%. Acceptable performance
criteria (CLIA 88) for measurement of calcium require
that laboratories be accurate to within 1.0 mg/dL of the
peer-group mean.
The intraindividual variation of calcium in blood has
been determined to be approximately less than 2.0%.
Desirable specifications for analytical imprecision
derived from studies of biological variation indicate an
assay imprecision of no greater than 1.0% and a total
error or no greater than approximately 2.5% [14].
12 Ladenson JH, McDonald JM, Goren M.
Multiple myeloma and hypercalcemia [case
conference]. Clin Chem 1979;25:1821-1825.
13 Herfarth K, Schmidy-Gayk H, Graf S, Maier A.
Circadian rhythm and pulsatility of parathyroid
hormone secretion in man. Clin Endocrinol
1992;37:511-519.
14 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biologic variation: pros, cons and
progress. Scand J Clin Lab Invest 1999;59:491-
500.

References
1 Weissman N, Pileggi VJ. Inorganic ions. In:
Henry RJ, Cannon DC, Winkelman JW, eds.
Clinical Chemistry: Principles and Technics.
2nd ed. New York: Harper & Row; 1974:646-
653.
2 Clark EP, Collips JB. A study of the Tisdall
method for the determination of blood serum
calcium with suggested modification. J Biol
Chem 1925;63:461-464.
3 Jackson JE, Breem M, Cheng C. Fluorometric
titration of calcium. J Lab Clin Med
1962;60:700-708.
4 Shirey TL. Development of a layered-coating
technology for clinical chemistry. Clin Biochem
1983;16:147-155.
5 National Committee for Clinical Laboratory
Standards (NCCLS). RS9-Calcium: proposed
summary of methods and materials credentialed
by the NRSCL council. Wayne, PA: NCCLS;
1989.
6 Cali JP, Mandel J, Moore L, Young DS. U.S.
Department of Commerce National Bureau of
Standards. A reference method for the
determination of calcium in serum. NPS Spec.
Pub. 260-36. Washington, DC: U.S.
Government Printing Office; 1972.
7 Brett EM, Hicks JM. Total calcium
measurement in serum from neonates:
limitations of current methods. Clin Chem
1981;27:1733-1737.
8 Hein M, Heil W, Wihold W. Storage of serum
or whole blood samples? Effects of time and
temperature on 22 serum analytes. Eur J Clin
Chem Clin Biochem 1995;33:231-238.
9 Leidtke RJ, Kroon G, Batjer JD. Centrifugal
analysis with automated sequential reagent
addition: measurement of serum calcium. Clin
Chem 1981;27:2025-2028.
10 Porter WH, Carrol JR, Roberts RE. Hemoglobin
interference with the DuPont automatic clinical
analyzer procedure for calcium. Clin Chem
1977;23:2145-2147.
11 Hass RG, Mushel S. Modified Dupont aca
calcium method for hemolyzed specimens. Am
J Clin Pathol 1982;77:216-219.
300
Calcium

Table 1: Methods of Calcium Analysis

Method 1: Precipitation by oxalate and redox titration
Principle of analysis:
Ca2+ + oxalate Ca oxalate (ppt)
Ca oxalate (ppt) + H2SO4 oxalate + CaSO4
_
2KMnO4 + 5 oxalate + 2H2SO4 70C K2SO4 + 2MnSO4 + 10CO2 + 8H2O
Comments: Historical; initial reference method
Method 2: Precipitation by colored anions; spectrophotometric
Principle of analysis:
Ca2+ + chloranilate Ca-chloranilate (ppt)
_
Ca-chloranilate (ppt) + EDTA OH Ca-EDTA + chloranilic acid (purple)
Comments: Historical; labor intensive, imprecise, many other dyes available
Method 3: Titration of fluorescent Ca2+ complex
Principle of analysis:
Ca2+ + calcein Ca-calcein (fluorescent)
EGTA + Ca-calcein Ca+-EGTA + calcein (decreased fluorescence)
Comments: Stat or small labs; small sample size, dedicated instrument
Method 4: Spectrophotometric measurement of Ca2+ complexes
a. Direct
b. Dialysis
Principle of analysis:
_ -
a. Ca2+ + o-cresolphthalein OH red complex (520 nm)
_
b. Ca2+ complex + H+ dialysisCa2+ in recipient stream
Ca2+ detected as in (a)
Comments:
a. Most common; early adapted to a variety of automated instruments; positive bias compared to atomic
absorption
b. On Technicon AutoAnalyzer
Method 5: Flame emission
Principle of analysis:
_ _
Ca2+ heat Ca0 heat Ca* Ca0 + photon 2e
Comments: Historical; poor sensitivity
Method 6: Atomic absorption
Principle of analysis:
_
Ca2+ 2e- Ca0
Photon + Ca0 Ca*
Comments: Reference method; excellent accuracy and sensitivity
Method 7: Indirect potentiometry
Principle of analysis: Calcium ionselective electrode utilized in conjunction with sodium reference electrode;
changes in electrical potential due to calcium referenced to sodium electrode
Comments: Generally shows better accuracy compared with dye-binding techniques, increasing usage
Method 8: Isotope-dilution mass spectrometry
Principle of analysis: Ca and known amount of Ca isotope; isolate Ca2+, and record ratio of two isotopes on
mass spectrometer
Comments: Definitive method; available in reference centers only
Ca*, Calcium atom in excited state; Ca0, calcium atom in ground state; EGTA, ethylene glyco-bis(-aminoethylether)-
N,N1-tetraacetic acid; ppt, precipitate.
301
Calcium

Table 2: Reaction Conditions for Calcium Analysis

Condition: Temperature
o-Cresolphthalein spectrophotometric*: 25C to 37C
Atomic absorption: 2300C
Condition: Sample volume
o-Cresolphthalein spectrophotometric*: 20 L
Atomic absorption: 50 L
Condition: Volume fraction
o-Cresolphthalein spectrophotometric*: 0.01
Atomic absorption: 0.02
Condition: Final concentration
o-Cresolphthalein spectrophotometric*:
o-Cresolphthalein: 0.06 mmol/L
8-Hydroxyquinoline: 9.6 mmol/L
Diethylamine: 193.4 mmol/L
Potassium cyanide: 3.8 mmol/L
Atomic absorption:
Lanthanum diluent: 1 g/L (serum); 5 g/L (urine)
Condition: Wavelength
o-Cresolphthalein spectrophotometric*: 575 nm
Atomic absorption: 422.7 nm
Condition: Reaction mode
o-Cresolphthalein spectrophotometric*: End-point
Atomic absorption: End-point
Condition: Time of reaction
o-Cresolphthalein spectrophotometric*: 30 sec
Atomic absorption: 12 sec
Condition: Linearity
o-Cresolphthalein spectrophotometric*: 150 mg/L
Atomic absorption: Highest standard, usually 120 mg/L
Condition: Interferences
o-Cresolphthalein spectrophotometric*: Gross lipemia
Atomic absorption: Calcium chelators

*From Baginski ES, Marie SS, Alcock NW et al. Calcium in biological fluids. In: Faulkner WR, Meites S, eds. Selected
Methods of Clinical Chemistry. Vol 9. Washington, DC: American Association for Clinical Chemistry Press; 1982.
302
Calcium

Figures

Figure 1: Absorption spectra of o-cresolphthalein reagent.

A, Complexed with calcium standard sample. Dashed line, reagent versus water; dotted dashed line, reagent and sample
versus water; solid line, reagent and sample versus reagent.
B, With various types of serum samples. Reaction was performed at pH 10.4 with 25 L of serum and either 2.00 mL of
blank base solution or reagent. a, Absorbance spectrum obtained with either clean hemolyzed, lipemic, or icteric serum and
the reagent; b, reagent blank; c, lipemic (creamy) serum and base solution; d, hemolyzed (deep red) serum; e, base-solution
curve for icteric (bilirubin level of 240 mg/L) serum and base solution; f, clear serum and base solution. Measured
maximum was 560 to 568 nm.

Procedure: Calcium by Atomic Absorption
Spectrophotometry
Principle
Calcium determinations by atomic absorption
spectroscopy are based on the fact that atoms of an
element in the ground, or unexcited, state absorb light
of the same wavelength as that emitted by the element in
the excited state. Each element has its own characteristic
absorption or resonance lines, and no two elements are
known to have an identical resonance line.
Calcium is determined in serum or urine after the fluid is
diluted sufficiently with lanthanum oxide solution to
avoid interference by chelating substances, especially
phosphate ions.
Reagents
Use specially cleaned atomic-absorption glassware and
deionized water (that is, free of calcium).
1. Stock lanthanum, 50 g/L (314 mmol/L).
Weigh 58.64 g of lanthanum oxide (La2O3), place in a
1-L flask, and add about 50 mL of deionized water to
wet the powder. Add 250 mL of concentrated HCl
slowly and cautiously under hood while swirling the
flask. Dilute to 1 L with deionized water. Store in dark at
room temperature. Stable for 6 months.
2. Working lanthanum diluent for serum, 1 g/L
(6.28 mmol/L). Dilute 20 mL of stock lanthanum to 1 L
with deionized water. Store in dark bottle at room
temperature. Stable for 6 months.
3. Working lanthanum diluent for urine, 5 g/L
(31.4 mmol/L). Dilute 100 mL of stock lanthanum to 1
L with deionized water. Store in dark bottle at room
temperature. Stable for 6 months.
4. Stock calcium standard, 1000 mg/L (25
mmol/L). Dissolve 250 mg of reagent-grade CaCO3 in
3 mL of 1 mol/L HCl. Dilute to 100 mL with deionized
water. Stable 6 months at room temperature in a closed
container.
5. Calcium blank solution (140 mmol/L Na, 5
mmol/L K, 0.1 mmol/L phosphate). Place 8.2 g of
NaCl, 0.373 g of KCl, and 0.014 g of Na2HPO4 into a 1-
L volumetric flask. Dissolve salts in about 250 mL of
deionized water, and dilute to mark with deionized
water. Stable for 1 month at refrigerator temperatures.
303
Calcium

Working Standards
Dilute to Volume With
Ca (mg/L) Stock Ca (mL) Calcium Blank Solution (mL)
50 5 100
100 10 100
120 12 100
150 15 100
200 20 100
6. Place instrument back in concentration mode.
Store at room temperature in glass flasks. Do 7. Place dirty burner head in 5% FL-70 to soak
not pipet from flasks. Pour aliquots daily into separate overnight.
containers.

Assay
Equipment: PerkinElmer Atomic Absorption
Spectrophotometer Model 460 (or other similar
instrument) with a single-slot burner head and calcium-
magnesium hollow-cathode lamp. Follow each
manufacturers directions for maintenance of burner,
optimization of lamp and flame by peaking the flame,
and airacetylene fuel mixture, as well as for the actual
analytical procedure.

Notes
1. The dilution (1:50) of serum or urine prevents
protein interference. The dilution can be
performed accurately and conveniently with a
semiautomatic dilutor (such as Micromedic
Systems, Inc., Philadelphia, PA).
2. Sera left overnight in plastic sample cups may
give low calcium values.
3. There is no appreciable variation in the
patients calcium levels throughout the day if
exercise is avoided, and there is no difference
between fasting and nonfasting serum calcium
levels.
4. There is no difference between venous and
capillary serum calcium levels.
5. Serum or plasma should not be allowed to
remain in prolonged contact with the
erythrocytes, since with time, the cells become
permeable to calcium.
6. Cerebrospinal fluid (CSF) can be analyzed for
calcium using the procedure for serum analysis.
Since CSF concentrations may be lower than
serum concentrations, use of a slightly lower
standard is advisable.

Daily maintenance to be performed before

instrument use:
1. Turn flame on, and aspirate 5% FL-70 (a
detergent supplied by Fisher Scientific Co.,
Fairlawn, NJ; 50-F-105) from probe for 20 min.
2. Turn flame off, and let cool.
3. Remove burner head, and pour 100 mL of
distilled water down neck of chamber (where
head was, water will come out waste line).
4. Position clean burner head, securing pin in
place and slipping wires over hooks on either
side of chamber; tighten burner head collar.
5. Set instrument for maximum absorbance
(peaking the flame), following
manufacturers instructions.
304
Cancer Antigen 125 (CA 125)
Cancer Antigen 125 (CA 125)
Hassan M.E. Azzazy
Name: Cancer antigen 125, CA 125
Molecular mass: >200,000 D
Chemical class: Glycoprotein
Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage i
The first CA 125 assay was developed by Centocor Inc.
and approved by the U.S. Food and Drug Administration
(FDA) for monitoring women with primary epithelial
invasive ovarian cancer. The Centocor CA 125 assay is a
sandwich immunoradiometric assay (IRMA) for
quantitative determination of CA 125 in serum; it became
commercially available in 1983. The assay utilizes OC 125
murine monoclonal antibody developed from cell line
OVCA 433, which was derived from an ovarian serous
carcinoma [1]. The OC 125 antibody was immobilized on
polystyrene beads and used to capture the CA 125 antigen.
125
I-Labeled OC 125 was used as the detector [2,3]. The
second generation of this assay (CA 125 II) utilizes OC
125 antibody for capture and M11 monoclonal antibodies
for detection of CA 125 antigen; the two antibodies bind
to non-overlapping epitopes of CA 125 [4,5]. It was
reported that the first-generation CA 125 assay can give
discordant [6] and even discrepant [7] results. On the other
hand, marker values obtained with second-generation
assays are reported to be more in agreement [8,9].
In addition to IRMA, enzyme immunoassays (EIA),
luminometric immunoassays (LIA), and microparticle
enzyme immunoassays (MEIA) were also developed for
detection of the CA 125 antigen [4]. Currently, several CA
125 immunoassays are available on automated open
random access analyzers, many of which are FDA
approved [10].
Reference and Preferred Methods
There is no reference method for CA 125. CA 125 II RIA
is considered the preferred method.
Specimen
Serum is preferred, although it is possible to use plasma in
some assays. Samples can be stored prior to testing at 4C
for 1 to 5 days or for longer periods (2 weeks to 3 months)
at 20C, depending upon which assay is used. Long-term
storage should be at 70C [11].
Interferences
Heparin and oxalate were found to interfere with CA 125
measurements [12]. As is the case with other
immunoassays, the presence of heterophilic antibodies in
the sample (e.g., following diagnostic or therapeutic use)
could interfere with the CA 125 immunoassay [11].
i with stage III and IV disease; CA 125 correlates with
CA 125
New method:
Fifth edition: Hassan M.E. Azzazy
Reference Intervals
The upper limit of CA 125 in a healthy population is 35
kU/L [11,13]. The reference interval of 0-35 kU/L is the
same for most CA 125 assays; however, results generated
by different assays are not interchangeable [4]. Therefore,
the same method must be used for serial monitoring of
patients, and laboratory reports should indicate the method
used.
Age, race, and menopause were found to affect CA 125
concentrations in healthy individuals. CA 125
concentrations appear to decline with aging and the
menopause. Menstrual cycle variations were also observed
with increases in the follicular phase. A 20% to 50%
variation with race in postmenopausal women has been
reported with CA 125 II measurement (second-generation
assay), with concentrations in Caucasian women higher
than in African and Asian women [14]. Increased
concentrations were detected in 28% of women with non-
gynecological cancers, in 5% of those with benign
diseases, and in 1% to 2% of normal, healthy individuals
[2,15,16].
In a conventional laboratory setting, imprecision of CA
125 assays can contribute significantly to variations of
results. The degree of result variability is directly related
to the absolute CA 125 value, and the relative variability is
fairly constant, with a median of 1.5% to 2.5% of the CA
125 concentration [17].
Interpretation
CA 125 is used as a marker of ovarian and endometrial
carcinomas. It is also increased in some gastrointestinal
tumors and lung and breast carcinomas. Several expert
groups and organizations have developed guidelines for
the clinical utility of CA 125 as a tumor marker for
ovarian cancer. These include the National Institutes of
Health (NIH) [18], National Comprehensive Cancer
Network (NCCN) [19], the European Group on Tumor
Markers (EGTM) [20], European Society for Medical
Oncology (ESMO) [21], and the National Academy for
Clinical Biochemistry (NACB) [11].
CA 125 concentrations exceed 35 kU/L in 80% of women
with epithelial ovarian cancer. Its level is elevated in 50%
of patients with stage I ovarian carcinoma, in 90% of
patients with stage II disease, and in over 90% of those
tumor size and staging [15].
305
Cancer Antigen 125 (CA 125)
Einhorn et al. [22] studied 100 patients who were
investigated for palpable adnexal masses using diagnostic
laparotomy, and 23 of these patients were found to have
malignant disease. Using a cutoff of 35 kU/L as a
predictive value for malignancy, CA 125 was found to
have 78% sensitivity, 95% specificity, 82% PPV, and 91%
NPV. However, owing to lack of specificity and
sensitivity for a single marker measurement, none of the
above expert groups recommended CA 125 for screening
asymptomatic women.
Based on current evidence, CA 125 is more widely
accepted and recommended as an adjunct for differential
diagnosis of suspicious pelvic masses, particularly in
postmenopausal women [18,20,23]. In postmenopausal
women, CA 125 concentration > 95 kU/L can discriminate
benign from malignant pelvic masses with a positive
predictive value of 95% [2].
Elevated CA 125 concentration (>35kU/L) after debulking
surgery and chemotherapy indicates the likelihood of a
residual disease. Although elevated postoperative
concentrations predict relapse, negative values do not
exclude disease presence.
CA 125 may also have a role in monitoring chemotherapy.
Serial measurements of CA 125 correlated with clinical
disease outcome in 89% of 531 patients [24]. There is a
general consensus among current guidelines to use CA
125 to monitor response to chemotherapy. The
Gynecologic Cancer Intergroup (GCIC) defines response
as a 50% reduction in CA 125 concentration compared to
that in a pretreatment sample [25,26]. Response should be
confirmed and sustained for at least 20 days. Patients can
only be evaluated if they have a CA 125 concentration in a
pretreatment sample that is twice the upper reference limit
and taken 2 weeks prior to initiation of therapy. Additional
samples are recommended at 2 to 4 weeks during
treatment and at intervals of 2 to 3 weeks during follow-
up. The same method should be used to monitor the
patient throughout, and patients who receive
immunotherapy (mouse antibodies) cannot be evaluated.
Serial measurement of CA 125 to aid in monitoring
therapeutic response is approved by the FDA.
Pre- and postoperative CA 125 concentrations may be of
prognostic significance [27-29]. After primary surgery and
chemotherapy, persistent elevations of CA 125
concentrations were associated with poor prognosis.
Patients who had preoperative CA 125 concentrations > 65
kU/L were reported to have a lower 5-year survival rate
and a 6.37-fold risk of death as compared to patients who
had CA 125 levels < 65 kU/L [24]. The half-life of CA
125 antigen was reported to have an additional prognostic
value. A half-life of < 20 days was associated with
improved survival as compared to half-life > 20 days [30].
Normalization of CA 125 levels after three cycles of
combination therapy also correlates with improved
survival.
It should be noted that CA 125 concentration is not
elevated in 10% to 20% of patients with advanced ovarian
cancer. For these patients, the use of radiological imaging
techniques and/or monitoring other tumor markers is
necessary.
Performance Goals
For CA 125 antigen, the within- and between-subject
coefficients of variation (CVs) were 24.7% and 54.6%,
respectively. The desirable analytical quality
specifications for imprecision, bias, and total error (as
derived from biological variation) for serum CA 125 were
12.4%, 15.0%, and 35.4%, respectively [31].
The EGTM indicate that assays for tumor markers,
including CA 125, should demonstrate interassay
variability under 10% and intraassay variability less than
5% [32]. None of the current CA 125 assays meet their
criteria. The current Clinical Laboratory Improvement
Amendments (CLIA) performance goal for measurement
of CA 125 is for laboratories to be within 3 standard
deviations of the peer-group mean. According to the 2007
College of American Pathologists (CAP) Survey, CV for
all CA 125 assays at a mean value of 107.3 ng/L (SD 14.5)
was 13.5%.
References
1 Bast RC, Feeney M, Lazarus H, Nadler LM,
Colvin RB, Knapp RC. Reactivity of a
monoclonal antibody with human ovarian
carcinoma. J Clin Invest 1981;68:1331-1337.
2 Bast RC, Xu FJ, Yu YH, Barnhill S, Zhang Z,
Mills GB. CA 125: the past and the future. Int J
Biol Markers 1998;13:179-187.
3 Bast RC, Klug TL, St John E, Jenison E, Niloff
JM, Lazarus H et al. A radioimmunoassay using a
monoclonal antibody to monitor the course of
epithelial ovarian cancer. N Engl J Med
1983;309:883-887.
4 Davelaar EM, van Kamp GJ, Verstraeten RA,
Kenemans P. Comparison of seven
immunoassays for the quantification of CA 125
antigen in serum. Clin Chem 1998;44:1417-1422.
5 Sakahara H, Kousaka T, Hattori N, Tomida K,
Yao Z, Hosono M et al. Dissociation in serum
CA125 concentrations measured by different
monoclonal antibodies. Gynecol Oncol
1994;52:301-305.
6 van Kamp GJ, Verstraeten AA, Kenemans P.
Discordant serum CA 125 values in commercial
immunoassays. Eur J Obstet Gynecol Reprod
Biol 1993;49:99-103.
7 Kenemans P, Bon GG, Kessler AC, Verstraeten
AA, van Kamp GJ. Multicenter technical and
clinical evaluation of a fully automated enzyme
immunoassay for CA 125. Clin Chem
1992;38:1466-1471.
8 Bonfrer JM, Baan AW, Jansen E, Lentfer D,
Kenemans P. Technical evaluation of three
second generation CA 125 assays. Eur J Clin
Chem Clin Biochem 1994;32:201-207.
306
Cancer Antigen 125 (CA 125)
9 Kenemans P, Verstraeten AA, van Kamp GJ, von
Mensdorff-Pouilly S. The second-generation CA
125 assays. Ann Med 1995;27:107-113.
10 U.S. Food and Drug Administration website.
<http://www.accessdata.fda.gov/scripts/cdrh/cfdo
cs/cfCLIA/search.cfm>
11 National Academy of Clinical Biochemistry:
Practice Guidelines and Recommendations for
Use of Tumor Markers. In: The Clinic, 2006.
Available at
<http://www.aacc.org/AACC/members/nacb/LM
PG/OnlineGuide/DraftGuidelines/TumorMarkers
/> Accessed 10.7.2007.
12 Klug TL, Bast RC Jr, Niloff JM, Knapp RC,
Zurawski VR Jr. Monoclonal antibody immuno-
radiometric assay for an antigenic determinant
(CA 125) associated with human epithelial
ovarian carcinomas. Cancer Res 1984;44:1048-
1053.
13 Duffy MJ, Bonfrer JM, Kulpa J, Rustin GJ,
Soletormos G, Torre GC et al. CA 125 in ovarian
cancer: European Group on Tumor Markers
guidelines for clinical use. Int J Gynecol Cancer
2005;15:679-691.
14 Pauler DK, Menon U, McIntosh M, Symecko
HL, Skates SJ, Jacobs IJ. Factors influencing
serum CA125II levels in healthy postmenopausal
women. Cancer Epidemiol Biomarkers Prev
2001;10:489-493.
15 Jacobs I, Bast RC Jr. The CA 125 tumour-
associated antigen: a review of the literature.
Hum Reprod 1989;4:1-12.
16 Fleisher M, Dinistrian A, Sturgeon C, Lamerz R,
Witliff J. Practice guidelines and
recommendations for use of tumor markers in the
clinic. In: Diamandis EP, Fritsche H, Lilja H,
Chan DW, Schwartz M, eds. Tumor Markers:
Physiology, Pathobiology, Technology, and
Clinical Applications. Washington, DC: AACC
Press; 2002:33-63.
17 Tso E, Elson P, Vanlente F, Markman M. The
real-life variability of CA-125 in ovarian
cancer patients. Gynecol Oncol 2006;103:141-
144.
18 National Institutes of Health Consensus
Conference. NIH Consensus Development Panel
on Ovarian Cancer. Ovarian cancer: screening,
treatment, and follow-up. JAMA 1995;273:491-
497.
19 Ozols RF. Update of the NCCN ovarian cancer
practice guidelines. Oncology (Williston Park)
1997;11:95-105.
20 Bonfrer J, Duffy M, Radke M, Segurado O, Torre
G, Van Dalen A et al. European Group on Tumor
Markers. Tumor markers in gynaecological
cancers: EGTM recommendations. Anticancer
Res 1999;19:2807-2810.
21 ESMO minimum clinical recommendations for
diagnosis, treatment and follow-up of ovarian
cancer. Ann Oncol 2001;12:1205-1207.
22 Einhorn N, Bast RC Jr, Knapp RC, Tjernberg B,
Zurawski VR Jr. Preoperative evaluation of
serum CA 125 levels in patients with primary
epithelial ovarian cancer. Obstet Gynecol
1986;67:414-416.
23 Chan DW, Shih LM, Sokoll LJ, Bast RC, Jr.
National Academy of Clinical Biochemistry
Guidelines for the use of tumor markers in
ovarian cancer: practice guidelines and
recommendations for use of tumor markers in the
clinic, 2006. Available at
<http://www.aacc.org/AACC/members/nacb/LM
PG/OnlineGuide/DraftGuidelines/TumorMarkers
/> Accessed 10.7.2007.
24 Meyer T, Rustin GJ. Role of tumour markers in
monitoring epithelial ovarian cancer. Br J Cancer
2000;82:1535-1538.
25 Rustin GJ. Can we now agree to use the same
definition to measure response according to CA-
125? J Clin Oncol 2004;22:4035-4036.
26 Rustin GJ, Quinn M, Thigpen T, du Bois A,
Pujade-Lauraine E, Jakobsen A et al. Re: New
guidelines to evaluate the response to treatment in
solid tumors (ovarian cancer). J Natl Cancer Inst
2004;96:487-488.
27 Sjovall K, Nilsson B, Einhorn N. The
significance of serum CA 125 elevation in
malignant and nonmalignant diseases. Gynecol
Oncol 2002;85:175-178.
28 Cooper BC, Sood AK, Davis CS, Ritchie JM,
Sorosky JI, Anderson B, Buller RE. Preoperative
CA 125 levels: an independent prognostic factor
for epithelial ovarian cancer. Obstet Gynecol
2002;100:59-64.
29 Gadducci A, Cosio S, Fanucchi A, Negri S,
Cristofani R, Genazzani AR. The predictive and
prognostic value of serum CA 125 half-life
during paclitaxel/platinum-based chemotherapy
in patients with advanced ovarian carcinoma.
Gynecol Oncol 2004;93:131-136.
30 Verheijen RH, von Mensdorff-Pouilly S, van
Kamp GJ, Kenemans P. CA 125: fundamental
and clinical aspects. Semin Cancer Biol
1999;9:117-124.
31 Westgard QC website.
<http://www.westgard.com/biodatabase1.htm>
32 European Group on Tumor Markers website.
<http://egtm.eu/1280.html>
307
Carbamazepine
Carbamazepine
Gus Koerbin and Julia M. Potter
Name: Carbamazepine, 5-carbamoyl-5H-dibenz[b,f]azepine
Clinical Significance: Refer to Chapter 47, Nervous System, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular formula: C15H12N2O
Molecular mass: 236.26 D
Merck Index: 1758
Chemical class: Tricyclic iminodibenzene
Carbamazepine
CONH2
Structure:
Principles of Analysis and Current Usage
Carbamazepine is the generic name for 5-carbamoyl-5H-
dibenz[b,f]azepinei. It is a derivative of the tricyclic
iminodibenzyl nucleus (Figure 1). The drug is used to
treat epilepsy. A metabolite, carbamazepine 10,11-
epoxide (CBZ-E), found in the plasma, is
pharmacologically active and is increased with
concurrent use of other anticonvulsive medication [1].
Methods for the measurement of carbamazepine in
biological fluids include spectrophotometry with
ultraviolet radiation [2], visible light [3], fluorometry,
often combined with thin-layer chromatography [4-6],
gas-liquid chromatography methods [7], and high-
performance liquid chromatography methods [8-13].
Thin-layer chromatography (TLC) (Table 1, Method 1)
quantitative ultraviolet spectrophotometric assays (Table
1, Method 2) and visible-light spectrophotometric
methods (Table 1, Method 3) are of historical interest.
i
Carbamazepine previously under Anticonvulsants
Previous and current authors of this method:
First edition: Steven J. Soldin
Methods edition: Elizabeth B. Solow
Second edition: Steven J. Soldin
Third edition: Paul Salm, Paul J. Taylor, Julia M.
Potter
Fourth edition: Paul Salm, Paul J. Taylor, Julia M.
Potter
Fifth edition: Gus Koerbin and Julia M. Potter
After TLC separations, fluorescence can be induced by
treatment of the chromatogram with 70% perchloric acid
and heat. An excitation wavelength of 358 nm and an
emission wavelength of 498 nm were used by
Christiansen [4]. Meilink [5] reported a technique using
ammonium cerium(IV) sulfate and phosphoric acid
reagent to induce fluorescence. For quantitative
ultraviolet spectrophotometric assays, extracted samples
were measured at the absorbance of the secondary peak
wavelength, 290 nm, rather than at the maximum
absorbance of 215 to 220 nm, to eliminate major
interferences from other commonly administered
anticonvulsants. The visible-light spectrophotometric
method of Herrman [3] extracted carbamazepine with
dichloroethane at a neutral pH. The extract was then
treated with sodium nitrite and nitric acid to produce a
soluble yellow compound whose absorbance at 400 nm
was proportional to the level of carbamazepine.
Spectrophotometric scans of carbamazepine and
carbamazepine epoxide are shown in Figure 2.
Immunoassay techniques are most commonly used today
for the quantitative measurement of carbamazepine. The
homogeneous enzyme-multiplied immunoassay
technique (EMIT) (Table 1, Method 4) employs a
derivative of carbamazepine. In this derivative, the
carbamazepine is attached to the enzyme glucose-6-
phosphate dehydrogenase. When antibody to the drug
binds to the conjugate, the enzymatic activity is
inhibited. Drug present in the patient sample competes
with the labeled drug for the antibody binding sites. The
more drug present in the sample, the less antibody is
308
Carbamazepine
available to react with the drug-enzyme complex, and
the greater the enzymatic activity observed [9,14]. A
homogeneous substrate-labeled fluorescence assay
(SLFIA) procedure (Table 1, Method 5) [15-17], a drug
derivatized with the fluor umbelliferone, attached to the
drug through a galactoside linkage, is used as the
indicator molecule. The drug derivative binds to an
antibody specific for the drug. In the presence of drug in
the patient sample, the galactoside derivative of the drug
is not bound by the antibody and is free to be cleaved by
the enzyme beta-galactosidase, present in the
homogeneous reaction mixture, to release umbelliferone.
The fluorescent umbelliferone formed is readily
measured by fluorometry. In the absence of drug, the
antibody binds to the labeled drug and prevents
cleavage, resulting in low fluorescence. In the
fluorescence polarization assay, FPIA, a fluorescent drug
derivative (usually fluorescein labeled) is bound by
antibody (Table 1, Method 6). The bound drugfluor has
restricted movement, allowing incident polarized light to
be re-emitted as highly polarized fluorescence. In the
presence of drug in the patient sample, more of the drug-
fluor remains unbound, allowing more rapid movement
and resulting in the emission of fluorescent light that is
less polarized. The decrease in polarized fluorescence
can be used to quantitate the concentration of
carbamazepine.
Gas-liquid chromatography has been widely used for
carbamazepine determination (Table 1, Method 7). Early
methods used derivatization techniques (Table 1,
Method 7a). More recent procedures, which use
deactivated column packings, do not require
derivatization of the drug to achieve good resolution.
Most GLC methods measure only the parent drug [18-
26], although the procedure of Morselli et al. [26]
measured both the parent drug and its epoxy metabolite.
To increase sensitivity further, the combination of GLC
and mass spectrometry has been used.
High-performance liquid chromatography (HPLC)
(Table 1, Method 8) is becoming increasingly popular
for the measurement of both carbamazepine and the
epoxide, since the current state of column technology
has alleviated the problem of thermal instability.
Detection is by ultraviolet spectrophotometry (212 and
254 nm), and several internal standards, such as
dihydrocarbamazepine and cyheptamide, have been used
to improve analysis. Rambeck described a liquid
chromatographic method for quantitating antiepileptic
drugs, including carbamazepine and its epoxide [12],
using a reversed-phase column requiring an analysis
time of 22 min. Eichelbaum and Bertilsson [10]
presented a high-performance liquid chromatography
method using a Carbowax 400-Corasil column and
monitoring the eluent absorbance at a nonspecific
wavelength of 254 nm. They reported that a minimum of
1 mL of plasma was required to measure the drug and its
metabolite concurrently. A high-performance liquid
chromatography method was developed by Sawchuk and
Cartier [13] for measuring both carbamazepine and the
epoxide simultaneously in a single extract using
cyheptamide as an internal standard.
Reference and Preferred Methods
There is no reference method for carbamazepine.
Ultraviolet spectrophotometry is not currently used for
the analysis of carbamazepine, since it is not specific for
the drug, and the extraction procedures are labor
intensive. Colorimetric procedures are not recommended
for the same reason. Fluorometric determination of
carbamazepine and its metabolites by TLC are also labor
intensive and subject to considerable imprecision. The
imprecision is greater than by other techniques, and the
level of technical expertise necessary is greater than can
be easily achieved by most laboratories.
Immunoassay procedures are very easy to perform and
are well suited for measurement of carbamazepine in the
therapeutic range. Immunoassay procedures are the
current methods of choice for laboratories performing
therapeutic drug monitoring, but consideration should be
given to assays that measure the metabolite CBZ-E as
well. According to survey data from the 2007 College of
American Pathologists Participant Summary Report,
only immunoassays are currently used for measurement
of carbamazepine. All of these methods are acceptable,
so methods for carbamazepine assay are determined by
the choice of instrumentation in the laboratory.
Although it has adequate sensitivity, gas-liquid
chromatography is not free from problems. The majority
of these problems are the result of decomposition
products from high assay temperatures or
chromatographic column conditions (Figure 3A).
Derivatization of the compound can minimize these
difficulties [19,20,22]. The addition of an internal
standard with a related chemical structure enhances the
accuracy of measurement. Figure 3B illustrates the
structure of possible standards. Newer technology and
deactivating column packings allows carbamazepine
determinations without derivatization and minimizes
breakdown products [25]. Using GLC in conjunction
with mass spectrometry detection, Palmer et al. [27]
obtained low-nanogram-range sensitivity. Other drugs
can be analyzed simultaneously, but the epoxide
metabolite of carbamazepine is unstable, and its
measurement is unpredictable with either the flame
ionization detector or the mass spectrometer.
High-performance liquid chromatography seems to be
the procedure that offers the most analytically accurate
estimation of carbamazepine, since it measures both
parent compound and the 10,11-epoxide. The precision
of HPLC assays is 4% to 5% in the therapeutic range. A
study of Mihaly et al. [9] showed a better correlation of
dosage with the serum 10,11-epoxide concentrations
than with the parent carbamazepine. The minimum
detectable quantity by HPLC for both the parent and the
metabolite is 0.8 ng [13].
Most reports demonstrate good correlations between the
assay systems EMIT, TDx, GLC, and HPLC for serum
values. This is true where the major component present
in the patient samples is carbamazepine. The correlation
may not be as good in the presence of metabolites.
309
Carbamazepine
Active CBZ-E is not routinely monitored, partly because
an HPLC method is needed to measure the metabolite
[28]. However, for laboratories without HPLC
technology, immunoassays will give adequate results for
most patients. The extent of cross-reactivity of CBZ-E
with common immunoassays has been demonstrated by
Hermida and Tutor [29].
Specimen
Either serum or plasma samples are acceptable for
analysis. There are no known interferences from
commonly used anticoagulants, commercial vacuum
tubes, or tubes with inert polymer gel barrier for serum
separation. The free fraction has been shown to be
higher in plasma (26%) than serum (19%) [30].
If a sample cannot be analyzed the same day as the blood
was drawn from the patient, short-term storage at room
temperature or normal refrigeration is satisfactory. When
the sample is frozen at 10C, no deterioration has been
observed during a 6-month period. Specimens stored on
gel-barrier tubes showed a decrease compared to
specimens not stored on gel [31].
Interferences
None of the commercially available immunoassays are
specific for the parent compound, and all cross-react to a
greater or lesser degree with the 10,11-epoxide. The
carbamazepine 10,11-epoxide cross-reacts with the
currently available polyclonal antibodies. The
immunoassays also cross-react with the tricyclic
antidepressants; however, since these drugs are present
in much lower concentrations than carbamazepine is,
this does not appear to be of analytical significance.
Hemolysis and lipemia have been shown to interfere in
some immunoassay procedures.
Carbamazepine Reference Interval
The effective plasma carbamazepine level is 4 to 12
mg/L (16.9 to 50.8 mol/L). Some patients seem to
benefit from lower levels [32,33]. Furthermore, the
evaluation is complicated by the presence of 10,11-
epoxide and drug interactions. McKauge et al. [34]
reported that over the usual concentration range of
plasma carbamazepine in 295 subjects, values
encountered for the epoxide level averaged 20% to 25%
of the parent drug levels. Hundt et al. [35] summarized 8
years of monitoring carbamazepine and its metabolites
and encouraged the routine assessment of these levels.
The relationship of age, dose, and associated therapy
complicates interpretations [36,37].
Interpretation
Carbamazepine has been widely used for the treatment
of certain types of epilepsy, trigeminal neuralgia, and
other clinical ailments, including depression and mania.
The pharmacological properties and the therapeutic use
of carbamazepine have been reviewed extensively
[38,39].
The double bond between positions 10 and 11 in the
tricyclic nucleus is somewhat unstable and provides the
major site of action for the biotransformation enzymes.
The biologically active metabolite carbamazepine 10,11-
epoxide (Figure 3A), is found in substantial
concentration in plasma. It has been shown to have both
anticonvulsant activity [40] and antineuralgic properties
[41]. The effectiveness and toxicity of carbamazepine
may best be correlated with the combined total plasma
levels of the parent drug and its major metabolite.
The apparent plasma half-life of carbamazepine has been
reported to range from 8 to 60 hours. The plasma half-
life of 10,11-epoxide is shorter than that of the parent
compound. Protein binding is reported to average 70% to
80% for the parent drug and 50% to 60% for the 10,11-
epoxide.
Toxic effects are frequent with concentrations over 12
mg/L. Cases of overingestion of carbamazepine and a
few postmortem cases have been reported [32].
Interaction with erythromycin was shown to increase
carbamazepine levels in epileptic children [42] and to
have associated toxicity.
McKauge, Tyrer, and Eadie [34] demonstrated a drug
interaction involving valproic acid and carbamazepine.
The co-administration of valproic acid and
carbamazepine resulted in elevated plasma
concentrations of the epoxide while leaving plasma
carbamazepine concentration unaltered. Levy et al. [43]
have also reported a drug interaction involving valproic
acid and carbamazepine in both man and rhesus monkey.
In man, they reported, carbamazepine levels were
reduced, and the ratio of 10,11-epoxide to
carbamazepine was increased. With the potential for
interaction with other drugs, measurement of both the
parent drug and its metabolite are indicated. Post et al.
[44,45] reported that levels of 10,11-epoxide in both
plasma and CSF were better correlated with the dose of
carbamazepine than were the levels of carbamazepine
itself.
Carbamazepine Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report shows
imprecision values (% coefficient of variation [CV]) for
measurement of acetaminophen range from 3.9% to
9.5% at concentrations of approximately 6.5 g/mL and
from 3.2% to 9.6% at about 13.5 g/mL for assays with
> 20 participants. Acceptable performance criteria for
measurement of carbamazepine (CLIA 88) requires that
laboratories be accurate to within 25% of the peer-
group mean.
Desirable specifications for analytical imprecision
derived from studies of biological variation indicate an
assay imprecision of no greater than 0.5 g/mL and 0.77
mg/mL at decision levels of 8.0 and 12.0 g/mL [46].
The 2007 CAP data show that there is a lack of
harmonization between commonly used carbamazepine
310
Carbamazepine
assays, and there are differences in concentration
between these assays of greater than 25%.
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29 Hermida J, Tutor JC. How suitable are currently
used carbamazepine immunoassays for
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Variations in protein binding of drugs in plasma
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37 Paxton JW, Aman MG, Werry JS. Fluctuations
in salivary carbamazepine and carbamazepine
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312
Carbamazepine

Tables
Table 1: Methods of Carbamazepine Analysis
Method 1: Thin-layer chromatography; qualitative
Principle of analysis: Drug extracted from matrix, chromatographed by TLC, and detected by reaction with
perchloric acid and heat
Comments: Rare; not as accurate as other methods
Method 2: Spectrophotometric; quantitative
Principle of analysis: Drug extracted from matrix, quantified by its ultraviolet absorption at specific
wavelengths, such as 290 or 255 nm
Comments: Historic; interference from other compounds
Method 3: Colorimetric; quantitative
Principle of analysis: Drug extracted from matrix, reacted with nitrous acid to make a yellow nitrogen derivative
measured at 400 nm
Comments: Rare; nonspecific colorimetric reaction
Method 4: Enzyme multiplied immunoassay technique (EMIT); quantitative
Principle of analysis: Patient drug competes for antibody with enzyme-labeled drug; enzyme activity
proportional to concentration
Comments: Common; simple; however, does not measure epoxide separately; hemolysis a potential problem
Method 5: Substrate-labeled fluorescence immunoassay (SLFIA); quantitative
Principle of analysis: Patient drug competes for antibody with drug-galactoside derivative; unbound drug
derivative hydrolyzed to fluor by exogenous galactosidase
Comments: Uncommon; as above
Method 6: Fluorescence polarization immunoassay; quantitative
Principle of analysis: Patient drug competes for antibody with drug fluorescein derivative; unbound drug has
lower polarized fluorescence than bound drug
Comments: Common
Method 7: Gas chromatography; quantitative
a. Gas-liquid derivatized
b. Gas-liquid underivatized
Principle of analysis:
a. Drug extracted into organic solvent, converted to a derivative, detected by flame ionization or
nitrogen detector
b. As above, special deactivated columns that do not react with drug are used; has been used with a mass
spectrograph detector
Comments:
a. Uncommon; labor intensive; drug and metabolite subject to thermal decomposition
b. Uncommon; labor intensive; metabolite subject to thermal decomposition
Method 8: High-performance liquid chromatography (HPLC); quantitative
Principle of analysis: Drug extracted, separated by chromatography, detected by ultraviolet absorbance
Comments: Common; detects parent drug and metabolite; method of choice

Figure 1: Carbamazepine and its precursors.

313
Carbamazepine

Figure 2: Spectrophotometric scans

A, Carbamazepine. B, Carbamazepine 10,11-epoxide. C, Dihydrocarbamazepine, internal standard.

Figure 3: A, Carbamazepine and its metabolites and breakdown products. B, Structures of potential
internal standards.
314
Carbamazepine

Figure 4: Chromatograms from HPLC instrumentation.

A, Plasma or serum blank. B, Plasma plus internal standard (IS). C, Drug-free plasma spiked with
carbamazepine (CBZ), 8 mg/L, and its 10,11-epoxide, 2 mg/L. D, Patient sample with both metabolite
and parent drug. Internal standard is at 6.6 min, carbamazepine is at 5.5 min, and its epoxide is at 2.6
min.

Figure 5: Chromatograms from GLC instrumentation.

A, Plasma blank. B, Plasma plus internal standard. C, Drug-free plasma spiked with carbamazepine, 4
mg/L. D, Patient sample with carbamazepine therapy. Internal standard is at 2.5 min, and the
carbamazepine is at 2.9 min. FID, Flame ionization detector.
315
Carbamazepine
Procedure: High-Performance Liquid
Chromatography
Principle
The method described below is based on an alkaline
solvent extraction with an analog dihydrocarbamazepine
included as an internal standard. The residue is
reconstituted in the mobile phase for HPLC analysis.
Detection and quantitation are achieved by ultraviolet
spectroscopy at 212 nm [8]. The lower limit of detection
for the drug or its metabolite is 0.1 g/mL without
modification of procedure. (See HPLC chromatograms,
Figure 4.)
Reagents
1. Saturated trisodium phosphate buffer. Add
190 g of hydrated trisodium phosphate (Na3PO4
12H2O) to 500 mL of distilled water. Heat to dissolve
the crystals. Cool to room temperature. Excess crystals
form, showing that the solution is saturated. Store at
room temperature for up to 2 years.
2. Benzene/ethyl acetate, 4:1 (NOTE: When
using benzene, follow usual safety precautions and work
under fume hood.)
3. Methanol. HPLC grade. Filter through
Millipore, type FH filter, with vacuum.
4. Stock carbamazepine standard, 2 g/L (8.46
mmol/L). Dissolve 10 mg of carbamazepine in 5 mL of
methanol. Stable for at least 6 months when stored
tightly sealed at normal refrigeration temperature of 4C.
5. Stock carbamazepine 10,11-epoxide, 1 g/L
(4.0 mmol/L). Dissolve 5 mg of dihydrocarbamazepine
in 5 mL of methanol. Stability is the same as described
above for the parent drug carbamazepine.
6. Stock dihydrocarbamazepine, 1 g/L (4.20
mmol/L). Dissolve 5 mg of dihydrocarbamazepine in 5
mL of methanol. Stable for at least 12 months when
stored at 4C.
7. Carbamazepine and epoxide plasma
standards.
Carbamazepine (33.9 mol/L) and epoxide (8.0
mol/L). To a 50-mL volumetric flask, deliver drug-free
plasma to approximately half-full level. Deliver 200 L
of the stock carbamazepine standard and 100 L of the
stock epoxide standard to the flask. Add each solution
slowly and carefully, with mixing, to prevent
precipitation of protein or coagulation, which would
hinder solubility of the compounds. Add a minute drop
of caprylic alcohol to prevent foam. Fill to the 50 mL
mark with plasma, and mix thoroughly. This standard
contains 8 g of carbamazepine and 2 g of the epoxide
per milliliter of plasma.
Carbamazepine (16.9 mol/L) and epoxide (4.0
mol/L). Similarly to drug-free plasma, in a 50-mL
volumetric flask, deliver 100 L of the stock
carbamazepine and 50 L of the epoxide. Fill to the
mark with plasma, and mix thoroughly. Use the
precautions described above to prevent foam and to
prevent protein precipitation. This standard contains 4
g of carbamazepine and 1 g of epoxide per milliliter
of plasma. Aliquot 1 mL volumes of the standards to
polystyrene tubes, and seal tightly for storage. Stable for
at least 6 months when frozen at 20C.
8. Extraction solvent with
dihydrocarbamazepine internal standard (4.20
mol/L). Prepare benzene/ethyl acetate, 4:1 (v/v), to
contain 1 mg of dihydrocarbamazepine per liter. Deliver
1 mL of the stock dihydrocarbamazepine to a 1-L
volumetric flask. Fill to the mark with benzene/ethyl
acetate, and mix well. This solvent now contains 1 g of
the internal standard per milliliter. When stored at room
temperature, this compound is stable for several months.
9. Control serum or plasma. Prepare drug-free
plasma or serum with carbamazepine and its epoxide so
that the concentrations are 10 mg/L (42.3 mol/L) for
the parent drug and 1.5 mg/L (6.0 mol/L) for the
epoxide metabolite. Aliquot 1-mL volumes into
polystyrene tubes and seal. Stable for at least 6 months
when frozen at 20C.
Assay
Equipment: A high-performance liquid chromatograph.
The instrument should be equipped with a variable
wavelength ultraviolet spectrophotometer. The column
should be a reversed-phase octadecyldimethylsilyl
(ODS) on 5 m spherical silica particles.
1. Label appropriate number of 5-mL, glass-
stoppered, conical tubes for standards, control,
and unknowns.
2. To each tube, add 0.5 mL of the respective
plasma or serum samples.
3. Add 250 L of saturated trisodium phosphate
buffer solution.
4. Add 2 mL of the extraction solvent containing
internal standard.
5. Mix with vortex-mixing device, and centrifuge at
500 g for 5 min.
6. Remove solvent layer to a conical 3 mL tube, and
evaporate to dryness at 40C to 60C with a flow
of nitrogen.
7. Dissolve residue in an appropriate volume of
diluent. For HPLC automatic injection system,
add 200 L of the mobile phase methanol/water,
45:55, v/v, and transfer to vials for 0.1 mL
volume. Using HPLC automatic sampling
system, inject 10 L (equivalent to 0.025 mL of
serum or plasma).
8. HPLC parameters:
Temperature. Ambient temperatures for column and
mobile phase are satisfactory, but if temperature control
devices are available, the column oven temperature is
40C, and the mobile phase reagent temperature is 30C.
Mobile phase. Methanol/water, 45:55, v/v
Flow rate. 2.5 mL/min
Ultraviolet wavelength detector. 212 nm
Attenuation of integrator. Set so that 200 ng of
carbamazepine gives a peak two thirds the full scale on
the chart recorder.
316
Carbamazepine
Chart speed. 0.5 cm/min
Carbamazepine: Figure 3 shows the absorbance
spectra for carbamazepine 10,11-epoxide and
dihydrocarbamazepine (internal standard). Examples of
typical chromatograms are illustrated in Carbamazepine:
Figure 4.
Calculations
1. Measure the peak height (in millimeters) of the
drug, carbamazepine 10,11-epoxide, and internal
standard.
2. Compute the ratio of the peak height (mm) of the
drug and metabolite versus the peak height of the
internal standard for the standards, control, and
unknowns.
Peak height ratio = Peak height (mm) of analyte
Peak height (mm) of internal standard
3. Plot standard curve from the peak height ratios of
standards. See Carbamazepine: Figure 5 for
representative illustration of plot for standard
curve. Calculate the amount of drug or
metabolite by comparing the peak height ratios
on the appropriate standard curve.
Notes
1. Absolute recovery was shown to average 95% for
carbamazepine and 83% for its 10,11-epoxide.
2. Reproducibility (CV) was 3.6% within-day for
carbamazepine and 1.2% for the epoxide.
Between-day reproducibility was shown to be
4% CV for carbamazepine and 6.3% CV for the
epoxide.
3. Although both the HPLC and GLC (below)
procedures are described using the
dihydrocarbamazepine as the internal standard,
any one of the three compounds illustrated in
Carbamazepine: Figure 2B are acceptable and are
eluted without problems with either the HPLC or
the GLC instruments.
Procedure: Gas-Liquid Chromatography
Principle
The method described below is based on an alkaline
solvent extraction with an analog dihydrocarbamazepine
included as an internal standard. The residue is
reconstituted in methanol for flame ionization gas-liquid
chromatography. (See GLC chromatograms, Figure 5.)
Reagents
1. Saturated trisodium phosphate buffer.
2. Benzene/ethyl acetate, 4:1
3. Methanol.
4. Stock carbamazepine standard, 2 g/L (8.46
mmol/L).
5. Stock carbamazepine 10,11-epoxide, 1 g/L
(4.0 mmol/L).
6. Stock dihydrocarbamazepine, 1 g/L (4.20
mmol/L).
7. Carbamazepine and epoxide plasma
standards. 33.9 and 8.0 mol, respectively, and 2, 16.9,
and 4.0 mol, respectively.
8. Extraction solvent with internal standard.
9. Control serum or plasma.
Assay
Equipment: Gas chromatograph equipped with a
hydrogen flame detector. The columns are 6 ft 2 mm
(inner diameter) silanized glass. The column packing is
2% SP-2510 DA on 100/1209 mesh Supelcoport.
The carrier gas is nitrogen, and gas flow rates are
as follows: nitrogen, 40 mL/min; hydrogen, 40
mL/min; and air, 250 mL/min.
The injection port temperature is 250C; the
detector temperature is 300C.
The column oven temperature is programmed as
follows: The initial temperature is 200C with
zero hold time; the rate of rise is 4C/min; the
final temperature is 260C.
1. Label an appropriate number of 5-mL, glass-
stoppered, conical tubes for standards, control, and
unknowns.
2. To each tube, add 0.5 mL of the respective
plasma or serum samples.
3. Add 250 L of saturated trisodium phosphate
buffer solution.
4. Add 2 mL of the extraction solvent containing
internal standard.
5. Mix with vortex-mixing device, and centrifuge
at 500 g for 5 min.
6. Remove solvent layer to a conical 3-mL tube
(Reliance Glassworks, Inc., Bensenville, IL 60106,
catalog no. R-2827-200), and evaporate to dryness at
40C to 60C with a flow of nitrogen. Reconstitute in
100 L of methanol, and inject 2 L.
7. Chromatograms are illustrated in
Carbamazepine: Figure 6 for GLC.
Calculations
1. Measure the peak height (mm) of the drug,
10,11-epoxide, and internal standard.
2. Compute the ratio of the peak height of the drug
and metabolite versus the peak height of the internal
standard for the standards, control, and unknowns.
Peak height ratio = Peak height (mm) of analyte
Peak height (mm) of internal standard
3. Plot standard curve from the peak height ratios
of standards. See Carbamazepine: Figure 5 for
representative illustration of plot for standard curve.
Calculate the amount of drug or metabolite by
comparing the peak height ratios on the appropriate
standard curve.
Notes
1. Absolute recovery was shown to be 89% for
carbamazepine with this GLC procedure.
2. Reproducibility was shown to have a CV of
1.9% within-day and a CV of 4.0% between days.
317
CA 15-3
CA 15-3
Gregory A. Hobbs i
Name: CA 15-3
Clinical significance: Post-surgical monitoring of recurrence of breast cancer. Refer to Chapter 53,
Neoplasia, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation
Molecular weight: 300,000 D to 400,000 D, depending on extent of glycosylation
Chemical class: Glycoprotein
Principles of Analysis and Current Usage
CA 15-3 is a glycoprotein encoded by the MUC1 gene.
It is present in healthy breast tissue, but at much higher
concentrations, and with altered glycosylation in breast
carcinoma [1]. CA 15-3 is determined by immunoassay.
Different assay designs are used, but there are two basic
approaches [2]. Sandwich-type assays utilize two
antibodies, usually 115D8 as the capture antibody and
DF3 as the signal antibody. 115D8 binds to the
carbohydrate portion of the molecule. DF3 binds to an
amino-acid sequence near the C terminus (sequence
DTRPAPGS)[2]. The Siemens IMMULITE, however,
uses Ma 695 as the capture antibody and Ma 552 as the
signal antibody. The competitive-type assays use a
single antibody. ADVIA Centaur, for example, uses a
purified antigen attached to a paramagnetic particle as a
capture method and the B27.29 as the signal antibody.
B27.29 binds to the amino-acid sequence SAPDTRPA,
thus distinguishing it from DF3. Assay designs include
enzyme-linked immunosorbent assay (ELISA) [3],
microparticle enzyme immunoassay (MEIA)[4,5], and
chemiluminescent microparticle immunoassay [4],
among others.
Reference and Preferred Methods
There is no reference method for CA 15-3. Centocor,
Inc. developed the first radioimmunoassay, and it has
served as the comparison standard in a number of studies
[7]. The assay is still available through Fujirebio
Diagnostics [8]. However, this method is no longer the
preferred method. The preferred methods are
immunoassays with various optical detection systems.
They include those mentioned above and listed in Table
1. As an example, the assay on the Abbott Laboratories
ARCHITECT instrument is discussed in detail at the end
of this methods chapter [6].
The performance of many of the above methods have
been evaluated and compared [9]. Though the methods
were largely comparable, significant deviations among
methods for some samples were noted. The authors
recommended redetermination of the baseline when
changing methods.
i
CA 15-3
New method:
Fifth edition: Gregory A. Hobbs
Specimen
Serum (collected in SST tubes) or plasma (collected in
EDTA or heparinized tubes) are the only acceptable
specimens.
Interferences
Patients who have received mouse monoclonal
antibodies for diagnosis or therapy may have either
falsely elevated or falsely depressed values for CA 15-3.
Likewise, patients who have been routinely exposed to
animals or animal serum products can develop
heterophilic antibodies which can interfere with reagent
immunoglobulins.
Reference Interval
Among healthy, female study subjects (N=396), 99%
had assay values of < 31.3 U/mL [6].
Interpretation
CA 15-3 is most commonly used to assess posttreatment
progress in breast cancer patients. It is not intended to be
used as a screening tool. Some workers claim that CA
15-3 concentrations have prognostic value [10,11].
CA 15-3 Performance Goals
When validating CA 15-3 assay, method performance
generally should meet or exceed claims of the
manufacturer. In particular, total imprecision (day to
day) should be evaluated and physicians informed of
what constitutes a significant deviation from previous
values (>2SD above a mean). If validating an assay to
replace an existing assay, a correlation with the existing
method versus the new method, using patient samples,
should be performed. A correlation coefficient r > 0.90
can be expected, though slopes may vary with method
[2]. Differences in slope, indicating possible bias, must
be communicated to clinicians who are following
patients long term. Bias must be taken into account in
the evaluation of such patients with respect to what
constitutes a significant shift in CA 15-3 values.
References
1 Duffy MJ, Shering F, Sherry F, McDermott E,
OHiggins N. CA 15-3: a prognostic marker in
breast cancer. Int J Biol Markers 2000;15:330-
3.
2 Klee GG, Schreiber WE. MUC1 gene-derived
glycoprotein assays for monitoring breast
318
2

cancer (CA 15-3, CA 27.29): are they

measuring the same antigen? Arch Pathol Lab
Med 2004;128:1131-5. Procedure [6]
3 MP Biomedicals. Breast Cancer Antigen CA
15-3 Enzyme Immunoassay Test Kit. MP Principle
Biomedicals, Orangeburg, NY 10962. The ARCHITECT CA 15-3 assay is a two-step
4 Abbott Laboratories Diagnostics Division. IMx immunoassay. In the first step, the sample, wash buffer,
System CA 15-3. May 2007. Abbott and paramagnetic microparticles coated with the
Laboratories, Abbott Park, IL 60064. monoclonal antibody 115D8 are combined. CA 15-3
5 Abbott Laboratories Diagnostic Division. present in the sample binds to the 115D8-coated
Abbott AxSYM System Tumor Markers CS 15- microparticles. After washing, anti-CA 15-3 antibody
3. January 2007. Abbott Laboratories, Abbott conjugated to acridinium is added. Pre-trigger (1.32%
Park, IL 60064. H2O2) and trigger (0.35 N NaOH) solutions are then
6 Abbott Laboratories Diagnostic Division. added. The resulting chemiluminescent reaction is
ARCHITECT System CA 15-3 September measured in relative light units (RLU). There is a direct
2004. Abbott Laboratories, Abbott Park, IL relationship between the amount of CA 15-3 in the
60064. sample and the RLU detected by the optical system.
7 Chell CD, Morris DL, Kish L, Goldblatt J,
Neaman I, Allard JW et al. Mulitcenter Materials Provided
evaluation of Bayer Immuno-I CA 15-3 assay. 2K44 ARCHITECT CA 15-3 reagent kit
Clin Chem 1998;44:765-72.
8 Fujirebio Diagnostics, Inc. CA 15-3 Materials Required But Not Provided
Radioimmunoassay. October 2002. Fujirebio ARCHITECT i System, 3K50 ARCHITECT assay CD-
Diagnostics, Malvern, PA 19365. ROM, CA 15-3 controls, CA 15-3 calibrators, multi-
9 Slev PR, Rawlins ML, Roberts WL. assay manual diluent, pre-trigger solution, trigger
Performance characteristics of seven automated solution, wash buffer, reaction vessels, sample cups,
CA 15-3 assays. Am J Clin Pathol septum, replacement caps, pipettes or pipette tips.
2006;125:752-7.
10 Park BW, Oh JW, Kim JH, Kim KS, Kim JH, Test Procedure
Lee KS. Perioperative CA 15-3 and CEA serum 1. Invert the microparticle bottle 30 times before
levels as predictor for breast cancer outcomes. loading reagent on ARCHITECT. Visually
Ann Oncol 2008;19:675-81. inspect the bottle to make sure microparticles
11 Al-azawi D, Kelly G, Myers E, McDermott are resuspended. If not, continue to invert until
EW, Hill AD, Duffy MJ, Higgins NO. CA 15-3 there is complete resuspension of
is predictive of response and disease recurrence microparticles.
following treatment in locally advanced breast 2. Remove and discard the cap. Wearing clean
cancer. BMC Cancer 2006;6:220. gloves, remove a septum from the bag.
12 Harris L, Fritsche H, Mennel R, Norton L, Squeeze the septum in half to confirm that the
Ravdin P, Taube S et al. American Society of slits are open Carefully snap the septum onto
Clinical Oncology 2007 update of the top of the bottle.
recommendations for the use of tumor markers 3. Order tests.
in breast cancer. J Clin Oncol 2007;25:5287- 4. Load the ARCHITECT CA 15-3 reagent kit on
5312. the ARCHITECT i System. Verify that all
necessary assay reagents are present. Ensure
that septa are present on all reagent bottles.
Table 1: Methods for the Measurement of CA 15-3 5. If using primary or aliquot tubes, use the
Ref. sample gauge to ensure sufficient patient
Int. specimen is present.
Vendor Instruments Principle (U/mL) 6. ARCHITECT CA 15-3 calibrators and controls
ADVIA competitive 035 should be mixed by gentle inversion prior to
Centaur immunometric 035 use.
Siemens IMMULITE 7. When calibrating, hold the bottles vertically,
and dispense 7 drops of each calibrator
Roche Elecsys immunometric 034 solution.
IMx immunometric 031.3 8. When adding controls, hold the bottles
Abbott AxSYM immunometric 031.3 vertically, and dispense 4 drops of each control.
ARCHITECT immunometric 031.3 9. Load samples.
Ortho Vitros ECi immunometric 035 10. Press RUN. The ARCHITECT performs the
following functions:
MP ELISA Plate
Moves the sample to the aspiration point
Biochemicals reader immunometric 035
Loads the reaction vessel (RV) into the
Fujirebio immunometric 0 5 process path
Aspirates and loads the sample into the RV
319
3

Advances the reaction vessel one position
Aspirates wash buffer and microparticles and
transfers them into the RV
Mixes, incubates, and washes the reaction
mixture
Adds conjugate to the RV
Mixes, incubates, and washes the reaction
mixture
Adds pre-trigger and trigger solutions
Measures chemiluminescent emission to
determine the quantity of CA 15-3
Aspirates contents of RV to liquid waste and
unloads RV to solid waste
Calculates the result
11. Calibration
Calibrators A, B, C, D, E, and F are tested in
duplicate. No further calibration is necessary unless
controls are out of range or a reagent kit with a new lot
number is used.
12. Quality controls
Quality control should be performed once
every 24 hours or according to laboratory procedure, if
that is more frequent.
13. Calculation
The ARCHITECT CA 15-3 assay utilizes a
four-parameter logistic curve fit data-reduction method
to generate a calibration curve.
Assay Range
The calibration range for the CA 15-3 assay on the
ARCHITECT is 0-800 U/mL. Specimens exceeding 800
U/mL are flagged with the code >800.0 and may be
diluted using either the Automated Dilution Protocol or
the Manual Dilution Procedure.
1. Automated Dilution Protocol
The system performs a 1:5 dilution of the
specimen and automatically calculates the concentration
of the sample before dilution and reports the result.
2. Manual Dilution Procedure
The suggested dilution for the ARCHITECT
CA 15-3 assay is 1:5.
For a 1:5 dilution, add 100 L patient
specimen to 400 L ARCHITECT I Multi-
Assay Manual Diluent. The operator must enter the
dilution factor in either the patient or control order
screen. All assays selected for that order will be diluted.
The dilution factor will be used by the system to
automatically calculate the concentration of the sample
before dilution. The diluted result should read greater
than 30 U/mL.
Interpretation
When monitoring breast cancer patients, changes
observed in serial CA 15-3 assay values should be
evaluated in conjunction with other clinical methods
used for monitoring breast cancer. The American
Society for Clinical Oncology has recommended the
following [12]:

CA 15-3 is not recommended for use as a screening test for breast cancer.
Screening
CA 15-3 is not recommended for diagnosing or staging of breast cancer
Diagnosis/Staging
CA 15-3 can be used in conjunction with diagnostic imaging, history, and physical exam
for monitoring patients with metastatic disease during active therapy.
Present data are insufficient to recommend the use of CA 15-3 alone for monitoring
response to treatment.
Monitoring response to
therapy When there is no readily measurable disease, increasing levels of CA 15-3 may be used to
indicate treatment failure.
Use caution while monitoring during the first 4 to 6 weeks of a new therapy to avoid
spurious early rises.

CA 15-3 is not recommended for monitoring patients for recurrence after primary breast
Surveillance cancer therapy.
320
Carbohydrate Antigen 19-9 (CA19-9)
Carbohydrate Antigen 19-9 (CA19-9)
Hassan M.E. Azzazy
Name: Carbohydrate antigen 19-9 (CA19-9)
Clinical significance: Refer to Chapter 53, Neoplasia, in 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Molecular mass: 200,000 to 1,000,000 D
Chemical class: Glycolipid
Principles of Analysis and Current Usagei
CA19-9 is a sialylated derivative of the Lewisa blood
group antigen (sialylated lacto-N-fucopentaose II
ganglioside; denoted Lexa) [1]. It is synthesized by
normal human pancreatic and biliary ductular cells, as
well as by gastric, colonic, and endometrial epithelia,
and exists in serum as a mucin.
CA19-9 was originally identified by a monoclonal
antibody produced from hybridomas obtained from a
mouse immunized with the human colon cancer cell line
SW 1116 [2]. In 1983, an immunoradiometric assay
(IRMA) for CA19-9 determination was developed which
used the anti-CA19-9 antibody as both the capture and
signaling antibody. The assays calibration curve covers
the range from 0 to 120 kU/L [3]. In 1991, a
chemiluminescent enzyme immunoassay was developed
to quantify several tumor markers including CA19-9 [4].
Currently, several automated CA19-9 assays are
available: ARCHITECT 12000 (Abbott Diagnostics),
ADVIA Centaur (Bayer Diagnostics), UniCel Dxl 800
(Beckman Coulter), IMMULITE 2000 (Diagnostics
Products), and Elecsys E170 (Roche Diagnostics).
ARCHITECT 12000 and ADVIA Centaur assays
employ the same CA19-9 antibodies.
Although measurement of CA19-9 is currently available
on several routine analytical systems, a recent study by
Passerini et al. concluded that the same analytical system
should be used during tumor follow-up to eliminate the
possibility of method-induced variation [5].
Reference and Preferred Methods
There is no reference method for CA19-9. Several
automated non-isotopic CA19-9 immunoassays are
currently available that have acceptable analytic
performance. There appears to be no reason to prefer one
assay over another, and selection is therefore based
ultimately on the type of the analyzer available in the
laboratory.
CA19-9 results of a given specimen vary when measured
with assays from different manufacturers, owing to
differences in methods, reagents, and antibodies used.
Efforts to harmonize assays, including the availability of
an international reference material, are needed.
i
CA19-9
New method
Fifth edition: Hassan M.E. Azzazy
Specimen
Different manufacturers may recommend either serum or
plasma for their individual assays. Serum samples can be
stored at 2C to 8C for 24 hours or frozen at 20C or
lower for long-term storage [6]. Plain and gel-containing
tubes may be used for measuring CA19-9 in sera stored
at 4C for no more than 24 hours from the time of blood
collection. Use of sera stored in tubes containing
thixotropic gel separator for > 24 hours is not
recommended [7].
Interferences
No chemical interferences or in-vivo effects have been
reported to influence CA19-9 measurements. However,
immunoassay results may be subject to interference from
heterophilic antibodies which are present in 30% to 40%
of patients and arise as a result of contacting the same
animal species used for production of diagnostic
antibodies. Human anti-mouse antibodies (HAMA) have
become a major source of interference since the use of
murine monoclonal antibodies to deliver
immunoscintigraphic or chemotherapeutic agents to
tumors. Immunoassays, especially single-step
immunometric ones, also suffer from hook effect that is
characterized by the generation of falsely low results in
samples containing super-high concentrations of the
analyte (beyond the upper limit of the assay).
Reference Intervals
The most commonly used cutoff value for CA19-9 is <
37 kU/L. CA19-9 levels were measured in 20,517
patients, including 90 with pancreatic cancer, 70 with
benign pancreatic disease, 152 with biliary cancer, 170
with benign biliary disease, and 20,035 asymptomatic
controls. In asymptomatic individuals, the mean CA19-9
level was 9.42 9.95 U/mL, whereas levels > 37 U/mL
were found optimal for discriminating pancreatic cancer
from benign pancreatic disease (sensitivity 77%,
specificity 87%) [8]. A recent study reported that CA19-
9 results of healthy volunteers were < 37 kU/L as
assayed using five automated CA19-9 immunoassays
[9].
Interpretation
CA19-9 is a marker of pancreatic and colorectal
carcinoma [10]. It is not a tumor-specific marker but a
tumor-associated marker. CA19-9 is elevated in all
forms of gastrointestinal cancers and other
adenocarcinomas. It is the most frequently elevated
marker in pancreatic cancer, but its concentration does
321
Carbohydrate Antigen 19-9 (CA19-9)
not correlate to tumor mass. It is useful for monitoring
pancreatic and colorectal cancers, and elevated levels
can indicate recurrence 1 to 7 months prior to detection
by radiographs or other clinical findings [6,11]. Elevated
levels have also been reported in acute and chronic
pancreatitis and cirrhosis.
CA19-9 is the most commonly used serum marker for
pancreatic cancer and has good utility for confirming
diagnosis of symptomatic patients [12,13]. CA19-9 has a
specificity range of 68% to 91% (median 82%) and
sensitivity of 70% to 90% (median 79%) [12,14].
The diagnostic usefulness of serum CA19-9 suffers from
a number of limitations. First, the sensitivity of CA19-9
is significantly low (55%) in pancreatic ductal
adenocarcinoma patients with small curative tumors (<3
cm) [11], and almost half of patients (48.4%) with small
pancreatic cancer do not show elevated CA19-9 levels
[15]. Second, CA19-9 antigen secretion depends on
Lewisa antigen status. The interpretation of CA19-9
levels may be difficult because ~ 7% of the population
are Lewis-negative and have no detectable levels of
CA19-9 regardless of the tumor burden. Third, CA19-9
is elevated in non-pancreatic gastrointestinal
malignancies and in a variety of benign diseases such as
biliary obstruction, cholecystitis, cholangitis, hepatic
cirrhosis, and acute and chronic pancreatitis. Based on
the previous limitations, CA19-9 has a limited role in the
diagnosis of pancreatic cancer and should be used to
complement accurate pancreatic imaging procedures
such as computed tomography (CT) and endoscopic
ultrasonography (EUS) [16,17].
CA19-9 measurement has a documented role in
monitoring gemcitabine chemotherapy for pancreatic
cancer. A decline of > 20% in CA19-9 levels (as
compared to baseline) after 8 weeks of therapy has been
shown to be a better indicator of response and survival
than CT imaging [18,19]. Serial CA19-9 measurements
have greater sensitivity and specificity for predicting
response than individual measurements. One study
showed that serial falls in CA19-9 had a sensitivity of
67% for predicting partial response, and serial elevations
had a sensitivity of 86% for predicting progressive
disease [20]. Another study concluded that kinetics of
CA19-9 serum concentration serve as an early indicator
of response to gemcitabine chemotherapy in patients
with inoperable pancreatic adenocarcinoma [18]. The
U.S. Food and Drug Administration (FDA) has approved
CA19-9 tests as aids in the management of patients with
confirmed pancreatic cancer and serial monitoring of
their response to therapy and disease progression.
However, tests should only be used in patients with
CA19-9 values above the cutoff at the time of diagnosis.
One study investigated the role of CA19-9
measurements in enhancing the effectiveness of staging
laparoscopy in patients with suspected pancreatic
malignancy [21]. The study included 159 patients who
had CT-predicted resectable disease and who had
undergone laparoscopic staging. The study determined
that laparoscopy could have been avoided in 40% of
patients by using a CA19-9 cutoff 150 kU/L, and the
use of serum CA19-9 levels would increase the
efficiency of laparoscopic staging in patients with
suspected pancreatic malignancy.
CA19-9 is basically ineffective for screening
asymptomatic populations [13]. In one study, C19-9
levels were measured in an asymptomatic Korean
population (n = 70,940). Only four cases of pancreatic
cancer were detected, along with 159 false positives,
yielding a positive predictive value of 0.9% [22].
Serum CA19-9 concentration provides an independent
prognostic value for overall patient survival. In a cohort
study of pancreatic cancer patients undergoing resection
(n = 347), the median survival time for patients with the
same tumor stage was significantly longer in patients
whose CA19-9 levels normalize after resection as
compared to those whose CA19-9 did not return to
normal levels [23]. In cases of colorectal cancer, C19-9
has a lower sensitivity than carcinoembryonic antigen
(CEA). However, a correlation between elevated
preoperative CA19-9 levels and adverse patient outcome
has been found. Based on the available data,
measurement of CA19-9 cannot be recommended for
patients with colorectal cancer, but it may provide some
independent prognostic information [24].
Performance Goals
For CA19-9, the within- and between-subject biological
variations were 16.0% and 102.0 %, respectively. The
desirable analytical quality specifications for total error,
imprecision, and bias, derived from biological variation,
were 39%, 8%, and 25.8%, respectively [25]. A recent
study investigated the effect of intraindividual biological
variation on C19-9 serial results. Using four blood
samples, each collected at a 2-week interval from each
healthy volunteer (n = 49; ages 18 to 60 years; 25 males
and 24 females), the calculated intraindividual and inte-
rindividual coefficients of variation (CVs) for CA19-9
(as measured by an immunoluminometric assay on the
ARCHITECT analyzer) were 27.2% and 62.2%,
respectively [26].
The recommendations of the European Group on Tumor
Markers and National Academy of Clinical
Biochemistry guidelines indicate that assays for tumor
markers, including CA19-9, should demonstrate
interassay variability under 10% and intraassay
variability less than 5% [27,28]. The current Clinical
Laboratory Improvement Amendments (CLIA)
performance goal for measurement of CA19-9 is for
laboratories to be within 3 standard deviations of the
peer-group mean. According to the 2007 College of
American Pathologists (CAP) Survey, CV for all CA19-
9 methods at mean values of 72 U/mL (SD 24.5) and
114.9 U/mL (SD 41.99) were 34% and 36.5%,
respectively. Current CA19-9 assays should be
standardized to reduce the large discrepancies commonly
observed among different methods.
References
1 Magnani JL, Nilsson B, Brockhaus M, Zopf D,
Steplewski Z, Koprowski H, Ginsburg V. A
322
Carbohydrate Antigen 19-9 (CA19-9)
monoclonal antibody-defined antigen associated
with gastrointestinal cancer is a ganglioside
containing sialylated lacto-N-fucopentaose II. J
Biol Chem 1982;257:14365-9.
2 Koprowski H, Steplewski Z, Mitchell K, Herlyn
M, Herlyn D, Fuhrer P. Colorectal carcinoma
antigens detected by hybridoma antibodies.
Somatic Cell Genet 1979;5:957-71.
3 Del Villano BC, Brennan S, Brock P, Bucher C,
Liu V, McClure M et al. Radioimmunometric
assay for a monoclonal antibody-defined tumor
marker, CA19-9. Clin Chem 1983;29:549-52.
4 Nishizono I, Iida S, Suzuki N, Kawada H,
Murakami H, Ashihara Y, Okada M. Rapid and
sensitive chemiluminescent enzyme
immunoassay for measuring tumor markers.
Clin Chem 1991;37:1639-44.
5 Passerini R, Riggio D, Salvatici M, Zorzino L,
Radice D, Sandri MT. Interchangeability of
measurements of CA19-9 in serum with four
frequently used assays: an update. Clin Chem
Lab Med 2007;45:928-9.
6 Wu AHB, ed. Tietz Clinical Guide to
Laboratory Tests. 4th ed. Philadelphia:
Saunders; 2006.
7 Banfi G, Parma P, Pontillo M. Stability of
tumor markers CA19.9, CA 125, and CA 15.3
in serum obtained from plain tubes and tubes
containing thixotropic gel separator. Clin Chem
1997;43:2430-2431.
8 Kim HJ, Kim MH, Myung SJ, Lim BC, Park
ET, Yoo KS et al. A new strategy for the
application of CA19-9 in the differentiation of
pancreaticobiliary cancer: analysis using a
receiver operating characteristic curve. Am J
Gastroenterol 1999;94:1941-6.
9 Laulu SL, Roberts WL. Performance
characteristics of five automated CA19-9
assays. Am J Clin Pathol 2007;127:436-40.
10 Lamrez R. CA19-9: GICA (gastrointestinal
cancer antigen). In: Sell S, ed. Serological
Cancer Markers. Totowa, NJ: Humana Press;
1992:309-339.
11 Steinberg W. The clinical utility of the CA19-9
tumor-associated antigen. Am J Gastroenterol
1990;85:350-5.
12 Okusaka T, Yamada T, Maekawa M. Serum
tumor markers for pancreatic cancer: the dawn
of new era? J Pancreas 2006;7:332-6.
13 Freelove R, Walling AD. Pancreatic cancer:
diagnosis and management. Am Fam Physician
2006;73:485-92.
14 Goonetilleke KS, Siriwardena AK. Systematic
review of carbohydrate antigen (CA19-9) as a
biochemical marker in the diagnosis of
pancreatic cancer. Eur J Surg Oncol
2007;33:266-70.
15 Egawa S, Takeda K, Fukuyama S, Motoi F,
Sunamura M, Matsuno S. Clinicopathological
aspects of small pancreatic cancer. Pancreas
2004;28:235-40.
16 European Group on Tumor Markers. Tumor
markers in gastrointestinal cancers: EGTM
recommendations. Anticancer Res
1999;19:2811-5.
17 DiMagno EP, Reber HA, Tempero MA.
American Gastroenterological Association.
AGA technical review on the epidemiology,
diagnosis, and treatment of pancreatic ductal
adenocarcinoma. Gastroenterology
1999;117:1464-84.
18 Ziske C, Schlie C, Gorschluter M, Glasmacher
A, Mey U, Strehl J et al. Prognostic value of
CA19-9 levels in patients with inoperable
adenocarcinoma of the pancreas treated with
gemcitabine. Br J Cancer 2003;89:1413-7.
19 Halm U, Schumann T, Schiefke I, Witzigmann
H, Mossner J, Keim V. Decrease of CA19-9
during chemotherapy with gemcitabine predicts
survival time in patients with advanced
pancreatic cancer. Br J Cancer 2000;82:1013-6.
20 Gogas H, Lofts FJ, Evans TR, Daryanani S,
Mansi JL. Are serial measurements of CA19-9
useful in predicting response to chemotherapy
in patients with inoperable adenocarcinoma of
the pancreas? Br J Cancer 1998;77:325-8.
21 Connor S, Bosonnet L, Alexakis N, Raraty M,
Ghaneh P, Sutton R, Neoptolemos JP. Serum
CA19-9 measurement increases the
effectiveness of staging laparoscopy in patients
with suspected pancreatic malignancy. Dig Surg
2005;22:80-5.
22 Kim JE, Lee KT, Lee JK, Paik SW, Rhee JC,
Choi KW. Clinical usefulness of carbohydrate
antigen 19-9 as a screening test for pancreatic
cancer in an asymptomatic population. J
Gastroenterol Hepatol 2004;19:182-6.
23 Safi F, Schlosser W, Falkenreck S, Beger HG.
CA19-9 serum course and prognosis of pan-
creatic cancer. Int J Pancreatol 1996;20:155-61.
24 Duffy MJ, van Dalen A, Haglund C, Hansson
L, Klapdor R, Lamerz R et al. Clinical utility of
biochemical markers in colorectal cancer:
European Group on Tumour Markers (EGTM)
guidelines. Eur J Cancer 2003;39:718-27.
25 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biological variation: pros, cons
and progress. Scand J Clin Lab Invest
1999;59:491-500.
26 Erden G, Barazi AO, Tezcan G, Yildirimkaya
MM. Biological variation and reference change
values of CA19-9, CEA, AFP in serum of
healthy individuals. Scand J Clin Lab Invest
2008;68:212-8.
27 European Group on Tumor Markers website.
<http://egtm.eu/1280.html>
28 National Academy of Clinical Biochemistry:
Practice Guidelines and Recommendations for
Use of Tumor Markers in the Clinic, 2005.
Available at
http://www.aacc.org/SiteCollectionDocuments/
NACB/LMPG/tumor/chp2_qreqs.pdf
323

Carbon Dioxide and Bicarbonate

Carbon Dioxide and Bicarbonate

William J. Korzun
Name: CO2, total CO2 content
Clinical significance: Refer to Chapter 29, Acid-Base Control and Acid-Base Disorders, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: CO2 or O=C=O
Molecular mass: 44.01 D
Merck Index: 1793
Chemical class: Gas, organic acid in soluble phase

i titrated with a solution of NaOH [2] (Table 1, Method

Principles of Analysis and Current Usage
2).
Total carbon dioxide in serum or plasma exists in three
major chemical forms: dissolved CO2 (3%), carbamino According to the 2007 College of American Pathologists
(CAP) Participant Summary, approximately 64% of
derivatives of plasma protein (33%), and bicarbonate
participating laboratories measure total carbon dioxide
(HCO3) anion (64%) [1]. Other quantitatively minor
by an enzymatic/spectrophotometric assay. This method
forms are carbonic acid (H2CO3) and carbonate ions
has been adapted to many automated analyzers. All CO2
(CO3). Several procedures that quantitate total carbon
forms are quantitatively converted to HCO3 by adding
dioxide content in serum or plasma involve acidification
alkali to the serum. The bicarbonate is then
of the sample to convert all carbon dioxide forms to CO2
enzymatically converted to oxaloacetic acid, which is
gas and measurement of the amount of gas formed. A measured by an NADH-consumption reaction that is
second category of procedures utilizes the enzymatic quantitated spectrophotometrically as follows (Table 1,
reaction of bicarbonate with phosphoenolpyruvate Method 4):
carboxylase coupled to a spectrophotometric indicator
reaction. Most assays which specifically measure
bicarbonate have reagents at an alkaline pH. This HCO3 + phosphoenolpyruvate PEPC
converts other forms of CO2 to bicarbonate, so these oxaloacetate + Pi
assays are also measuring total CO2.
Oxaloacetate + NADH + H+ MDH malate + NAD+
The historical reference method used the Natelson
Microgasometer to manometrically measure CO2 gas PEPC is phosphoenolpyruvate carboxylase, Pi is
liberated after acidification of serum. The CO2 gas was inorganic phosphate, and MDH is malate
then absorbed by alkali, and the residual pressure due to dehydrogenase. The decrease in absorption at 340 nm is
gases other than CO2 was measured. The difference is related to the serum concentration of CO2.
the specific CO2 content of the serum sample (Table 1,
Approximately 36% of laboratories participating in CAP
Method 1). An alternative reference method has been proficiency surveys employ a method that uses a PCO2
described by the IFCC Scientific Division Working electrode to quantitate the CO2 gas produced by
Group on Selective Electrodes, utilizing a semi-
acidification of the sample. Here again, the CO2 gas
automated extraction/titration device. Equal volumes of
specimen and 0.1 mol/L lactic acid are mixed in a diffuses across a silicon-rubber membrane and changes
temperature-controlled extraction vessel. The CO2 the pH of a bicarbonate electrode buffer. The pH change
is detected by a glass pH electrode within the PCO2
liberated from the specimen is then swept by a stream of
electrode assembly (Table 1, Method 3).
CO2-free air into a titration vessel containing a solution
of BaCl2 and a pH electrode. The CO2 reacts with Ba2+
In another method, total CO2 is calculated during a
and H2O to form BaCO3, releasing 2 moles of H+ for
each mole of CO2. The H+ ions are volumetrically blood-gas analysis (Table 1, Method 5). Using the
measured pH and PCO2 values, one can estimate the total
CO2 using the following equation:
i
Carbon Dioxide and Bicarbonate
Previous and current authors of this method:
Total CO2 (mEq/L) = PCO2 + [HCO3]
First edition: William J. Korzun, W. Gregory Miller
Methods edition: William J. Korzun, W. Gregory Miller
Second edition: William J. Korzun, W. Gregory Miller where (0.0301), the Bunson coefficient, is the solubility
Third edition: William J. Korzun, W. Gregory Miller coefficient of CO2, and the bicarbonate concentration
Fourth edition: William J. Korzun, W. Gregory Miller
Fifth edition: William J. Korzun
324
Carbon Dioxide and Bicarbonate
can be estimated from the Henderson-Hasselbalch
equation:
[HCO3] = PCO2 (antilog [pH pKa]) Cord blood [6]
where pKa = 6.10. Although this calculation can provide
only an indirect, unmeasured estimate of total CO2, it
correlates well with measured total CO2 values.
Reference and Preferred Methods
Although there are no reference methods for the
determination of bicarbonate or CO2 concentration, all of
the common automated CO2/bicarbonate methods
produce accurate and precise results when calibrated and
used according to the manufacturers instructions.
Typical within-lab precision for automated analyzers
using acidification/pH electrode or enzymatic methods is
2% to 4% coefficient of variation (CV). Methods which
can be calibrated using primary aqueous standards of
sodium carbonate are suitable as accuracy comparison
methods to validate performance of methods which use
serum-based secondary calibration materials.
Specimen
Either serum or heparinized plasma may be used. Other
anticoagulants cannot be used, because they disturb the
equilibrium between erythrocyte and plasma CO2. They
also lower the pH of the sample and accelerate the loss
of CO2 to ambient air. Whole blood cannot be used
because the heme-bound CO2 and carbamino-bound
CO2 vary with the hematocrit and oxygen saturation and
produce variable results. Samples collected in tubes
containing separator gel give the same results as tubes
without separator gel.
The major errors in total CO2 measurement are
associated with sample handling. Samples should ideally
be handled anaerobically, centrifuged at 37C, and
stored tightly stoppered before analysis. Once separated
from erythrocytes and kept stoppered, serum or plasma
total CO2 content is stable for several days at 4C.
Centrifugation at room temperature produces a decrease
in CO2 of approximately 0.5 mmol/L [3]. This is the
result of the shift of CO2 into the erythrocyte as the pH
decreases as the temperature of the blood drops from
37C to 25C. Loss of CO2 from uncapped tubes is
approximately 4 mmol/L after 1 hour [4]. CO2 loss can
be prevented by raising the pH of the sample to a range
of 9.0 to 9.3 by addition of NH4OH. Layering with oil
should not be performed because of the high solubility of
CO2 in oil.
Interferences
In general, most of the enzymatic total CO2 assays are
not affected by bilirubin concentrations up to 20 mg/dL
and hemoglobin up to 500 mg/dL [5]. The presence of
high triglyceride concentrations may cause light
scattering and cause a negative bias in some enzymatic
assays.
Carbon Dioxide Reference Interval
Arterial 1329 mmol/L
Venous 1528 mmol/L
Serum or plasma [7]
< 1 month 1727 mmol/L
16 months 1729 mmol/L
6 mo1 yr 1829 mmol/l
> 1 year 2131 mmol/L
Interpretation
Measurement of total serum CO2 is useful in
determining the acid-base status of a patient. The total
CO2 concentration is depressed in metabolic acidosis.
The degree of depression depends on the degree of initial
acidosis and subsequent compensatory mechanisms. In
respiratory acidosis, the total CO2 is mildly increased
because of an increased PCO2. However, if renal
compensation occurs, the bicarbonate levels rise, and
total CO2 also increases.
Patients with a respiratory alkalosis initially exhibit only
small decreases in total CO2. As renal compensation
occurs, the levels can become moderately depressed.
Metabolic alkalosis results in an increase in bicarbonate
levels and thus total CO2. The increase in total CO2 can
become much larger as a result of respiratory
compensation.
Although the physician can use total CO2 data to
diagnose and monitor a patients acid-base status, in
acute cases, this information is much less valuable than a
complete blood-gas analysis. Total CO2 analysis is
performed frequently because of historical rather than
clinical factors. Total CO2 was part of a battery of tests
available on the original Technicon SMA instruments.
Physicians are accustomed to having this test available,
and laboratories are used to providing it, though its
clinical utility is limited to occasional monitoring of
patients in renal failure or acute acidosis. The continued
availability of total CO2 as a component of routine
profiles may be questioned in the future.
Carbon Dioxide Performance Goals
Total CO2 content is not regulated under the Clinical
Laboratory Improvement Amendments of 1988 (CLIA
88). Carbon dioxide is difficult to include in
proficiency-testing material because of its volatility.
When included as part of a special diluent for freeze-
dried materials, there may be substantial variability
between labs due to loss of CO2 during handling of the
material.
Within-subject variation has been reported to be less
than 6% over periods greater than 1 month [8]. Precision
goals have been defined as a maximum standard
deviation of approximately 0.5 mmol/L at a decision
325

Carbon Dioxide and Bicarbonate

threshold of 20 mmol/L and approximately 0.7 at a

decision threshold of 30 mmol/L [9].

References
1 Davenport HW. The ABC of Acid-Base
Chemistry. 5th ed. Chicago: University of
Chicago Press; 1969:34.
2 Burnett RW, Covington AK, Fogh-Anderson N,
Kulpmann WR, Lewenstam A, Maas AHJ et al.
IFCC reference measurement procedure for
substance concentration determination of total
carbon dioxide in blood, plasma or serum. Clin
Chem Lab Med 2001;39:283-289.
3 Astrup P. A simple electrometric technique for
the determination of carbon dioxide tension in
blood and plasma, total content of carbon
dioxide in plasma, and bicarbonate content in
separated plasma at a fixed carbon dioxide
tension. Scand J Clin Lab Invest 1956;8:33-43.
4 Gambino SR, Schreiber H. The measurement of
CO2 content with the autoanalyzer. Am J Clin
Pathol 1966;45:406-411.
5 Young DS. Effects of Preanalytical Variables
on Clinical Laboratory Tests. 2nd ed.
Washington, DC: AACC press; 1997:3-793-
82.
6 Perkins SL, Livesey JF, Belcher J. Reference
intervals for 21 clinical chemistry analytes in
arterial and venous umbilical cord blood. Clin
Chem 1993;39:1041-1044.
7 Greeley C, Snell J, Colaco A, Beatey J, Bailey
J, Bjorn S et al. Pediatric reference ranges for
electrolytes and creatinine. Clin Chem
1993;39:1172.
8 Morris HA, Wishart JM, Horowitz M, Need
AG, Nordin BE. The reproducibility of bone-
related biochemical variables in post
menopausal women. Ann Clin Biochem
1990;27:562-568.
9 Fraser CG. Biological variation in clinical
chemistry. An update: collated data, 1988-1991.
Arch Path Lab Med 1992;116:916-923.
326

Carbon Dioxide and Bicarbonate

Tables

Table 1: Carbon Dioxide Methods

Method 1: Gas release; manometric or thermometric
Principle of Analysis: O2 is released from samples after addition of acid, and the gas pressure is measured
manometrically or by thermal conductivity.
Comments: Rarely used; the reference method, used for calibration purposes
Method 2: Gas release; titrimetric
Principle of Analysis: CO2 + acid CO2 gas
+ H2O + Ba+2 BaCO3 + 2 H+
CO2 gas
2 H2 O
2 H+ + 2 OH-
Comments: Rarely used; potential reference method
Method 3: CO2 electrode; ion-selective (pH) electrode
Principle of Analysis: After acidification of sample, CO2 diffuses across a silicone membrane into dilute
bicarbonate buffer. The resulting pH change, as measured by a pH electrode, is related to total CO2 content.
Comments: Common; same method by which PCO2 is measured in blood-gas analyzers
Method 4: Enzymatic; spectrophotometric
_
Principle of Analysis: HCO + PEP PEPC OX + P
3 i

OX + NADH + H+ MDHMAL + NAD+ ( A, 340 nm

Comments: Common; used on discrete analyzers such as Du Pont aca and Kodak Ektachem
Method 5: Calculation; ion-selective (pH and PCO2) electrode
Principle of Analysis: Total CO2 calculated from data obtained in blood-gas analysis; total CO2 = PCO2 +
[HCO3]
Comments: Common; although only an estimate, compares well with measured values

MAL, Malate; MDH, malate dehydrogenase; NADH, dihydronicotinamide adenine dinucleotide; OX, oxaloacetate; Pi ,
phosphate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase.

Figures

Carbon Dioxide: Figure 1

Schema of PCO2 electrode assembly.

327
Carcinoembryonic Antigens (CEA)
Carcinoembryonic Antigens (CEA)
Gregory A. Hobbs
Name: Carcinoembryonic antigens (CEA)
Clinical significance Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular mass: Colon CEA, 160,000 to 180,000 D
Ovarian CEA, 200,000 D
Breast CEA, 90,000 to 105,000 D
Chemical class: Glycoprotein
i
Principles of Analysis and Current Usage
Cancer-specific antigens of the digestive tract were
first discovered by Gold and Freedman in 1965 [1-
2]. They derive their name from the discovery that
they occur in the fetal gut as well as in neoplastic
tissues [2]. The first CEA-reacting molecule that
was extensively characterized by this group was
obtained from an ovarian carcinoma [3]. Later
reports indicated that colon CEA showed a lower
molecular mass range (160,000 to 180,000) than
the ovarian-derived molecule [4].
Although the collective term for these antigens,
CEA, has been used since their discovery, it has
long been known that CEA is a heterogeneous
group of molecules [5]. This is why not all CEA-
containing sera from cancer patients produce linear
dilution curves in parallel with CEA standard
curves [6], nor will the results obtained from one
CEA-ligand assay be identical with another.
Two similar high-molecular-mass glyco-proteins
have been examined extensively. CA 125 was
shown to better distinguish progression, regression,
or stabilization in patients with epithelial ovarian
cancer than CEA, suggesting a significant antigenic
difference between the two markers [7]. This was
shown directly with in-vitro interference studies in
which anti-CA 125 did not bind CEA above
background [8]. CA 19-9, also of interest in
colorectal cancer, has been purified and studied in
detail [9]. It was shown to have a different amino
acid and carbohydrate composition from CEA,
though its molecular mass was close to 200,000,
depending on the method used to measure it.
i By the early 1980s, RIA began to be displaced as
Carcino-embryonic antigen (CEA)
Previous and current authors of this method:
First edition: Not done
Methods book: Allyn H. Rule
Second edition: Allyn H. Rule
Third edition: Gregory A. Hobbs
Fourth edition: Gregory A. Hobbs
Fifth edition: Gregory A. Hobbs
In 1969, a radioimmunoassay (RIA) was developed
to measure perchloric acid (PCA)-extracted CEA in
sera obtained from patients with adenocarcinoma
of the colon [10]. RIA used to investigate CEA-
reacting molecules soon showed that many such
molecules could be identified from fetal gut
extracts obtained at different stages in utero. RIA
of saline extracts from patients with 20 primary
adenocarcinomas of the colon showed 8 to 12 CEA
varieties by isoelectric focusing [11]. It is now
known that three of these bands are cancer specific,
and four belong to a family of normal colon cross-
reacting antigens (NCA) which may also be
produced in excess during carcinogenesis. Other
CEA-reacting molecules are bound to serum
lipoproteins, membranes, and immune complexes.
A number of assays have used PCA or heat or both
to extract serum glycoproteins, including CEA, into
the fluid phase while precipitating 90% to 98% of
the other serum proteins (globulins and albumins)
[12,13]. Both methods inactivate proteolytic
enzymes, cause antigenic loss of different CEA
molecules, and remove proteins that would
otherwise cause nonspecific protein binding.
Measurement of CEA using radioimmunoassay
utilizes a monoclonal antibody complexed with a
plastic bead that reacts with the patient sample,
binding CEA. Simultaneously, a second
monoclonal antibody, specific for a different site
from the first, binds the CEA-Ab-bead complex.
The second antibody is labeled with 125I. After a
washing step, the bound counts are determined in a
gamma counter. The bound counts are proportional
to the plasma concentration of CEA, based on a
standard curve.
the method of choice by enzyme immunoassay
(EIA). In this method, an anti-CEA antibody is
irreversibly bound to a solid phase. These bound
antibodies selectively remove CEA from the fluid
phase. However, other serum proteins will also
attach to the positively charged immunoglobulins
or the negatively charged glycoproteins because of
nonspecific charges or hydrophobic interactions.
328
Carcinoembryonic Antigens (CEA)
These nonspecific proteins must be removed by a
series of washes before addition of a second anti-
CEA antibody, to which is attached a label. The
second antibody binds to additional antigenic
determinants on CEA to form a sandwich.
Nonspecific proteins are again removed by several
washes. In some assays, both CEA and the labeled
antibody can be added simultaneously in the first
step before washing. At this point, reactions can be
measured for color development. The color results
from the addition of a dye substrate in a second or
third step before spectrophotometric determination
of color. The color developed is directly
proportional to CEA content. The methods differ
only in the enzyme employed, with the former
using alkaline phosphatase with p-nitrophenyl
phosphate as a substrate and the latter horseradish
peroxidase with o-phenylenediamine as a substrate.
EIA was an improvement over RIA in a number of
ways. The newer method replaced the time-
consuming PCA extraction with heat [14], thus
eliminating the problems associated with removing
perchloric acid. A further improvement was the
introduction of monoclonal antibodies against CEA
[15], which dramatically improved the specificity
of the assay, eliminating the need for an extraction
step (current RIA also uses a monoclonal
antibody). The monoclonal EIA was particularly
less susceptible than (polyclonal) RIA to false
positives (non-malignancies) due to liver disease.
Conversely, an assay employing a combination of
polyclonal and monoclonal antibodies against CEA
was unable to improve discrimination between
malignant and nonmalignant causes of elevated
CEA [16]. Moreover, analytical problems such as
the hook effect have not disappeared with the
advent of monoclonal antibodies [15,17,18].
Currently, the most popular method for the
determination of CEA is an automated
modification of the two-step, monoclonal EIA
method. In this approach, the trapping antibody is
attached to a microparticle, which is retained on a
glass-fiber filter in the washing steps. Detection is
not by color but by the production of a fluorescent
product, which further improves the precision of
the assay at low concentration. Because it is
automated, such run-to-run factors as incubation
time and temperature and washing steps are more
consistent. This has resulted in improved analytical
performance [19]. This method is referred to as
microparticle enzyme immunoassay (MEIA). A
digest of the above methods, with their underlying
principles is presented in Table 1.
Methods using chemiluminescence as a means of
detection, either in an enzyme immunoassay or in a
direct immunometric assay [20,21], have also been
developed. This EIA approach differs from the
above EIA in that it uses as a substrate a phosphate
ester of adamantyl dioxetane which undergoes
hydrolysis in the presence of the sandwich-
associated alkaline phosphatase to produce an
unstable intermediate. This intermediate degrades
with the concomitant production of light, detected
by a luminometer. This continuous production of
light allows for multiple readings, which
potentially improves precision. The direct
immunometric method differs in that (1) the
trapping antibody is bound to a magnetic particle.
The bound CEA and complete sandwiches are
separated from unbound material by physical
separation of the particles in a magnetic field, and
(2) the second antibody is polyclonal and tagged
with acridinium. Thus no enzyme reaction is
required.
Reference and Preferred Methods
There is no reference method for CEA.
Since cancer patients may be monitored over long
periods of time, considerable thought must be
given before a laboratory changes to an alternative
kit or method. Because of the heterogeneity of
CEA, it is doubtful that all kits measure the same
entity, so the laboratory must work closely with
physicians ordering the test, justifying the change
with improved performance and/or reduced cost.
Careful correlations should be run between the
current method and the proposed one, and patient
samples should be measured and reported with both
methods for a mutually agreeable period of time.
The 2007 College of American Pathologists (CAP)
ligand general-participant summary report showed
that the chemiluminescence assays are the most
popular technique for measurement of CEA. These
techniques accounted for approximately 90% of all
assays. As was suggested above, there are
appreciable differences among methods as regards
accuracy (mean) and precision (coefficient of
variation, or CV). Differences in mean
measurements probably reflect antibody
differences. This is likely because although
antibodies differ among manufacturers, the
calibrators are all traceable, directly or indirectly,
to the original Gold and Freedman isolate of CEA.
However, this is not necessarily true of CAP survey
material. This material may possess a different set
of epitopes which will vary in affinity for different
antibodies. Thus the CEA measurements in a given
sample may differ considerably among methods
employing antibodies of various specificities.
Specimen
At one time, EDTA plasma was the preferred
sample for CEA analysis because of interference
from plasmin, a calcium-dependent proteolytic
enzyme. However, more recent methods can
329
Carcinoembryonic Antigens (CEA)
measure CEA with equal accuracy in plasma or
serum. For consistency of results, it is preferred
that the same matrix be used for the one patient.
CEA is stable for up to 1 week when stored at 4C
[22].
Interferences
Specimens with moderate hemolysis or lipemia or
bilirubin concentrations up to 30 mg/dL typically
do not interfere with most CEA methods. The
presence of human anti-mouse antibodies may
result in falsely increased or decreased values.
Freeze/thaw cycles have been found to not affect
CEA concentrations [23].
CEA Reference Interval
A value of 2.5 ng/mL has been used to distinguish
healthy patients from diseased [24]. Commonly,
the reference interval for smokers is 0 to 5 ng/mL,
though this may vary considerably from study to
study [17,25]. Sensitivity, specificity, and
predictive value in selected studies shows various
diagnostic sensitivities, specificities, and predictive
values that have been published [26]. It is clear that
selection of method and cutoff concentrations
profoundly influence the diagnostic power of CEA
immunoassay.
Interpretation
Unfortunately, CEA has proven to not be specific
for colon cancer in numerous clinical trials
[24,27,28] using CEA-RIA. CEA has found its
most important use in the detection of recurrence of
colorectal cancer after resection. One study [29]
showed a significant increase in 5-year survival
rates when resections were directed by elevated
CEA as opposed to other indicators. Others have
found CEA to be elevated before clinical diagnosis
of recurrence, especially if a critical value of 7.5
ng/mL was used [30]. A study comparing CEA
with computerized tomography (CT) reported that
CEA provided the first indication of recurrence in
78% of patients, and 100% of recurrences could be
detected with a combination of CEA, symptoms,
and physical examination [31]. They concluded
that CEA remains the most accurate indicator of
recurrence. The clinical usefulness of preoperative
CT scans may be enhanced if they are used only
after a CEA value of 6.1 ng/mL [32]. Though
monoclonal antibodies have improved the
specificity of CEA for carcinomas, one report [33]
has criticized the use of CEA in monitoring
patients with resected colon cancer. These workers
argue that monitoring patients with CEA (and
CEA-directed surgery) does not result in
significantly improved survival and thus does not
justify the large (overall) expense of CEA. This
would seem to be more an indictment of the
treatment for colorectal cancer, which is poor, than
for any means of detection. CEA is not thought to
be useful as a mass screening tool [34], though one
Chinese study recommends it for persons over the
age of 50 in their population [35]. Currently, the
most useful application of CEA is in the detection
of liver metastases from colorectal cancer [36].
The use of CEA ligand assays in monitoring the
therapy of other types of cancer is a more dubious
practice. Though CEA is effective at distinguishing
patients with various cancers from disease-free
controls, it is not selective for various tissues [37]
and shows poor diagnostic sensitivity for cancers
other than colon cancer [28]. Measurement of CEA
cannot be the sole factor used for the diagnosis of
disease; it must be used together with clinical
findings.
Carcinoembryonic Antigen Performance Goals
At present, CEA is not regulated for proficiency
testing. The current target for acceptable
performance is that laboratories be within 3
standard deviations of the peer-group mean. The
CV of most methods is less than 8% at a CEA
concentration of approximately 12 ng/mL. One
study found the intraindividual variation of CEA in
healthy individuals to be approximately 10% [38].
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tumor-specific antigens in human colonic
carcinomata by immunologic and
absorption techniques. J Exp Med
1965;121:439-462.
2 Gold P, Freedman SO. Specific
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1965;122:467-481.
3 Krupey J, Gold P, Freedman SO.
Physicochemical studies of the
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4 Krupey J, Wilson T, Freedman SO, Gold
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5 Rule AH, Ball HO, Rajarajan B, Diaz S.
An immunochemical comparison of colon,
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annual meeting of the International
Society for Oncodevelopmental Biology
and Medicine, Houston, Texas, Oct. 4,
1984.
6 Vrba R, Alpert E, Isselbacher KJ.
Immunological heterogeneity of serum
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7 Bast RC, Klug TL, St. John E, Jenison E,
Niloff JM, Lazarus H et al.
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8 Bast RC, Feeney M, Lazarus H, Nadler
LM, Colvin RB, Knapp RC. Reactivity of
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9). Cancer Res 1988;48:1505-1511.
10 Thompson DMP, Krupey J, Freedman SO,
Gold P. The radioimmunoassay of
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11 Rule AH, Kirch ME. Gene activation of
molecules with carcinoembryonic antigen
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12 Lo Gerfo P, Krupey J, Hansen JH.
Demonstration of an antigen common to
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14 Smith AM, Macdonald DJ. Enzyme
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15 Hedin A, Carlsson L, Berglund A,
Hammarstom S. A monoclonal antibody-
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16 Buchegger F, Phan M, Rivier D, Carrel S,
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17 Liu Y-S V, Tobias RJ, Zurawski VR. A
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18 Hobbs GA, Hess PP, Watson M, Valdes
R. A hook effect detected in a two-step
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19 Chen I-W, Barnes E, Sperling M.
Evaluation of a fully automated
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Masnyk IJ, Go VL, Aamodt R. Effect of
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1987:710-712
27 National Cancer Institute/American
Cancer Society. Joint investigation: a
collaborative study of a test for
carcinoembryonic antigen in the sera of
patients with carcinoma of the colon and
rectum. Can Med Assoc J 1972;107:25.
28 Laurence DJ, Stevens U, Bettelheim R,
Darcy D, Leese C, Turberville C et al.
Role of plasma carcinoembryonic antigen
in diagnosis of gastrointestinal, mammary,
and bronchial carcinoma. Br Med J
1972;3:605-9.
29 Minton JP, Hoehn JL, Gerber DM. Results
of a 400-patient carcinoembryonic antigen
second-look colorectal cancer study.
Cancer 1985;55:1284.
30 Carlsson U, Stewnius J, Ekelund G,
Leandoer L, Nosslin B. Is CEA analysis of
value in screening for recurrences after
331
Carcinoembryonic Antigens (CEA)
surgery for colorectal carcinoma? Dis
Colon Rectum 1983;26:369-73.
31 Sardi A, Agnone CM, Nieroda CA,
Mojzisik C, Hinkle G, Ferrara P et al.
Radioimmunoguided surgery in recurrent
colorectal cancer: the role of
carcinoembryonic antigen, computerized
tomography, and physical examination.
South Med J 1989;82:1235-44.
32 Holt AD, Kim JT, Murrell Z, Huynh R,
Stamos MJ, Kumar RR. The role of
carcinoembryonic antigen as a predictor of
the need for preoperative computed
tomography in colon cancer patients. Am
Surg 2006;72:897-901.
33 Moertel CG, Fleming TR, Macdonald JS,
Haller DG, Laurie JA, Tangen C. An
evaluation of the carcinoembryonic
antigen (CEA) test for monitoring patients
with resected colon cancer. JAMA
1993;270:943-7.
34 Tumor Marker Expert Panel. Clinical
practice guidelines for the use of tumor
markers in breast and colorectal cancer. J
Clin Oncol 1996;14:2843-2877.
35 Colorectal Cancer Cooperating Group.
Beijing Society of Gastroenterology.
Comparative analysis of patients with
clinically diagnosed colorectal cancer and
those detected by mass screening. Chin J
Dig Dis 2005;6:26-30.
36 Duffy MJ. Carcinoembryonic antigen as a
marker for colorectal cancer: is it
clinically useful? Clin Chem 2001;47:624-
630.
37 Chevinsky AH. CEA in tumors of other
than colorectal origin. Semin Surg Oncol
1991;7:162.
38 Sltormos G, Schiler V, Nielsen D,
Skovsgaard T, Dombernowsky P.
Interpretation of results for tumor markers
on the basis of analytical imprecision and
biological variation. Clin Chem
1993;39:2077-83.
39 Primus FJ, Freeman, JW, Goldenberg DM.
Immunological heterogeneity of
carcinoembryonic antigen: purification
from meconium of an antigen related to
carcinoembryonic antigen. Cancer Res
1983;43:679.
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Carcinoembryonic Antigens (CEA)

Table 1: Methods of Analysis for Carcinoembryonic Antigens (CEA)

Method 1: Indirect radioimmunoassay (RIA); competitive-binding assay
a. Acid extraction
b. Acid removal
1. Dialysis
2. Chromatography
3. Ultrafiltration
Principle of analysis:
a. Plasma is extracted to remove 95% of all other proteins and release CEA from
complexes. After centrifugation and removal of all acid, antibody first reacts with cold CEA for 30
minutes at 45C and then with 125I-CEA for 30 minutes at 45C. Heat speeds the reaction; sequential
addition gives better low-end sensitivity.
b.
1. Dialysis allows acid to diffuse totally out of cellophane pores after many changes of
water and a final buffer step.
2. CEA is separated from acid based on molecular mass differences; CEA comes out in
first fraction.
3. CEA is retained on top of filter or inside of bag while acid is removed. Serum CEA
sticks to filter.
Comments:
a. Historical; more CEA is available to measure. Assay is sensitive to pH, ions, and
nonspecific protein quench.
b.
1. Method is cheap but tedious and labor intensive.
2. Columns are moderately expensive and must be carefully washed, or they can
become contaminated.
3. Expensive equipment; CEA values are significantly lower.
Method 2: Indirect RIA (heat extraction); competitive binding
Principle of analysis: Heat (70C) causes most serum proteins to precipitate out; after centrifugation,
assay performed on supernatant as in Method 1.
Comments: Historical; complex CEA is not released to supernatant.
Method 3: Direct RIA assay; competitive binding
Principle of analysis: Plasma is directly placed into distilled water with antibody, and assay proceeds
similarly to that in Method 1.
Comments: Historical; used to monitor patients with CEA 20 ng/mL, owing to lack of sensitivity in
low range; sensitive to ion, pH, and protein effects.
Method 4: Solid-phase extraction; sandwich immunoassay
a. One-step assay
b. Two-step assay
Principle of analysis: Antigen binds to a monoclonal anti-CEA fixed on a bead, microparticle, or
solid surface. A second anti-CEA labeled with 125I (RIA) or conjugated with an enzyme (EIA).
a. Trapping antibody, sample, and signal antibody together at the same time. One wash step
removes unbound conjugate.
b. Two wash steps; first to remove unbound material from solid phase, then a second wash to
remove unbound conjugated anti-CEA.
Comments: Plasma or serum; most widely used. Extraction step eliminated. Highly sensitive but still
susceptible to interferences. EIA incubation time consuming.
a. Timing of incubation important in EIA
b. First wash reduces potential for nonspecific interference. Most systems semi-automated;
washes and incubations automated.
333
Carcinoembryonic Antigens (CEA)

Procedure: Direct Radioimmunoassay of CEA further absorption.

(This is a homebrew method described in the earlier Add 50 L of the plasma fraction of the A-positive
edition of Methods in Clinical Chemistry and is to be blood per milliliter of the above-treated antiserum,
used as an example only.) incubate overnight at 0C to 4C and for 30 minutes at
Principle 37C. Centrifuge at 29,000 g for 20 minutes at 0C to
This is a competitive binding assay using 125I- 4C. Save the supernatant for further absorption.
labeled CEA and a second antibody to separate
antibody-bound labeled CEA from unbound. The CEA Prepare a normal tissue absorbent by homogenizing
molecules are not extracted before analysis. fresh human liver, lung, spleen, and colon. Mince each
Reagents tissue, suspend in saline, and wash free of hemoglobin
1. Water. Use deionized distilled water (DW) or by repeated centrifugation. Homogenize the tissue with 2
distilled water that is both prefiltered and postfiltered. mL of saline for each gram of tissue. Centrifuge the
2. CEA-free plasma (NHP). Obtain plasma suspension at 29,000 g. The resulting pellet will appear
samples from healthy persons, assay, and pool those to be a greenish gel. Combine equal volumes of the
with less than 2 ng of CEA/mL. Alternatively, outdated preparations and add to an equal volume of the
citrated plasma from A-positive blood donors may be previously absorbed antiserum. Incubate for 30 min at
used. This should be extensively dialyzed at 0C to 4C 37C and for 60 min at room temperature. Centrifuge at
against many changes of 0.05 M NaCl during a 24-hr 29,000 g for 30 min, and discard the pellet. Repeat the
period, centrifuged at 29,000 g for 30 min to remove centrifugation procedure. The absorbed antiserum is
aggregated proteins, and tested for the presence of CEA. stored at 20C. Stable for at least 3 years.
Only those plasmas with values less than 2 ng/mL 4. Radiolabeled CEA. 125I-CEA may be
should be used. The NHP is frozen in 40-mL aliquots purchased from Roche Diagnostics, Nutley, NJ.
and then thawed before use, and the unused reagent is 5. CEA standards. Controls and secondary
discarded. antibody may be purchased separately from Roche
3. Antibody to CEA. Obtain a commercially Diagnostics.
available antiserum, such as the one sold by Dako. These 6. Goat anti-rabbit serum antisera. Can be
preparations usually have antibodies to other proteins purchased from many commercial suppliers.
and should be absorbed before use.
Alternatively, prepare antisera from CEA Anti-CEA antibody titration
purified from an adenocarcinoma of the colon. Mark 16 100 mm glass tubes in duplicate as
Homogenize the carefully trimmed cancer in 2 mL of follows:
0.15 M NaCl for each milligram of tumor. Remove the 1. Total counts: TC
insoluble material by centrifugation at 29,000 g for 30 2. Nonspecific binding: NSB CEA
minutes. Further purify the CEA by adding an equal antisera diluted 100-fold with distilled
volume of 1.2 M perchloric acid to the supernatant to water
precipitate proteins. Centrifuge the mixture at 29,000 g 3. 100 L
for 30 min at 4C. Dialyze the resultant supernatant 4. 50 L
extensively to remove the perchloric acid. Purify the 5. 25 L
antigen further by sepharose-6B column 6. 10 L
chromatography. The CEA fractions are concentrated by CEA antisera diluted 2000-fold with distilled
lyophilization and stored at 20C. Elicit an antibody water
response in rabbits using four biweekly injections of 10
7. 100 L
to 100 g of protein with Freunds adjuvant. Test for
8. 50 L
antibody response by use of Ouchterlony plates.
9. 25 L
Both commercial and individually prepared antisera 10. 10 L
should be absorbed with normal human tissues (such as CEA antisera diluted 40,000 fold with distilled
lung, liver, and spleen) as described below before use. water
11. 100 L
Obtain fresh red blood cells of the A-positive blood type, 12. 50 L
and wash thoroughly in 0.15 M saline. Do not refrigerate 13. 25 L
before use. Decomplement the antisera by heating to 14. 10 L
56C for 10 min in a water bath before absorption.
Dilute the packed red blood cells to 30% volume to 1. Each tube, except for TC, contains 5.0 mL of
volume with the antisera. Incubate for 60 min at room distilled water and 25 L of dialyzed CEA-
temperature, centrifuge at 1500 g for 30 min at room free normal human plasma (NHP) before anti-
temperature, and remove the supernatant fraction for CEA addition: TC contains 0.5 mL of distilled
334
Carcinoembryonic Antigens (CEA)
water but no NHP. Vortex mix the tubes after each
addition.
2. Add antibody diluted in distilled water to
each tube (except for NSB) as indicated
above, and incubate for 30 min in a water
bath set at 45C.
3. Remove tubes, add 50,000 counts of 125I-
CEA in 50 L, and again incubate for 30
min at 45C.
4. Remove from water bath, add 1 mL of
appropriate diluted second solution of goat
anti-rabbit antibody, and let tubes sit for
15 min at room temperature before
centrifugation at 1500 to 2000 g for 15
min.
5. Decant supernatants and count tubes in a
gamma-ray scintillation counter at 80%
efficiency.
Calculation
Standard curves are constructed and read as cpm
versus ng of CEA or B/B0 versus ng of CEA. If
one is comparing different monoclonal or organ-
specific anti-CEA reagents, the latter calculation
should be made. If possible, choose the antibody
concentration for future assays that yields 35% to
50% binding or at least 10,000 counts of 125I-
CEA.
If monoclonal or in-house polyclonal antibodies
are being assessed, they should be absorbed with
normal spleen, liver, and lung at 30% w/v for 30 to
60 min at 37C and overnight at 0C to 4C as
previously described to remove antibodies to cross-
reacting antigens found on normal neutrophils and
normal colon. If all or part of the CEA reactivity is
lost after absorption, the antibodies were not
cancer-specific reagents suitable for clinical use.
Stock anti-CEA at 1:100 (etc.) can be made in
the same normal serum of the species used to raise
anti-CEA antibodies and frozen at 20C. These
titrations and direct CEA procedures have been
used to measure CEA obtained from tumors,
meconium, fetal guts, or ascites fluids [11,39].
Assay
Equipment: a gamma-ray scintillation counter and
refrigerated centrifuge capable of 1500 to 2000 g
force.
1. Setup
a. Label 16 100 mm glass test tubes in
duplicate as follows:
(1) Total counts: TC
(2) Nonspecific binding: NSB
(3) Maximum binding: I0
(4-8) Standards: I5, I10, I25, I50, and
I100
(9-12) Controls: 5, 10, 25, and 50 L of
a CEA sample diluted in distilled water to contain
approximately 800 ng/mL.
(13, etc.) Samples: 1, 2, . . .
b. To all tubes except TC, add 5 mL of
distilled water. Add 500 L of distilled water to
TC tube.
c. To all standards, including NSB and I0, to
all control tubes, and to samples that do not contain
plasma or serum, add 25 L of normal human
plasma (NHP).
NOTE: Since CEA tends to adhere to the sides of
glass tubes, it is essential to complete steps 1b and
1c before adding standard or samples to tubes.
d. To standard tubes (tubes 4 through 8), add
the indicated volumes of standard containing 125
ng of CEA/mL.
5 L for I5 = 0.625 ng/mL
10 L for I10 = 1.25 ng/mL
25 L for I25 = 3.12 ng/mL
50 L for I50 = 6.25 ng/mL
100 L for I100 = 12.5 ng/mL
e. To control tubes, add the appropriate
amount of in-house CEA preparation to yield
final concentrations of approximately 1, 2, 4, and 8
ng/mL.
f. Samples: Add appropriate amounts of
sample (10 to 100 L) so that results fall within the
standard curve. For samples above 500 ng of
CEA/mL, dilute 10-fold with distilled water.
2. To all tubes except TC and NSB, add 50
L of diluted CEA antiserum. Vortex mix.
3. Incubate tubes in a water bath at 45C for
30 min.
4. To all tubes, add 50 L of diluted 125I-
CEA. Vortex mix.
5. Return to water bath for 30 min.
6. To all tubes except TC, add 1 mL of
second goat/anti-rabbit antibody suspension. Make
sure it is thoroughly mixed into the reaction
mixture by vortex mixing.
7. Let stand at room temperature for 15 min,
and then centrifuge for 15 min at 1500 to 2000 g at
a temperature of 4C to 25C.
8. Decant carefully, and count in gamma-ray
scintillation counter.
Calculations
Prepare standard curve on semilogarithmic graph
paper using counts per minute (cpm) on the vertical
axis and nanograms of CEA (ng of CEA) on the
horizontal logarithmic axis. Read unknown counts
per minute off of the standard curve, and multiply
by correct dilution factor to obtain results, in ng of
CEA/mL.
Notes
1. The direct assay of CEA is exceptionally
sensitive to ions, pH, colloidal metals, bacterial
products, and organic molecules. The water should
be filtered before use, since bacteria can grow in
distilled-water systems. Additionally, improperly
deionized tanks may leak resins. In troubleshooting
problems with the CEA assay, one must thoroughly
check the water as a possible cause of problems.
335
Carcinoembryonic Antigens (CEA)
2. Many problems arise in the control of
CEA values. Calibrators must be independent of
the current laboratory methods, whether they are
internally devised or obtained from an external
commercial source. Additionally, since cancer
cures are considered to be obtained after a 5-year
interval, standards must be available in sufficient
quantity to ensure long-term quality control. This is
particularly true if one contemplates changing CEA
methodologies, manufacturers, or source materials.
Because of the heterogeneity of CEA-reacting
molecules, it is well known that CEA values
obtained from one method cannot be obtained with
a second method, and a correction factor cannot
be applied between different standards or different
assays. Above all, one should not obtain reference
CEA from the same company from which one
obtains reagents, because the ranges of the
reference material will be established by the same
assay used to evaluate the reagents. It will be
impossible to determine if assay results have been
drifting over the years.
How should one proceed? Obtain your own
hospital or laboratory standards. First realize that
CEA has traditionally been quantitated in
immunochemical reactivity in nanograms of CEA
per milliliter. This has no reality in terms of
nanograms of CEA per milligrams of protein. The
carbohydrate-to-protein ratio may vary from 1:1 to
1:4 in different preparations. CEA purified from
different tumors will have different immunological
reactivities because of the different number of
CEA-reacting antigenic sites: that is, ovarian CEA
> colon CEA > breast CEA. However, in terms of
serum CEA: colon CEA > breast CEA > ovarian
CEA. Additionally, spiking commercially obtained
CEA into CEA-free plasma will give spurious
effects because of the variability of these
preparations, lyophilization aggregation,
nonspecific heterologous reactions, and deleterious
matrix effects [5].
Our first recommendation is that the investigator
obtain a fluid containing an exceptionally high
amount of CEA from a patient with colon cancer.
Dilute this in distilled water to achieve a final
concentration that falls below the high end of the
standard curve with the addition of the appropriate
amount of CEA-free plasma required for that assay.
This latter addition controls nonspecific protein
quench. CEA at 0.5, 2.0, 4.0, and 8.0 ng/mL should
be used for assessment of low-end sensitivity.
These CEA values should parallel the standard
curve. In our laboratory, our group uses an ascites
fluid that contains 16,000 ng of CEA/mL, as
measured by perchloric acid extraction. Aliquots of
0.5 mL of 1:100 (perchloric acid assay) and 1:200
(direct assay) dilutions are frozen and used monthly
for monitoring of CEA assays, along with five
previously analyzed CEA-containing plasma
samples. Then 100, 50, 20, and 6 L of these
standards are delivered into the appropriate assay
tubes along with the appropriate amounts of CEA-
free plasma normally required for that assay, that
is, 0.5 mL of plasma for perchloric acid extraction
or 50 to 200 L for various direct or unextracted
CEA ligand assays. The small amount of extra
volume (water) does not alter the CEA measured
by any of the currently available commercial CEA
assays, nor does it affect the protein matrix (ions,
pH, or salt) if deionized distilled water is used as
diluent.
My second recommendation is that the reference
CEA preparation prepared in the laboratory be
experimentally tested by a reference laboratory
whose CEA standard has been experimentally
calibrated against early Phil Gold and Roche-CEA
preparations from the past 17 years. If the
laboratory chooses to purchase reference controls
in quantity, these also should be sent out to a well-
standardized, qualified laboratory.
Finally, standardized CEA reference serum should
be run once monthly with the assay of your choice
or at any time when CEA values (or curves) seem
to drift. Sufficient standards should be obtained to
last for a 6-year period. However, in the fifth year,
new reference control materials should be
calibrated against old standards so that continuity
in monitoring patient samples is ensured.
3. In patients with bladder cancer, CEA-
reacting substances are found in urine. If an RIA is
used to measure urinary CEA, 25 L of fresh
normal urine must be added to each point of the
standard curve while 25 L of the urine obtained
from the cancer patient and 25 L of NHP must be
in the unknown sample or samples.
4. Ascites fluids that contain heparin may
nonspecifically quench the CEA-RIA, even after
excessive dialysis, and thus their use is not advised.
However, if an ascites fluid comes from a patient
with exceptionally high CEA and is needed for
research purposes (or CEA standard), heparin can
be removed using Sephadex G-100 column
chromatography. CEA is eluted in the void volume
while heparin (9000 to 20,000 D) is retained in the
stationary phase.
5. If ascites, pleural effusion fluids, or spinal
cord fluid is tested for CEA content, the 25 L of
NHP may be omitted from the assay procedure. For
very dilute CEA samples, for example, those from
peritoneal washes, purification steps, and so on, the
addition of 25 L of NHP in the assay is very
necessary. If doubt exists, add the NHP. Do not
extract CEA from dilute solutions. Enzyme
immunoassays are not advised for research uses,
that is, purification of CEA, because of their
limited specificities and high expense.
336
Carcinoembryonic Antigens (CEA)
6. The direct method is sensitive to high salt
or protein levels, acid, base, and different ions. If
dilutions of CEA-containing samples, used for
research purposes, do not yield linear curves that
parallel the standard curve, they should be dialyzed
against dilute saline (0.03 M) or distilled water. It
is also important to dilute protein samples that have
more than 100 to 120 mg of protein/mL because
nonspecific protein binding will result.
7. Normal CEA-free plasma (NHP) is used
to investigate nonspecific binding and to coat the
inner surface of glass tubes. Previous directions
must be carefully followed because all the citrate
must be removed before the CEA assay. The use of
A-positive plasma is suggested so that only anti-B
plasma heterospecific antibodies will be present.
These are not likely to interact with plasma
samples, since more than 90% of the people on this
continent have A-positive or O-positive plasma.
Thus the various assays for CEA serve many
clinical and research purposes. With good quality
control, both the RIA and EIA measurement of
CEA yield dependable clinical data.
337
Catecholamines (Plasma)

Catecholamines (Plasma)
Brett McWhinney
Name: Catecholamines: epinephrine (E), adrenaline, adrenalin norepinephrine (NE),
noradrenaline, levarterenol dopamine (DA), dihydroxyphenylalanine (DOPA),
dihydroxylphenylactic acid (DOPAc), dihydroxyphenylglycol (UI)(PG)
Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Epinephrine Norepinephrine Dopamine
Structural formula: See below See below See below
Molecular formula: C9H13NO3 C8H11NO3 C8H11NO2
Molecular mass: 183.20 D 169.18 D 153.18 D
Merck Index: 3543 6504 3422
Chemical class: Catechol derivatives
Dihydroxypheny- Dihydroxyphenyl- Dihydroxyphenyl-
alanine acetic Acid glycol
Molecular formula: C9H8O4 C8H5O4 C8H7O7
Molecular mass: 197.2 D 168.1 D 170.2 D
Merck Index:
Chemical class: hydroxyphenyl

Norepinephrine Epinephrine Dopamine

i with 1 (E) or 2 (NE) moles of ethylenediamine, with the
Principles of Analysis and Current Usage
The earliest chemical methods for the analysis of plasma
catecholamines were fluorometric, involving the
oxidation and reduction of the catecholamines to
fluorescent products. The two most widely used
fluorometric assays were the trihydroxyindole (THI) and
ethylenediamine (EDA) methods [1,2,3]. In the THI
method (Table 1, Method 1), epinephrine (E) and
norepinephrine (NE) are oxidized by a suitable oxidizing
agent such as ferricyanide, and an intramolecular
cyclization then occurs, forming the corresponding
adrenochrome. At an alkaline pH, the adrenochromes
rearrange to form adrenolutin and noradrenolutin. The
latter two compounds fluoresce at 505 nm after
excitation at 400 nm, allowing quantitation of total
catecholamines (NE + E).
In the ethylenediamine method (Table 1, Method 2), E
and NE are oxidized, and the oxidation products react
i
Catecholamines (Plasma)
Previous and current authors of this method:
First edition: Steven J. Soldin
Methods edition: Lawrence E. Webb, Lawrence A.
Kaplan
Second edition: Not updated
Third edition: Not updated
Fourth edition: Theodore Dashman, Stanley Samuels
Fifth edition: Brett McWhinney
elimination of water and hydrogen ions to form
fluorescent products. Because the two fluorescent
products derived from E and NE are different, E and NE
levels can be estimated by measurement of the
fluorescence of the final reaction at two different
wavelengths, 510 nm and 580 nm, after excitation at 420
nm. The fluorescence of the product derived from
epinephrine is half the fluorescence derived from the
norepinephrine product at 510 nm, whereas at 580 nm
the ratio is reversed; simultaneous equations can be
established to estimate E and NE levels.
Both the THI and EDA methods require some sample
purification to separate the catecholamines from
interfering compounds. The purification steps typically
include alumina and cation-exchange column
chromatography and often include a liquid-liquid
extraction.
A method used frequently to quantitate plasma
catecholamines is the radioenzymatic assay (Table 1,
Method 3). The radioenzymatic assay is based on the
method of Peuler and Johnson [4]. This rather laborious
procedure radiolabels the plasma catecholamines by
methylating them in the following reaction:
3H-SAM + epinephrine/norepinephrine
COMT3H-metanephrine/3H-normetanephrine
37C, 60'
338
Catecholamines (Plasma)
3H-SAM is [3H-methyl]-S-adenosyl-L-methionine, and
COMT is catechol-O-methyltransferase (EC 2.1.1.6).
The catecholamines are extracted by a liquid-liquid
extraction, and the methylated derivatives
(metanephrine, normetanephrine, and 3-
methoxytyramine [from dopamine]), labeled and
unlabeled, are separated by thin-layer chromatography
on silica gel. The metanephrine, normetanephrine, and 3-
methoxytyramine zones are identified by their Rf values
and are scraped off and placed in vials. They are then
eluted from the silica gel, and the 3-methoxytyramine
eluate is counted directly. The metanephrine fractions
are then oxidized to 3H-vanillin, which is extracted into
scintillation fluid and counted. To assess recovery of the
final 3H-vanillin from each sample and to assess any
interference in the methylation reaction, each sample
must be processed in duplicate, one sample containing
added norepinephrine and epinephrine internal standards
(usually 100 pg each), while the other is unspiked. The
internal standard is used to assess the extent of the
enzymatic reaction and correct for interferences.
A method that is becoming the standard for
catecholamine analysis employs high-performance liquid
chromatography (HPLC) with electrochemical detection
(Table 1, Method 4) [5,6,7]. HPLC procedures use a
fairly rapid and simple batch type of alumina extraction
step to partially purify the catecholamines [8]. Cation-
exchange chromatography on Dowex 50 and selective
absorption on boric acid gels [7] have also been used to
partially purify the catecholamines [9]. The alumina
HClO4 extract is analyzed directly, usually by reversed-
phase HPLC (most often, C18 columns), with the mobile
phase usually consisting of an acid buffer (phosphate,
phosphate-citrate, or monochloroacetic acid), small
amounts of a polar organic solvent (methanol), and a
paired-ion agent (such as heptane- or octanesulfanate).
Cation-exchange stationary phases have also been
employed in HPLC analysis [10,11]. The radioenzymatic
and HPLC methods are the most commonly used
methods for the measurement of plasma catecholamines.
Reference and Preferred Methods
There is no current reference method for the analysis of
plasma catecholamines; however, HPLC with
electrochemical detection appears to give the best
specificity and sensitivity for the low levels seen in
patients. The fluorometric methods lack sensitivity for
plasma catecholamines. The purification steps required
to remove interfering compounds only add to the
tediousness of this method. The reported precision of the
radioenzymatic procedures seems acceptable, and the
assay has the capability of reasonable accuracy and
sensitivity. The major problems with the assay derive
from material present in the plasma interfering in the
methylation reaction [12]. One of the reasons for
introducing the final step, oxidizing the radiolabeled
metanephrine and normetanephrine to 3H-vanillin, was
to selectively extract the vanillin and increase the
specificity of the assay. Highly purified COMT
preparations are not readily available, and inhibitory
substances present in the enzyme preparations can also
interfere in the methylation reaction. These inhibitors in
both plasma and enzyme cause a large variability in the
production of the 3H-labeled O-methylated products.
Suggestions for removing these inhibitors include the
addition of EGTA to bind inhibitory Ca2+ in plasma, the
precipitation of EGTA-Ca2+ and protein from plasma
before analysis using HClO4, and the more extensive
purification of COMT [13,14]. More purified COMT
preparations contain a smaller quantity of interfering
enzymes such as L-aromatic amino acid decarboxylase
[15] and monoamine oxidase [13].
Additional problems with the radioenzymatic assay can
result from the use of inappropriate Michaelis-Menten
conditions in the critical transmethylation step [14]. If an
insufficient concentration of methyl donor (3H-SAM) is
present in the reaction mixture, inaccurate data can
result. This problem is exacerbated by the addition of
internal standard, as is recommended in the
commercially available assay. This only lowers the ratio
of 3H-SAM to catecholamines and does not allow proper
comparison of reaction rate in the unspiked and spiked
(internal standard) samples [14,16]. A 10-fold excess of
SAM to catecholamine concentration in the reaction
mixture is suggested [14]. The large variability in the
amount of labeled added internal standard recovered
prompted one group to suggest that an internal standard
not be run on each patient, but that instead a mean net
standard value obtained from many patients be assumed
[17]. This procedure, however, only circumvents the real
problems of individual interferences, which should be
corrected. The use of a partial purification of the
catecholamines by an alumina extraction before the
methylation reaction [14], the use of purified COMT
preparation [12,14], and the use of higher levels of SAM
in the reaction mixture [14] seem to be more appropriate
suggestions.
The HPLC procedures also have deficiencies. The
review of methods of plasma catecholamine analysis by
Hjemdal [15] showed that although the various HPLC
techniques seemed to be appropriate, with generally
excellent correlation between HPLC and radioenzymatic
procedures, the ability of individual laboratories to
recover added analyte or to demonstrate good precision
was limited. The fluorometric trihydroxyindole
procedure was inferior to the HPLC assays. The
technology of the HPLC electrochemical procedures has
advanced dramatically in recent years. Improvements in
column technology, better control of column
temperature, and the introduction of the triple-electrode
coulombic detection system has enhanced the
quantitation of NE, E, DA, and other catecholamine
metabolites [7,15]. Thus an HPLC electrochemical
procedure is the recommended method for measurement
of plasma catecholamines.
339
Catecholamines (Plasma)
Specimen
Sample collection and storage must be rigorously
controlled. Heparinized plasma is the preferred sample.
During transport from patient to the laboratory, blood
samples should be maintained at 4C. After removal of
the cellular matter, plasma is immediately analyzed or
stored by freezing at 70C to 80C. However, if the
sample is to be forwarded to a distant location, it should
be frozen and shipped in solid CO2.
Specimen Collection
During collection, the patient should be supine and
motionless because movement, even from supine to
erect, may result in a two- to threefold variation in
plasma catecholamine levels. Also, patients should not
drink coffee for 24 hours prior to blood collection.
Caffeic acid from coffee may interfere with
catecholamine resolution on HPLC. Smokers should be
warned not to smoke for a minimum of 1 to 2 hours
before blood collection because of the stimulatory
effects of nicotine.
To ensure a stable metabolic status of the patient during
blood collection for catecholamine analysis, a two-step
procedure is recommended.
1. Insert a venous catheter percutaneously in the
forearm or antecubital region of the arm. The patient is
then requested to lie quietly for 20 to 30 min.
2. Blood is drawn into a prechilled (4C),
heparinized 5-mL Vacutainer. The filled tube is
immediately transported to the laboratory at 4C.
Normotensive
Norepinephrine <30400 pg/mL (<0.1772.36 nmol/L)
Epinephrine <30100 pg/mL (<0.1640.546 nmol/L)
Hypertensive
Norepinephrine <30700 pg/mL (<0.1774.14 nmol/L)
Epinephrine <30120 pg/mL (<0.1640.655 nmol/L)
Using the method described by Holmes et al. [6], the following levels of plasma catecholamines were found in healthy
individuals.
Catecholamine pg/ml
Norepinephrine 348 10
Epinephrine 48 2
Dopamine 15 2
Dihydroxyphenylalanine 1596 47
Dihydroxyphenyl acetic acid 1304 59
Dihydroxyphenylglycol 1168 55
Interpretation
The catecholamines are produced by the adrenal medulla
(mostly E) and peripheral nerves (NE and dopamine).
The plasma levels of the analytes are usually increased
in persons with hypertension. Plasma levels are even
higher in patients with pheochromocytomas. Since the
plasma catecholamine assay is very difficult to perform
properly, it is not advocated by most clinicians as the
primary diagnostic test for pheochromocytomas. In
addition, because plasma catecholamine levels reflect
Caution: Collection of blood from children is
particularly difficult because of their normal fear of
venipuncture and their restlessness; for this reason,
catecholamine levels from excited children may be
misleading.
Plasma is separated within 30 min of sample collection
by centrifugation (3000 g, 20 min, 4C). Aliquots (1.2
mL) are quickly transferred to self-capped centrifuge
tubes, and 10 mg of sodium metabisulfite is added. The
plasma is immediately analyzed or is stored at 70C to
80C.
Interferences
The fluorometric methods are not sufficiently specific,
especially with the number of commonly occurring
interfering drugs. These include methyldopa, ampicillin,
vitamin B complexes, and caffeine. Nicotine does not
interfere directly with measurement of dopamine,
epinephrine, or norepinephrine by HPLC, but smoking
within 2 hours of blood collection may stimulate
catecholamine release, and an artifactually increased
result may be obtained.
Plasma Catecholamine Reference Intervals
Using the HPLC-EC method described previously [18],
(Table 1, Method 2), the following levels of plasma
catecholamines were found in healthy individuals.
nmol/L
2.06 0.06
0.26 0.01
0.10 0.01
8.10 0.24
6.07 0.35
6.87 0.32
only very recent catecholamine synthesis, plasma
catecholamine levels can be within the reference interval
in cases of tumors whose catecholamine excretion is
highly episodic. Plasma catecholamine measurements
should be performed for those patients with borderline
elevations (above their reference intervals) of urinary
HMMA (vanillylmandelic acid or VMA),
metanephrines, or catecholamines.
340
Catecholamines (Plasma)

A useful challenge test employing plasma norepinephrine by reverse-phase liquid

catecholamines is the clonidine suppression test. chromatography. Anal Chem 1981;53:156-159.
Clonidine normally will cause a suppression of plasma 9 Frayn KN, Maycock PF. Sample preparation
catecholamine levels. Failure of the plasma with ion-exchange resin before liquid
catecholamine levels to decrease after a dose of chromatographic determination of plasma
clonidine is highly suggestive of a pheochromocytoma. catecholamines. Clin Chem 1983;29:1426-
1428.
10 Allenmark S, Hedman L, Sderberg, A.
Plasma Catecholamine Performance Goals Microanalysis of catecholamines in human
The interlaboratory precision and accuracy for plasma by high-performance liquid
laboratories employing reversed-phase HPLC is chromatography with amperometric detection
generally poor, with an overall % coefficient of variation as compared with a radioenzymatic method.
(CV) precision of < 20%. The most reliable HPLC Microchem J 1980;25:567-575.
analyses appear to be those employing cation-exchange 11 Eriksson BM, Persson BA. Determination of
stationary phases (overall % CV precision of < 10%), a catecholamines in rat heart tissue and plasma
method not widely used in the United States, where C18 samples by liquid chromatography with
reversed-phase seems most popular. In this study by electrochemical detection. J Chromatogr
Hjemdahl, the overall interlaboratory precision and 1982;228:143-154.
accuracy of the laboratories using the COMT procedure 12 Hussain MN, Benedict CR. Radioenzymatic
was similar to that achieved by the laboratories using an microassay for simultaneous estimations of
HPLC method. In a comparison between an HPLC dopamine, norepinephrine, and epinephrine in
procedure using a C18 column and a radioenzymatic plasma, urine, and tissues. Clin Chem
1985;31:1861-1864.
assay [19], a definite bias was shown to exist; the
13 Ben-Jonathan N, Porder NC. A sensitive
radioenzymatic results were higher than the results
radioenzymatic assay for dopamine,
obtained by HPLC.
norepinephrine and epinephrine in plasma and
tissues. Endocrinology 1970;98:1497-1507.
References
14 Mason L, Weinkove C. Kinetics of enzymatic
1 Manger, WM, Steinsland, OS, Nahas, GG,
O-methylation: its value in assays for
Wakim KG, Dufton S. Comparison of improved
catecholamines in plasma. Clin Chem
fluorometric methods used to quantitate plasma
1984;30:24-27.
catecholamines. Clin Chem 1969;15:1101-
15 Hjemdahl P. Interlaboratory comparison of
1123.
plasma catecholamine determinations using
2 Martin LE, Harrison C. An automated method
several different assays. Acta Physiol Scand
for determination of noradrenaline and
1984;527:43-54.
adrenaline in tissues and biological fluids. Anal
16 Johnson GA, Kupiecki RM, Baker CA. Single
Biochem 1968;23:529-545.
isotope derivative (radioenzymatic) methods in
3 Victorio JK, Baukal A, Wolff FW. New
the measurement of catecholamines.
automated fluorometric methods for estimation
Metabolism 29(suppl)1980;1106-1113.
of small amounts of adrenaline and
17 Ellis JP Jr, Burns JW. Modified handling of
noradrenaline. Anal Biochem 1968;23:513-528.
internal catecholamine standards in
4 Peuler JD, Johnson GA. Simultaneous single
radioenzymic assays. Clin Chem 1983;29:144-
isotope radioenzymatic assay of plasma
147.
norepinephrine, epinephrine, and dopamine.
18 Kaplan, LA. Catecholamines, plasma. In: Pesce
Life Sci 1977;21:625-636.
AJ, Kaplan LA, eds. Methods in Clinical
5 Premel-Cabic A, Allain P. Simultaneous liquid
Chemistry. 4th ed. St Louis: Mosby; 1987:944.
chromatographic determination of norepi-
19 Causon RC, Brown MJ, Boulous PM, Perret D.
nephrine, 3,4-dihydroxyphenylethyleneglycol,
Analytical differences in measurement of
3-droxyphenylalanine and 3,4-dihydroxy-
plasma catecholamines [letter]. Clin Chem
phenylacetic acid. J Chrom 1988;434:187-190.
1983;29:735-737.
6 Holmes C, Eisenhofer G, Godstein DS.
20 The procedure is based on the method described
Improved assay for plasma dihydroxy-
in a personal communication with C. Holmes
phenylacetic acid and other catechols using
and G. Eisenhofer and Reference 6.
high performance liquid chromatography with
electrochemical detection. J Chrom
1994;653:131-138.
7 Imai Y, Ito S, Maruta K, Fujita K. Simultaneous
determination of catecholamines and serotonin
by liquid chromatography after treatment with
boric acid gel. Clin Chem 1988;34:528-530.
8 Davis GC, Kissinger PT. Strategies for
determination of serum or plasma
341
Catecholamines (Plasma)
Tables
Table 1: Methods of Plasma Catecholamine Analysis
Method 1: Trihydroxyindole (THI); fluorometric
Principle of analysis: Partially purified
epinephrine and norepinephrine are oxidized to
corresponding adrenochromes which, at
alkaline pH, rearrange to adrenolutin and
noradrenolutin. Quantitation of fluorescence is
at 505 nm after excitation at 400 nm.
Comments: Historical; measures total
catecholamines (E and NE); requires one or
more purification steps
Method 2: Ethylenediamine (EDA); fluorometric
Principle of analysis: Partially purified
epinephrine and norepinephrine are oxidized to
products that are allowed to react with 1 (E) or
2 (NE) moles of ethylenediamine to form
fluorescent derivatives. Quantitation of
fluorescence of E (510 nm) and NE (580 nm) is
accomplished after excitation at 420 nm; using
differential equations, one can individually
quantitate E and NE.
Comments: Historical; can quantitate E and
NE; requires one or more purification steps
Procedure: Catecholamine Analysis in Plasma by
High-Performance Liquid Chromatography with
Electrochemical Detection [20]
Principle
Plasma catecholamines, partially purified by an alumina
extraction, are fractionated by HPLC on C18 reversed-
phase columns and quantitated by electrochemical
detection.
Reagents (Stock Standard Solutions Table above)
1. Acid-washed alumina. Brockman activity 1,
60-325 mesh, obtained from Fisher Scientific Co.,
Pittsburgh, PA, stored at 90C and at time of use, cooled
to ambient temperature.
2. Extraction buffer, Tris buffer (1 mol/L, pH
8.6) containing EDTA (53.7 mmol/L). Weigh 121 g
of Tris base and 20 mg EDTA-Na2, and dissolve in
approximately 800 mL type I water. Adjust to pH 8.6
with HCl, and dilute to 1000 mL. Stable for 2 months at
4C to 8C.
3. Wash buffer, sodium bicarbonate (200
mmol/L) containing EDTA (26.8 mol/L) and
sodium metabisulfite (26.3 mol/L). Weigh 1.6 g
sodium bicarbonate, 10 mg of EDTA-Na2, and 5 mg
sodium metabisulfite; dissolve in approximately 80 mL
of type I water, and dilute to 100 mL. Prepare fresh
daily.
4. Mobile phase, monobasic phosphate (100
mmol/L, pH 3.4) containing EDTA (134.3 mol/L), 1-
octanesulfonic acid (184.9 mol), and acetonitrile
(3%). Place 13.8 g sodium monobasic phosphate, 50 mg
EDTA-Na2, and 40 mg sodium 1-octanesulfonic acid in
a 1-L volumetric flask and dissolve in approximately
Method 3: Radioenzymatic; isotopic-enzymatic
Principle of analysis: Epinephrine and
norepinephrine are enzymatically converted to
their corresponding tritiated methylated
derivatives (metanephrines). These are
extracted and separated by thin-layer
chromatography (TLC). The individual
compounds are extracted from the stationary
phase of the TLC plate and converted to 3H-
vanillin, and the tritium label is measured.
Radioactivity present is directly proportional to
concentration of E and NE.
Comments: Laborious method; difficult to
control variation in extent of reaction; problems
with purity of enzyme reagent.
Method 4: High-performance liquid chromatography
(HPLC); chromatographic
Principle of analysis: Catecholamines are
extracted from plasma by alumina and are
directly analyzed by ion-paired, reversed-phase
HPLC. Detection and quantitation are by an
electrochemical detector.
Comments: Commonly used; requires a fair
amount of technical expertise for reliable
analysis.
900 mL of type I water. Adjust pH to 3.4, and dilute to
1000 mL. Add 30 mL acetonitrile to the buffer; mix. The
mobile phase is filtered and degassed by filtering
through a 0.22 m microfiltration system (Kimble Glass
Inc., Vineland, NJ). Prepare mobile phase daily.
5. Elution solution (ES), phosphoric acid (40
mmol/L) plus acetic acid (200 mmol/L), 1:4, v/v.
Phosphoric acid (40 mmol/L). Prepare an intermediate
stock solution of 100 mmol/L phosphoric acid by
diluting 3.4 mL phosphoric acid (85%) to 500 mL type I
water. Prepare 40 mmol/L phosphoric acid by diluting 4
mL of 100 mmol/L phosphoric acid to 100 mL with type
I water. Stable 1 year at room temperature if covered.
Acetic acid (200 mmol/L). Prepare an intermediate stock
solution of 1 M acetic acid by diluting 5.75 mL glacial
acetic acid (99.5%) to 500 mL with type I water. Prepare
200 mmol/L acetic acid by diluting 20 mL of the 1 M
acetic acid to 100 mL with type I water. Stable 1 year at
room temperature if covered.
To prepare the eluting solvent, add 40 mL of 40 mmol/L
phosphoric acid to 160 mL of 200 mmol/L acetic acid.
Prepare daily.
6. Stock Standards
Norepinephrine (NE) (1 mg/mL). Dissolve 19.0 mg
norepinephrine bitartrate per 10 mL of elution solution.
Epinephrine (E) (1 mg/mL). Dissolve 18.9 mg
epinephrine bitartrate per 10 mL of elution solution.
Dopamine (DA) (1 mg/mL). Dissolve 12.4 mg dopamine
hydrochloride per 10 mL of elution solution.
342
Catecholamines (Plasma)
Dihydroxyphenylalanine (DOPA) (1 mg/mL). Dissolve
10 mg dihydroxyphenylalanine free base in 10 mL of
elution solution.
Dihydroxylphenylglycol (DHPG) (1 mg/mL). Dissolve
10 mg dihydroxylphenylglycol free base in 10 mL of
elution solution.
Dihydroxyphenylacetic acid, (DOPAC) (1 mg/mL).
Dissolve 10 mg dihydroxyphenylacetic acid free acid in
10 mL of elution solution.
7. Internal standard. Dihydroxybenzylamine
(DHBA) (1 mg/mL). Dissolve 14.1 mg
dihydroxybenzylamine hydrobromide per 10 mL of
elution solution.
8. Working standards (100 ng/mL). Dilute
separately 0.1 mL of each of the stock standards of the
standard solution to 10 mL with elution solution. Then
dilute each solution 1:100 with ES. Aliquot 1.0 mL of
these solutions (100 ng/mL) in polypropylene tubes, and
store at 70C to 80C. 1.
9. Combined standard
Amine mixture: Add 100 L of each of the NE,
EPI, DA working standards and 100 L of elution
solution to a 500 to 1000 L polypropylene tube. The
concentration of each component of the mixture is 25
ng/mL.
Test standard. To a separate tube, add 100 L of the
amine mixture plus 100 L each of the working
standards of DOPA, DOPAC, and DHPG, 200 L of
internal standard, and 300 L of elution solution. Total
volume of combined standard solution is 900 L. When
90 l of the extract of the test standard is analyzed by the
HPLC system, this is equivalent to injecting 250 pg of
NE, EPI, and DA and 1000 pg of DOPA, DOPAC, and
DHPG.
Assay
Equipment:
Column: Axxion Chromatography ODS 5 m reverse
phase C18 4.6 mm 25 cm (obtained from Thompson
Inst. Co., Springfield, VA). Column temperature (16C
to 17C) was controlled using a refrigerated water
circulator (Fisher Scientific, Pittsburgh, PA). The
coolant (1:1 mixture of Prestone antifreeze and Type I
water) circulates through a glass jacket (Column
Engineering, Ontario, CA) surrounding the column.
Pump: Model 510 dual-head pump (Waters, Milford,
MA).
Pre-Column Filter: with a C18 insert (Waters,
Milford, MA).
Injector: Model 7161 injector (Rheodyne, Cotita, CA).
Centrifuge: Eppendorf Model 5415C microcentrifuge
(Brinkman Instruments, Westbury, NY) with fixed-angle
rotor to accept 1.5-mL centrifuge tubes.
Calibrated Spatula: Volumetric spatula, 10 mg (The
Nest Group, Southboro, MA).
Detector: Amperiometric or coulombic electrochemical
detector. The procedure described here utilized a
coulombic electrochemical detector (Model 5100A,
ESA, Inc. Chelmsford, MA) with triple electrode system
(Models 5021 and 5011; ESA, Inc.). The first electrode
in the series was set at 300 mV, the second at 160 mV,
and the third at 350 mV.
Data System: Millennium 2.1 data system (Waters,
Milford, MA).
Procedure
Blanks and standards for plasma specimens
1. BlankTo a polypropylene centrifuge tube add
1.0 mL type I water and 10.0 L of DHBA (internal
standard, IS).
2. Recovery standardsTo a polypropylene
centrifuge tube, add 1 mL type I water and 90 L of test
standard solution.
3. Extraction standardsTo a separate
polypropylene centrifuge tube, add 1 mL of type I water
and 90 L of test standard solution.
Plasma
Centrifuge fresh or thawed plasma (3000 g,
10 min, 4C) Transfer 1.0 mL to a 2.0-mL
polypropylene centrifuge tube.
2. Add 20 L internal standard to each plasma
specimen.
3. Add 10 mg alumina from the volumetric spatula
to blanks, extraction standards, QC sample, and test
plasma.
4. Add 400 L of extraction buffer, pH 8.6, to
each tube.
5. Mix contents on a rotary or orbital shaker for 20
to 30 min.
6. Centrifuge in a microcentrifuge for 1 minute.
7. Aspirate supernatant, but leave a few mL of
solution above the pellet.
8. Add 1.0 mL of wash buffer to the alumina
pellet. Wash by inversion with wrist action, centrifuge as
in step 6 above, and aspirate as in step 7 above. (Wash 1)
9. Wash the pellet with wash buffer as in step 8.
(Wash 2)
10. Wash the pellet a third time with wash buffer,
but at this stage, attempt to remove the total volume of
supernatant. (Wash 3)
11. Add elution solution (100 L) to the lumina
pellet. Vortex for 2 minutes.
12. Centrifuge for 1 minute as described in step 6
above. Transfer the eluent to the centrifuge tube with a
0.2-m filter insert (Gelmar Instrument Co., Ann Arbor,
MI).
13. Repeat step 11 using 50 L elution buffer.
Repeat step 12, and combine supernatants.
14. Centrifuge combined eluents at 16,000 g for 1
minute. Discard filter. The sample is now ready for
chromatography. Either begin the analysis or store
eluates at 70C overnight. Attempt to complete the
analysis within a working day.
15. Inject 90 L of each sample eluate or combined
standard onto the HPLC column for analysis.
Calculations
Calculate using the method of area ratios, that is, the
peak area of the catecholamine to the peak area of the
343
Catecholamines (Plasma)

internal standard. A standard curve for each component Catecholamine ratio (Ru) = ___Unknown area___
is obtained from peak-area ratios. Internal standard area

Area ratio (R): Catecholamine (pg/mL plasma) =

Standard ratio (Rs) = Recovery standard area Ru Concentration of standard
Internal standard area Rs
Recovery (%) = RE 100% = % recovery
Extraction ratio (RE) = Extracted standard area Rs
Internal standard area

temperature. When thawed, immediately process each

Notes QC sample as if it were a patient sample. QC samples
are included in each HPLC run.
Limit of Detection and Reportable (Linear) Range:
Catecholamine Blanks are included in the analysis to be certain that the
Limit of Linear Range (pg) HPLC system is operating satisfactorily. If peaks other
than the internal standard are observed, stop analysis and
Detection (pg) check system for contamination. Determine recovery
Dihydroxyphenyl glycol (DHPG) 20 each day of analysis. If recovery is less than established
1002000 value, do not report data; check reagents and system for
Norepinephrine (NE) 20 deterioration.
125500 Notes
Dihydroxyphenylalanine (DOPA) 20 a. If an autosampler is available, it is suggested
1001000 that about 30 samples should be the maximum number
Epinephrine (EPI) 20 of samples per run. The suggested sample sequence is:
50250 First and last injection is the reagent blank. Second and
Dopamine (DA) 25 penultimate injection is the recovery standard. Third and
50500 third-from-last injection, the extraction standard. Fourth
Dihydroxyphenylacetic acid (DOPAC) 50 and fourth-from-last injection the QC sample. Add
502000 plasma sample in order of priority between the fourth
and fourth-from-last QC samples.
Precision: b. If an autosampler is not available, it is
Within-run: A CV of about 5% is achievable. suggested that 8 samples be the maximum number of
Between-run: A CV of about 5% is achievable. patient samples in one run. The suggested sample
sequence is: first, reagent blank; second, recovery
standard; third, extraction standard; and fourth, QC
Quality Control: sample. Add plasma samples after the QC sample in
Plasma: Obtain outdated plasma from the blood bank, order of priority.
and store 1.2-mL aliquots containing 10 mg sodium
metabisulfate at 80C. At the time of analysis, remove
two tubes from storage, and thaw to ambient
344
Catecholamines (Urine)

Catecholamines (Urine)
Brett McWhinney

Name: Epinephrine, E, 4-[1-hydroxy-2-(methylamino)ethyl]-1,2-benzenediol, adrenaline

Norepinephrine, NE, 4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, noradrenaline
Dopamine, DA, 4-(2-aminoethyl)-1,2-benzenediol
Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.

Epinephrine Norepinephrine Dopamine

Structural formula: See below
Molecular formula: C9H13NO3 C8H11NO3 C8H11NO2
Molecular mass: 183.20 169.18 153.18
Merck Index: 3567 6536 3428
Chemical class: Amino acid metabolite, dihydroxyphenyl amino derivative

Norepinephrine Epinephrine Dopamine

i relatively independent of the diet. Ingestion of bananas

Principles of Analysis and Current Usage
Chemically, catechols are benzene rings with two
adjacent hydroxy groups. In catecholamines, the amine
function is in a side chain, as shown in Figure 1 for
norepinephrine, epinephrine, and dopamine. These are
the three catecholamines most commonly measured in
urine. In neutral or alkaline solutions, the dihydroxy
group is easily oxidized, with the loss of two electrons
and two protons as shown in Figure 2.
Catecholamines are found in urine both in the free and
conjugated form, primarily as sulfates. The excretion
rate of conjugated urinary norepinephrine and
epinephrine is about three times that of the free
catecholamines [1,2]. The conjugates can be hydrolyzed
by being boiled in acid before sample extraction and
included in the total catecholamines measured. Most
studies on the clinical significance of catecholamines
have been done with free catecholamine measurements,
since these are more closely related to the tumor load of
pheochromocytoma than measurements of conjugated
catecholamines [3]. Since dietary catechols are
extensively conjugated before reaching the systemic
circulation, free catecholamine measurements are also
i
Catecholamines (Urine)
Previous and current authors of this method:
First edition: Not done
Methods edition: Not done
Second edition: Not done
Third edition: Not done
Fourth edition: Lawrence E. Webb
Fifth edition: Brett McWhinney
or synthetic vanilla has been reported to increase the
excretion of conjugated catecholamines [4]. For these
reasons, the hydrolysis step is usually omitted, and only
the free catecholamines are measured.
All urine catecholamine methods require careful sample
purification because of the many closely related amines
and catechols found in urine. Currently, ion-exchange
chromatography, adsorption chromatography, or a
combination of both of these techniques is most often
used to isolate the catecholamines. For ion-exchange
purification, the urine sample is adjusted to a neutral pH
and passed through a cation-exchange resin. The amines
are positively charged and are retained by the resin.
Amino acids such as 3,4-dihydroxyphenylalanine (dopa)
are not retained, since at a pH above 3, they are
uncharged zwitterions. The amines are then eluted with
strong acid or with a solution of high ionic strength.
Additional specificity can be conferred by eluting with
boric acid, which forms a complex with cis-diol groups
as shown in Figure 3. The complexed catecholamines in
the eluate are stable for several days [5]. Alumina is used
to separate catechols from noncatechols by adsorption of
the former onto alumina at pH 8.6. The catechols are
eluted with acid. A small amount of alumina is mixed
with the sample and buffer, the supernatant is removed
by aspiration, and the catecholamines are eluted with
only 200 to 300 L of acid, providing a convenient
preconcentration of the catecholamines before
quantitation. Purification on a column of immobilized
boric acid offers advantages of rapidity, specificity, and
high recovery [6,7], and this technique may become
345
Catecholamines (Urine)
more common in the future now that pre-prepared
columns are commercially available.
Low detection limits are an absolute necessity for
catecholamine analyses, since samples may have
concentrations as low as 1 ng/mL. The sensitivity of
fluorometric or electrochemical detectors is required.
Ultraviolet detection, though used in early high-
performance liquid chromatographic (HPLC) methods
[8], is not sufficiently sensitive.
Current methods of analysis can be conveniently
classified according to their capabilities of separating
norepinephrine, epinephrine, and dopamine from each
other in the purified sample. The older methods do not
make this separation and measure only the total
catecholamines, or else they differentiate norepinephrine
and epinephrine indirectly. Norepinephrine and
epinephrine are distinguished either by their differing
rates of oxidation at pH 3 and pH 6 or by their differing
fluorescence intensities at two different excitation and
emission wavelengths. The latter procedure is easier to
do, but the former provides better discrimination [5].
HPLC methods, on the other hand, separate
norepinephrine, epinephrine, and dopamine
chromatographically and measure each of these
compounds as it appears in the chromatographic eluate.
The older methods (Table 1, Method 1) depend on the
conversion of norepinephrine and epinephrine into
derivatives called fluorophores which emit a
characteristic fluorescence greater than the native
fluorescence of the parent amine. The most commonly
used of these methods is the trihydroxyindole (THI)
method [1,5,9,10], so called because the fluorophore
contains three hydroxyl groups and an indole ring. The
reaction sequence and associated chemical structures for
epinephrine are shown in Figure 4. After isolation,
usually by a cation-exchange column, norepinephrine
and epinephrine are oxidized with potassium
ferricyanide to noradrenochrome and adrenochrome,
respectively. These rearrange in the alkaline medium to
form adrenolutins. Ascorbic acid must be added and the
adrenolutin fluorescence measured quickly (405 nm
excitation, 495 nm emission), because these derivatives
are easily oxidized. The method is summarized in Table
1, Method 1a. Dopamine is not included in the reaction.
This catecholamine can be detected separately using
periodate as the oxidant [11,12]. More recently,
norepinephrine, epinephrine, and dopamine have been
measured simultaneously using iodine as an oxidizing
agent; differential analysis of these three catecholamines,
however, requires measurement of fluorescence at three
different wavelengths (excitation/emission: 310/365,
386/480, 410/500 nm) after oxidation at two different
pHs [7].
In the ethylenediamine (EDA) method (Table 1, Method
1b), the adrenochrome formed from epinephrine is
condensed with 1 mole of ethylenediamine to form a
fluorophore [13], as shown in Figure 5. The
condensation of norepinephrine with ethylenediamine is
more complex and less well understood. One
representation of the reaction sequence is shown in
Figure 6. The catecholamines are isolated by a cation-
exchange column or alumina [14,15,16]. The eluate is
mixed with ethylenediamine, the condensation products
are extracted with isobutanol, and the fluorescence is
measured (Table 1, Method 1b) using 435 nm as the
exciting wavelength and 560 nm as the emitting
radiation.
HPLC methods can be divided into HPLC-F and HPLC-
EC procedures according to whether fluorometric (F) or
electrochemical (EC) detection is used (Table 1,
Methods 2a-2c). The catecholamines are isolated and
concentrated by ion exchange preceded or followed by
alumina adsorption chromatography. The HPLC
separation of norepinephrine, epinephrine, and dopamine
is done by ion-exchange or reversed-phase ion-pair
chromatography. The microparticulate silica stationary
phase is bonded with sulfonic acid groups for ion
exchange or an alkyl organic group for reversed-phase
separations. In the reversed-phase system, an ion-pair
reagent, such as the sodium salt of heptylsulfonic acid, is
added to the mobile phase. There are several theories of
the separation mechanism [17,18]. The simplest
approach for interpretation of the reversed-phase
chromatography is to liken the column to a dynamic ion
exchanger. The hydrophobic part of the ion-pair reagent
is attracted to the reversed-phase column in a dynamic
exchange process. The column then presents negatively
charged sites to the analyte and acts as a cation-exchange
column.
Adequate sensitivity can be obtained with HPLC-F
methods using native fluorescence (200 nm excitation,
300 nm emission or 280 nm excitation, 320 nm
emission) [19] (Table 1, Method 2a). Fluorophores can
be formed by precolumn [20] or postcolumn [21,22]
derivatization, though the derivatization step is not
required for urine assay applications. The THI reaction
(Figure 6) has been used in a postcolumn derivatization
system for the detection of norepinephrine and
epinephrine by monitoring of fluorescence at 485 nm,
with excitation at 395 nm [21]. Derivatization with o-
phthalaldehyde and 2-mercaptoethanol (Figure 7) has
been applied to the analysis of norepinephrine,
dopamine, and other biogenic amines (340 nm
excitation, 480 nm emission) [20], but secondary amines
such as epinephrine do not react.
Electrochemical detectors are most commonly operated
in the amperometric mode, which means that the
electrode is maintained at a constant operating potential
[17]. This potential forces oxidation of the ortho-
dihydroxyl group, yielding an o-quinone (Table 1,
Method 2c). The electrons from this reaction pass into
the electrode, and the resulting current is measured as
each oxidizable analyte passes the detector. Lower
detection limits can be attained than with HPLC-F
methods that measure native fluorescence.
Reference and Preferred Methods
There is no established reference method at this time.
Selection of catecholamine methods requires a good
346
Catecholamines (Urine)
understanding of the advantages of the available sample
purification techniques. Adsorption of catechols on
alumina must be done at pH 8.6, and catecholamines are
very susceptible to oxidation at this pH. With ion-
exchange methods, the catecholamines are adsorbed at a
neutral or acidic pH, at which they are relatively stable.
With the weak carboxylic acid ion-exchange resin and
boric acid eluent, recoveries are consistently greater than
90%. The same column can be used to separate both
catecholamines and metanephrines. The metanephrines
are eluted with ammonia after elution of the
catecholamines.
Neither the ion-exchange nor the alumina methods are
completely specific for catecholamines. For the HPLC
methods, which depend on detection of the underivatized
catecholamines, ion exchange is usually used with
alumina to obtain the required specificity and sensitivity
[19]. Ion exchange has been used alone [23,24], but the
dilution of the sample by boric acid eluent has made it
difficult to obtain good precision for epinephrine
concentration within the healthy reference interval.
Where HPLC equipment is not available, the
trihydroxyindole (THI) method is most commonly used
and is the method of choice. The EDA method is used in
some of the older plasma catecholamine assays, but it is
generally agreed that it lacks the specificity required for
urine assays [14,16,25,26]. The sensitivity of the assay is
difficult to describe because it is dependent on the
fluorometer used and on sample elution volumes. With
filter fluorometers, as little as 0.01 g of norepinephrine
can be detected at a sensitivity adequate for human urine
[5]. The THI method presented here is commercially
available in a convenient kit form that provides the
carboxylic acid resin in the form of prefilled
minicolumns. The differentiation of norepinephrine and
epinephrine is not presented here; this separation and
quantitation should be performed by HPLC methods.
References may be consulted for the details of other
differential quantitation methods [5,9,25].
HPLC methods offer many advantages over the
traditional methods, which do not employ this
technology. Derivatization and the strict time limitations
of the associated reactions are not required. Sensitivity
can be extended to levels lower than 1 ng/mL of urine, if
required. Norepinephrine, epinephrine, and dopamine
are easily separated from each other and from
interferences, including drugs such as -methyldopa
(Aldomet), isoproterenol, quinidine, and propranolol
[17,19,24,27].
The THI and HPLC methods correlated fairly well, and
values from normal urine determined by the two
methods were in close agreement [19,24,27]. Correlation
of the THI and HPLC data was not so good for urines
from hypertensive patients who had stopped taking
drugs. This finding indicated that minor amounts of
drugs or drug metabolites may interfere with the THI
analysis [24]. When norepinephrine and epinephrine
values for normal urines were calculated from the THI
data by solution of a simultaneous equation, the THI
epinephrine values were twice the epinephrine values
determined by HPLC [24], in agreement with an earlier
study [28].
The method of choice for the routine measurement of
catecholamines in urine appears to be one of the HPLC
procedures. The THI method is not recommended for
differentiation and quantitation of norepinephrine and
epinephrine, but the THI method can be used to
determine the sum of these two catecholamines. If
possible, medications should be discontinued during the
urine collection period when the THI method is used.
Both ion-exchange and reversed-phase HPLC columns
have been used successfully; the reversed-phase columns
with ion-pair reagents have the advantage of predictable
and convenient adjustment of retention times by simply
varying the concentrations of ion-pair reagent and
organic modifier.
There is a good correlation between electrochemical and
fluorescence detection methods [19]; the choice between
them is a matter of personal preference and availability
of the technique in a particular laboratory. If plasma
catecholamines are also determined, electrochemical
detection is advantageous because it has sufficient
sensitivity to measure down to the low picogram per mL
concentration required for this assay. Fluorometric
detectors are simple to set up and operate, and freedom
from baseline noise and small interfering peaks is easily
obtained.
The methods presented here use fluorometric and
electrochemical detection. Both detectors may be used
simultaneously. The excitation and emission
wavelengths for the fluorescence method are optimized
for a Kratos Model 970 fluorometer with a deuterium
lamp. Fluorometers with xenon lamps may also be used
with 285 nm excitation [19].
Specimen
A 24-hour urine specimen is required. A 1983 study [29]
has confirmed that catecholamines deteriorate rapidly in
untreated urine at room temperature and that the simplest
effective treatment is acidification with hydrochloric
acid. Refrigeration offers an extra measure of security,
though this precaution may be impossible in the hospital
room. Once collected and properly preserved, urine may
be frozen.
It is essential that the urine be kept acidic during the
entire collection. At least 10 to 15 mL of 6 mol/L HCl
should be added to the collection vessel before the start
of the collection period, with the amount of acid added
adjusted according to the expected urine volume. The
addition of 4 mL of 5 mol/L sulfuric acid per 400 mL of
urine has been shown to be adequate [15]. Boric acid
should not be used as a preservative. When the
collection is completed, the pH should be less than 3.
Guidelines for Collection of Urine HMMA (VMA),
HVA, Catecholamines, and Metanephrines
Urine catecholamines and metabolites are unstable at a
pH above 3.0, so it is necessary to add HCl prior to
347
Catecholamines (Urine)

starting the urine collection. However, since excess HCl method, when used with a carboxylic acid ion-exchange
will interfere with the analysis of VMA and HVA (not resin and boric acid eluent, has generally good accuracy,
cats and mets) the urine collection should be commenced though drugs such as -methyldopa (Aldomet),
with a minimum of acid. This is particularly important in isoproterenol, and quinidine interfere [5]. Substances in
the case of pediatric patients being screened for urine may cause a quenching of fluorescence, which also
neuroblastoma, because low 24-hour urine volumes can interferes.
be expected. The following is a list of recommendations
for the amount of acid required per volume of urine: It should be recognized that a particular drug may
interfere in an individual HPLC system [28]. However,
an interfering substance can often be detected simply
Expected Urine Volume Add from the appearance of the HPLC chromatogram [27],
whereas the recognition of interferences in the THI
<100 mL 0.5 mL 6 N HCl methods is much more difficult and time consuming
100250 mL 3 mL 6 N HCl or [30].
1.5 mL conc HCl
250750 mL 6 mL 6 N HCl or Urine Catecholamines Reference Intervals
3 mL conc HCl Reference interval values should be established by each
7501250 mL 10 mL 6 N HCl or individual laboratory. Urinary dopamine excretions of
5 mL conc HCl 100 and 115 g/24 hr have been reported as upper limits
>1250 mL (most adults) 15 mL 6 N HCl or of normal [6,7]. Reference intervals established with the
10 mL conc HCl THI method have been generally found to agree with
those determined more recently by HPLC methods
[19,27]. Moyer et al. [27], in an extensive population
Interferences study using an HPLC method, found no significant age
The presence of catechols in bananas may cause falsely or sex differences in adult normal values, but excretion
increased concentrations in individuals eating bananas rates for children were much lower than in adults. Earlier
immediately prior to or during urine collection. The THI studies [31,32,33] based on the THI method also found
lower ranges for children, as follows:
Total* Urine Catecholamines Reference Intervals
Age (years) Norepinephrine Epinephrine
Newborn 0.573.3 g/day 0-0.77 g/day
1 5.415.9 0.14.3
15 8.130.8 0.89.1
615 19.071.1 1.310.5
>15 34.487.0 3.513.2
*Total urine catecholamines is the sum of free and conjugated catecholamines in urine.

However, excretion rates expressed in terms of excretion per kilogram of body weight were found to be higher than in
adults [32].

The reference intervals for free catecholamines in adults, as measured by HPLC, are as follows:

Free (Unconjugated) Urine Catecholamines Reference Intervals

Norepinephrine Epinephrine Dopamine
(g/24 h) (g/24 h) (g/24 h)
1580 020 65400

In a population study using an HPLC method with electrochemical detection, no significant age or sex differences were
evident in adult reference intervals [27]. Excretion rates for children were much lower than in adults.

Interpretation the nerve endings at the synapse, with only small

Circulating catecholamines are derived from either the amounts appearing in peripheral blood. Most (80%) of
adrenal medulla or peripheral nerves [34]. The adrenal the circulating norepinephrine and epinephrine and
medulla is the source of almost all the plasma essentially all the dopamine are excreted in the urine as
epinephrine, along with small amounts of the sulfate (predominantly) or glucuronide conjugates
norepinephrine. Norepinephrine and dopamine are [35].
released from the axonic terminals of adrenergic nerves
as a neurotransmitter. Catecholamines synthesized in the The most frequent use for urine catecholamine analysis
brain do not cross the blood-brain barrier. is the diagnosis of pheochromocytomas.
Norepinephrine is rapidly retaken up or metabolized by Pheochromocytomas are tumors derived from
348
Catecholamines (Urine)
chromaffin cells distributed throughout the body. Most
of these cells and thus most (90%) of the
pheochromocytomas are located in the adrenal medulla.
Most of the extra-adrenal tumors are located in the
abdomen. Only 10% of pheochromocytomas are
bilateral, except in cases of familial
pheochromocytomas, when the rate exceeds 50% [36].
Although pheochromocytomas are relatively uncommon
(only 1 in 200 hypersensitive patients), it is important to
detect these tumors. Removal of a pheochromocytoma
will usually cure uncontrollable and sometimes fatal
hypertension. In addition, some pheochromocytomas are
malignant (less than 5%), but they metastasize slowly.
The diagnosis of a pheochromocytoma can alert the
physician to other associated endocrine disturbances that
are part of a familial multiple endocrine neoplasia
syndrome (MEN II) [36].
A urinary catecholamine analysis is usually requested for
patients whose hypertension is poorly controlled by the
usual drug and diet regimens. The existence of a
pheochromocytoma as an organic cause of the
hypertension must be ruled out for the reasons listed
above. The hypertension in these patients is usually
continuous but may be intermittent. Widely fluctuating
or paroxysmal blood pressure changes are noted in three-
quarters of the patients known to have
pheochromocytomas [36]. This finding is considered to
reflect the intermittent release of catecholamines from
the tumors themselves. Frequently associated clinical
symptoms, including headache, excessive perspiration,
and palpitations, also tend to occur as paroxysmal
events, lasting from minutes to days. The presence of all
these symptoms has a clinical diagnostic sensitivity for
pheochromocytoma of 90.3% and a specificity of 93.8%.
The absence of all four signs has reported a negative
predictive value of 99.9% [37].
It is because of the paroxysmal nature of catecholamine
release that a 24-hour urine collection is considered the
preferred specimen, since the catecholamine excretion is
averaged over a long period. Shorter timed collections
(12 hours or random [spot] urines) have been
suggested, for which the catecholamine excretion rate
would be normalized to creatinine excretion (milligrams
of catecholamine per gram of creatinine). However, this
technique is not widely used. Greater than 90% of
patients with pheochromocytoma will have a urine
excretion of catecholamines more than twice the upper
limit of normal [38]. Most others will require
consecutive urine analyses to demonstrate a pattern of
elevated catecholamine excretion. The measurement of
at least one other indicator of catecholamine synthesis
and excretion is advocated. The most frequently
measured analyte is HMMA or vanillylmandelic acid
(VMA). The repeated elevation of several parameters of
catecholamine excretion can be used as biochemical
evidence for the presence of a pheochromocytoma.
Rarely, the measurement of the urinary catecholamines
and their metabolites will not establish a clear diagnosis
of pheochromocytoma, although the clinical indications
are very suggestive of the disease. In these cases, the
measurement of plasma free metanephrines would be
recommended [39].
Urine Catecholamine Performance Goals
Studies suggest that the precision of the THI method
(coefficient of variation [CV] 10% to 15%) is less than
that of the HPLC method [19,24,27]. Long-term
precisions (%CV) for norepinephrine and epinephrine
measured in urine by HPLC were found to be 3.3% at a
concentration of 48 g/L and 5.3% at a concentration of
13 g/L, respectively. This better precision for the
HPLC method can be attributed to at least two factors:
(1) the better sensitivity of the HPLC method and (2) the
addition of an internal standard to each urine processed
by the HPLC method. The internal standard is used to
correct for variation in catecholamine recovery.
References
1 Crout JR. Catecholamines in urine. In: Seligson
D, ed. Standard Methods of Clinical Chemistry.
Vol 3. New York: Academic Press; 1961:62-80.
2 Khane Z, Esser AH, Kline NS, Vestergaard P.
Estimation of conjugated epinephrine and
norepinephrine in urine. J Lab Clin Med
1967;69:1042-1050.
3 Causon RC, Brown MJ. Catecholamine
measurements in phaeochromocytoma: a
review. Ann Clin Biochem 1982;19:396-404.
4 Crout JC, Sjoerdsma A. The clinical and
laboratory significance of serotonin and
catecholamines in bananas. N Engl J Med
1959;261:23-26.
5 Sandhu RS, Freed RM. Catecholamines and
associated metabolites in human urine. In:
Seligson D, ed. Standard Methods of Clinical
Chemistry. Vol 7. New York: Academic Press;
1972:231-246.
6 Speek AJ, Odink J, Schrijver J, Schreurs WHP.
High-performance liquid chromatography
determination of urinary free catecholamines
with electrochemical detection after
prepurification on immobilized boric acid. Clin
Chim Acta 1963;128:103-113.
7 Oka K, Sekiya M, Osada H, Fujita K, Kato T,
Nagatsu T. Simultaneous fluorometry of urinary
dopamine, norepinephrine, and epinephrine
compared with liquid chromatography with
electrochemical detection. Clin Chem
1982;28:646-649.
8 Mell LD, Gustafson AB. Urinary free
norepinephrine and dopamine determined by
reverse-phase high-pressure liquid
chromatography. Clin Chem 1977;23:473-476.
9 Catecholamines by Column Test: Instruction
Manual. Bio-Rad Laboratories, Richmond, CA
94804.
10 Sobel C, Henry R. Determination of
catecholamines (adrenalin and noradrenalin) in
urine and tissue. Am J Clin Pathol 1957;27:240-
245.
11 Anton AH, Sayre DF. The distribution of
dopamine and DOPA in various animals and a
method for their determination in diverse
349
Catecholamines (Urine)
biological material. J Pharmacol Exp Ther
1964;145:326-366.
12 Routh JI, Bannow RE, Fincham RW, Stoll JL.
Excretion of L-dopa and its metabolites in urine
of Parkinsons disease patients receiving L-
dopa therapy. Clin Chem 1971;17:867-871.
13 Natelson S, Lugovoy J, Pincus J. A new
fluorometric method for the determination of
epinephrine. Arch Biochem 1949;23:157-158.
14 Natelson S. Techniques of Clinical Chemistry.
3rd ed. Springfield, IL: Charles C Thomas;
1971:237-244.
15 Weil-Malherbe H. The chemical estimation of
catecholamines and their metabolites in body
fluids and tissue extracts. Methods Biochem
Anal 1971;Suppl:119-152.
16 Manger WM, Steinsland OS, Nahas GG,
Wakim KG, Dufton S. Comparison of improved
fluorometric methods used to quantitate plasma
catecholamines. Clin Chem 1969;15:1101-
1123.
17 Urinary Catecholamines by LCEC. LCEC
Application Note No. 15. Bioanalytical Systems
Inc., Purdue Research Park, West Lafayette, IN
47906.
18 Krstulovic AM, Dziedzic SW, Bertani-Dziedzic
L, DiRico DE. Plasma catecholamines in
hypertension and pheochromocytoma
determined using ion-pair reversed-phase
chromatography with amperometric detection:
investigation of the separation mechanism and
clinical methodology. J Chromatogr
1981;217:523-537.
19 Anderson GM, Young JG, Jatlow PE, Cohen
DJ. Urinary free catecholamines determined by
liquid chromatography-fluorometry. Clin Chem
1981;27:2060-2063.
20 Davis TP, Gehrke CW, Gehrke CW Jr,
Cunningham TD, Kuo KC, Gerhardt KO et al.
High-performance liquid chromatographic
analysis of biogenic amines in biological
materials as o-phthalaldehyde derivatives. J
Chromatogr 1979;162:293-310.
21 Yamatodani A, Wada H. Automated analysis
for plasma epinephrine and norepinephrine by
liquid chromatography, including a sample
cleanup procedure. Clin Chem 1981;27:1983-
1987.
22 Yui Y, Fujita T, Yamamoto T, Itokawa Y,
Kawai C. Liquid-chromatographic
determination of norepinephrine and
epinephrine in human plasma. Clin Chem
1980;26:194-196.
23 Jackman GP. Differential assay for urinary
catecholamines by use of liquid
chromatography with fluorescence detection.
Clin Chem 1981;27:1202-1204.
24 Schleicher ED, Kees FK, Wieland OH.
Analysis of total urinary catecholamines by
liquid chromatography: methodology, routine
experience and clinical interpretations of
results. Clin Chim Acta 1983;129:295-302.
25 Anton AH, Sayre DF. A study of the factors
affecting the aluminum oxide-trihydroxyindole
procedure for the analysis of catecholamines. J
Pharmacol Exp Ther 1962;138:360-378.
26 Viktora JK, Baukal A, Wolff FW. New
automated fluorometric method for estimation
of small amounts of adrenalin and noradrenalin.
Anal Biochem 1968;23:513-528.
27 Moyer TP, Jiang NS, Tyce GM, Sheps SG.
Analysis for urinary catecholamines by liquid
chromatography with amperometric detection:
methodology and clinical interpretation of
results. Clin Chem 1979;25:256-263.
28 Jones TA, Walwick ER. Interference of Tylenol
with liquid chromatography of urinary
catecholamines. Clin Chem 1981;27:1951.
29 Giles HG, Meggiorini S. Stability of
catecholamines in urine. Clin Chem
1983;29:595.
30 Jacobs SL, Sobel C, Henry RJ. Specificity of
the trihydroxyindole method for determination
of urinary catecholamines. J Clin Endocrinol
Metab1961;21:305-314.
31 Grenburg RE, Gardner LE. The excretion of
free catecholamines by newborn infants. J Clin
Endocrinol Metab 1960;20:1207-1214.
32 Voorhees ML. Urinary catecholamine excretion
by healthy children. 1. Daily excretion of
dopamine, norepinephrine, epinephrine, and 3-
methyl-4-hydroxymandelic acid. Pediatrics
1967;39:252-257.
33 Meites S. Pediatric Clinical Chemistry.
Washington, DC: American Association for
Clinical Chemistry Press; 1977:66-67.
34 Krakoff LR. Pheochromocytoma and clinical
hypertension: pathogenesis and diagnosis.
Cardiovasc Res Rep 1982;3:199-206.
35 Kuchel O, Buu NT, Hamet P, Larochelle P,
Bourque M, Genest J. Essential hypertension
with low conjugated catecholamines imitates
pheochromocytoma. Hypertension 1981;3:347-
355.
36 Cryer PE. Diseases of the adrenal medullae and
sympathetic nervous system. In: Felig P, Baxter
JD, Broadus AE, Frohman LA, eds.
Endocrinology and Metabolism. New York:
McGraw-Hill; 1981:529-545.
37 Gambino SR. Laboratory diagnosis of
pheochromocytoma. Lab Rep Physicians
1982;4:75-79.
38 Sjoerdsma A, Engelman K, Waldman TA et al.
Pheochromocytoma: current concepts of
diagnosis and treatment. Ann Intern Med
1966;65:1302-1326.
39 Eisenhofer G. Biochemical diagnosis of
pheochromocytoma: is it time to switch to
plasma-free metanephrines? J Clin Endocrinol
Metab 2003;88:550-552.
350
Catecholamines (Urine)

Tables
Table 1: Methods of Urine Catecholamine Analysis
Method 1: Fluorescence derivatives; total (NE, E) or NE, E individually without prior chromatographic separation
a. Trihydroxyindole (THI)
b. Ethylenediamine (EDA)
Principle of analysis: Formation of derivatives, which are quantitated fluorometrically. Differential analysis of
NE and E by oxidation at pH 3 and pH 6 or by fluorescence at two different excitation and emission wavelengths.
a. Isolation by weak cation-exchange resin, followed by oxidation to fluorescent adrenolutins:
NE, E _ferricyanideadrenochromes _ascorbic acidadrenolutins
alkaline pH
pH 6.5
405 nm excitation, 495 nm emission (436 nm excitation and 520 nm emission for differential assay)
b. Isolation by adsorption on alumina or by cation-exchange chromatography. Oxidation to
adrenochromes followed by condensation with EDA to form fluorophores, which are extracted into isobutanol.

NE, E ___ adrenochromes ____EDA fluorophores

pH 6 condensation

Comments: Does not include dopamine

a. Common when HPLC not available; generally good accuracy but some drugs interfere
b. Historical; limited specificity
Method 2: High-performance liquid chromatography (HPLC); HPLC separation and quantitation of NE, E, and DA
a. Native fluorescence (HPLC-F)
b. HPLC with fluorescent derivatives
c. Electrochemical (HPLC-EC)
Principle of analysis: Isolation by cation-exchange chromatography and alumina adsorption; HPLC separation
by cation-exchange or ion-pair chromatography
a. Monitor eluate at 200 nm excitation, 300 nm emission or 280 nm excitation, 320 nm emission
b. Precolumn or postcolumn derivatization, such as THI formation (method 1a)
c. With amperometric detector, monitor eluate at 0.65 volts and detect current from the reaction (Figure
2)
Comments: Good sample cleanup required
a. Common where HPLC available; good sensitivity; excellent specificity
b. Rare; excellent sensitivity; not required for urines
c. Common where HPLC available; excellent sensitivity; good specificity

DA, Dopamine; E, epinephrine; NE, norepinephrine.

351
Catecholamines (Urine)

Table 2: Reaction Conditions for Catecholamine Analysis THI Method

Temperature 20C to 25C

pH 6.5 0.02
Final concentration of reagent ZnSO4: 0.29 mmol/L
components K3FE(CN)6: 0.26 mmol/L
Sodium ascorbate: 3.6 mmol/L
NaOH: 0.69 mol/L
Phosphate buffer: 0.17 mol/L
Volume of sample 5 mL
Fraction of sample volume 0.30
Sample Urine
Linearity (depending on At least 0 to 300 g/L
fluorometer used)
Time of reaction Add ferricyanide: 0 min
Add alkaline ascorbate: 2 min
Read fluorescence within 20 min
Excitation wavelength 405 nm
Emission wavelength 495 nm
Major interferences Drugs, such as -methyldopa (Aldomet),
isoproterenol, quinidine
Precision (between-day) Normal X = 30 g/L, CV = 15%
Abnormal X = 250 g/L, CV = 8%

CV, Coefficient of variation.

Table 3: Chromatographic Conditions for Urine Catecholamine Analysis by HPLC

Condition: Temperature
Comment: Ambient
Precision (with fluorometric method) (between-day): Norepinephrine
Low normal (X = 15 ng/mL), CV = 8.1%
Abnormal (X = 499 ng/mL), CV = 1.8%
Condition: Flow rate
Comment: 2.5 mL/min
Precision (with fluorometric method) (between-day): Epinephrine
Normal (X = 16 ng/mL), CV = 16.9%
Abnormal (X = 497 nm/mL), CV = 1.9%
Condition: Excitation wavelength
Comment: 200 nm
Precision (with fluorometric method) (between-day): Dopamine
Low normal (X = 36 ng/mL), CV = 11.9%
Abnormal (X = 505 ng/mL), CV = 3.1%
Condition: Emission wavelength
Comment: 300 nm
Condition: Electrode
Comment: Glassy carbon
Condition: Applied potential
Comment: 0.65 volts versus Ag/AgCl electrode
Condition: Sample
Comment: Urine
Condition: Linearity
Comment: 0-80 ng/mL
Condition: Detectability limit
Comment: 1 ng/mL (lower with electrochemical detector)
Condition: Interferences
Comment: No interferences from drugs such as -methyldopa (Aldomet), isoproterenol, or quinidine;
methyltyrosine can interfere with NE, whereas L-dopa medication is broken down into dopamine

CV, Coefficient of variation; NE, norepinephrine and epinephrine.

352
Catecholamines (Urine)

Table 4: Excretion Rates (Range in g/24 hr) of Free Catecholamines for Children 0 to 15 Years of Age
Age (years)
Catecholamine 0-1 1-2 2-4 4-7 7-10 10-15
Norepinephrine 0*-10 1-17 4-29 8-45 13-65 15-80
Epinephrine 0-2.5 0-3.5 0-6.0 0.2-10 0.5-14 0.5-20
Dopamine 0-85 10-140 40-260 65-400 65-400 65-400

*0 implies not detected.

Figures

Figure 1: Three catecholamines

commonly measured in urine.

______________________________________________________________________________

Figure 2: Oxidation of o-dihydroxy groups of the

catechol core, with formation of quinone derivative.

__________________________________________________________________________________
353
Catecholamines (Urine)

Figure 3: Formation of cis-diol complexes

between boric acid and catecholamines.

__________________________________________________________________

Figure 4: Sequence of reactions

converting catecholamines to
trihydroxyindole derivatives, the
adrenolutins.
In this example, epinephrine is
first converted to adrenochrome
and then oxidized to adrenolutin.
354
Catecholamines (Urine)

Figure 5: Sequence of reactions

converting epinephrine to a
fluorescent condensation
product with ethylenediamine.

Figure 6: Sequence of reactions converting norepinephrine to a fluorescent condensation product with

ethylenediamine.

____________________________________________________________________________
355
Catecholamines (Urine)

Figure 7: Reaction of o-phthalaldehyde with norepinephrine and 2-mercaptoethanol to form a fluorescent isoindole
product.
Only primary amines, such as norepinephrine and dopamine, will undergo this reaction. (Modified from Davis TP et al.
High-performance liquid chromatographic analysis of biogenic amines in biological materials as o-phthalaldehyde
derivatives. J Chromatogr 1979;162:293-310.)

Figure 8: Example of standard curve for trihydroxyindole fluorescence assay for catecholamines.
Corrected (relative, Rel.) fluorescence at 495 nm (excitation at 405 nm) versus concentration of catecholamine.

Figure 9: Fluorescence spectra of trihydroxyindole derivatives of catecholamines.

A, Norepinephrine; B, epinephrine; C, a typical urine.

A, Activation spectrum; B, blank; F, fluorescence spectrum; IS, internal standard S, standard; U, urine. (From Jacobs SL et
al. Specificity of the trihydroxyindole method for determination of urinary catecholamines. J Clin Endocrinol Metab
1961;21:305-314.)
356
Catecholamines (Urine)

Figure 10: Excitation spectra for norepinephrine.

A, epinephrine, B, and dopamine, C, in 0.1 M perchloric acid.

Figure 11: Example of chromatographic pattern of

catecholamine standard (40 ng/mL): relative detector
response versus reaction time (in min).
DA, Dopamine; DHBA, dihydroxybenzylamine; E,
epinephrine; ECD, electrochemical response; FLUOR,
fluorescence response; NE, norepinephrine. Vertical
arrows on horizontal axes, Time of injection; numbers on
chromatogram, retention times of corresponding peaks.
357
Catecholamines (Urine)

Figure 12: Example of

chromatographic pattern for a
patient sample. Abbreviations as for
Figure 11.

Figure 13: Example of

standard curve for
fluorescence HPLC assay for
catecholamines.
Peak area ratio
(analyte/DHBA) versus
concentration (20, 40, 80, and
200 ng/mL standards). ( )
and , Norepinephrine; (- - - - -
) and , epinephrine; ()

and , dopamine.
358
Catecholamines (Urine)
Procedure: Trihydroxyindole Method
Principle
The urine sample is adjusted to pH 6.5 and poured
through a column filled with a weak cation-exchange
resin. After the catecholamines are eluted with boric
acid, norepinephrine and epinephrine are oxidized by
ferricyanide in the presence of zinc ions to form
adrenochromes. In alkaline medium, the adrenochromes
rearrange to form adrenolutins. Ascorbic acid is added to
destroy excess oxidizing agent and to prevent further
oxidation of the adrenolutins. The fluorescence of the
adrenolutins is measured at 495 nm, with excitation at
405 nm. Quenching in samples prepared from urine may
reduce a fluorometric reading relative to that obtained in
an aqueous standard [1,5]. Standards are therefore
prepared in a urine matrix to make their fluorescence
characteristics as close as possible to those of the
specimens (Table 2).
Reagents
The procedure is based on Catecholamines by Column
Test Kit (Bio-Rad Laboratories, Richmond, CA 94804),
which includes columns and all reagents. Instructions are
included here for those who prefer to prepare their own
reagents. All water used must be deionized, and reagents
should be reagent grade.
1. Disodium ethylenediaminetetraacetic acid
(Na2EDTA), 1 g/L (2.7 mmol/L). Dissolve 1 g of
Na2EDTA in 1 L of water. Stable 1 year at room
temperature.
2. Sodium hydroxide (5 mol/L, 20%). Dissolve
20 g of NaOH pellets in 100 mL of water. Stable 1 year
at room temperature.
3. Sodium hydroxide (0.5 mol/L). Dilute 5 mL
of 5 mol/L NaOH to 50 mL with water. Stable 1 year at
room temperature.
4. Hydrochloric acid (0.1 mol/L). Dilute 0.85
mL of concentrated HCl to 100 mL with water. Stable 1
year at room temperature.
5. Boric acid, 40 g/L (0.65 mol/L). Dissolve 40 g
of boric acid in 1 L of water. The crystal form dissolves
more readily than the powder form. Stable 1 year at
room temperature.
6. Phosphate buffer (1.0 mol/L, pH 6.5).
Dissolve 6.38 g of Na2HPO4 and 7.52 g of KH2PO4 in
approximately 75 mL of water with warming. Cool.
Adjust pH to 6.5 0.02. Make volume up to 100 mL
with water. Stable 1 month at 4C. Discard if solid
material forms.
7. Zinc sulfate (ZnSO47H2O), 2.5 mg/mL (8.7
mmol/L). Add 10 mL of water to 25 mg of
ZnSO47H2O and mix well. Stable 1 week at 4C.
8. Potassium ferricyanide, 2.5 mg/mL (7.6
mmol/L). Add 10 mL of water to 25 mg of K3Fe(CN)6
and mix well. Stable 1 week at 4C. If crystals form
under refrigeration, allow to stand at room temperature
until crystals dissolve. If the solution turns from pale
yellow to green, it should be discarded.
9. Ascorbic acid. Weigh out 100 mg quantities
and store at room temperature in tightly sealed vials.
Stable 1 month.
10. Ascorbic acid solution, 20 mg/mL (0.11
mol/L). Prepare immediately before use by dissolving
100 mg of ascorbic acid in 5 mL of water.
11. Alkaline ascorbic solution (4.1 mol/L NaOH,
21 mmol/L sodium ascorbate). Prepare immediately
before use by mixing 18 mL of 5 mol/L NaOH with 4
mL of 20 mg/mL ascorbic acid.
12. Standard curve. Should be obtained with each
set of determinations.
Normal urine. Use a urine known from a
previous run to have low blank and test readings with the
fluorometer used for the assay. Stable 4 months at 4C at
pH 3 0.1.
Stock standard (norepinephrine 100 mg/L).
Dissolve 60.8 mg of norepinephrine hydrochloride in
500 mL volumetrically with 0.1 mol/L HCl. Stable at
least 6 months at 4C.
1 mg/L standard. Dilute 1 mL stock to 100 mL
volumetrically with 0.1 mol/L HCl. Prepare on day of
run.
For the curve, run 0, 100, 200, 300 g/L
standards by mixing the 1 mg/L standard and normal
urine as follows:
Standard mL of 1 mg/ mL of mL of
(g/L) standard normal urine
0 0 5
100 0.50 4.5
200 1.00 4.0
300 1.50 3.5
Prepare these standards immediately before the analysis.
Linearity of the standard curve will exceed the
concentration of the highest standard; the linearity
should be checked with the fluorometer used for the
assay.
13. Controls. Controls are available commercially,
or they may be prepared from urine characteristic of the
specimens to be analyzed. To 100 mL of urine that has
been acidified to a pH less than 3, add 1 mL of 5 g/L
sodium metabisulfite and 1 mL of 50 g/L Na2EDTA. If
particulate matter is present, filter under vacuum through
coarse filter paper. Add norepinephrine standard for
controls in the abnormal range. Mix well. Place aliquots
in scintillation vials. Store in freezer (20C). Stable for
about 6 months.
14. Columns. These are filled with the weak
carboxylic acid ion-exchange resin Bio-Rex 70 and are
included in the Bio-Rad kit. Store in refrigerator and use
before expiration date. Columns can be prepared directly
from the Bio-Rex 70 resin itself [5,17,19]. However, the
mesh size of the bulk Bio-Rex 70 is not so closely
controlled as it apparently is for the prepacked columns,
and good flow characteristics through the columns may
be more difficult to obtain than with the prepacked
columns.
Assay
359
Catecholamines (Urine)
Equipment: (1) Fluorometer, either a filter type such as
Turner Model 111 (G.K. Turner Associates, Inc., Palo
Alto, CA 94303) or a diffraction grating instrument; (2)
stopwatch.
1. Set fluorometer at 405 nm excitation
wavelength and 495 nm emission wavelength
with appropriate settings or filters.
2. Pipet 5 mL of urine into a 50-mL beaker.
(Excessively turbid urines should be filtered or
centrifuged first.) Prepare each standard in a
separate 50-mL beaker.
3. To each beaker add 15 mL of 1 g/L Na2EDTA
and mix well.
4. Adjust the pH of the contents of each of the
beakers to 6.5 0.2 by adding 0.5 mol/L NaOH
dropwise and monitoring the pH with a pH
meter.
5. Prepare the resin columns, one for each sample
and one for each standard. Shake or rotate
gently to resuspend the resin, and then place
each column upright to allow the resin to settle.
Remove the caps, snap off the tips, and allow
the columns to drain.
6. Pour each sample and standard into a separate
column. Allow to drain completely, and discard
the eluate. Wash each column with 2 full
reservoirs (20 mL) of water, and discard the
eluate.
7. Place the column in a clean, appropriately
labeled 16 150 mm test tube, add 10.0 mL of
40 g/L boric acid to each column, collect each
eluate in its test tube, and mix.
NOTE: At this point, the samples are stable for
at least 96 hr if refrigerated.
8. Pipet from each tube two separate 3.5-mL
aliquots into two separate 16 100 mm test
tubes, one appropriately labeled blank and the
other test. To each tube add 1.0 mL of
phosphate buffer and mix.
9. Prepare the alkaline ascorbate solution, and add
1.0 mL of it to each tube labeled blank. Mix
well.
10. To each blank tube, add 0.2 mL of the zinc
sulfate solution and 0.2 mL of the K3Fe(CN)6
solution. Mix well.
NOTE: The reaction must be halted
immediately by quick and thorough mixing, or the
blanks will have a high value. The reaction is stopped
when the yellow of the K3Fe(CN)6 disappears. The
solutions must be clear. The following steps (steps 11
and 12) must be done in carefully timed sequence
without interruption. The additions at 15-sec intervals
allow 8 samples to be run as a batch in 2 min.
11. To each of the tubes labeled test: from step 8,
add 0.2 mL of the zinc sulfate solution and 0.2
mL of the K3Fe(CN)6 solution in sequence at
15-sec intervals, mixing well after the addition
of the second solution.
12. Precisely 2 min after the addition of zinc sulfate
and K3Fe(CN)6 solutions to the first tube, add
1.0 mL of alkaline ascorbate reagent to the first
tube, mix, and then continue adding 1.0 mL of
alkaline ascorbate reagent to each of the
remaining tubes in order at 15-sec intervals.
13. Read all tubes on fluorometer within 20 min.
Calculations
Calculate the net fluorescence reading for each unknown
and standard by subtracting the blank reading from the
corresponding test reading. Let
RF100 = F100 F0
RF200 = F200 F0
RF300 = F300 F0
where RF100, RF200, and RF300 are the relative
fluorescence readings of the 100, 200, and 300 g/L
standards; F100, F200, and F300 are the fluorescence
readings of these standards; and F0 is the fluorescence
reading of the 0 g/L standard.
Prepare a standard curve by plotting RF100,
RF200, and RF300 versus concentration. Use the curve
and the net fluorescence reading of the unknown sample
to find its concentration in micrograms per liter of urine.
A typical standard curve is given in Figure 8. Then:
g of catecholamines per 24 h = g/L
24-hr volume in liters
Interferences
Drugs such as -methyldopa (Aldomet), isoproterenol,
and quinidine interfere. A high blank reading indicates
possible interfering substances.
Spectrophotofluorometric examination of activation and
fluorescence spectra may be required to reveal
occasional instances of falsely high results caused by
interferences [24]. The fluorescence spectrum of the
trihydroxyindoles is given in Figure 9.
Catecholamines-Urine Reference Interval
click here
Procedure: High-Performance Liquid
Chromatography
Principle
The urine sample is adjusted to pH 6.5 and poured
through a column filled with a weak cation-exchange
resin. After the catecholamines are eluted with
ammonium sulfate, they are absorbed on alumina at pH
8.6. The catechols are eluted with acid and separated on
a reversed-phase column, using an ion-pair reagent in the
mobile phase. The eluted peaks are detected by
measurement of fluorescence at 300 nm after excitation
at 200 nm or by measurement of the current produced by
oxidation of the o-dihydroxy group (Table 3). The
excitation maximum at 280 nm (Figure 10) may also be
used with emission at 320 nm.
Reagents
The reagents are stable for a year unless otherwise noted.
All water used must be deionized, and reagents should
be reagent grade.
360
Catecholamines (Urine)
1. Norepinephrine hydrochloride (Sigma
Chemical Co. or equivalent). Store it desiccated in
freezer.
2. L-Epinephrine (Sigma or equivalent). Store
it desiccated in refrigerator.
3. Dopamine hydrochloride (Sigma, or
equivalent)
4. 3,4-Dihydroxybenzylamine hydrobromide
(DHBA)
5. Phosphoric acid (0.015 mol/L). Dilute 1 mL
of 85% phosphoric acid to 1 L with water.
6. Phosphoric acid (0.3 mol/L). Dilute 10 mL of
85% phosphoric acid to 500 mL with water.
7. Sulfuric acid (0.7 mol/L). Dilute 20 mL of
concentrated sulfuric acid to 500 mL with water.
8. Ammonium sulfate (2 mol/L). Place 132 g of
ammonium sulfate in a 500-mL volumetric flask, and
dilute to volume with deionized water. Stable when
stored in refrigerator.
9. Sodium metabisulfite (5 g/L, 26.3 mmol/L).
Dissolve 1 g of sodium metabisulfite in 200 mL of
water. Prepare fresh daily.
10. Disodium ethylenediaminetetraacetic acid,
Na2EDTA (50 g/L, 149 mmol/L). Dissolve 5 g of
Na2EDTA in 100 mL of water.
11. Tris-EDTA buffer, pH 8.6 (3 mol/L). Add
about 120 mL of water to 72 g of Trizma base (Sigma
Chemical Co. or equivalent) and 10 g of Na2EDTA. Stir
until dissolved. The solution may require warming for
complete dissolution. When the solution is at room
temperature, adjust the pH to 8.6 with concentrated HCl.
Transfer to a 200-mL volumetric flask, and dilute to
volume with deionized water. Stable for 4 to 6 months
when stored in refrigerator. Warm to room temperature
before using. Check pH before use.
12. Phosphate buffer, pH 7 (85.2 mmol/L).
Dissolve 9.64 g of Na2HPO4, 2.36 g of KH2PO4, and
20 g of Na2EDTA in about 950 mL of deionized water.
Adjust to pH 7.0 with 2 mol/L NaOH. Transfer to a
1000-mL volumetric flask, and dilute to volume with
deionized water. Store in refrigerator. Warm to room
temperature and check pH before use. Discard after 3
months, or earlier if bacterial growth appears.
13. Acid-washed alumina. Acid-washed alumina
is prepared according to the procedure of Anton and
Sayre [25]. Add 100 g of neutral alumina (Sigma,
activity grade 1, or equivalent) to 500 mL of 2 mol/L
HCl in a 1000-mL beaker. Cover with a watch glass, and
heat for 45 min at 90C to 100C, with continuous and
rapid stirring. Remove from heat and allow 1.5 min for
the heavier particles of alumina to settle. Discard the
supernatant and the finer particles of alumina. Wash the
precipitate twice with fresh 250-mL portions of 2 mol/L
HCl at 70C for 10 min, discarding the supernatant and
the finer alumina particles each time. Wash the
precipitate with 500 mL of 2 mol/L HCl at 50C for 10
min. Decant the HCl. Wash the precipitate repeatedly
(about 20 to 25 times) with fresh 200-mL portions of
deionized water, decanting the finer particles each time
until the pH of the supernatant is 3 to 4. Transfer the
alumina to an evaporating dish; heat at 120C for 1 h and
then at 200C for 2 hr. Store in a tightly sealed bottle in
a desiccator. Stable at least 1 year.
14. Mobile phase. Dissolve 19.05 g of KH2PO4,
800 mg of 1-heptanesulfonic acid sodium salt (HPLC
grade) and 74 mg of Na2EDTA in about 1850 mL of
deionized water. Adjust the pH to 4.0 with 85%
phosphoric acid. Transfer the solution to a 2000-mL
volumetric flask, add 80 mL acetonitrile (HPLC grade),
and dilute to volume with deionized water. Stable 1
week when stored in a refrigerator. Immediately before
using, filter through a 0.45 m filter and degas by
stirring under vacuum for 5 to 10 min.
15. Standards. The working standards are
included with each set of determinations as described in
the procedure.
Stock catecholamine standards (100 mg/L as
free base)
Norepinephrine. Dissolve 60.8 mg of
norepinephrine hydrochloride in 500 mL volumetrically
with 0.015 mol/L phosphoric acid.
Epinephrine. Dissolve 50.0 mg of epinephrine
in 500 mL volumetrically with 0.015 mol/L phosphoric
acid.
Dopamine. Dissolve 61.9 mg of dopamine
hydrochloride in 500 mL volumetrically with 0.015
mol/L phosphoric acid.
These standards are stable for at least 6
months when stored tightly sealed in amber glass bottles
in a refrigerator.
Combined working standard (1 mg/L
norepinephrine, epinephrine, and dopamine). Pipet 1.0
mL of each stock standard into a 100-mL volumetric
flask, and dilute to volume with 0.015 mol/L phosphoric
acid. Prepare fresh daily.
Norepinephrine working standard (1 mg/L).
Dilute 1.0 mL of norepinephrine stock standard to 100
mL with 0.015 mol/L phosphoric acid. Stable as a
chromatographic marker for at least 6 months when
stored in a refrigerator.
Stock internal standard (100 mg/L). Dissolve
79.1 mg of DHBAHBr in 500 mL volumetrically with
0.015 mol/L phosphoric acid. Stable for at least 6
months when stored tightly sealed in a refrigerator.
Working internal standard (1 mg/L DHBA).
Dilute 1 mL of DHBA stock standard to 100 mL with
0.015 mol/L phosphoric acid. Prepare fresh daily.
16. Controls. Controls are available commercially
but, in my experience, have not given satisfactory
chromatograms, partly because these controls contain
very little epinephrine. Controls satisfactory for HPLC
assay may be prepared from urine collections. To 100
mL of urine which has been acidified to a pH less than 3,
add 1 mL of 5 g/L sodium metabisulfite and 1 mL of 50
g/L Na2EDTA. If particulate matter is present, filter
under vacuum through coarse filter paper. Add
catecholamine standards as required for controls in the
abnormal range. Mix well. Place aliquots in scintillation
vials. Store in freezer at 20C. Stable for about 6
months.
17. Columns. Columns are filled with the weak
carboxylic acid ion-exchange resin Bio-Rex 70 and are
included in Catecholamines by Column Kit (Bio-Rad
361
Catecholamines (Urine)
Laboratories, Richmond, CA 94804) or can be ordered
separately (Bio-Rad). Columns can be prepared directly
from the Bio-Rex 70 resin itself [2,14,16] (click here).
However, the mesh size of the bulk Bio-Rex 70 is not so
closely controlled as it apparently is for the prepacked
columns, and good flow characteristics through the
columns may be more difficult to obtain than with the
prepacked columns.
Assay
Equipment: (1) HPLC equipped with either a
fluorometric detector (Kratos Analytical Instruments
Model FS970 or equivalent) or an electrochemical
detector (Bioanalytical Systems Model LC-4 or
equivalent) with glassy carbon electrode, (2) integrating
recorder, (3) du Pont Zorbax ODS column, 7 m particle
size (4.6 mm inner diameter 25 cm) or equivalent, (4)
rotary mixer or reciprocal shaker, (5) microcentrifuge
capable of 500 g.
Extraction of Standards and Samples
1. Prepare the catecholamine isolation columns,
one for each sample and standard. Shake the
columns, or rotate them gently to resuspend the
resin, and then place each column upright to
allow the resin to settle.
2. Transfer a 5-mL aliquot of each sample to a 30-
mL beaker.
3. For low, middle, and high standards, transfer
100, 200, and 400 L, respectively, of the
combined working standard to separate 30-mL
beakers. These standards correspond to 20, 40,
and 80 ng of norepinephrine, epinephrine, and
dopamine per milliliter.
4. Add 200 L of the working DHBA internal
standard and 15 mL of pH 7 phosphate buffer to
each beaker.
5. Adjust the pH to 6.50 0.02 using 2 mol/L
NaOH.
6. Pour each sample and standard onto a separate
column. Allow to drain completely. Discard
eluate.
7. Add 10 mL water. Allow to drain completely.
Discard eluate.
8. Add 1.5 mL of 0.7 mol/L sulfuric acid. Allow
to drain completely. Discard eluate.
9. Elute the catecholamines into test tubes with 4
mL of 2 mol/L ammonium sulfate.
10. Add 100 L of 5 g/L sodium metabisulfate to
each tube and mix.
11. NOTE: Complete this step for each sample
individually, one at a time. Add 0.5 mL of 3
mol/L Tris/EDTA buffer to each tube.
Immediately adjust the pH to 8.60 0.03 using
2 mol/L NaOH. Pour into a 5-mL conical
reaction vial containing approximately 50 mg
of acid-washed alumina. Put the vial on a rotary
mixer or reciprocal shaker.
12. Rotate or shake each vial for at least 15 min.
13. Allow alumina to settle, and remove
supernatant by aspiration.
14. Wash the alumina twice with aliquots of about
2 mL of water. Agitate the alumina thoroughly
by squirting the water vigorously from a wash
bottle into the reaction vial. Allow the alumina
particles to settle, and remove the water by
aspiration after each wash. Remove as much
water as possible after the second wash without
removing any alumina.
15. Add 300 L of 0.3 mol/L phosphoric acid to the
alumina to elute the catecholamines. Vortex
mix gently, and let stand for 5 min. Transfer the
supernatants to polypropylene centrifuge tubes.
Centrifuge at 500 g in a microcentrifuge for at
least 5 min. Transfer the supernatants to
separate tubes for injection into the HPLC.
Stable for at least 1 week when stored tightly
sealed in a refrigerator.
HPLC Operation
1. Set fluorometer at 200 nm excitation
wavelength, and insert 300 nm emission filter.
Set range at 0.1 A full scale. For the
electrochemical detector, set the applied
potential to +0.65 to +0.75 volts versus the
Ag/AgCl electrode. The chromatographic
conditions and detector settings are summarized
in Table 3.
2. Pump at least 10 mL of deionized water
through the column.
3. Equilibrate the column with at least 100 mL of
mobile phase. Set flow rate to 2.5 mL/min.
4. Inject 10 L of the norepinephrine working
standard, and note the norepinephrine retention
time. If necessary, add acetonitrile to the mobile
phase to decrease retention, and reinject the
norepinephrine working standard until the
retention time is about 2.5 min.
5. Calibrate the assay with the 80 ng/mL
combined standard (see step 3 of Extraction of
Standards and Samples), using peak areas and
the internal standard procedure (see
Calculations). For studies of small changes
from the normal range, calibrate with the 40
ng/mL combined standard.
6. Inject from 20 to 50 L of standards and
samples. Increase the injection volume for
samples with low concentrations of
catecholamines.
7. At the end of the run, pump at least 20 mL of
deionized water through the column and all
other components of the HPLC system, and
then pump at least 20 mL of acetonitrile
through the column. Typical chromatograms of
standards and samples are given in Figures 11
and 12.
Calculations
Measure peak areas, and calculate concentrations of
norepinephrine, epinephrine, and dopamine by the
internal standard procedure.
362
Catecholamines (Urine)

__Au__
Cu = _ADHBA,u_ Cs
__As__
ADHBA,s

where Au = peak area of the unknown

ADHBA,u = DHBA peak area of the unknown
As = peak area of the standard
ADHBA,s = DHBA peak area of the standard
Cu = concentration of the unknown (ng/mL)
Cs = concentration of the standard (ng/mL)

A typical standard curve is presented in Figure 13.

Many chromatographic data processors will do these

calculations automatically after the calibration step is
completed. For each catecholamine:

g of catecholamine per 24 hr = g/L 24-hr volume in

liters

Interferences
Drugs such as -methyldopa (Aldomet), isoproterenol,
and their metabolites are eluted after the catecholamines
and do not interfere. L-Dopa is metabolized to dopamine
and will cause an elevation in this analyte. -Methyl-L-
tyrosine (Demser) can be eluted close to norepinephrine
in many chromatographic systems. This drug blocks
tyrosine decarboxylase and is frequently given to control
catecholamine synthesis by persons with suspected
pheochromocytomas.

Notes
1. Many laboratories follow a procedure in which
the order of the sample cleanup procedures is
reversed; that is, the sample is first added to
alumina and then eluted with 50 mM acetic
acid, 5 mM sodium bisulfite. This material is
then added to the Bio-Rex column, where the
catecholamines are eluted with boric acid (40
g/L).
2. Sodium octyl sulfate can also be used as the ion
pair.
3. A useful alternative mobile phase that may
perform better on some manufacturers columns consists
of 28.3 g of monochloroacetic acid, 9.35 g of NaOH, 1.5
g of disodium EDTA, and 210 mg of sodium octyl
sulfate in 2 L of water, pH 3.00 to 3.05. The amounts of
octyl sulfate and organic solvent (such as acetonitrile)
may vary as the column ages so that the proper retention
times and resolution are maintained.
 363
Cerebrospinal Fluid (CSF) Protein Quantitation
Cerebrospinal Fluid (CSF) Protein Quantitation
Danyel H. Tacker and Anthony O. Okorodudu
Name: CSF Proteins
Clinical significance: Central nervous system (CNS) disease, trauma Refer to Chapter 47,
Nervous System, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: N/A See information for individual proteins
Molecular mass: N/A See information for individual proteins
Merck Index: N/A
Chemical class: Protein
Principles of Analysis and Current Usage i
Cerebrospinal fluid (CSF) is the fluid in the ventricles of
the brain, in the space between the arachnoid and the pia
mater, and surrounding the spinal cord. It is an ultrafiltrate
of plasma that passes through the blood-brain barrier
(BBB) in the choroid plexus [1,2]. Selective transport and
diffusion of proteins, electrolytes, and other molecules and
bloodborne factors occurs at the level of this highly
selective form of capillary endothelium. Thus CSF has
protein constituents and an electrophoretic pattern similar
to that of plasma/serum but at vastly lower concentrations
in normal health states. In disease states, the permeability
and function of the BBB can be compromised, increasing
the protein concentrations of CSF relative to
plasma/serum.
The choroid plexus is also the site of in-situ production
and secretion of various proteins, peptides, and hormones
in healthy and disease states. Thus alterations in the
protein concentration of CSF relative to plasma/serum, as
well as the appearance of proteins unique to the CSF fluid,
can give an indication of the general health of the central
nervous system (CNS) [2]. Finally, the appearance of
CSF-specific proteins in non-CSF body fluids can
indicate CNS trauma, which can be helpful in diagnosis.
Like serum, CSF is a complex solution including ions,
nutrients, and proteins. The entry of ions present in CSF
(H+, K+, Ca2+, Mg2+, bicarbonate, etc.) is specifically
regulated by ion channels in the membranes composing the
BBB. However, nutrients and wastes (i.e., glucose, urea,
creatinine) diffuse freely through the BBB. Bilirubin is
typically not present in CSF in measurable quantities [1,2].
i
CSF Proteins
Previous and current authors of this method:
First edition: Gayle Birkbeck
Methods edition: Gayle Jackson
Second edition: Not updated
Third edition: Not updated
Fourth edition: Gayle Jackson
Fifth edition: Danyel H. Tacker, Anthony O.
Okorodudu
The proteins present in CSF, or which are measured in
CSF, include:
Albumin, which passively diffuses through the
BBB. Albumin is the predominant protein in the
plasma, as well as the CSF, and has a primary
function in CSF as a transporter of a variety of
compounds [1-4].
Immunoglobulins, which are usually excluded
from the CSF by the BBB. Immunoglobulins that
appear in CSF have two potential sources: (1)
massive failure of the BBB and leakage into the
CSF from the plasma or (2) intrathecal production
in cases of CNS infection [1-3,5-8].
Transthyretin/prealbumin, which is synthesized
and secreted in situ. Transthyretin is a shuttle
protein and transporter of thyroid hormones; it
complexes with retinol-binding protein to aid in
transport of vitamin A/retinols [1-3,5].
2-Transferrin, which is a desialated form of
transferrin found only in the CNS and functions
in iron complexation and maintenance of metal
ion redox status. 2-Transferrin can be detected
long (months and sometimes years) after CNS
trauma in collected body fluids [1-3,5,9,10].
C-Reactive protein (CRP), which is usually found
in very low concentrations or is altogether absent
from CSF. CRP is an acute-phase reactant and
appears to have a role in modulating
inflammatory response [1,2,5,11].
2-Macroglobulin, which is normally excluded
owing to its size. 2-Macroglobulin is a protease
inhibitor and an acute-phase protein. Presence of
this protein in the CSF usually only occurs when
the integrity of the BBB has been heavily
compromised [1-3,5].
-Amyloid and tau ( proteins, which are
normally absent from CSF. -Amyloid is a
cleavage product of amyloid precursor protein
(function is not currently described), which forms
extracellular amyloid plaques when not properly
removed from sites of production. Protein
 364
Cerebrospinal Fluid (CSF) Protein Quantitation
normally associates with tubulin to stabilize
microtubule structures present in neurons;
hyperphosphorylation of protein causes it to
form massive complexes, which result in
pathological plaques that are neurotoxic. Both of
these proteins are associated with
neurodegeneration [2].
2-Microglobulin, which is normally present in
low concentrations and is largely an
investigational marker in the CSF. 2-
Microglobulin composes part of the major
histocompatibility complex I protein complex,
which is responsible for non-self recognition
(primarily of viruses and tumor antigens). 2-
Microglobulin is amyloidogenic [1-3,5,12,13].
Protein 14-3-3, which is normally absent from
CSF. Protein 14-3-3 is a general name for a
highly conserved class of intracellular signaling
and regulatory proteins. Presence of 14-3-3
protein in the CSF is observed in spongiform
encephalopathy (e. g., Creutzfeldt-Jakob disease)
[14].
Myelin basic protein, which is normally absent
from CSF. MBP was originally isolated from
myelin sheaths. Its presence in the CSF is
associated with multiple sclerosis [5,7,15,16].
Fibronectin, which is normally present in low
concentrations. Fibronectin complexes with
integrins to stabilize the extracellular matrix of all
tissues. Presence of increased fibronectin in CSF
is associated with trauma and disruption of the
extracellular matrix of the CNS [1,2,5,17].
CSF has a number of functions in the CNS [1,2]. Firstly,
CSF surrounds the brain in the subarachnoid space, fills
the ventricles, and surrounds the spinal cord, providing a
fluid layer of insulation against mechanical trauma to the
fragile tissues of the CNS. Since it completely surrounds
these tissues, CSF also gives tissues ready access to
nutrients, hormones, and other factors within the CNS
compartment. Further, the ions present in CSF allow for
maintenance of the ambient ionic charge needed for
efficient CNS function. Finally, because there is no lymph
drainage in the CNS, the CSF provides an important route
for the excretion of CNS waste.
The constant production of CSF in the CNS also aids in the
maintenance of intracranial pressure and homeostasis and
delays alterations of these when acute changes in
peripheral pressure and homeostasis occur. In short, CSF
provides protection against abrupt pathological change
because of its isolated location and highly regulated
composition.
Reference and Preferred Methods
Reference Method: N/A Method used is dependent on
the protein and type of analysis.
Preferred & Other Methods: Specific for each protein;
see below.
Total Protein: [1-3,5,18-21]
Accurate measurement of CSF protein is difficult because
CSF proteins are present in small quantities and because
qualitative differences exist between the amounts of
immunoglobulin and the amounts of albumin present in
patient specimens. Methods used for CSF total protein
measurements can be sensitive but can give inaccurate
results when the albumin-to-gamma globulin ratio
changes.
A. Preferred Method: Trichloroacetic Acid (TCA) +
Nephelometric or Turbidimetric Analysis. The TCA
precipitates the protein, which is then read using
nephelometric or turbidimetric techniques. Light
scatter or absorbance is proportional to protein
concentration. Comments: Simple and fast, but
requires the largest sample volume. Alternate
precipitating agents include benzalkonium chloride
and benzethonium chloride.
B. Preferred Method: Sulfosalicylic Acid (SSA): 3%
Sulfosalicylic and 7% Sodium Sulfate; Total Protein,
Turbidimetric. The assay principle is based on
precipitation of protein, with the resultant turbidity
measured in a spectrophotometer. Comments: CSF
(500 L), commonly used; sulfosalicylic acid alone
results in greater turbidity with albumin than with
globulin. The same phenomenon is seen with
sulfosalicylic acid-sodium sulfate in combination.
C. Historical Method: Biuret Method/Colorimetric
Analysis. Copper ions in the Biuret solution interact
with amide groups (e.g., lysine) on amino acid
chains. Albumin has the best binding/detection.
Comments: Rare; low color yield restricts usage to
very sensitive instruments with rate-measurement
capability.
D. Historical Preferred Method: Lowry Method. Two-
step assay that incorporates the Biuret reaction with a
second, Folin-Ciocalteu reagent step (whereby
molybdate and tungstate bind to tryptophan and
tyrosine residues on the protein). This dual reaction
gives greater sensitivity but preferentially binds to
albumin. Comments: CSF (200 L), rarely used; time
consuming; influenced by endogenous phenols and
drugs.
E. Other Method: Coomassie Brilliant Blue Dye
Binding; Total Protein, Chemical,
Spectrophotometric. In the assay, Coomassie Brilliant
Blue G-250 binds to protein, with a resultant color
change from brownish orange to an intense blue
color, and the absorbance of the dye shifts from 465
to 595 nm. Comments: CSF (25 to 100 L),
infrequently used; problem with standardization;
different proteins show variability in sensitivity and
variation in standard curves.
 365
Cerebrospinal Fluid (CSF) Protein Quantitation
Specific Proteins: [1-8,11-19,22-37]
A. Immunonephelometry light scatter is proportional
to protein-antibody complex concentration.
Typically, the target protein is selectively bound by a
specific antibody, and the resulting antigen-antibody
complex increases the light scatter of the sample. The
scattered light is detected at an angle >180 degrees
from the incident light beam passing through the
sample.
B. Immunoassay (IA) protein-specific antibodies bind
target proteins in the CSF sample. Depending on the
type of IA, tagging of the detection antibody, and
other analytical specifications, the signal-to-
concentration relationships vary. Examples of IA
used in the above-referenced publications include:
1. IA after high-performance affinity
chromatography (for ultra-low, specific IgG
detections)
2. Enzyme IA an example is fibronectin, but any
enzyme-linked specific antibody used for
detection falls in this category. Enzyme-linked
detection antibodies react with a substrate added
after the detection antibody has complexed with
the target protein and excess has been washed
away. The reaction with the substrate produces a
signal (typically colorimetric) that is
proportional to the target protein concentration
and rate-limited by the presence of the enzyme
after the washing steps.
3. Radio IA an example is 2 microglobulin, but
new and emerging assays often start with RIA
and then progress to other IA techniques. RIA is
typically competitive in nature, whereby a
radioactive, tagged target protein is added to the
sample. The tagged protein and the native
protein compete for antibody binding (typically
to a solid substrate). After thorough washing, the
radioactivity on the substrate is measured,
usually via scintillation counting. Owing to the
nature of competitive IA, concentration of the
target protein in the sample is typically inversely
proportional to the signal/scintillation detected.
4. Electrochemiluminescence (ECLIA) and related
immunoassay technologies are used for
measuring specific proteins. A concentration step
is usually necessary for CSF protein quantitation.
Depending on the size of the target, the detection
can either be proportional/indirect, or inversely
proportional/competitive. For most proteins, the
target is large enough that the assay is indirect.
Using this approach, a trapping antibody is
bound to a substrate (often a flat detection
surface or a paramagnetic bead), and the target
protein in the sample is first incubated with the
trapping antibody. A second antibody (the
detection antibody, which is tagged with a
chemiluminescent compound) is added to the
mixture for a second incubation. Excess sample
is washed away or removed in a flow cell, and a
substrate for the chemiluminescent compound is
applied. After electrical stimulation of the
sample, the chemiluminescent compound is
excited and gives off photons of light. These
photons are counted, and the amount of signal is
proportional to target protein concentration.
C. High-performance liquid chromatography
(HPLC) with coulometric or other detection
method is typically used for peptides and
hormones present in very low concentrations.
With HPLC, processed samples are injected into a
column under high pressure and carried by a
liquid phase. Depending on the column, the liquid
phase, and the protein chemistry, fractions of
proteins in the sample will elute at different times.
These fractions can be subsequently analyzed in a
variety of ways. Use of internal standards and in-
run calibrators can allow for quantitation.
Electrophoretic Patterns (Semiquantitative): [3,7-
11,25,36,38-40]
A concentration step is necessary for this procedure.
Polyacrylamide and other gel techniques are used for
electrophoretic separation of protein bands in serum and
CSF. In short, CSF is applied to an agarose gel and
separated in an electric field through a liquid buffer phase.
Dye-binding techniques (utilizing bromocresol purple,
Coomassie Brilliant Blue, and other dyes) are used for
visualization and densitometric quantitation. Densities of
bands are used to express protein fraction concentration
based on their percentages of total protein, usually
measured via nephelometric or turbidimetric detection
(above).
The most common application of CSF electrophoresis is
for the visualization of oligoclonal bands in the gamma
region of the gel. Banding in the gamma region suggests
that in-situ expression of monoclonal antibodies is taking
place and occurs in a variety of CNS diseases, including
multiple sclerosis and malignancy.
Specialized/Emerging/Research Techniques: [15,34,38]
A. Enzyme-linked immunosorbent assay (ELISA) and
related methods are used for specialized testing of
specific and uncommon proteins. ELISA is a
common first step in research analysis and often
expands to automated IA. IAs were discussed
previously.
B. Specialized detections using Western blot are usually
limited to research but are possible as long as specific
antibodies are available. In Western blotting, proteins
are extracted from the sample and concentrated. The
concentrated protein extract is electrophoretically
separated using an agarose gel preparation then
 366
Cerebrospinal Fluid (CSF) Protein Quantitation
incubated with specific antibodies (which are
typically linked to enzymes). Colorimetric detection
is then possible, as is semiquantitation using
densitometric analysis of protein bands.
Specimen
Preparation: [2]
There are many side effects associated with the collection
of CSF (typically via lumbar puncture, also called a spinal
tap) that are undesirable and even life-threatening. The
risks associated with CSF collection must be weighed
against the need for diagnostic information, and volume of
collection must be kept to an absolute minimum for the
testing required. Patients should remain calm. Opening
pressure should be between 90 and 180 mm of water (for
normal adults) but is dependent on postural changes and
elevations, as well as patient physiological status and age
[2]. An opening pressure greater than 200 mm of water in
a relaxed patient suggests serious disease and limits CSF
collection volume to 2.0 mL.
Draw/Collection: [2,5]
When opening pressure allows for full collection, three
tubes are typically drawn. The first tube is used for
chemistry and immunology (unless traumatic; bloody
specimens will affect protein measurements and
determinations), the second tube is for culturing, and the
third tube is for cell counting/differentials. Tubes may be
substituted or added, but volumes must be kept to a
minimum. Maximal volume drawn is 20 mL in an adult
(total CSF volume typically 90 to 150 mL), and about 3
mL in a neonate (total CSF volume typically 10 to 60 mL).
Collection of blood samples for comparisons and index
calculations is optimal; when collected 2 hours before the
CSF collection, the best comparisons are obtained
(because of the delayed equilibrium of the CSF).
Storage: [2]
CSF degradation begins within 1 hour, so all efforts should
be made to promptly store the sample tubes properly. All
samples should be centrifuged upon receipt. If analysis
cannot take place immediately, store CSF at 4C for < 72
hours. Freezing CSF at 20C keeps constituents stable for
up to 6 months. Freezing CSF at 70C keeps constituents
stable indefinitely. Blood samples should be handled and
stored normally.
Patient Risks Involved: [2]
The most common untoward effect of lumbar puncture is
headache. Infections, meningitis, and death have occurred
as a result of lumbar puncture procedures.
Interferences [1,2]
The location and specific functions of CSF make it
particularly susceptible to diurnal variation and hormonal
factors, which can affect specific protein levels. Such
influences should be considered accordingly when
analyzing CSF.
Depending on the assay used for CSF analysis, a number
of factors could cause interference. For example,
colorimetric techniques employing spectrophotometric
detection can be susceptible to interference from
hemolysis, icterus, uremia, drugs with spectral activity,
and high ammonium concentrations (for Biuret and Lowry
reactions). Also, other conditions or therapies (i.e.,
dextran, contrast media, and high concentrations of
immunoglobulins) that increase turbidity of the CSF can
affect nephelometric and turbidimetric analysis.
CSF Proteins Reference Intervals
Reference Intervals: Please consult Table 1: Available
Reference Intervals for CSF Proteins
Calculations: [1-3,20]
A. Albumin Index
Albumin Index = CSF albumin (mg/dL)__
Serum albumin (mg/dL)
Interpretation: For adults, an albumin index < 9 is
normal. An index of 9 to 14 reflects slight
impairment of the BBB. An index of 14 to 30
reflects moderate impairment of the BBB, and an
index > 30 reflects severe impairment of the
BBB.
B. IgG Ratio
IgG Ratio = CSF IgG (mg/dL)__
Serum IgG (mg/dL)
Interpretation: For adults, an IgG ratio of 3.0 to
8.7 is normal. IgG ratios > 8.7 reflect BBB
impairment.
C. CSF IgG Index
CSF IgG Index =
[CSF IgG (mg/dL) / serum IgG (g/dL)]
[CSF Alb (mg/dL) / serum albumin (g/dL)]
Interpretation: For adults, a CSF IgG index < 0.8
is normal. A CSF IgG index > 0.8 suggests
intrathecal IgG production.
NOTE: With additional time-sensitive information
about the samples used, IgG synthesis rates may
be calculated.
Interpretation and Clinical Utility [1,2,14,22,38]
Interpretation of CSF Electrophoresis:
Normally, CSF samples show an evident
transthyretin/prealbumin band (typically 2% to 7% of total
sample protein content), followed by bands for albumin
 367
Cerebrospinal Fluid (CSF) Protein Quantitation
(56% to 76%), 1 (2% to 7%), 2 (4% to 12%), and 1/2 Neoplastic Disease:
(8% to 18%) globulins. The region (3% to 12% of total
sample protein content) should be faintly staining and
diffuse.
If the region shows multiple distinct bands, this is called
an oligoclonal banding pattern. Oligoclonal bands are
typically associated with demyelinating disease. However,
if the region shows only a single band, this pattern is
typically associated with infectious disease, such as
meningitis.
When CSF electrophoresis is performed alongside a serum
sample from the same patient, collected at generally the
same time as the CSF sample, then an assessment of BBB
integrity can be made. Typically, the CSF lane on the
electrophoresis will be faint when compared to the serum
lane. If the CSF lane appears to be equal in density or
darker than the serum lane, then the BBB is probably
compromised. Calculation of the CSF IgG index and/or the
albumin index should always accompany CSF
electrophoretic analysis; the calculations give a more exact
assessment of BBB integrity than the density of sample
staining.
Disease-Specific Interpretation:
Infection:
In CNS infection, the BBB is compromised, and increased
CSF protein levels relative to plasma protein/serum levels
are observed. Other indicators, such as elevation of CRP
and immunoglobulins, aid in diagnosis. Electrophoresis of
CSF can reveal oligoclonal banding patterns of
intrathecally produced immunoglobulins.
CNS Trauma:
Subarachnoid hemorrhage and other CNS trauma increases
BBB permeability. Total protein will be elevated in the
CSF specimen relative to plasma protein concentrations,
and typically absent components such as bilirubin and
methemoglobin are detectable.
In cases of rhinorrhea or otorrhea, the presence of 2-
transferrin is suggestive of CSF leakage outside the CNS.
Transthyretin detection is only useful in this type of
analysis if present in very high concentrations, since
transthyretin is also seen in plasma.
Demyelinating Disease:
The intrathecal production of immunoglobulins in the
choroid plexus occurs in demyelinating diseases such as
multiple sclerosis and produces oligoclonal banding
patterns on electrophoresis. The ratio of IgG in the CSF
relative to that of the plasma will be elevated, and specific
studies for myelin basic protein in the CSF are typically
positive.
Different neoplasms produce different alterations of CSF
protein content, the description of which is beyond the
scope of this summary. Markers used in diagnosing
neoplasm in CSF fluid include 2-microglobulin,
fibronectin, myelin basic protein, and others.
Other CNS Disease:
Terminal spongiform encephalopathies are typically
associated with elevations in Protein 14-3-3. Imaging and
case-relevant symptomatic information are usually used
for diagnosis.
Alzheimers disease diagnosis is made with imaging
results, case-relevant symptomatic information, and
prediction of early risk of disease by determining the ratio
of protein to 2-amyloid protein in CSF.
CSF Proteins Assay Performance Goals
Total protein analysis of CSF employs techniques also
used for serum and plasma protein quantitation. This is a
benefit to CSF protein quantitation in that the available
assays are largely automated and come with reliable
control materials. Typically, automated versions of the
SSA and TCA techniques show coefficients of variation
(CVs) 5% and wide measuring ranges.
Immunoassay performance will vary according to the
detection technique measured. For automated IA assays,
CVs tend to be slightly wider than the serum/plasma
version of the assay. Depending on the availability of a
reference material for a given protein, degrees of assay
standardization, and thus the resulting assay performance,
will vary.
Electrophoretic assay performance is largely based on
satisfactory visualization of all relevant bands when
compared to control samples and serum samples
accompanying the CSF sample on the gel. When analysis
is semiquantitative and tied to band density as a percent of
total sample protein concentration, then cumulative
statistics can be generated and performance assessed. CVs
10% for major bands and 20% for minor bands are
generally acceptable.
HPLC technique performance, when made
semiquantitative through the use of internal and assay
controls, varies according to the targets being detected and
the degree of automation used in sample preparation and
assay.
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23 Coore J, Ambler J. Potential problem with
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25 Dinesen B, Nielsen S. A sensitive enzyme
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27 Javors MA, Bowden CL, Maas JW. 3-methoxy-4-
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Gironelli L, Voci L, Bianchi F. Laser
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30 Nerenberg ST, Prasad R. Radioimmunoassays for
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Cerebrospinal Fluid (CSF) Protein Quantitation
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31 Phillips TM, More NS, Queen WD, Holohan TV,
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gamma globulin. II. Radioimmunoassay
determination of levels of post-gamma globulin
and beta 2-microglobulin. Clin Chim Acta
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33 Pressac M, Vignoli L, Aymard P.
Immunoturbidimetric assay of transthyretin by
random-access analysis. Clin Chem 1988;34:176.
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determination of albumin in the nanogram range:
assay in cerebrospinal fluid and comparison with
radial immunodiffusion. Clin Chim Acta
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35 Tourtellotte WW, Tavolato B, Parker JA, Comiso
P. Cerebrospinal fluid electroimmunodiffusion.
An easy, rapid, sensitive, reliable, and valid
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36 Tourtellotte WW, Tavolato BA, Parker JA,
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37 Vatassery GT, Krezowski AM, Sheridan MA.
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39 Sorjonen DC, Cox NR, Swango LJ.
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Reference Intervals. 7th ed. Washington DC:
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 370

Cerebrospinal Fluid (CSF) Protein Quantitation

Tables and Figures

Table 1: Available Reference Intervals for CSF Proteins

Analyte Method Range (mg/dL)

1-Acid glycoprotein Radial immunodiffusion (RID) 0.28-0.54

Albumin [4,18,24,28,34] IA techniques, turbidimetric, Biuret/colorimetric 0-45 (3 mo 4 y)

10-30 (>4 y)
2-HS-glycoprotein Enzyme immunodiffusion (EID) 0.17

IgA [30] Radioimmunoassay (RIA) (in mg/dL by age):

15-20 y: 0.07 0.04
21-40 y: 0.07 0.03
41-60 y: 0.10 0.03
61-87 y: 0.11 0.06
IgD RIA (in U/mL by age):
15-20 y: 3.56 2.0
21-40 y: 3.02 1.3
41-60 y: 2.96 0.88
61-87 y: 3.20 0.9
IgG RIA (in mg/dL by age):
15-20 y: 3.5 2.0
21-40 y: 4.2 1.4
41-60 y: 4.7 1.0
61-87 y: 5.8 1.6
IgM RIA (in mg/dL by age):
15-20 y: 0.020 0.009
21-40 y: 0.016 0.003
41-60 y: 0.017 0.004
61-87 y: 0.017 0.005
2-Macro-globulin Laser nephelometry 0.11-0.45 mg/dL

2-Microglobulin [12,13] Nephelometry 1.5 0.2 mg/L

2-Transferrin [9,10] Electrophoresis, immunofixation electrophoresis, CSF, positive

Western blot
Myelin basic protein [7,15,16] RIA, ELISA <2.5 ng/mL

Total protein [1,2,41] Turbidimetry, nephelometry, Biuret/colorimetric (in mg/dL)

Adult 18-32
Pediatric:
Premature: 15-130
Full-term at birth: 40-120
<1 month: 20-80
>1 month: 15-40
Transthyretin/prealbumin Nephelometry, turbidimetry, densitometry ~2% of total protein
[23,33] concentration

NOTE: Reference intervals will vary by the method used and should be established in-house. In newborns, CSF proteins are
higher, owing to high permeability of the newborns BBB. Reference intervals for specific proteins have been published and
should be established or verified accordingly.
 371

Cerebrospinal Fluid (CSF) Protein Quantitation

Procedure: Sulfosalicylic Acid Turbidimetric Assay 2.
Principle: Protein is precipitated as a fine, white
precipitate by the addition of sulfosalicylic acid. The
resulting turbidity is determined spectrophotometrically at
430 nm.
Reagents
1. Sulfosalicylic acid, 30 g/L (118 mmol/L).
Dissolve 30 g of sulfosalicylic acid in 800 mL of
distilled water, and bring to 1 L. Store in a brown
bottle. Stable for 1 year at room temperature.
2. Control. Ortho Diagnostics CSF control (Ortho
Diagnostics, Raritan, NJ).
Assay
Equipment: Spectrophotometer capable of reading
at 430 nm.
1. Add 1.0 mL of 3% sulfosalicylic acid to three 3.
test tubes; one for an instrument blank, one for
the Ortho standard, and one for the patient.
2. Add 0.2 mL of Ortho Diagnostics CSF control to
the standard tube (click here). 4.
3. Add 0.2 mL of CSF to the patients tube. Add 0.2
mL of saline solution to blank tube.
4. If the spinal fluid is strongly colored, prepare a
patient blank using 0.2 mL of CSF and 1.0 mL of
saline. Notes
5. Cover all test tubes with plastic cups or Parafilm, 1.
and gently invert several times.
6. Let stand 10 min at room temperature.
7. Set absorbance at zero on the spectrophotometer
at 430 nm, using the instrument blank of
sulfosalicylic acid plus saline.
8. Invert test tube gently to mix, introduce sample
into cuvette, and read absorbance (A) of all
standards and patient samples. 2.
9. Dilute samples 1:2 with saline, and rerun if
concentration is greater than 2000 mg/L.
Calculations
1. Correct absorbance of samples with blank:
To prepare the standard curve, dilute a control
serum of 70 g/L of total protein with saline so that
5 dilutions with protein concentration between
100 and 1500 mg/L are prepared as follows:
Stock Volume Total Volume
Concentration (mg/L) (mL)
2.0 100
1.5 100
1.0 100
0.5 100
0.2 100
0 100
Do not use a serum control with elevated lipid or
bilirubin content.
Standard curve. Construct the standard curve,
plotting absorbance at 430 nm versus protein
concentration. This should be a straight line that
passes through the origin.
The standard curve needs to be run only when
there is a change in the spectrophotometer (i.e.,
bulb change) or that lot of quality control material
or the quality control sample does not give the
expected value.
The first drop of spinal fluid should be added
slowly to the sulfosalicylic acid reagent. If a
dense cloud forms immediately, the protein
concentration is very high, and dilution will be
necessary. In this case, the rest of the 0.2-mL
sample should not be added to the acid but instead
returned to the original tube. The spinal fluid
should be diluted with saline.
If red blood cells are present in the spinal fluid,
the fluid must be centrifuged before protein
analysis.

Apatient Apatient blank = Acorrected patient

2. Read the corrected absorbance from the standard

curve (see below) to obtain the protein
concentration.
Controls and Standards
1. Since sulfosalicylic acid produces different
degrees of turbidity with equal concentrations of
different types of CSF proteins, it is necessary to
construct a curve using a standard that has the
same albumin-globulin ratio as the spinal fluid
being tested. Usually spinal fluid and serum have
a very similar albumin-globulin ratio, so a normal
serum control with the proper dilutions can be
used to construct the curve. The albumin-globulin
ratio of the standard should be between 1.0 and
1.5.
 372

Ceruloplasmin

Ceruloplasmin
Ahmed Zakaria

Name: Ceruloplasmin: iron(II)-oxygen oxidoreductase (ferroxidase)

Clinical significance: Refer to Chapter 42, Trace Metals, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular weight: 132,000 D
Enzyme number: EC 1.16.3.1
Merck Index: 14: 2006
Chemical class: Glycoprotein

Principles of Analysis and Current Usage i molecular oxygen, while ferric iron thereby becomes
Ceruloplasmin is a single polypeptide synthesized in the available for binding to and transport by transferrin.
liver [1]. It contains six to eight atoms of copper in its
structure [2], carries up to 90% of copper in plasma, and
performs ferroxidase (EC 1.16.3.1.) or iron (II):oxygen
oxidoreductase activity essential for normal iron
homeostasis. Ceruloplasmin catalyzes the oxidation of
ferrous iron Fe (II) to ferric iron Fe (III), which is carried
in the plasma by transferrin [3].

Holmberg and Laurel isolated and purified ceruloplasmin

from plasma and established its molecular mass, copper
content, and oxidase activity [4]. The purified protein has
an intense blue color and imparts a green color to serum
when the albumin is abnormally low. Human
ceruloplasmin is a 132-kDa protein, and the subunit-like
fragments that are found during its preparation are formed
because of autolytic degradation [2]. Human
ceruloplasmin is a single polypeptide chain and contains
1046 amino-acid residues and four glucosamine
oligosaccharides [5,6].
Numerous assays were developed based on the activity of
the ceruloplasmin oxidase, using polyamine or polyphenol
substrates such as p-phenylenediamine, N,N-
dimethylphenylenediamine, in addition to ferrous iron,
benzidine, and others. However, the measurements by
these assays are relatively nonspecific. The principle of
ceruloplasmin oxidase assays is based on the conversion of
substrate to the oxidized colored product that is measured
photometrically. Ceruloplasmin utilizes the electron
chemistry of bound copper ions to couple iron oxidation
with the four-electron reduction of dioxygen (Scheme 1).
The reduced form of ceruloplasmin slowly reduces
i colored Fe (II) complex was measured photometrically at
Ceruloplasmin
Previous and current authors of this method:
First edition: Not done
Methods edition: Gayle Jackson
Second edition: Not updated
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Ahmed Zakaria
In 1967, Curzon and Speyer developed a colorimetric
method utilizing p-phenylenediamine as a substrate [7]
(Table 2, Method 1), whereas Schosinsky et al. used an o-
dianisidine dihydrochloride as a substrate (Table 2,
Method 2) [8]. Winkles et al. developed an automated
assay for ferroxidase using a centrifugal analyzer [9]. They
showed that the oxidase activity of ceruloplasmin towards
Fe (II) has physiological significance both in iron
metabolism and as a serum antioxidant. Furthermore, the
ferroxidase activities correlated significantly with
ceruloplasmin and copper concentrations.
Erel described an automated measurement of serum
ceruloplasmin ferroxidase activity in which Fe (II) ions
were used as the substrate in acetate buffer pH 5.8 [10].
The ceruloplasmin catalytic activity was determined by
oxidation of ferrous ions to ferric ions and a chromogen, 3-
(2-pyridyl)-5,6-bis(2-[5-furylsulfonic acid])-1,2,4-triazine
was added at the end of the incubation period. This
chromogen forms a colored complex with the remaining
nonoxidized ferrous ions but not with ferric ions. The
590 to 610 nm. The difference in the ferrous ion
concentration before and after the enzymatic reaction
indicated the amount of oxidized ferrous ion [10]. The
range for serum ceruloplasmin ferroxidase activity in
healthy persons was 198 to 1107 U/L.
 373

Ceruloplasmin

The enzymatic methods measure the oxidase activity of The antisera used in most of the immunochemical methods
ceruloplasmin by using either p-phenylenediamine or o- were significantly different in their antibody activity [13].
dianisidine dihydrochloride as the substrate. The p- The reason is in part due to ceruloplasmin degradation or
phenylenediamine method is relatively simple and has loss of copper from its structure, which subsequently
been utilized in clinical laboratories since the early 1960s. produces variable antigenicity if used as an immunogen.
Nevertheless, the method is not specific for ceruloplasmin
for several reasons. Catalytic oxidation of the substrate by The variation in antiserum specificity makes it difficult to
free metals such as cupric and ferrous ions occurs, causing standardize ceruloplasmin measurement between
falsely elevated levels. The reaction is inhibited by high laboratories.
concentrations of blood urea nitrogen (>1 g/L), uric acid
(>200 mg/L), or bilirubin (163 mg/L) [8]. The oxidation of Specimen
p-phenylenediamine is decreased by ascorbic acid and Serum is the preferred specimen. EDTA and citrate are not
increased by exposure to visible light. The enzymatic acceptable if enzymatic methods are used. Assays should
assays are also subject to negative interference by certain be performed on fresh specimens. If this is not possible,
anions and organic compounds [7]. Since the o-dianisidine serum should be frozen until the assays are performed. No
is more stable and forms a more stable product than p- fasting or other preparation is usually needed [14].
phenylenediamine, the use of o-dianisidine as a substrate is Ceruloplasmin is stable for 3 days at 4C and 4 weeks at
the preferred method, based on the oxidase activity 20C. Ceruloplasmin is susceptible to degradation or
measurements. The direct enzymatic methods were oxidative modification, which alters its reactivity with
preferred until the development of immunochemical antibodies.
methods, which were considered specific and easy to
perform in hospital laboratories (Table 2, Methods 3 and Interferences
4). The immunochemistry methods are subject to interfering
substances, including hemoglobin (500 mg/dL), lipids
Reference and Preferred Methods (1000 mg/dL), and bilirubin (30mg/dL). Increasing
There is no specific reference method for ceruloplasmin. triglyceride may contribute to the depression of
Immunoturbidimetry or immunonephelometry are the ceruloplasmin concentration up to 50% [15,16].
preferred methods for quantitative determination of human
ceruloplasmin concentration on automated instruments Ceruloplasmin Reference Interval
[11]. Methods based on nephelometric and turbidimetric The reference intervals for ceruloplasmin are shown below
assays demonstrate acceptable imprecision compared to [14]:
the earliest immunochemical assays, radial
immunodiffusion, and rocket laurel Table 1: Ceruloplasmin Reference Intervals
immunoelectrophoresis [12]. In the immunochemical Age Concentration (mg/L)
methods, a specific antibody is used against ceruloplasmin. Cord blood (term) 50330
<5 months 150560
Recently, more specific anti-human ceruloplasmin 56 months 260830
antibodies have become available, and a variety of manual, 736 months 310900
automated immunoturbidimetric and >312 years 250450
immunonephelometric assays are commercially available
on several manufacturers platforms. >1219 years Male 150370
Female 220500
Pesce and Bodourian described a kinetic nephelometric Adults, Male 220400
assay for the measurement of ceruloplasmin in serum [12].
Adults, Female (No 250600
The reaction rate was not affected by temperature changes
contraceptive)
between 30C and 37C or pH between 6.0 and 8.4.
(Contraceptive estrogen) 270660
Ceruloplasmin concentrations measured by the centrifugal
(Pregnant ) 3001200
analyzer were compared to results obtained by
nephelometric, radial immunodiffusion, and an enzymatic
procedure using p-phenylenediamine as the substrate [12].
The correlation coefficients were 0.93, 0.88, and 0.86,
respectively. They attributed the bias between the Normal adult plasma concentration is reached 3 to 6
nephelometric and the RID techniques to the different months after birth. Females taking oral contraceptives
antisera specificities, since the antisera were from different show higher concentrations of ceruloplasmin when
sources. Unlike the enzymatic procedures, measurement of compared with age-matched females [17]. Pregnant
ceruloplasmin with immunochemical methods does not females have ceruloplasmin concentrations that are
require rigorously controlled temperature or pH. approximately twice those of nonpregnant women. Aging
 374
Ceruloplasmin
does not appear to result in any significant changes in
ceruloplasmin concentrations when compared with healthy
young adults [18].
Interpretation
Ceruloplasmin is synthesized in the liver. Its primary
function is to transport copper, and in addition, it plays an
important role in iron transport, metabolism, and
homeostasis. Its major function may be to facilitate the net
flux of iron between cells and tissues
[19,20]http://www.jbc.org/cgi/ijlink?linkType=ABST&jou
rnalCode=sci&resid=279/5351/714. As a result of
defective copper transport, ceruloplasmin concentrations
are usually but not always reduced in Wilsons disease
[21]. However, in Wilsons disease, copper is accumulated
in tissues including liver (hepatolenticular degeneration),
brain, and cornea [21]. Decreased ceruloplasmin levels
have been observed with nephrosis, tropical and
nontropical sprue, Menkes syndrome (the defect is
secondary to poor absorption and utilization of dietary
copper), scleroderma of the small bowel, primary biliary
cirrhosis, and primary biliary atresia [22].
Ceruloplasmin is an acute-phase reactant, and blood levels
may increase in infections or inflammatory disease [23].
High levels occur in pregnancy, with estrogens, and with
oral contraceptive use when the agent contains estrogen as
well as progesterone [24].
Excessive therapeutic zinc may block intestinal absorption
of copper and lead to a copper deficiency syndrome
characterized by hypochromic microcytic anemia with
leukopenia/neutropenia and very low concentrations of
ceruloplasmin [25].
Ceruloplasmin is also associated with diabetes and
cardiovascular disease. It is involved in promoting LDL
oxidization and inhibits nitric oxide (NO) production [26].
Ceruloplasmin Performance Goals
Both the Clinical Laboratory Improvement Amendments
of 1988 (CLIA 88) and biological variability may be used
to determine the performance goal of an analytical assay.
The intraindividual and interindividual biological variation
of ceruloplasmin concentration is 5.8% and 11.1%,
respectively. Therefore, the desired performance for
ceruloplasmin assays is defined by the biological variation
imprecision estimate as 2.9% (50% of intraindividual
variation, which is 5.8%). Using these criteria, the total
error allowable in ceruloplasmin measurement is estimated
to be 7.8% [27]. The overall ceruloplasmin performance
ranges are considered to be well within the capability of
most automated immunoassays currently available in
clinical laboratories. The analytical performance of the
immunonephelometric methods is summarized in Table 3.
The data is from the 2007 College of American
Pathologists surveys.
References
1 Ryden L. Single-chain structure of human
ceruloplasmin. Eur J Biochem 1972;26:380-386.
2 Kingston IB, Kingston BL, Putnam FW.
Chemical evidence that proteolytic cleavage
causes the heterogeneity present in human
ceruloplasmin preparations. PANS, USA
1977;74:5377-5381.
3 Osaki S, Johnson DA, Frieden E. The possible
significance of the ferrous oxidase activity of
ceruloplasmin in normal human serum. J Biol
Chem 1966;241:2746-2751.
4 Holmberg CG, Laurell CB. Investigations in
serum copper III. Ceruloplasmin as an enzyme.
Acta Chem Scand. 1951;5:476-480.
5 Dwulet FE, Putnam FW. Complete amino acid
sequence of a 50,000-dalton fragment of human
ceruloplasmin. PNAS, USA 1981;78:790-794.
6 Takahashi N, Ortel TL, Putnam FW. Single-chain
structure of human ceruloplasmin: the complete
amino acid sequence of the whole molecule.
PANS USA 1984;81:390-394.
7 Curzon G, Speyer BE. Inhibitors of
ceruloplasmin. Biochem J 1967;105:243-250.
8 Schosinsky KH, Chavarria M, Lehmann HP.
Measurement of ceruloplasmin from its oxidase
activity in serum by use of o-dianisidine
dihydrochloride. Clin Chem 1974;20:1556-1563.
9 Winkles J, Joens AF, Winyard P, Blacke DR,
Lunec J. An automated method for the kinetic
measurement of ferroxidase activity. Ann Clin
Biochem 1988;25:250-254.
10 Erel O. Automated measurement of serum
ferroxidase activity. Clin Chem 1998;44:2313-
2319.
11 Blirup-Jensen S. Protein standardization III:
method optimization. Basic principles for
quantitative determination of human serum
proteins on automated instruments based on
turbidimetry or nephelometry. Clin Chem Lab
Med 2001;39:1098-1109
12 Pesce MA, Bodourian SH. Nephelometric
measurement of ceruloplasmin with a centrifugal
analyzer. Clin Chem 1982;28:516-519.
13 Buffone GJ, Brett EM, Lewis SA, Iosefsohn M,
Hicks JM. Limitations of immunochemical
measurement of ceruloplasmin. Clin Chem
1979;25:749-751.
14 Roberts WL, McMillin GW, Burtis CA, Burns
DE. Tietz Textbook of Clinical Chemistry. 4th ed.
Philadelphia: Saunders; 2006.
15 Bornhorst JA, Roberts RF, Roberts WL. Assay-
specific differences in lipemic interference in
native and Intralipid-supplemented samples. Clin
Chem 2004;50:2197-2201
16 IMMAGE Immunochemistry Systems:
Beckman Chemistry Information Package Insert;
2005.
 375
Ceruloplasmin
17 Milne DB, Johnson PE. Assessment of copper
status: affect of age and gender on reference
ranges in healthy adults. Clin Chem 1993;39:883- 23
887.
18 Gaggiotti G, Orlandoni P, Ambrosi S, OnoratoG, 24
Piloni S, Amadio L et al. The influence of age and
sex on nutritional parameters in subjects aged 60
years and over. Arch Gerontol Geriat
1995;12:117-128. 25
19 Mukhopadhyay CK, Attieh ZK, Fox PL. Role of
ceruloplasmin in cellular iron uptake. Science
1998;279:714-717.
20 Attieh ZK, Mukhopadhyay CK, Seshadri V,
Tripoulas NA, Fox PL. Ceruloplasmin ferroxidase 26
activity stimulates cellular iron uptake by a
trivalent cation-specific transport mechanism. J
Biol Chem 1999;274:1116-1123.
21 Percival SS, Harris ED. Copper transport from
ceruloplasmin: characterization of the cellular 27
uptake mechanism. Am J Physiol Cell Physiol
1990;258:C140-C146.
22 Wallach J. Interpretation of Diagnostic Tests: A
Handbook Synopsis of Laboratory Medicine. 4th
Table 2: Methods Summary
Method Principle of Analysis:
1 Colorimetric. Ceruloplasmin oxidizes p-
phenylenediamine in the presence of oxygen to form a
purple product with an absorption maximum at 530 nm
2 Colorimetric. o-Dianisidine dihydrochloride is oxidized
by ceruloplasmin at pH 5 in the presence of oxygen; the
product formed is solubilized in sulfuric acid, 9 mol/L,
and measured at 540 nm
3 Radial immunodiffusion. Specific antibody to
ceruloplasmin forms precipitin ring in agarose medium
4 Immunonephelometric. Specific antibody to
ceruloplasmin forms complex that scatters light
ed. Boston/Toronto: Little, Brown & Company;
1986.
Denko CU. Protective role of ceruloplasmin in
inflammation. Agents Actions 1979;9:333-343.
Carruthers ME, Hobbs CB, Warren RL. Raised
serum copper and ceruloplasmin levels in subjects
taking oral contraceptives. J Clin Pathol
1966;19:498-500.
Irving JA, Mattman A, Lockitch G, Farrell K,
Wadsworth LD. Element of caution: a case of
reversible cytopenias associated with excessive
zinc supplementation. Can Med Assoc J
2003;169:129-131.
Mukhopadhyay C, Ehrenwald E, Fox P.
Ceruloplasmin enhances smooth muscle cell and
endothelial cellmediated low density lipoprotein
oxidation by a superoxide-dependent mechanism.
J Biol Chem 1996;271:14773-14778.
Fraser GC. Biological Variation: From Principle
to Practice. Washington, DC: AACC Press; 2001.
Comments:
Historical; substrate requires special
precautions and is unstable and
nonspecific
Serum; preferred chemical
oxidation
Serum; simple immunochemical
method; less frequently used
Serum; requires specific
instrumentation; currently used
 376

Ceruloplasmin

Table 3: Analytical Performance of Ceruloplasmin Assays

METHOD SURVEY College of American Pathologists Survey 2007

NEPHELOMETRY No. of Mean SD CV Median Low High

Labs mg/dL Value Value

Beckman Array S2B 11 33.8 1.3 3.7 34.0 32.0 36.0

S2C 12 3.6 0.5 14.4 4.0 3.0 4.0

Beckman Immage S2B 99 34.4 1.8 5.1 35.0 31.0 38.0

S2C 105 3.4 0.5 14.5 3.0 3.0 4.0

Dade Behring S2B 74 38.2 2.3 6.1 38.0 31.0 44.0

Nephelometer systems S2C 75 3.9 0.4 10.2 4.0 3.0 5.0

TURBIDIMETRIC

Roche COBAS Integra S2B 21 43.0 2.8 6.5 43.0 38.0 50.0
S2C 12 4.7 0.6 14.0 0.0 4.0 6.0

Roche Hitachi/Cobas c S2B 18 40.8 1.1 2.7 41.0 39.0 43

S2C 5 - - - 5.0 4.0 8.0

Olympus AU system S2B 5 - - - 40.0 36.0 42

377

Chloride

Chloride
William J. Korzun
Name: Chloride
Clinical significance: Refer to Chapter 28, Physiology and Pathophysiology of Body Water and
Electrolytes, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Atomic symbol: Cl
Atomic mass: 35.453 D
Merck Index: 2062
Chemical class: Inorganic anion
NRSCL Definitive and
Reference Methods: NCCLS RS10-9

i principle, one can determine the absolute amount of

Principles of Analysis and Current Usage
Chloride is the major anion in extracellular body fluids
and is measured in serum, plasma, urine, sweat, and
occasionally other body fluids. The most common
method in current use is ion-selective electrode (ISE),
representing greater than 99% of College of American
Pathologists (CAP) Comprehensive Chemistry survey
participants in 2007. Amperometric/coulometric titration
and mercuric nitrate/thiocyanate methods accounted for
the non-ISE-based methods in use.
silver ions generated from the number of coulombs
(current time) produced and Faradays constant
(96,487 coulombs per equivalent). In practice, the time
required to titrate a chloride standard solution or
unknown sample with a constant current is measured.
The unknown concentration is then calculated according
to the following formula:
Chloride conc. (standard) = Chloride conc. (unknown)
Titration time (standard) Titration time (unknown)

Ion-selective electrode methods are used in most
automated chemistry analyzers, ranging from large,
multichannel instruments to compact, physicians-office
lab equipment. The measurement technology (Table 1,
Method 1) and limitations are essentially the same as
described for sodium and potassium. The ion-selective
sensing element is typically silversilver chloride or
silver sulfide. ISE methods using direct, undiluted
specimens and using indirect, diluted specimens are both
used for chloride measurement.
The coulometric titration method is used in some
analyzers and is also the basis for the reference method
for chloride. Silver ions, generated at a constant voltage
from a silver electrode, react with chloride ions to form
insoluble silver chloride (Table 1, Method 2) [1,2]. The
end-point is detected amperometrically by a second pair
of electrodes that specifically measure the free silver
ions that result when all chloride ions are consumed. In
i and a reproducible detection of the end-point. It is
Chloride
Previous and current authors of this method:
First edition: W. Gregory Miller
Methods edition: F. Phillip Anderson, W. Gregory
Miller
Second edition: F. Phillip Anderson, W. Gregory
Miller
Third edition: F. Phillip Anderson, W. Gregory
Miller
Fourth edition: F. Phillip Anderson, W. Gregory
Miller
Fifth edition: William J. Korzun
Methodological precision in coulometric titration is
limited by the rate of mass transfer of silver ions from
the generating electrode to the bulk solution. The time
needed to detect the end-point once all the Cl is
consumed is called the blank time and depends on the
rate of mixing in the vessel and the rate of Ag+
formation. For maximum precision, the blank time
should be a small fraction of the total titration time. At
very high chloride concentrations (400 mmol/L under
typical test conditions), the physical bulk of precipitate
interferes with mass transfer and obscures detection of
the end-point. Dirty electrodes will cause poor
reproducibility of results, and some workers recommend
cleaning with ammonium hydroxide and nitric acid
solutions between each titration [2]. Gelatin in the
original procedure and polyvinyl alcohol, which is more
stable, in current procedures prevent reduction of silver
chloride at the indicating electrodes and promote
uniform deposition of excess silver ions on the indicator
cathode. This results in a smooth amperometric current
necessary for the titration mixture to have an acid pH to
prevent the formation of poorly soluble basic silver salts.
Nitric acid and acetic acid in the titration reagent ensure
an acid pH. Nitric acid also provides ionic conductivity,
and the acetic acid sharpens the end-point by reducing
the slight solubility of silver chloride [1].
The mercuric thiocyanate method employs the
quantitative displacement of thiocyanate by chloride
from mercuric thiocyanate and subsequent formation of
a red ferric thiocyanate complex, which is measured
378
Chloride
colorimetrically at 525 nm (Table 1, Method 3) [3]. The
sensitivity and linear range of the reaction are adjusted
by addition of an excess of mercuric ions as mercuric
nitrate. The chloride will first combine with the free
mercury ions (colorless) and then displace any
thiocyanate from mercuric thiocyanate to produce a
colored product measured at 525 nm (Figure 1). This
approach limits the minimum detectable chloride to 80
mmol/L but greatly improves the sensitivity (absorbance
change per millimole) in the clinically important serum
range of 80 to 125 mmol/L. The ferric thiocyanate
reaction is very temperature sensitive, and a constant
temperature must be maintained to obtain accurate
results. Failure to do so can result in a significant drift in
some automated methods.
A less common spectrophotometric method that has been
used by some automated analyzers is based on the
reaction between ferric nitrate and chloride ions to form
a ferric chloride complex which absorbs light at 340 nm
(Table 1, Method 4). The definitive method for chloride
analysis is isotope dilutionmass spectrometry using
samples spiked with 37Cl (Table 1, Method 5) [4].
Reference and Preferred Methods
Coulometric titration is both an American Association
for Clinical Chemistry (AACC) selected method [2] and
the National Institute of Standards and Technology
(NIST) reference method for serum chloride [4] because
of its precision and its freedom from interferences (see
Procedure: Serum Chloride by Coulometry). Using the
NIST procedure, 14 evaluating laboratories achieved an
average standard deviation of approximately 0.5
mmol/L, with a maximum bias of 0.5 mmol/L, analyzing
seven serum pools over a chloride range of 79.2 to 116.8
mmol/L. Coulometric titration, ion-selective electrode,
and mercuric-ferric thiocyanate methods all yielded
clinically identical results [5].
Specimen
Serum, heparinized plasma, urine, and other body fluids
are acceptable. Serum, plasma, and other fluids should
be promptly separated from cells to avoid shifts in the
ionic equilibrium with metabolism and pH changes.
Chloride in serum, plasma, urine, and other fluids is
stable for at least 1 week at room, refrigerator, or freezer
temperatures. Some ion-selective analyzers can measure
chloride activity in whole blood, and such measurements
should be made within 2 hours to avoid ionic shifts in
electrolytes. Samples collected in gel-barrier tubes do
not show any difference in chloride when compared with
samples collected in tubes not containing gel.
Sweat for chloride measurement is collected after
iontophoretic delivery of pilocarpine to skin sweat
glands to stimulate sweating. The area of skin that must
be stimulated depends on the volume of sweat needed
for analysis. Ion-selective electrode methods require less
volume than coulometric titration methods. In any event,
the rate of sweating must be greater than 1 g/m2/min to
obtain reliable results [6]. Lower sweating rates will
produce unreliable results because chloride
concentration varies with sweating rate and because the
quantity of chloride may be insufficient for accurate
analytical measurement. Children under 2 to 3 weeks of
age may have unusually high sweat electrolyte
concentrations. It is therefore advisable to delay the
sweat test until after this time [7]. Sweat specimens
should always be collected in duplicate. This procedure
is important in the diagnosis of cystic fibrosis.
Interferences
All the methods for chloride will show positive
interference from other halides. The only clinically
important interfering halide is bromide, which is
administered in some drug preparations. Bromide and
chloride do not react equivalently in all analytical
systems. Bromide ion showed a reactivity equivalent to
2.3 chloride ions with one ion-selective electrode
instrument, an equivalency to 1.6 chloride ions with the
continuous-flow mercuric thiocyanate method, but an
equivalency to 1 chloride ion with the coulometric
titration method [8]. Since one would like to be able to
detect bromism, a system that is highly responsive to
bromide may be desirable.
Ion-selective, electrode-based methods are generally not
affected by bilirubin or triglycerides. Gross hemolysis
may, however, result in a negative bias as a result of
dilution. The ferric thiocyanate procedure is subject to
interference from bilirubin, hemoglobin, and lipemia if
the assay does not include a protein-separation step, such
as dialysis. However, procedures using small sample
volumes (0.01 mL of serum to 1 mL of reagent) show no
interference from bilirubin up to 220 mg/L or from
triglycerides up to 6 g/L [3].
Chloride Reference Interval
Serum or Plasma, 101 to 111 mmol/L.
These values were determined as the central 95% range
obtained from analysis of 1205 (596 males, 609 females)
healthy, ambulatory persons 1 to 78 years old. No
variation with sex or age was observed. An automated
mercuricferric thiocyanate method, which was
calibrated against the coulometric titration method using
primary aqueous standards, was used. Reference
intervals obtained with use of ISE-based methods are
generally lower by 2 to 3 mmol/L. A slight diurnal
variation in chloride has been described; maximum
values occur from noon to 2:00 pm, and lowest values
are seen at night. Chloride concentrations may decrease
slightly following meals because of the role of chloride
in gastric acid production.
Urine, 110 to 250 mmol/24 h.
Urine chloride levels are strongly influenced by dietary
intake.
Sweat [6,7,9,10]
Normal 0 to 40 mmol/L
Indeterminate for cystic fibrosis 40 to 60 mmol/L
Suggestive of cystic fibrosis >60 mmol/L
379
Chloride
Reliable sweat chloride measurements depend on an
adequate rate of sweating being achieved, as discussed
earlier. A repeat determination of sweat chloride (new
collection and analysis) should always be made to
confirm a positive finding. Intermediate values do not
indicate heterozygote status. Adults have a more variable
sweat composition than children and can have sweat
chloride values up to 70 mmol/L.
Interpretation
Blood chloride concentration changes in a variety of
electrolyte disturbances, often paralleling changes in
sodium concentrations. Thus chloride levels increase in
dehydration and are decreased in overhydration,
congestive heart failure, Addisons disease, and other
disturbances of sodium-water balance. Primary
decreases in chloride are seen after gastric suction,
vomiting, and diarrhea.
Chloride Performance Goals
The current targets for acceptable performance for
chloride measurements are reported values that are 5%
of the peer-group mean. Coulometric titration at normal
serum concentrations of chloride gives within-
laboratory, between-day coefficients of variation (CV) of
1% to 1.5% for automated methods and 2% to 2.5% for
manual methods. Analysis with automated ion-selective
electrode or mercuric thiocyanate methods gives a CV of
1%. The manual coulometric titration method and any of
the automated methods are recommended for routine
analysis of serum. Because of the wide range of chloride
concentrations possible in urine and sweat, chloride
analyses are best done with a coulometric method. Ion-
selective electrode methods are also satisfactory within
the manufacturers documented reportable range.
Mercuric thiocyanate methods are generally not suitable
for urine or sweat because of the limited range of
concentration sensitivity.
Chloride concentrations are relatively stable in healthy
individuals. Intraindividual variability was 1.2% in
healthy individuals over a 20-week period [11].
References
1 Cotlove E. Chloride: Standard Methods of
Clinical Chemistry. Vol 3. New York:
Academic Press; 1961:81-92;
2 Dietz AA, Bond EE. Chloride, coulometric-
amperometric methods. In: Faulkner, WR,
Meites S, eds. Selected Methods of Clinical
Chemistry. Vol 9. Washington, DC: American
Association for Clinical Chemistry; 1982:149-
152.
3 Levinson SS. Chloride, colorimetric method.
In: Faulkner, WR, Meites S, eds. Selected
Methods of Clinical Chemistry. Vol 9.
Washington, DC: American Association for
Clinical Chemistry; 1982:143-148.
4 Velapoldi RA, Paule RC, Schaffer R, Mandel
TJ, Gramlich JW. Standard reference materials:
a reference method for the determination of
chloride in serum. NBS Special Pub No. 260-
67. Washington, DC: U.S. Government Printing
Office; 1979.
5 Geisinger KR, Geisinger KF, Wakely PE Jr,
Batsakis JG. Serum chloride: a CAP survey.
Am J Clin Pathol 1980;74:546-551.
6 Clinical Laboratory Standards Institute. Sweat
testing: specimen collection and quantitative
analysis. Approved Guideline CLSI C34-A2.
Villanova, PA: CLSI; 2000.
7 Hammond KB, Johnston EJ. Sweat test for
cystic fibrosis. In: Faulkner WR, Meites S, eds.
Selected Methods of Clinical Chemistry. Vol 9.
Washington, DC: American Association for
Clinical Chemistry; 1982:347-351.
8 Elin, RJ, Robertson, EA, Johnson, E. Bromide
interferes with determination of chloride by
each of four methods. Clin Chem 1981;27:778-
779.
9 Gibson LE, Cooke RE. A test for concentration
of electrolytes in sweat in cystic fibrosis of the
pancreas, utilizing pilocarpine by iontophoresis.
J Pediatr 1959;55:545-549.
10 Warwick WJ, Hansen L. Measurement of
chloride in sweat with the chloride-selective
electrode. Clin Chem 1978;24:050-2053.
11 Gonzalez-Revalderia J, Garcia-Bermejo S,
Menchen-Herreros A, Fernandez-Rodriguez E.
Towards narrower analytical goals in routine
laboratory work. Clin Chem 1991;37:596.
380

Chloride

Table 1: Methods of Chloride Measurement

Method 1: Ion-selective electrode; quantitativepotentiometric end-point, or kinetic
Principle of analysis: Ag/AgCl(s); test solution | reference electrode
Comments: Most frequently used, good accuracy and precision; manual or automated
Method 2: Coulometric titration; quantitativetitration, end-point
Principle of Analysis: Ag+ + Cl AgCl()
Comments: Reference method, highly accurate; manual or automated
Method 3: Mercuric/ferric thiocyanate; quantitativeend point
Principle of analysis: 2 Cl + Hg(SCN)2 HgCl2 + 2(SCN)
3 (SCN) + Fe3+ Fe(SCN)3 (red); Amax, 525 nm
Comments: Less frequently used, good accuracy and precision; manual or automated
Method 4: Ferric chloride; quantitativeend point
Principle of analysis: Fe3+ + Cl (FeCl)2+
Comments: Uncommon
Method 5: Isotope dilution; mass spectrometric
Principle of analysis: Dilution of common isotopes with 37Cl
Comments: Definitive method; used in research or reference laboratories

Figure 1

Difference spectra of mercuric ferric thiocyanate complexed with chloride. Reagent versus water, long dashes; chloride
(100 mmol/L) plus reagent versus water, short dashes; chloride (100 mmol/L) plus reagent versus reagent, solid curve.
381
Chloride
Procedure: Serum Chloride by Coulometry
Principle
This coulometricamperometric titration is based on the
method of Cotlove [1]. The silver ions generated by the
instrument combine with the chloride in the test sample.
The end-point is detected by a second pair of electrodes
which measure the free silver ions present when all the
chloride ions have reacted.
Reagents
1. Nitric acetic acid reagent (0.1 mol/L HNO3 +
1.76 mol/L acetic acid). To 800 mL of distilled water,
add 6.4 mL of concentrated nitric acid and 100 mL of
glacial acetic acid. Mix. Stable for 6 months at room
temperature.
2. Polyvinyl pyrrolidone (PVA) solution, 18
g/L. 1.8 g of powdered PVA is placed in 100 mL of
cool distilled water. Heat just to boiling with stirring to
dissolve PVA. Cool. Use immediately to prepare
acid/PVA solution.
3. Acid/PVA solution. Add the 100 mL of PVA
solution to the 900 mL of nitric acetic acid reagent. Mix.
This is stable at room temperature for 6 months.
4. Chloride standard, 100 mEq/L of Cl. Place
5.845 g of dry NaCl in a 1-L volumetric flask, and add
approximately 900 mL of distilled water to dissolve the
salt. Bring volume to 1 L with distilled water and mix
well. Stable for 6 months at room temperature.
Specimen
Reagents and Materials
Assay
Equipment: The assay is performed in our
laboratory with a Labconco Digital Chloridometer
Model 442-5000. Different instruments will require
slightly different protocols, which will be found in the
individual instrument operation manuals.
1. Preparation of sample. Dispense 4.0 mL of
acid/PVA solution into vial. Pipet 0.1 mL of
standard, sample, or control into appropriate
vials, and rinse pipet in acid/PVA solution.
Mix. For blanks, use 4.0 mL of acid/PVA
solution. NOTE: Rinsing the pipet is absolutely
necessary to avoid viscosity errors between
aqueous standards and serum.
2. Daily setup. With the instrument off, verify
that the generator electrode is the same length
and thickness as the other electrodes. If not,
feed additional silver wire from the spool, clip
off the thinned-out tip so that the fresh wire is
at the same length as the other electrodes.
Tighten the binding post/electrical connection,
and polish all of the electrodes. Rinse
thoroughly with deionized water and blot dry.
Make sure that no liquid is bridging any of the
electrodes at their common mounting post.
3. Reagent blank time delay adjustment. Turn the
instrument on. Set the TITRATION switch to
AUTO. Fill each of six vials with 5.5 mL of
acid/PVA solution. Raise each vial, in turn, into
position. Record each of the blank times,
average the last four values, and enter that
number on the BLANK thumbwheel switch.
4. Titration. Raise a vial containing 5.5 mL of
acid/PVA solution and 0.1 mL of chloride
standard into position, and press the
TITRATION switch. The reagent blank time
will automatically be subtracted, and the value
displayed will be in meq/L. If replicate
determinations of the standard are not within +1
meq/L of the assigned value, use the
COMPENSATE switch and knob on the back
of the instrument to adjust the calibrator. If the
calibration of the instrument is adjusted with
the COMPENSATE switch, all specimens in
the run must be analyzed with the switch in that
position.
To analyze controls and patient specimens,
raise a vial containing 5.5 mL of acid/PVA
solution and 0.1 mL of specimen into position.
When the titration is complete, the displayed
reading will be the chloride concentration in the
specimen.
Calculations
For the chloridometer utilized in the above
procedure, no calculation is required. Rinse electrodes
after each sample.
For titrators without direct mEq/L readout, set
test switch to high. Run standard, control, and patient
samples, recording time in seconds for each. Calculate
the mEq/L as follows:
Time unknown time blank mEq/L Std = mEq/L of unknown
Time standard time blank
Example:
Titration time of standards: 42.3, 42.7 sec
Titration time of blanks: 1.7, 2.1 sec
Titration time of unknown: 41.2 sec
41.2 1.9 100 = 96.8 = 97 mEq/L
42.5 1.9
382
Cholesterol
Cholesterol
John R. Burnett and Ken Robertson
Name: Cholesterol
Clinical significance: Refer to Chapter 37, Coronary Artery Disease: Lipid Metabolism, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C27H46O
Molecular mass: 386.64 D
Merck Index: 2181
Chemical class: Sterol, lipid
H3C
CH3
CH3
CH3
CH3
Structure: HO
i rest in the free form. This has some analytical implications,
Principles of Analysis and Current Usage
Cholesterol is a sterol compound that is found in all
animal tissues and serves many important physiological
functions, including being a substrate for the synthesis of
bile acids and steroid hormones; it is an important
component in cell membranes. Because cholesterol
appears to be involved in atherosclerosis, cholesterol
measurement is one of the most common laboratory tests
used today [1].
The history of this famous organic compound goes back to
the 19th century. Liebermann [2], in 1885, first described the
color reaction of sulfuric acid with a solution of cholesterol
in acetic anhydride. Four years later, Burchard [3] reported
that a more intense blue-green color is produced when acetic
anhydride and sulfuric acid are added to a solution of
cholesterol in chloroform (Table 1, Method 1). Since that
time, this Liebermann-Burchard reaction has been widely
used as a colorimetric reaction for the estimation of
cholesterol in biological fluids. Literally hundreds of
cholesterol methods have been reported since then. For a
historical rsum and authoritative critique of cholesterol
methods, consult review articles by Zak and co-workers [4-
9], Tonks [10], Naito and David [11], Naito [12-14], and
Martnek [15]. The College of American Pathologists (CAP)
Check Sample exercise (Special Topics No. ST-104) written
by Werner et al. [16] is an excellent technical review.
Total cholesterol measurement includes both the esterified
and free forms of the sterol. In serum or plasma, two thirds
of the total cholesterol exists in the esterified form, with the
i
Cholesterol
Previous and current authors of this method:
First edition: Herbert K. Naito
Methods edition: Herbert K. Naito
Second edition: Herbert K. Naito
Third edition: Herbert K. Naito
Fourth edition: Herbert K. Naito
Fifth edition: John R. Burnett, Ken Robertson
since in some chemical reactions, the color development
with the ester cholesterol is greater in intensity than that with
free cholesterol. This in turn can lead to a large positive bias.
In some enzymatic reactions, hydrolysis of longer chain
cholesteryl esters, such as cholesteryl arachidonate, is not
complete, resulting in a negative bias. For this reason,
understanding the chemistry of the various cholesterol
methods and recognizing their limitations is of utmost
importance in the selection of the method to be used in the
laboratory. Most often, compromises must be made amongst
simplicity, speed, convenience, accuracy, and precision. The
result of these compromises up to now has been an assay that
still poses many problems for most laboratories and produces
results of questionable reliability [17].
Classification of Cholesterol Methods
Single-Step Methods
In these direct assays, there is no sample preparationthat
is, no isolation and purification of the steroid or steroids.
Thus direct procedures are those carried out on serum or
plasma samples without any prior solvent extraction steps.
These methods are simple and rapid and require little
manipulation of the sample and therefore are suitable for
automation. However, depending upon the chemical
reaction, these procedures are likely to exhibit both positive
and negative errors resulting from the presence of proteins,
bilirubin, hemoglobin, vitamins A, C, and D, steroid
hormones, uric acid, turbidity, and differences in the
chromogenicity of free cholesterol and ester cholesterol.
Many automated procedures are based on direct methods.
They are usually based on such acceptable methods as
Zlatkis et al. [18], Huang et al. [19], Pearson et al. [20], and
enzymatic procedures [21-42].
Two-Step Methods
In two-step assays, an organic-phase extraction step is
introduced before measurement of cholesterol and other
chemically related steroids. This pretreatment step removes
many nonspecific chromogens that might interfere with the
assay, with the relative chromogenic response of each
interfering substance being dependent upon the chemistry
383
Cholesterol
involved. Since no saponification step is involved in these
methods, the deleterious effect of differential color response
between free and ester cholesterol still remains, especially in
Liebermann-Burchard reactions. However, for most routine
clinical work, this two-step procedure has been well
accepted, with the values obtained corresponding closely
with that of the Abell et al. method [43]. The correlation is
even closer when a calibration factor is added to correct for
free and ester cholesterol differential color development. The
methods of Zak and Ressler [9], Carr and Drekter [44], Bloor
[45], and Chiamori and Henry [46] are examples of two-step
procedures.
Three-Step Methods
Three-step procedures involve, in addition to extraction of
cholesterol, a saponification step that hydrolyzes the fatty-
acid moiety from the cholesterol ester. Consequently, one
measures only free cholesterol. The CDC-proposed reference
method [47] for cholesterol, a modification of the method of
Abell et al. [43], belongs in this classification.
Four-Step Methods
Four-step methods go a step further than the three-step
method of extraction, saponification, and color development.
The total extractable steroids are purified for cholesterol
determination by the addition of a saponin, digitonin. The
reactive site on the C-3 position on the
cyclopentanoperhydrophenanthrene ring in cholesterol is a
hydroxyl group that is esterified by the digitonin. This causes
the complex to be precipitated. The addition of the digitonin
step also eliminates the effect of interfering nonspecific
chromogenic constituents. Empirically, four-step procedures
should be more accurate and precise than any other
procedure, with the possible exception of enzymatic
cholesterol determinations. However, unless extreme
precautions are taken, multiple steps may also mean multiple
errors. For instance, whereas digitonin precipitation
enhances the accuracy of the method, the digitonin must be
completely decomposed and removed or it will cause
additional color development and thus positive error.
The Schoenheimer and Sperry [48] and Sperry and Webb
[49] methods are four-step procedures for cholesterol
determinations. These two methods also employ
Liebermann-Burchard reagents. Although the Abell et al.
[43] method may be more tailored to serum or plasma
cholesterol analysis, four-step methods may be more suitable
for tissue extracts [11], since certain tissues may have
appreciable noncholesterol and cholesterol esters that may
contribute to the inaccuracy of the final development of
color.
Methods of Analysis
Literally hundreds of cholesterol methods have been
published, usually as modifications of the following
reactions: (1) Liebermann-Burchard, (2) iron-salt-acid, (3) p-
toluenesulfonic acid, or (4) enzymatic end-point.
Liebermann-Burchard Reaction
Among the nonenzymatic colorimetric reactions for
cholesterol, the Liebermann-Burchard (L-B) procedure
(Table 1, Method 1) is perhaps the most widely used. The L-
B reaction generally is carried out in a strong acid medium
sulfuric acid, acetic acid, and acetic anhydride. In the
chemical reaction, cholesterol goes through a stepwise
oxidation, with each step yielding a cholestapolyene
molecule, which has one more double bond than the
compound from which it was derived. The initial L-B step
involves protonation of the OH group in cholesterol and
subsequent loss of water to give the carbonium ion 3,5-
cholestadiene, which is the first step in the color reaction.
Sequential oxidation of this allylic carbonium ion by SO
yields a cholesta-hexenesulfonic acid chromophoric
compound with absorbance maxima (Amax) of 610 and 410
nm. This chemical reaction for cholesterol determination has
undergone many modifications through the years and has
included the measurement of either free or esterified
cholesterol or both. Digitonin, a saponin, reacts with the free
OH (at the C-3 position) group of the A ring of the sterol to
form an insoluble complex, cholesterol digitonide. The
measurement of the sterol content in the supernatant solution
provides an estimate of esterified cholesterol. To obtain free
cholesterol, one subtracts the esterified value from the total
(free plus ester) cholesterol value.
Iron-Salt-Acid Reaction
In 1953, Zlatkis and Boyle [18] proposed a new colorimetric
procedure for cholesterol, dependent on the magenta color
produced when a solution of ferric chloride in concentrated
sulfuric acid is added to a solution of cholesterol in glacial
acetic acid. The color developed in this reaction is more
intense and more stable than that developed in the L-B
reaction. This reaction (Table 1, Method 3) involves acetic
acidsulfuric acid in the absence of acetic anhydride. In this
reaction, however, Fe3+ must be added to obtain the desired
chromogen. As in the L-B procedure, the initial step is the
protonation of the OH group in the cholesterol molecule and
subsequent loss of water to form the carbonium ion (3,5-
cholestadiene). Serial oxidation of this allylic carbonium ion
by Fe3+ yields a tetraenylic cation with an absorbance
maximum of 563 nm. The iron-salt-acid procedures are
about sevenfold more sensitive than the L-B methods. This
increased sensitivity may be attributed largely to the
stabilizing effect on enylic cation formation at higher H2SO4
concentrations. In general, increasing the H2SO4
concentration would be expected to improve the stability of
each of the carbonium ions formed in the stepwise oxidation
of the sterol, thereby making it much more likely for one to
observe carbonium-ion formation in the iron-salt-acid
reaction than in the L-B reaction.
Measurements of serum cholesterol by the FeCl3-H2SO4
reaction were automated by Levine and Zak [50] and by
Block and co-workers [51].
In 1969, a micromethod for serum cholesterol, which
requires only 20 L of serum, was described by Jordan and
Knoblock [52]. In this technique, cholesterol is precipitated
as a dextran sulfate complex to avoid interference from
bilirubin. The precipitate is dissolved in glacial acetic acid,
and ferric chloride reagent is then added. Even greater
sensitivity can be achieved by fluorometric detection of the
384
Cholesterol
FeCl3-H2SO4 chromogen [53]. An automated fluorometric
technique for serum cholesterol was described by Robertson
and Cramp [54].
p-Toluenesulfonic Acid Reactions
These methods are based on reactions of cholesterol with p-
toluenesulfonic acid (p-TSA), acetic anhydride, glacial acetic
acid, and H2SO4 (Table 1, Method 4).
Enzymatic End-Point Reactions
Developments over the past 30 years have been toward more
rapid, direct analyses of cholesterol in serum or whole blood.
Pretreatment of samples increases the possibility for error
and increases turnaround time. These demands have led to
the most important development in cholesterol
measurements, which is the introduction of enzymatic
techniques.
Enzymatic techniques (Table 1, Method 5) for determining
cholesterol have emerged to compete with the classical L-B
reaction and have become the most popular method for
cholesterol analysis. The original work used preliminary
alkaline saponification of the sample to produce only free
cholesterol [9,38,44]. In the next step, cholesterol oxidase,
an enzyme almost entirely specific for cholesterol, was
added. This caused the breakdown of cholesterol to cholest-
4-en-3-one and hydrogen peroxide, after which several
different reaction systems have been used to produce a final
chromogen.
Subsequent developments led to the technique of enzymatic
hydrolysis of cholesteryl esters by employing cholesterol
esterase. The commercial kits on the market now offer a total
enzymatic procedure, utilizing the enzyme cholesterol
esterase to replace the chemical saponification. Cholesterol
esterase is specific for cholesteryl esters, splitting the esters
into free cholesterol and free fatty acids. The cholesterol
oxidase reaction follows, with the same enzyme reaction as
described above. The amount of color produced is directly
proportional to the amount of serum cholesterol. Because of
the increased use of enzymatic methods, a more extensive
discussion is provided here.
The first chemical step in the enzymatic methods for
cholesterol uses the enzyme cholesterol esterase to hydrolyze
the cholesteryl esters present in the serum to free cholesterol
and free fatty acids:
_
Cholesteryl esters + H2O cholesterol esterase
cholesterol + free fatty acids 1 the H2O2 produced by
As discussed previously, the second step uses the enzyme
cholesterol oxidase in the presence of oxygen to oxidize the
cholesterol (both the free cholesterol found in the serum and
the free cholesterol generated in step 1) to cholest-4-en-3-
one and H2O2:
_
Cholesterol + O2 cholesterol oxidase
cholest-4-en-3-one + H2O2 2
In this reaction, cholesterol concentration can be determined
by amperometric measurement of the rate of oxygen
depletion.
Other assays make use of the ability of H2O2 to oxidize
compounds to produce colored species that can be measured
spectrophotometrically:
_
2H2O2 + phenol + 4-aminophenazone peroxidase
quinone imine dye + 4H2O 3a
_
Acetaldehyde + NADP+ aldehyde dehydrogenase
acetate + NADPH + H+ 3b
or
_
H2O2 + methanol catalase formaldehyde + H2O 4
Formaldehyde + acetylacetone 3,5-diacetyl-1,4-dihydrolutidine 5
or
_
H2O2 + ethanol catalase acetaldehyde + H2O 6
peroxidase
H2O2 + homovanillic acid
2,2-dihydroxy-3,3-dimethyloxy-biphenyl-5,5-diacetic acid 7
or
_peroxidase
H2O2 + o-dianisidine chromophore + 2H2O 8
Reaction 3a (Trinders reaction), which forms the quinone
imine dye (absorbance maximum 500 to 525 nm), is the
basis of the majority of the methods currently on the market
[21,24,27,37,39,55]. Other indicator systems that have been
used in equation 3 include 4-aminophenazone plus 2-
hydroxy-3,5-dichlorobenzenesulfonic acid and 3,3,5,5-
tetramethylbenzidine.
The action of catalase in conjunction with methanol and
acetylacetone to produce dihydrolutidine (reaction 4 and
reaction 5) is measured at 405 nm. Reaction 5, monitored at
405 nm [22,30,35], has the advantage of not being subject to
bilirubin interference and has been developed as a rate
procedure to improve on the original time-consuming end-
point assay.
Reaction 3b is monitored at 340 nm as an end-point reaction
[56].
Additional methods that measure cholesterol by analysis of
cholesterol oxidase include:
(a) Polarography [38,57] employing electrodes specific
for hydrogen peroxide
(b) Action of peroxidase to oxidize homovanillic acid
to a fluorescent compound measured at 470 nm [33]
(reaction 7)
(c) the condensation with o-dianisidine in the presence
of peroxidase to produce a colored compound
which is measured at 450 nm [28,58] (reaction 8)
385
Cholesterol
According to a 2007 CAP Chemistry Participant Summary,
99% of the participants in the Chemistry Proficiency Survey
used an enzymatic method to measure total cholesterol [59].
High-Performance Liquid Chromatography
For many years, high-performance liquid chromatography
(HPLC) has been studied as a technique for the measurement
of cholesterol in serum, particularly in the research field.
One hindrance, however, has been that cholesterol with only
a single double bond is a poor absorber of ultraviolet light,
making detection difficult. Rather than trying to perform
these measurements at < 210 nm, one suggestion to
overcome this problem is to produce benzoyl derivatives of
the cholesterol, allowing detection at 228 nm possible
[60,61].
It has also been reported that the 4-3-cholestanone produced
after treatment with cholesterol oxidase is detectable at 241
nm [62].
Other work has looked at the derivatization of cholesterol
with 2-[2-(isocyanate)ethyl]-3-methyl-1,4-naphthoquinone,
or at the use of fluorescent detection following HPLC. Such
techniques do, however, take additional time, and each
derivatization step may introduce further error into the
results. At this juncture, these sensitive methods, although of
use for research purposes, are not suitable for the routine
clinical laboratory, where automation is required [63].
Reference and Preferred Methods
Definitive Methods
Definitive techniques permit measurement of the
concentration of a substance in a biological sample and
give results accepted as the nearest attainable to true
values. The Joint Committee for Traceability in
Laboratory Medicine (JCTLM) [64] recognizes two
isotopic dilution/gas chromatographymass spectrometry
(ID/GC-MS) techniques as definitive methods. They are
the methods of (a) Deutsche Gesellschaft fur Klinische
Chemie (DGKC) and (b) National Institute of Standards
and Technology (NIST)previously NBS. Such
techniques have coefficients of variation (CV) no greater
than 0.5% and a total uncertainty of no greater than 1%
(Cohen et al. [65]). Schaffer et al. [66] compared the
Karolinska Institute and NIST ID-MS methods for
cholesterol and found a 0.2% mean difference between
the two methods. The Karolinska Institute standard was
found to contain lathosterol (5--cholest-7-en-3-ol-3[]).
The NBS method appeared to be more precise, probably
because of the more complex and time-consuming
protocol for sample preparation and MS. For more
information on definitive cholesterol methods, consult
Cohen et al. [65] and Wolthers et al. [67]
The only method currently classified by JCTLM as a
Reference Method is the University of Ghents ID/GC/MS
method [64].
The Centers for Disease Control and Prevention (CDC,
Atlanta, GA) has proposed a modification of the procedure
of Abell et al. [24,27,29,30,34,39,43] for use as a reference
method for cholesterol [47]. This method includes hydrolysis
of the cholesteryl esters, extraction with petroleum ether, and
color development with acetic acidacetic anhydride
H2SO4 reagent. With proper equipment and good technical
skills, this procedure can be used to produce accurate,
precise results [68].
In practice, however, although the CDC method could be
used in most laboratories for calibrating their methods, it is
time consuming and requires particular expertise. Similarly,
ID/GC/MS techniques are not practical for routine
laboratories. In reality, only the enzymatic techniques
capable of being automated are suitable for use in most
working clinical chemistry laboratories.
Liebermann-Burchard Methods
The L-B reaction has been one of the most used cholesterol
chemistries. However, its wide usage does not automatically
ensure reliable cholesterol analysis. Much depends on
whether cholesterol (more specifically cholesteryl ester) is
hydrolyzed and purified before analysis. For example, 7-
dehydrocholesterol and 7--cholesterol give about two and
four times more color, respectively, with the L-B reaction
than cholesterol itself does. Also, the color intensity
produced by cholesteryl esters is greater than that produced
by free cholesterol. Thus the total cholesterol is
overestimated unless saponification is carried out first, as in
the methods of Schoenheimer and Sperry [48], Sperry and
Webb [49], and Abell et al. [43]. Because of these biases,
any procedure that uses an L-B type of reagent and does not
include saponification and purification (for example, with
digitonin) steps will give falsely elevated concentrations for
total cholesterol when pure cholesterol is used as a standard.
The Bloor method [45] is an example of a method yielding
results with a large positive bias. Microgram quantities of
vitamin A (retinol) give large positive errors by this method.
Enzymatic End-Point Methods
The enzymatic procedures are direct methods amenable to
automating. The primary advantages of the enzymatic
methods are that one avoids the use of the harsh reagents
required by the chemical determinations, and the
interferences of other blood constituents are greatly reduced
because of the specificity of the enzymes. A few cholesterol
analogs may interfere, but the magnitude of the interference
seen in the enzymatic assays is much less than the
interference seen with chemical measurements or is greatly
reduced. The option to determine the free and esterified
cholesterol is available with these methods.
A few precautions are necessary when one is using the
enzymatic kits that have quinone imine dye as the
chromogen. Blanking can cause problems with turbid
samples for manual procedures. For automated procedures,
all-glass tubing must be used after the point of the formation
of the quinone imine dye complex, since Tygon tubing will
absorb the dye, slowly releasing it in subsequent samples and
causing carryover problems.
Some variation in results of different methods may be caused
by measurement at different wavelengths. For example, a
range of 460 to 560 nm has been proposed for the Trinder-
based reaction (reaction 3), with 500 nm being the maximum
386
Cholesterol
absorbance of the dye. Commonly suggested wavelengths
for taking measurements are 505, 520, and 525 nm.
The problem of incomplete ester hydrolysis and thus of
standardization is the primary outstanding problem with
enzymatic methods for measuring cholesterol. This
incompleteness of hydrolysis may be generally true of all
cholesteryl esters, but one study indicates that it may be the
result of the specificity of the esterase [62]. For example, of
the various commercially available enzymatic cholesterol
reagents with aqueous preparations of cholesteryl esters,
those that have arachidonic acid or acetate as esters appear to
demonstrate limited reactivity. The species source of the
cholesterol esterase may also affect the specificity of the
reaction. Siedel et al. [31,32] and Deeg and Ziegenhorn [69]
reported a new commercial enzymatic preparation for
cholesterol with an esterase from a microbial source. It
reportedly had improved hydrolytic activity, virtually
completely hydrolyzing the cholesteryl esters (99.5%), as
verified by thin-layer chromatography. Siedel et al. [31,32]
found that the new reagent (Monotest Cholesterol, High
Performance, BMD, Inc., Houston, TX), with improved
lipolytic efficiency, yielded results that were identical to
those measured with a candidate reference procedure
involving alkaline cholesterol ester saponification.
The esterases may be the source of much of the difference
seen between results obtained by various enzymatic methods
and results obtained by reference procedures, with the
magnitude of the bias ranging from 4% to 6% of the total
cholesterol content [60-63,70,71]. To help circumvent this
analytical problem, secondary serum calibrators are required
for the assays to compensate for the incomplete cholesterol
esterase reaction.
A properly calibrated enzymatic method is certainly the
preferred method for routine laboratory analysis. Enzymatic
methods can be as accurate and precise as the Abell-Kendall
reference method. Since these methods are readily available
as kits, this technique is not presented in detail. A reference
L-B method is presented, since this may be used for the
initial calibration of an enzymatic method.
Specimen
Patient Preparation
Determinations of lipid constituents in plasma or serum are
usually performed on blood drawn from patients fasting for
12 to 16 hours. The fasting sample is essential for
triglyceride analysis, since triglyceride concentrations
increase as soon as 2 hours postprandially and reach a
maximum at 4 to 6 hours. Nonfasting samples are not
suitable for analysis, since elevated results caused by normal
assimilation of food cannot be distinguished from elevated
results resulting from abnormal lipid metabolism or inborn
errors of metabolism. Like total cholesterol, HDL cholesterol
concentrations change very little between fasting and
nonfasting conditions. In subjects in whom
hypertriglyceridemia is induced by nonfasting conditions,
interference with some HDL cholesterol assays becomes an
issue, causing a positive bias. In most healthy individuals,
the 12-hour fasting serum triglyceride concentration is about
80 to 100 mg/dL (0.9 to 1.1 mmol/L) and nonfasting is 140
to 180 mg/dL (1.6 to 2.0 mmol/L)not enough to cause
interference problems with HDL cholesterol measurements
(i.e., incomplete precipitation of LDL, VLDL, and
chylomicrons). However, with markedly
hypertriglyceridemic samples, that is, greater than 1,000
mg/dL (11.3 mmol/L), interference can occur with
colorimetric assays. Ethanol consumption causes acute but
transient elevations in serum triglyceride concentrations,
which are evident in carbohydrate-sensitive
hypertriglyceridemic persons. Therefore, it would be
advisable to request that the patient refrain from drinking
alcohol for 72 hours before the day of blood drawing.
Blood-Drawing Techniques
It is well recognized that plasma volume increases and the
concentrations of nondiffusible plasma components decrease
when a standing subject assumes a recumbent position, a
result of redistribution of water between the vascular and
extravascular compartments. A significant reduction in total
plasma cholesterol has been measured after 5 min, and
decreases of as much as 10% to 15% have been recorded 20
min after a recumbent position is assumed. The effect on
cholesterol concentration when the subject changes from a
standing position to a sitting position is also significant
though somewhat smallerabout 6% after 10 to 20 min.
If a tourniquet is used, it should be removed before blood
sampling, since prolonged use of a tourniquet has been
reported to increase lipid values. Serum cholesterol
concentrations were found to increase an average of 10% to
15% after 5 min of occlusion. From a practical standpoint,
errors of this magnitude would not normally be encountered,
since the tourniquet is usually removed within 30 to 60 sec,
and the changes occurring during this period are
insignificant. Increases of 2% to 5% have been observed
after about 2 min; these increases may be seen if difficulties
arise during sampling or if blood is taken by an
inexperienced technician.
Choice of Plasma or Serum
If phlebotomy is properly performed, either plasma or serum
is usually suitable for total cholesterol, triglyceride, or
phospholipid determinations. Because the plasma and serum
cholesterol values differ by 3% to 5%, the NCEP Laboratory
Standardization Panel recommended that if plasma is used,
the cholesterol values should be multiplied by 1.03 to
convert the values equivalent to serum values [72]. Plasma is
usually preferred when lipids and lipoproteins are being
chemically analyzed. If plasma is chosen, the suggested
anticoagulant is solid EDTA (1 mg/mL of blood), and the
blood cells should be separated as soon as possible (within 2
hours).
Certain anticoagulants, such as fluoride, citrate, and oxalate,
cause rather large shifts of water from the red blood cells to
the plasma, which result in the dilution of plasma
components. Heparin, on the other hand, causes no
detectable change in red-blood-cell volume and decreases
total cholesterol concentration by only 1% or less. Although
EDTA causes slightly greater changes than heparin does in
cholesterol concentration, it is generally preferred for
lipoprotein analysis for several reasons. EDTA retards the
387
Cholesterol
auto-oxidation of unsaturated fatty acids and cholesterol by
chelating heavy metal ions such as Cu2+. Oxidation leads to
alterations in the physical properties of the lipoproteins,
gross denaturation, and apparent degradation of lipoproteins.
In addition, EDTA can reduce changes that can occur as a
result of contamination by phospholipase Cproducing
bacteria. Such contamination is associated with spurious
increases in total glyceride concentration that are caused by
the production of partial glycerides from some
phospholipids. It is also associated with the artifactual
appearance of chylomicron-like particles and decreases in -
, -, and pre-lipoproteins. These changes are minimized by
the addition of EDTA, which is an antioxidant and an
inhibitor of phospholipase C activity.
It is good practice to perform the lipid analysis as soon as
possible. If analysis is substantially delayed after the sample
is drawn, alterations in lipoprotein composition can result
from the exchange of cholesterol esters and triglycerides
between HDL and the other lipoproteins. Several of these
processes can occur simultaneously and influence results.
Their cumulative effects can either be additive (they may
increase the error of the analysis) or compensatory (the
processes may have opposite effects on the results
minimizing the error), depending on the exact procedure.
Storage of Samples
Two of the common variables of sample handling that
influence measured lipid and lipoprotein values are the
length of time and the conditions under which samples are
stored before analysis. It is generally recommended that
plasma be stored in the liquid state when it is to be used for
lipid and, particularly, lipoprotein analysis or for lipoprotein
electrophoresis studies. In addition, the determinations
should be performed promptly. In practice, however, delays
can occur for a variety of reasons, and it may even be
necessary to analyze frozen samples. Several groups of
workers have measured lipoprotein levels in samples that
have been stored at different temperatures for varying
periods of time, frozen and unfrozen [15,73]. The
measurements were made with the analytical ultracentrifuge,
and the results generally indicated that the levels of all
lipoproteins may decrease with storage. No definitive studies
have been made determining the optimum freezing
temperature and the length of storage permissible at this
temperature. Cooper [74] reported that when specimens are
stored in the liquid state at 4C for longer than 1 week, high-
density lipoprotein (HDL) cholesterol values, reproducibility
of the test, and resolution of lipoprotein patterns decrease.
The HDL cholesterol values were found to be more stable
when the specimens were stored at 60C than when the
specimens were stored at 20C or 5C over 10 months. At enzymatic method. Haeckel and Perlick [75] reported that
present, the general consensus is that freezing at 60C
provides the longest stable storage and may allow for
reproducible results even after a year or more. Freezing at
20C will keep samples stable for a few months if self-
defrosting freezer units are avoided, since samples stored in
these units periodically undergo thawing and freezing,
causing a breakdown of the lipid and lipoprotein
components. If the samples are to be used in a few days,
refrigeration at 4C is sufficient. However, even at
refrigerated temperatures, spontaneous hydrolysis of the
triglycerides to free glycerol and fatty acids takes place and
effectively reduces the true triglyceride concentration. In
addition, the sharpness of the lipoprotein bands decreases
with time when electrophoresis is performed. Therefore, if
more than short-term storage is anticipated, serum and
plasma samples (but not whole blood) should be frozen to
prevent the spontaneous hydrolysis and oxidation of the
unsaturated lipids, which occurs during storage, especially at
warmer temperatures.
Stored serum or plasma samples must be adequately mixed
before use. Lipids will layer by density within the sample
when stored and give different results, depending on the
layer from which an aliquot is obtained. Simple inversion of
the tube that contains the serum or plasma is not sufficient,
particularly if the sample was frozen. Instead, the sample
should be carefully mixed on a vortex mixer for 5 to 10 sec.
Particular attention should be given to those samples that
have appreciable amounts of standing chylomicrons. One
can obtain accurate lipid results only when the chylomicrons
are dispersed evenly through the sample before taking an
aliquot for lipid analysis. If pronounced chylomicronemia
exists, the sample should be diluted with saline solution so
that positive-displacement errors do not occur with the
excessive macromolecules.
If an organic solvent extraction method is used, the extract
can be stored at 4C for short periods (a few days) if one is
concerned only with total cholesterol, triglyceride, or
phospholipid analyses. On the other hand, if one is interested
in fatty acid profiles, the extract should be frozen
immediately to inhibit auto-oxidation. Care must be taken
when one is storing the purified lipid extracts. Cork or
rubber stoppers or caps with paper, cork, or rubber liners
should be avoided when one is storing lipid extracts in
organic solvents. It is recommended that tubes specifically
designed for low-temperature storage should be used. Screw-
capped tubes with Teflon-lined caps are good storage vials if
a good seal is formed. Evaporation of the solvent can be a
problem if care is not taken to ensure the selection of a good
storage container. A well-sealed container or vial also helps
to keep oxygen out of the sample and prevent oxidation of
the unsaturated lipids. If the samples are to be stored in this
form for a long period, it is best to overlay the sample extract
with nitrogen before the vial is sealed.
Interferences
Because enzymatic techniques are the only ones in routine
use, only interferences relating to these will be discussed.
Only a few interferences have been reported for the
the coupled reactions of cholesterol esterase, cholesterol
oxidase, catalase, and aldehyde dehydrogenase (reaction 6
and reaction 3b) are not inhibited by bilirubin. In contrast,
enzymatic analyses of serum cholesterol, by action of
peroxidase with 4-aminoantipyrine and phenol, are inhibited
by bilirubin [39,40]. Miner-Williams [76] has reported that
certain surfactants can cause inhibition of cholesterol
oxidase activity.
388
Cholesterol
Although ascorbic acid (vitamin C) is known to interfere
with the Trinder reaction, in practice its effect in cholesterol
assays is not great. Martinello et al. [77] have shown that
oral doses as high as 4 g do not interfere significantly, owing
to the serum concentrations not reaching high enough levels
and also to the fact that by the time the sample is normally
assayed, the ascorbic acid has been oxidized. Many
cholesterol assays are performed on fasting samples, making
it less likely that vitamin C has been consumed shortly
before the sample is collected and assayed. However, mega-
doses (e.g., 15 g) or large intravenous dosages (e.g., 2 g IV)
may interfere, especially if the assay is performed soon after
collection. This interference could be a potential problem
with point-of-care assays.
Cholesterol Reference Interval
The 95th-percentile limit for normal total cholesterol values,
once advocated by Fredrickson et al. [78] for lipoprotein
phenotyping, is shown in Table 2.
Although it is customary to use normal limits or reference
intervals to determine abnormal values for most blood
constituents, the determination of abnormal cholesterol
levels is an exception to the rule. Prior to 1988, most clinical
laboratories and practicing physicians used the 95th-
percentile reference intervals when evaluating whether a
sample had normal or abnormal cholesterol values. These
nomograms are based on sampling apparently normal
persons in the U.S. population and arbitrarily defining
hyperlipidemia as being present when the plasma cholesterol
or triglycerides, or both, are above the 95th-percentile value
for the population to which the persons belong. For example,
a 40-year-old man would have been considered normal
with a total cholesterol of 260 mg/dL (6.20 mmol/L).
Unfortunately, because of the way we have defined normal
in the past, many test results do not correlate very well with
disease states or health-risk conditions on an individual
basis. Thus, the normal range may not be a healthy level.
Interpretation
There is a well-established curvilinear relationship between
cholesterol and risk of atherosclerotic CAD. CAD risk is
approximately 2% to 3% higher for each 1% increase in total
cholesterol concentration. However, most patients with CAD
have total cholesterol concentrations that are average for
their society. The measurement of total cholesterol includes
both atherogenic particles (mainly LDL) and
cardioprotective particles (HDL) which are independently
related to CAD risk. The association of LDL cholesterol is
therefore stronger than that for total cholesterol and is further
marked when accompanied by triglyceride elevation.
Furthermore, a ratio of total cholesterol to HDL cholesterol
is considered a better indicator of CHD risk than total
cholesterol alone. In epidemiological studies, the risk has
been shown to increase exponentially when the ratio is > 4.5.
Screening for dyslipidemia is indicated for all adults up to 75
years of age, regardless of CAD risk status, and for adults
older than 75 years of age who have multiple CAD risk
factors [79]. The current guidelines for classification of
serum lipids are shown in Table 3. Treatment approaches for
patients with dyslipidemias are based upon the number of
CAD risk factors and the concentrations of both LDL
cholesterol and HDL cholesterol. Current recommendations
for treatment approaches based on risk of CAD and LDL
cholesterol levels are shown in Table 4 [80].
Cholesterol Performance Goals
The current Clinical Laboratory Improvement Amendments
(CLIA) target for acceptable performance for cholesterol
measurements is 10% of the peer-group mean. Precision
goals based on the work of Fraser et al. [81] indicate that a
precision goal of a maximum standard deviation of 5.4
mg/dL at a cholesterol concentration of 200 mg/dL is
appropriate (a CV of 2.7%). The intraindividual variation in
cholesterol in healthy adults has been found to range from
approximately 4% to 8% over a 5-month period [82].
The NCEP Laboratory Standardization Panel recommended
laboratory standards for precision and accuracy of total
cholesterol measurements in the laboratory [72]. For the
interim goal for precision and accuracy, they recommended
5.0% CV or less and an average overall bias not to exceed
5.0% from the reference method values (which is
equivalent to 9.5% on a single measurement). They also
recommended that precision and accuracy should decrease to
3.0% CV or less and 3.0% or less, respectively. The 2007
CAP Chemistry Participant Summary [59] precision data
indicates that participants perform well against these criteria,
with all method CVs across the CAP range of values (106
to 268 mg/dL) being 2.8% to 3.1%. Some users of enzymatic
techniques are achieving CVs < 2% across that range.
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interference in the measurement of serum
biochemical parameters: In vivo and in vitro
studies. Clin Biochem 2006;39:396-403.
78 Fredrickson DS, Levy RI, Lees RS. Fat transport in
lipoproteinsan integrated approach to
mechanisms and disorders. N Engl J Med
1967;276:148-156.
79 Anderson RJ, Bergman DA, Davidson J, Palumbo
PJ, Rettinger HI, Rubenstein HA et al. The
American Association of Clinical Endocrinologists
medical guidelines for clinical practice for the
diagnosis and treatment of dyslipidemia and
2000;6:162-213.
80 Summary of the second report of the National
Cholesterol Education Program (NCEP) expert
panel on detection, evaluation, and treatment of
high blood cholesterol in adults. JAMA
1993;269:3015-3023.
81 Fraser CG, Peterson PH, Ricos C, Heackel R. In:
Heackel R, ed. Evaluation of Methods in Laboratory
Medicine. New York: VCH Publishers; 1993:87-99.
82 Fraser CG. Biological variation in clinical
chemistry. An update: collated data, 1988-1991.
Arch Pathol Lab Med 1992;116:916-923.

Table 1: Methods for Serum Cholesterol Analysis

Method 1: Liebermann-Burchard (L-B); one-, two-, three-, or four-step method
Principle of analysis: Cholesterol extracted and reacted with strong acid (sulfuric acid) and acetic anhydride to form
colored cholestahexaenesulfonic acid molecule (Amax, 410 nm); nonesterified cholesterol precipitated by digitoxin,
and remaining cholesterol measured and free cholesterol calculated; Total Esterified = Free
Comments: Very common method; total cholesterol reaction overestimates concentration of esterified cholesterol;
unstable color
Method 2: Abell et al. [7]; three-step method
Principle of analysis: Cholesterol extracted with zeolite, esters chemically hydrolyzed (saponification), and total
cholesterol measured by L-B reaction
Comments: Considered current reference method; laborious
Method 3: Iron-salt-acid; two-step method
Principle of analysis: Similar to reaction conditions of Method 2, except Fe3+ ions are added to yield tetraenylic
cation (Amax, 563 nm)
Comments: Not frequently used; sevenfold more sensitive than L-B method; free and esterified cholesterol give
same color; no need to hydrolyze esters
Method 4: p-Toluenesulfonic acid (p-TSA); three-step method
Principle of analysis: Similar to method 3; p-TSA reacts with cholesterol derivative to form chromophore (Amax,
550 nm)
Comments: Rarely used; free and esterified cholesterol give same color
Method 5: Enzymatic end-point; one-step method
Principle of analysis:
_
a. Cholesterylesters cholesterol esterase cholesterol + fatty acids
_
b.* Cholesterol + O2 cholesterol oxidase cholest-4-en-3-one + H2O2
_
c. H2O2 + 4-aminophenazone (or other dye) peroxidase oxidized dye
(Amax, 500 nm) + H2O2
Comments: Most common method; accurate and easily automated; future reference method
*Can monitor reaction by following O2 consumption with oxygen electrode.

Table 2: Historical Reference Intervals for Total Serum Cholesterol [79]

Age Men* Women* Suggested Normal Limits
(years) mg/dL mg/dL mg/dL mmol/L
0-19 172 34 179 33 120230 (3.105.95)
20-29 183 37 179 35 120240 (3.106.21)
30-39 210 33 295 37 140270 (3.626.98)
40-49 230 55 217 35 150310 (3.888.02)
50-59 240 48 251 49 160330 (4.148.53)

*Total cholesterol in mg/dL in mean standard deviation (values in parentheses are in mmol/L).
Conversion Factors: mg/dL 0.0259 = mmol/L mmol/L 38.6 = mg/dL
392

Cholesterol

Table 3: Classification of Adults Based on Total and HDL Cholesterol

Total Cholesterol
Desirable <200 mg/dL
Borderline high 200-239 mg/dL
High >240 mg/dL
HDL Cholesterol
Low <40 mg/dL
High >60 mg/dL

Table 4: Treatment Approach Based on CAD Risk Factors and LDL Cholesterol Level (mg/dL)
Nutrition Therapy, Drug
Setting Physical Activity Therapy Goal

CAD risk factors

<2 >160 >190 <160
>2 >130 >160 <130
With atherosclerotic disease >100 >130 <100
With type II diabetes mellitus >100 >130 <100
Note: Subtract one risk factor when HDL cholesterol is 60 mg/dL.
393

Cholinesterase

Cholinesterase
Danyal B. Syed
Name: Cholinesterase, acylcholine acylhydrolase, cholinesterase 2 (ChE 2),
RBC cholinesterase, AChE; Pseudocholinesterase, butyrylcholinesterase,
cholinesterase I (ChE 1), plasma cholinesterase, CHS
Clinical significance: Refer to Chapter 52, Diseases of Genetic Origin, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Enzyme number: EC 3.1.1.7 (Acetylcholinesterase, RBC cholinesterase, acetylcholine
acetylhydrolase)
EC 3.1.1.8 (Pseudocholinesterase, butyrylcholinesterase, acylcholine
acylhydrolase, plasma cholinesterase)
Molecular mass: 360,000 D
Number of units: Four identical subunits
Chemical class: Enzyme, protein
Known enzyme forms: RBC, brain, liver
Chromosomal location [1,2]: Acetylcholinesterase (3.1.1.7) = 7q22;
Butyrylcholinesterase ( 3.1.1.8) = 3q26.1-26.2

Biochemical reaction:

Principles of Analysis and Current Usage

i less specific for this substrate than is the red-blood-cell
The polymorphic serine esterases that catalyze the
hydrolysis of the acyl-esters to respective alcohols and
carboxylates are called cholinesterases. They differ in
their substrate specificity, sensitivity to certain
inhibitors, and their localizations at both cellular and
subcellular levels. There are two types: (1)
acetylcholinesterase (EC 3.1.1.7), also called RBC
cholinesterase, is a true cholinesterase found in the
blood and neural synapses; and (2) pseudocholinesterase
(EC 3.1.1.8), also referred to as plasma or serum
cholinesterase (and CHS, cholinesterase I, and
butyrylcholinesterase), which is synthesized by the liver
and is present in plasma [3-5]. Its true physiological
function is unknown. The two types differ in their
respective substrate preferences. Although
pseudocholinesterase can hydrolyze acetylcholine, it is
i
Cholinesterase
Previous and current authors of this method:
First edition: Mary Ellen King
Methods book: Mary Ellen King
Second edition: Mary Ellen King
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Danyal B. Syed
enzyme acetylcholinesterase, and so its function may be
to hydrolyze other choline esters. Cholinesterase activity
is usually measured for one of two reasons. The most
common reason is the determination of a decrease in
enzymatic activity as an indicator of exposure to
organophosphorus compounds, including many
pesticides. The second reason is to identify the presence
of inherited abnormal variants of cholinesterase.
Variants are identified by assaying both total activity and
the extent of inhibition by either dibucaine or fluoride;
some of these variants lead to prolonged apnea in
patients receiving the anesthetic succinylcholine or
mivacurium. Measurement of acetylcholinesterase
activity can be performed during amniotic fluid analyses
for neural tube defects to confirm an elevated amniotic-
fluid alpha-fetoprotein (AFP) level.
Since pesticide poisoning can present acutely, an assay
for cholinesterase must be simple enough to be
performed stat but accurate enough for determination
of cholinesterase phenotyping. The common test
principle for measuring cholinesterase activity is based
on the use of thiocholine esters. These substrates are
known to be unstable in aqueous solutions. Serum
acylcholinesterase, although active against most of the
thioesters of choline, is best determined by using
butyrylthiocholine as a substrate, which is stable in
394
Cholinesterase
aqueous solution for 30 days [6]. Cholinesterase activity
is usually measured by one of the three methods
described below:
1. Spectrophotometric. This method uses
propionylthiocholine or acetylthiocholine as substrate
(Table 1, Method 1). 5,5-dithiobis(2-nitrobenzoic acid)
(DTNB) is added to react with the released thiocholine
to form the yellow compound, 5-thio-2-nitrobenzoic acid
(absorption maximum, 410 mm) [7,8]. The reactions
may be followed as a rate or end-point procedure.
2. Titrimetric [9-13]. These methods follow the release
of hydrogen ions from a choline ester. One titrimetric
method (Michel method) directly measures hydrogen-
ion release by monitoring pH changes over a 1- to 1-
hour period (Table 1, Method 2). A second titrimetric
colorimetric method quantifies the amount of hydrogen
ion released by the hydrolysis of acetylcholine by
following the color change of a phenol red indicator over
a 30-min incubation time (Table 1, Method 3). The color
change is related to the moles of substrate converted.
3. Electrometric [14-16]. This is a special type of
titrimetric method usually referred to as an electrometric
procedure. It uses highly automated titration
instrumentation to continuously add base to neutralize
the hydrogen ions released from acetylcholine by
cholinesterase (Table 1, Method 4). This instrument
maintains the enzyme reaction at constant pH, and the
amount of base added is directly related to the number of
moles of acetylcholine hydrolyzed on a mole-per-mole
basis.
The presence of true acetylcholinesterase activity in
amniotic fluid can be determined by a quantitative
[17,18] or qualitative assay [19,20]. In either case, an
inhibitor of acetylcholinesterase activity is used. The
most frequently used inhibitor is 1,5-bis(4-
allyldimethylammoniumphenyl)pentan-3-one dibromide
(BW 284C51). For a quantitative assay, the enzyme
activity can be measured by Method 1 in Table 1. The
qualitative assay for acetylcholinesterase employs
polyacrylamide gel electrophoresis to separate
isoenzymes of cholinesterase [19,20]. After
electrophoresis, the gels are incubated with
acetylthiocholine in the presence and absence of the
acetylcholinesterase inhibitor BW 284C51. The
thiocholine formed by the reaction reacts with copper
sulfate, also present in the reaction mixture, to form a
white cupric thiocholine precipitate. The zone of
acetylcholinesterase activity is identified by its
disappearance when the acetylcholinesterase inhibitor is
present.
Reference and Preferred Methods
Currently there is no formal reference method for the
determination of cholinesterase activity. Some 3 decades
ago, propionylthiocholine/DTNB method was selected to
be the method for measuring cholinesterase activity by
the American Association of Clinical Chemistry
(AACC) [7]. In 1992, a national German procedure
utilized butyrylthiocholine as substrate [6]. The most
commonly used procedure in Italy and many other
countries employs the use of butyrylthiocholine as
substrate and DTNB as the chromogenic agent. More
recently it has been proposed [21] that the substrate
succinyldithiocholine/DTNB system be considered as a
basis for a candidate reference procedure for
standardization of the cholinesterase assays within the
work of the IFCC Committee on Reference Systems on
Enzymes [22].
These proposals were based upon the evaluation of six
different methods for the purpose of providing
comparison between methods for their ability to identify
succinyldicholine-sensitive patients. Cholinesterase
activity was measured using a total of 131 patients sub-
grouped according to cholinesterase phenotypes with
different sensitivity to succinyldicholine. By determining
the phenotypes based on dibucaine and fluoride
numbers, and by performing the DNA analysis,
correlation between the phenotype classification and
genotype was confirmed. The tested methods
significantly differed in their ability to discriminate
between the subjects with and without
succinyldicholine-sensitive phenotypes, as exhibited by
the area under the receiver operating curves (ROC) as
follows (21):
Methods Substrate Area under ROC
1. Succinyldithiocholine/
5,5-dithio-bis-nitrobenzoate (DTNB) 0.97
2. Propionylthiocholine / DTNB 0.94
3. Butyrylthiocholine (BTC) 0.90
4. Benzoylcholine 0.90
It was concluded that the succinyldithiocholine/DTNB
assay is the method of choice for selecting subjects
sensitive to succinyldicholine treatment and thereby
developing apnea. At present, most automated
instruments prefer the use of butyrylthiocholine as
substrate for determining the cholinesterase activity.
With the Vitros analyzer [23], butyrylthiocholine is also
used as substrate, but the hydrolysis product,
thiocholine, reduces the chromogen, potassium
hexacyanoferrate III (potassium ferricyanide), to
hexacyanoferrate II [24]. The rate of the loss of color, as
measured by reflectance spectrophotometry, is
proportional to the cholinesterase activity in the sample.
The thio-substrates are normally unstable and must be
prepared fresh prior to use. The use of polar solvents to
enhance the stability of BTC has recently been patented
[25]. In an earlier patented invention [26], p-
hydroxybenzoyl choline is used as substrate in the
presence of p-hydroxybenzoic acid hydroxylase enzyme.
The cholinesterase hydrolysis product p-hydroxybenzoic
acid is acted upon by p-hydroxybenzoic acid
hydroxylase enzyme in the presence of NADPH, which
is oxidized to NADP+ and measured by the decrease in
extinction coefficient at 340 nm. The reaction can also
be monitored by measuring the loss of oxygen using an
oxygen detector system and has been fully automated.
As is evident, this enzyme-coupled reaction to measure
the cholinesterase enzymatic activity avoids the use of
395
Cholinesterase
thioester compounds altogether as substrates.
Colorimetric assays using substrates as described above
are also available for several automated chemistry
systems. Dibucaine inhibition assays are also available,
or the automated cholinesterase assay can be modified in
the laboratory to measure the inhibition [27-30].
The temperature optimum is 37C to 40C for both
cholinesterase and acetylcholinesterase activity. The
Q10 (temperature activity coefficient) is 1.3 for
acetylcholinesterase and 1.5 for cholinesterase. A
standardized temperature of 37C has been
recommended for assessing cholinesterase sensitivity to
dibucaine and fluoride. However, some of the variant
forms of cholinesterase have temperature maxima as low
as 32C or as high as 51C, and both fluoride and
dibucaine inhibition numbers decline as the
measurement temperature is raised above 25C [3].
Described below is a simple colorimetric method based
on the procedure of Ellman, requiring only a
spectrophotometer and 10 min of analysis time. This
method has been published by the American Association
for Clinical Chemistry [7]. The substrate
propionylthiocholine iodide is used, since it gives lower
blank readings and 27% greater activity at 37C than
butyrylthiocholine iodide. Easier discrimination among
some common cholinesterase phenotypes is observed
with use of this substrate. Between-run coefficients of
variation (CVs) of 3% are reported [7,8]. The reaction
conditions are summarized in Table 2.
The Michel method [9] has served as a standard method
during the collection of most of the clinical data of
pesticide exposure and is still widely used. The
procedure is slow (10 min of equilibration plus 1 hour of
incubation), and good accuracy and precision depend
heavily on excellent temperature control and careful pH
measurement [4]. A 1C change produces a 5.5% change
in cholinesterase activity and a 3% change in
acetylcholinesterase activity. Newman and Que Hee [31]
investigated cholinesterase results using the Michel
method and recommended use of a reagent blank rather
than correction tables for improved accuracy. They also
found better inter-run precision with use of a reagent
blank (CV = 6%) than with use of the correction table
(CV = 10%).
The electrometric procedure, in which the neutralization
of the reaction is monitored by an automatic titrator,
requires a specialized instrument dedicated to the assay
but offers the advantage of being a rapid, rate method
[14,15]. CVs of 4% to 5% are reported [16]. The
instrument can also be used to measure
acetylcholinesterase.
Because of its ease of adaptation to most laboratory
spectrophotometers, the Ellman method is recommended
if cholinesterase is not available on automated
equipment.
Qualitative gel electrophoresis is the method of choice
for detection of amniotic fluid acetylcholinesterase [32].
Since this test is used to confirm the suspicion of a
neural tube defect after an elevated amniotic fluid AFP
level is found, results with high clinical specificity are
required. The British collaborative report showed that
the mere presence of acetylcholinesterase activity was
sufficient evidence to confirm an elevated AFP result.
Specimen
No special patient preparation is needed. Either serum or
heparinized plasma may be used for cholinesterase
assay. Cholinesterase is stable for up to 80 days at room
temperature and for 3 years if frozen at 20C [3].
Samples should be stored and shipped cold to avoid wide
extremes of temperature. Samples can be drawn into
either a plain red-top tube or a speckled-red-top tube
with gel as serum separator, or for plasma into a green-
top tube with heparin as anticoagulant, employing
normal phlebotomy procedure. Samples should be
centrifuged and serum or plasma separated quickly from
the clot or cells, avoiding hemolysis. If samples are
frozen, repeated freeze/thaw cycles should be avoided.
Samples can also be collected onto filter paper using the
dried-blood-spot method. This sample collecting method
is especially useful in field studies to evaluate
cholinesterase activity in young children to detect and
monitor organophosphate pesticide exposure in children
living in agricultural communities. The amount of
activity in dried blood has been determined to be less
than the activity in plasma or serum but exhibits
significant correlation: Spearman r = 0.6 (p = 0.01) [33].
Samples believed to contain a reversible inhibitor should
be collected on ice, kept cold, and assayed as soon as
possible to limit in-vitro destruction of the inhibitor.
Sample-to-reagent dilution and incubation times should
be minimized in the assay to limit reactivation of the
enzyme during assay [4].
Interferences
Moderate hemolysis will not interfere if the serum is
well centrifuged to remove red-blood-cell ghosts [7].
Lipemia or icterus (bilirubin up to 20 mg/dL) reportedly
does not interfere. The presence of oxalate or fluoride
has been reported to significantly decrease measured
cholinesterase activities [34]. Similarly citrate
anticoagulant, heavy metals, borate, some serum
separators, insecticides, organophosphates, carbamates,
neuromuscular relaxants, phenothiazines, both anabolic
and glucocorticosteroids, oral contraceptives, estrogens,
radiographic agents such as iopanoic acid, streptokinase,
testosterone, and many other such compounds have been
observed to decrease cholinesterase activity. Plasma
cholinesterase activity has been noted to decrease after
plasmapheresis and increase after transfusion in patients
who have abnormally low cholinesterase activity [35].
396
Cholinesterase
Cholinesterase Reference Intervals
Each laboratory should establish its own reference range
based upon population, instrument and methodology
used. Generally it is between 4 and 19 U/mL or 4,000
and 19,000 U/L. Cholinesterase values in neonates and
infants younger than 6 months are 40% to 50% of adult
levels [36]. Young adult females, 18 to 35 years of age,
have cholinesterase levels 64% to 74% of adult male
values. Older females and males (70 to 80 years of age)
have values not significantly different from those of
young adult females [37]. Cholinesterase activity
decreases during pregnancy [38].
Acetylcholinesterase levels in neonates are 63% of adult
levels [39] and rise gradually to adult levels by 1 year of
age [40]. There are no male/female differences in
acetylcholinesterase values in adults [37].
The most common phenotype (U, usual) is not
associated with a prolonged response to succinylcholine
and is inhibited approximately 84% by dibucaine and
80% by fluoride (see Table 3) [41]. Other phenotypes
that can occur include dibucaine-resistant A (atypical)
and fluoride-resistant F (fluoride). The so-called silent
phenotypes, S1 and S2, display little or no cholinesterase
activity in the homozygous state. The homozygous
variant forms are usually not inhibited more than 20% to
50% by the relevant inhibitor.
Acetylcholinesterase is present in fetal serum but is not
normally detected in amniotic fluid. The intraindividual
variation for cholinesterase activity in healthy adults has
been reported to be less than 7% [3,4,5,7].
The cholinesterase activity level in cerebrospinal fluid
(CSF) has been observed to be 1/20 to 1/100 of the
corresponding serum/plasma activity. In clinical
conditions where bleeding into CSF takes place, the CSF
cholinesterase enzymatic activity has been demonstrated
to increase to 1/4 to 1/2 of corresponding plasma activity
level [42].
Interpretation
Plasma cholinesterase (CHS) is found in plasma, liver,
brain, kidney, intestine, and pancreas. In contrast, RBC
cholinesterase (acetylcholinesterase) is found in red
blood cells, nerve tissue, skeletal muscle, and placenta.
The two enzymes have different Kms for acetylcholine
and succinylcholine, as tabulated in Table 4 [43,44].
Some cholinesterase variants are unable to break down
succinylcholine, a muscle relaxant used in anesthesia,
causing prolonged apnea and paralysis. It is important to
determine if a cholinesterase variant is the cause of an
unusual response to succinylcholine so that the drug can
be avoided or its dosing modified if the patient needs
anesthesia in the future. The frequency of response is
cited in Table 5 [5].
Measurements of RBC acetylcholinesterase (AChE) and
plasma cholinesterase (CHS) are performed to monitor
worker exposure to certain toxic pesticides, principally
organophosphates and carbamates. These compounds
have sufficient structural similarity to acetylcholine to
bond to both cholinesterase enzymes; organophosphates
phosphorylate and the carbamates acetylate a serine
residue of the enzymes active sites. The stability of
these bonds depends on their sensitivity to hydrolysis;
half-lives of the various inhibitor-enzyme complexes
range from minutes to weeks. The carbamates, which
form bonds that hydrolyze with relative ease in vivo and
in vitro, are referred to as reversible inhibitors. Other
compounds containing quaternary or tertiary amines or
other positively charged atoms may also reversibly
inhibit cholinesterases; these include physostigmine and
sulfonium and arsonium compounds [4].
The laboratory picture resulting from pesticide poisoning
is dependent on the pesticide involved and the level and
time frame of exposure. Some compounds preferentially
inhibit cholinesterase; others inhibit acetylcholinesterase
more strongly. Pesticides that preferentially inhibit
cholinesterase after low-level exposure will often inhibit
acetylcholinesterase as well if a longer exposure occurs
[45]. For example, a person exposed to slight to
moderate levels of a cholinesterase inhibitor will first
have a drop in cholinesterase activity as the enzyme is
inactivated in vivo. If the low-level exposure continues,
red cell acetylcholinesterase may also decline. This same
pattern of low cholinesterase and low
acetylcholinesterase activities could, however, be found
after a single, recent, moderate to severe exposure to the
same pesticide. When the exposure to the inhibitor stops,
cholinesterase activity returns to normal before
acetylcholinesterase activity, because regeneration of
cholinesterase through liver synthesis of the enzyme
proceeds faster than bone-marrow replacement of the red
blood cells, which contain acetylcholinesterase. A
person recovering from pesticide exposure that resulted
in depressed cholinesterase and acetylcholinesterase
levels will have normal levels of cholinesterase with low
levels of acetylcholinesterase for a time, the same picture
that another person would have after a mild exposure to
a pesticide preferentially inhibiting acetylcholinesterase.
A considerable decline in the activities of either or both
enzymes can occur before clinical symptoms appear. For
example, values as low as 10% of original levels may be
seen in a person exposed to low levels of pesticide over a
long period of time [45]. However, mild illness can
occur while either or both enzyme levels are within
population-reference intervals, particularly after short-
term moderate exposure to pesticide. In nearly all severe
poisonings, both acetylcholinesterase and cholinesterase
activities drop sharply.
Because of the relatively wide population reference
intervals for cholinesterase and acetylcholinesterase, it is
best to interpret a persons enzyme activity levels in
relation to his or her personal reference values. This is
particularly important in the evaluation of possible low-
level pesticide exposure, where only a small decline in
one or both enzymes may occur. A worker should have
397
Cholinesterase
at least two baseline measurements made of
cholinesterase and acetylcholinesterase before beginning
work with pesticides. Intraindividual CVs average 7% to
11% for each enzyme, with maximum fluctuations of
20% to 25% in healthy persons [45]. If either enzyme
activity drops 20% to 30% or more on a subsequent
analysis, pesticide exposure should be considered. Drops
of 50% to 75% are clear danger signals that poisoning
has occurred, and hospitalization may be required [46].
Other conditions associated with reduced cholinesterase
activity include liver disease, malnutrition, acute
infection, and some anemias. The presence of these
conditions must be considered in the evaluation of
enzyme changes. Acetylcholinesterase levels reflect the
number and age of the red blood cell population, with
the highest levels in the youngest erythrocytes [4].
Because the circulating levels of cholinesterase and
acetylcholinesterase are controlled by different
physiological mechanisms, a drop in both enzyme levels
points very strongly to exposure to an inhibitor such as a
pesticide.
The presence of both true acetylcholinesterase activity
and elevated AFP levels in amniotic fluid is considered
presumptive evidence of an open neural tube defect in a
fetus. The British Collaborative Acetylcholinesterase
Study [32] showed that 99.5% of all pregnancies
associated with an anencephaly and open spina bifida
had positive acetylcholinesterase results by gel
electrophoresis. Pregnancies associated with exomphalos
(75%), miscarriage (47%), and other serious congenital
malformations (50%) also had positive
acetylcholinesterase results. Only 6.4% of pregnancies
not associated with serious fetal malformations or
miscarriage had positive acetylcholinesterase results.
However, all but one of the false-positive cases were
associated with significant contamination of blood,
either of fetal or unspecified origin.
The involvement of cholinesterase in triglyceride
metabolism and correlation of its activity with plasma
LDL-cholesterol [47], HDL-cholesterol [47-51], and
triglyceride concentration has been reported. It has also
been shown to exhibit significant association with the
metabolic syndrome, as well as diabetes. This
connection between the cardiovascular risk factors,
metabolic syndrome, and cholinesterase activity has
recently been confirmed [52]. These studies established
heritability for plasma cholinesterase activity in the
general population and identified two chromosomal
locations that contribute to this heritability between the
cardiovascular risk factors, metabolic syndrome, and
cholinesterase activity.
It has been suggested that the low-activity K variant has
a causative role in Alzheimers disease [53-55].
Biochemical studies have also indicated that
acetylcholinesterase (AChE) induces amyloid fibril
formation, resulting in the formation of highly toxic
AChE-amyloid Abeta complexes, triggering more
neurodegeneration than those of the Abeta peptide alone
both in vitro and in vivo.
The fact that AChE is able to enhance amyloid
formation, the suggestion that the AChE activity
associated with amyloid formation is exhibited by a 3.5
kDa peptide (H peptide) containing a tryptophan of the
enzyme peripheral binding site [56-58], and that such an
effect is sensitive to drugs that block this AChE activity
of the H peptide, has resulted in the use of AChE
inhibitors such as donepezil, rivastigmine, and
galantamine, which are now commonly used
therapeutically in the treatment of mild to moderate
Alzheimers disease, possibly to reduce the
neurodegeneration and neurotoxicity [59]. However, the
level of efficacy of these drugs is still being debated.
Cholinesterase Performance Goals
Reportable Range (Dynamic Range, Analytical
Range): varies from instrument to instrument, based on
assay method, temperature and substrate. Based on the
butyrylthiocholine/ferricyanide method, as
recommended by the German Society for Clinical
Chemistry, the lower limit of reportable range
(sensitivity) is generally between 10 and 200 U/L (0.01
to 0.2 U/mL), and the upper limit is 11,000 to 25,000
U/L (11.0 to 25.0 U/mL). Samples with cholinesterase
activity greater than the upper limits of the dynamic
range must be diluted and reanalyzed according to
instrument manufacturers recommendations or a
laboratorys own protocol.
Precision: The within-run precision varies from
instrument to instrument and method to method, and
generally ranges from 0.5% to 2.0 %, whereas between-
day CV within a laboratory ranges from 1.4% to 5%.
The cholinesterase (pseudocholinesterase) activity is not
a regulated analyte according to the Clinical Laboratory
Improvement Amendments of 1988 (CLIA 88), and the
CAP criteria for acceptable performance in external
proficiency testing is peer group 30%.
References
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Taylor P. The human gene
encoding acetylcholinesterase is located on the long
arm of chromosome 7.
Am J. Hum Genet 1992;51:170-177.
2 Soreq H. The Multileveled regulation of the human
cholinesterase genes
and their protein products. Biological Sciences,
Biochemistry, Report
A372772. Jerusalem, Israel: Hebrew University;
September 30, 1993.
3 Brown SS, Kalow W, Pilz W, Whittaker M. The
plasma cholinesterases: a
new perspective. Adv Clin Chem 1981;22:1-123.
4 Willis JH. Blood cholinesterase: assay methods
and considerations. Lab Management
1982;20:53-64.
5 Lehmann H, Liddell J. The cholinesterase
variants. In: Stanbury JB, Wyngaarden JB, and
398
Cholinesterase
Fredrickson DS, eds. The Metabolic Basis of
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6 Working Group on Enzymes. Proposal of
standard methods for the determination of
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7 Dietz AA, Rubenstein, HM, Lubrano T.
Colorimetric determination of serum
cholinesterase and its genetic variants by the
propionylthiocholine-dithiobis (nitrobenzoic
acid) procedure. In: Cooper GR, King JS, eds.
Selected Methods for the Small Clinical
Chemistry Laboratory. Vol. 8. Washington, DC:
American Association for Clinical Chemistry;
1977:41-46.
8 Price EM, Brown SS. Scope and limitations of
propionylthiocholinesterase in the
characterization of cholinesterase variants. Clin
Biochem 1975;8:384-390.
9 Michel HO. An electrometric method for the
determination of red blood cell and plasma
cholinesterase activity. J Lab Clin Med
1949;34:1564-1568.
10 Witter RF, Grubbs LM, Farrior WL. A
simplified version of the Michel method for
plasma or red cell cholinesterase. Clin Chim
Acta 1966;13:76-78.
11 Caraway WT. Photometric determination of
serum cholinesterase activity. Am J Clin Pathol
1956;26:945.
12 Crane CR, Sanders DC, Abbot JN.
Cholinesterase use and interpretation of
cholinesterase measurements. In: Sunshine I,
ed. Methodology for Analytical Toxicology.
Cleveland, OH: CRC Press; 1975:86-104.
13 Ellin RI, Vicario P. A pH method for measuring
blood cholinesterase. Arch Environ Health
1975;30:263-265.
14 Nabb DP, Whitfield F. Determination of
cholinesterase by an automated pH stat method.
Arch Environ Health 1967;15:147-152.
15 Aldrich FD, Walker GF, Patnow CA. A
micromodification of the pH-stat assay for
human blood cholinesterase. Arch Environ
Health 1969;19:617-620.
16 Serat WF, Van Loon AJ, Lee MK.
Microsampling techniques for human blood
cholinesterase analysis with the pH stat. Bull
Environ Contam Toxicol 1977;17:542-550.
17 Hay DL, Ibrahim GF, Horacek I. Rapid
acetylcholinesterase screening test for neural
tube defect. Clin Chem 1983;29:1065-1069.
18 Hullin DA, Laurence KM, Elder GH, Roberts
A, Newcombe RG. Amniotic fluid
cholinesterase measurement as a rapid method
for the exclusion of fetal neural-tube defects.
Lancet 1981;1:325-326.
19 Smith AD, Wald NJ, Cuckle HS, Stirrat GM,
Borrow M, Lagercrantz H. Amniotic fluid
acetylcholinesterase as a possible diagnostic test
for neural-tube defects in early pregnancy.
Lancet 1979;1:685-688.
20 Barlow RD, Cuckle HS, Wald NJ. A simple
method for amniotic fluid gel-
acetylcholinesterase determination, suitable for
routine use in the antenatal diagnosis of open
neural tube defects. Clin Chim Acta
1982;119:137-142.
21 Andrea Mosca A, Bonora R, Ceriotti F,
Franzini C, Lando G, Maria Cristina Patrosso
MC et al. Italian Society of Clinical
Biochemistry and Clinical Molecular Biology
(SIBioC) Working Group on Enzymes. Assay
using succinyldithiocholine as substrate: the
method of choice for the measurement of
cholinesterase catalytic activity in serum to
diagnose succinyldicholine sensitivity. Clin
Chem Lab Med 2003;41:317-322.
22 Panteghini M, Ceriotti F, Schumann G,
Siekmann L. Establishing a reference system in
clinical enzymology. Clin Chem Lab Med
2001;39:795-800.
23 Ortho-Clinical Diagnostics. Test methodology:
CHE, Cholinesterase. Part No. MP2-91, CAT
No. 126 2096. Rochester, NY: Ortho-Clinical
Diagnostics, Inc; 1996.
24 Kenedel Bttger. Klin Wschr. 1967;45:325-327.
25 Weber F, Gottschalk N, Kytzia H-J, Muecke M,
Nagel R. Stabilized cholinesterase substrate
solution. Application Number 11/566788.
Filing date 12/05/06. Roche Diagnostics
Operations, Inc. US patent 20070178546.
Available at
<http://www.freepatentsonline.com/70178546.h
tml>
26 Motonaga H Naito, Shino M. Test Lab Co Ltd
(JP). Method for determination of
cholinesterase activity. Patent Number 4565780
pub 1986. Available at
<http://www.freepatentsonline.com/4565780.ht
ml>
27 Holownia P, Newman DJ, Bruno C. Automated
dibucaine number measurement with DuPont
Dimension ES and AR analyzers. Clin Chem
1995;41:664-667.
28 Naccarato WF. DuPont Imaging and Medical
Products, Diagnostics Division. New
application of the pseudocholinesterase method:
determination of dibucaine and fluoride
number. DuPont technical bulletin IPDP-2156.
Wilmington, DE: DuPont Imaging; 1980:1-4.
29 Denblaauwen DH, Poppee WA, Tritscher W.
Cholinesterase (EC 3.1.1.8) with butyryl iodide
as substrateage-dependent and sex-dependent
reference values with special reference to
hormonal effects and pregnancy. J Clin Chem
Biochem 1983;21:381-386.
30 Cattozzo G, Franzini C, Rettodini M. Dibucaine
number measured with the Ektachem system
[tech brief]. Clin Chem 1993;39:1545-1546.
31 Newman MA, Que Hee SS. Interconversion and
comparison of the results of three methods for
399

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acetyl cholinesterase in serum. Clin Chem Butyrylcholinesterase activity and metabolic

1984;32:308-310. syndrome in obese patients. Clin Chem Lab
32 Wald NJ, Cuckle HS. Report of the Med 2005;43:285-288.
Collaborative Acetylcholinesterase Study: 48 Abbott CA, Mackness MI, Kumar S, Olukoga
amniotic fluid acetylcholinesterase AO, Gordon C, Arrol S et al. Relationship
electrophoresis as a secondary test in the between serum butyrylcholinesterase activity,
diagnosis of anencephaly and open spina bifida hypertriglyceridaemia and insulin sensitivity in
in early pregnancy. Lancet 1981;2:321-327. diabetes mellitus. Clin Sci (Lond) 1993;85:77-
33 Hilborn ED, Padilla S. A dried blood spot 81.
method to evaluate cholinesterase activity in 49 Annapuma V, Senciall I, Davis AJ, Kutty KM.
young children. Arch Environ Health Relationship between serum
2004;59(9):467-70. pseudocholinesterase and triglycerides in
34 Kramer S, Watson LL, Lodato DT, Widsor- experimentally induced diabetes mellitus in
Long K, Gilmore JM, Augustyn JM. Assay of rats. Diabetologia 1991;34:320-324.
pseudocholinesterase on the dimension clinical 50 Kutty KM, Payne RH. Serum
chemistry system. Clin Chem 1991;37:913. pseudocholinesterase and very low density
35 Evans RT, MacDonald R, Robinson A. lipoprotein metabolism. J Clin Lab Anal
Suxamethonium apnoea associated with 1994;8:247-250
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36 Zsigmond EK, Downs JR. Plasma Beynen AC, Donker AJ, Heine RJ. Is
cholinesterase activity in newborns and infants. pseudocholinesterase activity related to markers
Can Anaesth Soc 1971;18:278-283. of triglycerol synthesis in type II diabetes
37 Shanor S, Van Hess GR, Baart N, Erdos EG, mellitus? Clin Sci (Lond) 2001;101:29-35.
Folders MD. The influence of age and sex on 52 Valle A, OConnor DT, Taylor P, Zhu G,
human plasma and red cell cholinesterase. Am J Montgomery GW, Slagboom PE, Martin NG,
Med Sci 1961;242:357-361. Whitfield JB. Butyrylcholinesterase: association
38 Howard J, East N, Chaney J. Plasma with the metabolic syndrome and identification
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Environ Health 1978;33:277-279. 2006;52:1014-1020.
39 Kaplan E, Herz F, Hsu KS. Erythrocyte 53 Lehmann DJ, Johnston C, Smith AD. Synergy
acetylcholinesterase activity in ABO hemolytic between the genes for butyrylcholinesterase K
disease of the newborn. Pediatrics 1964;33:205- variant and apolipoprotein E4 in late-onset
211. confirmed Alzheimers disease. Hum Mol
40 Kaplan E, Tildon JT. Changes in red cell Genet 1997;6:1933-1936.
enzyme activity in relation to red cell survival 54 Panegyres PK, Mamotte CD, Vasikaran SD,
in infancy. Pediatrics 1963;32:371-375. Wilton S, Fabian V, Kakulas BA.
41 Brock A. Immunoreactive plasma Butyrylcholinesterase K variant and
cholinesterase (E.C. 3.1.1.8) substance Alzheimers disease. J Neurol 1999;246:360-
concentration, compared with cholinesterase 70.
activity concentration and albumin: inter- and 55 Ghebremedhin E, Thal DR, Schultz C, Braak H.
intraindividual variations in a healthy Age-dependent association between
population group. J Clin Chem Clin Biochem butyrylcholinesterase K-variant and Alzheimer
1990;28:851-856. disease-related neuropathology in human
42 Kambam JR, Horton B, Parris, WC, Hyman brains. Neurosci Lett 2002;320:25-28.
SA, Berman, LM, Sastry BVR. 56 De Ferrari GV, Canales MA, Shin I, Weiner
Pseudocholinesterase activity in human LM, Silman I, Inestrosa NC. A structural motif
cerebrospinal fluid. Anaesth Analg of acetylcholinesterase that promotes amyloid
1989;68:486-488. -peptide fibril formation. Biochemistry
43 Shukuya R. On the kinetics of the human blood 2001;40:10447-10457.
cholinesterase. J Biochem 1953;40:135-140. 57 Inestrosa NC, Sagal JP, Colombres M.
44 Foldes FF, van Hees GR, Shanor SP, Baart N. Acetylcholinesterase interaction with Alzheimer
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45 Hayes WJ. Pesticides Studied in Man. Amyloid-cholinesterase interactions.
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47 Alcantara VM, Oliveira LC, Rea RR, Suplicy 630.
HL, Chautard-Freire-Maia EA.
400

Cholinesterase

Tables
Table 1: Cholinesterase Methods Summary
Method 1: Colorimetric; spectrophotometric, kinetic or end-point
Principle of analysis:

Comment: Serum or plasma; recommended method

Method 2: pH change; pH measurement
_
Principle of analysis: Acetylcholine CHS choline + acetate + H+
Comments: Serum or plasma; wide interlaboratory variation of results
Method 3: Colorimetric; pH dye color change, end-point
Principle of analysis:

Comments: Serum or plasma; wide interlaboratory variation of results

Method 4: Electrometric; titrate acid released, kinetic or end-point
Principle of analysis:
_
Acetylcholine CHS choline + acetate + H+
H+ + added base H2O and constant pH
Comments: Serum or plasma; requires automated pH titration
DTNB, 5,5-Dithiobis(2-nitrobenzoic acid).

Table 2: Conditions for Cholinesterase Analysis

Temperature 37C
Wavelength 410 nm
pH 7.6
Final concentration of reagent PCTI: 2 mmol/L
DTNB: 0.253 mmol/L
Phosphate: 25 mmol/L
Dibucaine: 0.03 mmol/L
Fluoride: 4 mmol/L
Sample volume 10 L
Fraction of sample volume 0.002 (dilution corrected)
Reaction time 10 min
Coefficient of variation 3%4
Linearity 0 through normal range

PCTI, Propionylthiocholine; DTNB, 5,5-dithiobis(2-nitrobenzoic acid).

401

Cholinesterase

Table 3: Reference Values for Genetic Variations of Cholinesterase*

Phenotype Activity at 37C, Percent inhibition by
mol/min/mL Dibucaine Fluoride
U 8.44 1.78 83.6 1.3 79.7 1.2
A 1.90 0.61 19.9 2.7 84.0 1.8
AD 1.30 0.37 20.7 4.1 82.3 3.4
S1 0.03 0.01 5.3 4.3 35.7 6.1
S2 0.18 0.05 67.6 4.3 67.6 1.7
F 3.57 71.8 53.6
AF 3.65 0.47 60.2 3.1 68.3 1.6
FS 3.47 76.7 64.9
UA 5.84 1.76 72.7 3.1 80.0 1.6
UF 5.99 1.26 79.8 1.2 73.0 1.7
US 4.61 0.57 84.4 0.8 79.3 1.4

*Derived from Dietz AA, Rubenstein HM, Lubrano T. Colorimetric determination of serum cholinesterase and its genetic
variants by the propionylthiocholine-dithiobis (nitrobenzoic acid) procedure. In: Cooper GR, King JS, eds. Selected
Methods for the Small Clinical Chemistry Laboratory. Vol 8 Washington, DC: American Association for Clinical
Chemistry; 1977.
Values are mean standard deviation.
Phenotypes associated with prolonged apnea in response to succinylcholine administration.

Table 4: Michaelis-Menten Constants for Cholinesterases

Substrate CHS (Cholinesterase) AChE (Acetylcholinesterase)
Acetylcholine 1.2 to 1.8 10-3 M[31,35] 3.7 10-4 M[31]
Succinylcholine 1. 10-3 M[35] Does not hydrolyze succinylcholine

Table 5: Prolonged Response to Succinylcholine

Phenotypes Estimated Frequency Sensitivity to Succinylcholine
Homozygote
UU Normal population 1/3200 moderately sensitive
AA 1/2800 All very sensitive
FF 1/300,000 All very sensitive
SS 1/140,000 All very sensitive

Heterozygote
UA 1/26 1/500 moderately sensitive
UF 1/280 1/200 moderately sensitive
US 1/190 Not known
AF 1/29,000 All very sensitive
AS 1/20,000 All very sensitive
FS 1/200,000 All very sensitive
402

Cholinesterase

Figures
Cholinesterase Figure 1: Polyacrylamide gel electrophoreses assay for acetylcholinesterase.

Polyacrylamide gel electrophoresis assay for acetylcholinesterase performed as described in the text. A, Amniotic fluid B
incubated in presence of substrate plus inhibitor. Notice absence of second band at arrow position. B, Amniotic fluid
incubated in presence of substrate. Notice second band at arrow position. Presence of a cholinesterase activity at second
position (arrow) is inhibited by BW 284C51, evidence that this is true acetylcholinesterase activity. An elevated amniotic
alpha-fetoprotein (>3 SD from mean) plus the presence of true acetylcholinesterase activity is strong evidence for the fetus
having a neural tube defect. C, Fetal calf serum that has a cholinesterase activity (arrow) that migrates slightly slower than
true acetylcholinesterase activity found in amniotic fluid. It is not inhibited by BW 284C51.

Procedure: Colorimetric Assay for Cholinesterase
Principle
Propionylthiocholine is used as a substrate for
cholinesterase in the following coupled reactions:
_
Propionylthiocholine + H2O EC 3.1.1.8 propionic
acid + thiocholine
Thiocholine + DTNB oxidized thiocholine + 5-thio-2-
nitrobenzoic acid
Phosphate buffer pH 7.6 is used, and the rate of reaction
is followed at 410 nm, an absorption maximum of 5-
thio-2-nitrobenzoic acid. In addition to total activity,
dibucaine and fluoride inhibitions (measured separately)
are determined when cholinesterase phenotyping is
requested. Only glass containers may be used, since
inhibitors may be extracted from some plastics.
Reagents
1. Phosphate buffer, 0.036 mol/L, pH 7.6.
Prepare a solution of 0.036 M Na2HPO4 by dissolving
4.73 g of the salt in approximately 800 mL of distilled
H2O and diluting to 1 L. Prepare a 0.1 M solution of
KH2PO4 by dissolving 13.6 g of the salt in a final
volume of 1 L of distilled water. Mix 50 mL of KH2PO4
with 950 mL of Na2HPO4 to achieve a final pH of 7.6.
Stable for 3 months at 4C to 8C. Check weekly for
bacterial growth and pH and stability.
2. Propionylthiocholine iodide solution (PCTI),
20 mmol/ L. Place 606 mg of PCTI in a 100-mL
volumetric flask, and dissolve with approximately 80
mL of distilled water. Bring volume to 100 mL with
distilled water. Stable for 1 day at ambient temperature.
3. 5,5-Dithiobis(2-nitrobenzoic acid) (DTNB),
167 mg/L (0.421 mmol/L). Weigh out 167 mg of
DTNB, dissolve, and dilute to a final volume of 1 L with
0.036 M phosphate buffer. Stable for 12 months when
stored in a brown glass bottle at 4C.
4. Dibucaine HCl (Nupercaine Hydrochloride,
Ciba), 0.3 mmol/L. Place 57 mg into a 500-mL
volumetric flask, dissolve in distilled water, and dilute to
a final volume of 500 mL with distilled water. Stable for
1 year at room temperature.
5. Sodium fluoride, 40 mmol/L. Weigh out 84
mg of NaF, dissolve in approximately 40 mL of distilled
water, and dilute to a final volume of 50 mL. Stable for 1
day at room temperature.
6. Quinidine sulfate, 14 mmol/L (5 g/L). Weigh
out 500 mg, dissolve in approximately 90 mL of distilled
water, and dilute to a final volume of 100 mL. Stable for
1 year at room temperature.
7. Working substrate reagents. Prepare 3 mL
of each solution per test sample.
1. Dilute PCTI with an equal volume of distilled
water.
403
Cholinesterase
2. Dilute PCTI with an equal volume of dibucaine
solution.
3. If NaF is used, dilute PCTI with an equal
volume of sodium fluoride solution.
Assay
Equipment: Spectrophotometer or photometer set at 410
nm, with bandpass 10 nm, and 37C water bath.
This procedure measures the activity of
cholinesterase and the effect of two inhibitors, dibucaine
and fluoride.
Assay for Total Activity
1. Prepare two test tubes for each sample or
control, one labeled test and the other blank.
2. Add 3 mL of DTNB buffer to each test tube,
and allow to equilibrate 5 minutes in a 37C water bath.
3. Add 1 mL of working PCTI substrate diluted
with water to each tube labeled test.
4. Add 1 mL of a 1:100 dilution of serum.
(Samples with low activity should be reanalyzed with a
smaller dilution, such as 10-fold or 50-fold.) Dilute the
serum with distilled water shortly before the assay, since
the cholinesterase may slowly lose activity after dilution.
5. After exactly 3 min, add 1 mL of quinidine
reagent to stop the reaction.
6. Prepare blank readings by reversing steps 2 and
5; that is, first add quinidine reagent to the blank tube,
and then serum, and then add the substrate solution.
7. Read absorbance of each test reaction against
the corresponding blank within 5 min.
Assay for Dibucaine or Fluoride Inhibition
1. Same as for step 1 above.
2. Add 3 mL of DTNB buffer to each test tube,
and allow to incubate 5 min in a 37C water bath.
3. Add 1 mL of working substrateinhibitor
solution, that is, PCTI diluted with either dibucaine or
NaF.
4. Follow steps 4 to 7 of the assay for total
activity.
Calculations
The cholinesterase activity is expressed in international
units per milliliter (IU/mL = mol/min/mL) at 37C and
calculated from the following equation:
U/mL = x Aunknown = 14.71 Aunknown y 13.6 z
where x is the total reaction volume (in milliliters), y is
the volume of undiluted sample used, Aunknown is the
reaction absorbance corrected for the blank, 13.6 is the
millimolar absorptivity of the 5-thio-2-nitrobenzoate for
a 1-cm cuvette, and z is the number of minutes of
incubation. Inhibition by dibucaine or fluoride is
calculated as follows:
Percent inhibition =
100 % U/mLwith inhibitor 100%
U/mLwithout inhibitor Adjust volume to 50 mL with preincubation indicator
Procedure: Qualitative Analysis for Amniotic Fluid
Acetylcholinesterase
Principle
Elevated levels of true acetylcholinesterase activity in
amniotic fluid have been associated with a high
probability of fetal open neural tube defects. Positive
identification of the activity can be determined by
polyacrylamide gel electrophoresis. Different bands of
cholinesterase activity are separated after electrophoresis
at pH 8.1. The activities are identified by incubation of
the gels with substrate (acetylthiocholine) and an
indicator (copper sulfate). Cholinesterases will cleave
the substrate to acetic acid and thiocholine. The
thiocholine reacts with Cu2+ to form complexes that
form a white precipitate at the positions in the gel to
which the enzyme activities have migrated. The true
serum acetylcholinesterase activity is defined by its
ability to be inhibited by the specific inhibitor, BW
284C51that is, 1,5-bis(4-
allyldimethylammoniumphenyl)pentan-3-one dibromide.
Reagents
1. Polyacrylamide 7.7% in buffer preservative.
Gels purchased as part of AlkPhor System from
TechAmerica Diagnostics, San Marcos, CA (Also
available from Quantimetrix Corp., Redondo Beach CA,
USA).
2. Tris-glycine buffer, pH 8.1.
3. Stock. To a 1-L volumetric flask, add 29 g of
glycine and 6 g of tris(hydroxymethyl)aminomethane.
Dissolve salts in 800 mL of distilled water, and adjust
pH to 8.1 with 3 M HCl. Adjust volume to 1 L. Store at
4C to 8C. Stable for 1 year.
Working buffer. Prepare a 1:10 dilution by
using 100 mL of stock Tris-glycine buffer and 900 mL
of distilled water. Check that pH is 8.1 (+0.1); adjust if
necessary. Discard after use.
4. Preincubation indicator solution, pH 6.5
(final concentration of components in parentheses).
To a 500-mL volumetric flask, add:
Na2SO4 (1.06 mol/L) 75.3 g
Sodium maleate (0.07 mol/L) 4.834 g
CuSO4 (0.004 mol/L) 0.500 g
Glycine (0.02 mol/L) 0.750 g
MgCl2 (0.003 mol/L) 0.305 g
Dissolve salts in approximately 450 mL of
distilled water. Adjust pH to 6.5 with 3 M
NaOH, and adjust volume to 500 mL with
additional distilled water. Stored at room
temperature. This reagent is stable for 4 weeks
if no bacterial growth is evident. Check pH
before use and adjust to 6.5 if necessary.
5. Indicator with acetylthiocholine. To a 50-mL
volumetric flask, add 0.1 g of acetylthiocholine iodide.
solution. Prepare fresh.
6. Stock acetylcholinesterase inhibitor (BW
284C51) solution (10-3 M). To prepare this solution,
404
Cholinesterase
wear a surgical gown and mask and disposable plastic
gloves. Work in a hood. Take a 25-mg vial of BW
284C51 (purchased from Sigma Chemical Co., St. Louis,
MO, A9013) and dissolve in 44 mL of distilled water.
Store in refrigerator for 3 months.
7. Indicator with acetylthiocholinesterase
inhibitor. To a 50-mL volumetric flask, add 500 L of
acetylcholinesterase inhibitor 10-3 mol/L. Bring to
volume with preincubation indicator solution. Prepare
fresh.
8. Indicator with acetylthiocholinesterase
inhibitor and acetylthiocholine. To a 50-mL
volumetric flask, add 500 L of stock
acetylcholinesterase inhibitor plus 0.1 g of
acetylthiocholine. Bring to 50 mL final volume with
preincubation indicator solution. Prepare fresh.
WARNING: Acetylcholinesterase inhibitor is extremely
toxic, and great care should be used when handling.
Never pipet by mouth, and wash hands thoroughly after
use.
Assay
Equipment: Any commercially available system for
column gel electrophoresis.
1. Remove gels from storage container, shaking
off excess buffer. Two tubes will be needed for each
patient and a positive control (aliquots of a positive
amniotic fluid should be stored in 70C freezer). One
tube will be needed for a negative control (normal
amniotic fluid). (Each patient and the positive control
will have separate tubes for the inhibitor.)
2. Insert gel tubes, unfilled end up, in the upper
bath of the electrophoretic chamber. The top of the gel
tubes should be flush (level) with the top of the silicone
tube adaptor. Be careful not to touch the end of the gel.
Plug all unused tube holes with plastic stoppers.
3. Place 800 mL of the working buffer in the
lower bath of the electrophoretic chamber and 200 mL of
buffer in the top portion. Be certain that no bubbles have
become trapped at the bottom of the gel tubes. Squirt
some buffer (using a Pasteur pipet) into the top of the
tubes to free any bubbles.
4. Place upper bath over lower bath. Place cover
over upper bath, and plug in the two white plugs over the
metal prongs.
5. Turn the power on and pre-run the gels at a
constant voltage of 200 volts. Electrophorese the gels for
2 hours.
6. At the end of 2 hours, turn the power off.
Remove the lid from the upper portion of the bath.
Apply 75 L of amniotic fluid to each tube. Introduce
the tip slowly through the buffer until it touches the gel
surface. Carefully layer the sample onto the gel.
7. Replace the cover lid, and plug in the two white
plugs.
8. Turn on power and set power at a constant
voltage of 200 volts. Electrophorese at 200 volts for 2
hours.
9. When electrophoresis is complete, remove the
gels from the tubes using a large syringe with a long,
thin needle. Fill the syringe with distilled water.
10. Remove each tube. Gently insert the needle into
the tube, moving it alongside the walls while expelling
water. Be careful not to gouge the gels. Place each gel in
a 25 75 mm test tube. Label each tube appropriately.
11. To one of each patient and positive control
tubes, add 5 mL of the preincubation indicator solution.
Also incubate the negative control in this solution. Let
stand for 30 min.
12. To the other set of patient and positive control
tubes, add 5 mL of the indicator + acetylcholinesterase
inhibitor solution. Cap and let stand for 30 min.
13. Pour substrate out of all tubes. Pipet any
remaining substrate (with a Pasteur pipet) from bottom
of tube, being careful not to gouge the gels. Wear
disposable gloves, taking care not to let the inhibitor
solution come into contact with your skin. Flush contents
down sink.
14. To the set of patients and positive and negative
controls that had the preincubation indicator solution,
add 5 mL of indicator and acetylthiocholine solution. Let
stand overnight.
15. To the set of patients and positive control that
were preincubated with indicator + acetylcholinesterase
inhibitor, add 5 mL of indicator + acetylcholinesterase
inhibitor + acetylthiocholine solution. Let stand
overnight.
Evaluation (Figure 1)
Zones of positive acetylcholinesterase activity will be
seen in the gels incubated with acetylthiocholine as
distinct bands, with the distance of migration similar to
that of the positive control. The gels incubated with the
acetylcholinesterase inhibitor will show no bands in the
same area.
Tubes negative for acetylcholinesterase activity
will show no banding pattern in either tube at this
position.
Notes
1. Amniotic fluid that is highly positive for AFP
should be stored in small aliquots at 70C. Amniotic
fluid with normal levels of AFP should also be frozen.
These will serve as positive and negative controls,
respectively, for acetylcholinesterase activity.
2. If an amniotic fluid positive for
acetylcholinesterase cannot be obtained, fetal calf serum
can be used instead. Fetal calf serum contains a
cholinesterase activity that migrates just slightly slower
than the true acetylcholinesterase found in amniotic
fluid. The fetal calf serum activity is not inhibited by
BW 284C51.
3. Amniotic fluid that is contaminated with fetal
blood may give false-positive results for
acetylcholinesterase activity. Fetal blood has this
activity. If the fluid is obviously bloody, a cell count and
a stain should be performed to determine the number of
fetal cells present. If there are over 60 million fetal cells
per milliliter, positive results should be interpreted with
caution. A repeat amniotic fluid specimen might be
appropriate.
405
Cytomegalovirus (CMV)
Cytomegalovirus (CMV)
Terry Pry and Greg Maine
Name: Cytomegalovirus (CMV)
Clinical significance: Refer to Chapter 7, Immunological Reactions, and Chapter 8, Immunochemical
Techniques, in the 5th edition of Clinical Chemistry: Theory, Analysis, and Correlation.
Principles of Analysis and Current Usage
Human cytomegalovirus (CMV) is a member of the
Herpesviridae family. CMV infections are very
common, with 40% to 100% of the general population
showing prior exposure by serology [1]. The majority of
persons with acute CMV will be asymptomatic. CMV is
capable of infecting and damaging many different cell
types, such as salivary glands, pancreas, adrenals, lung,
liver, eye, ear, placenta, gastrointestinal tract, heart,
ovaries, skin, and various components of the central
nervous system. The ability of CMV to infect white
blood cells facilitates dissemination of virus within the
host. As with other herpes viruses, CMV shares a
characteristic ability to remain dormant within the body
over a long period, reactivating and shedding when the
hosts immune system is suppressed or compromised.
Complications of primary CMV infection in
immunocompetent persons are rare except in newborns.
Intrauterine transmission of primary CMV infection
(approximately 40%), especially during the first
trimester, has the potential to cause significant fetal
damage and subsequent severe pathology in newborns.
CMV is the most common congenital infection,
occurring in approximately 1% of all live births [2].
CMV is also associated with significant disease and may
be life threatening in immunocompromised individuals.
Among the herpesviruses, CMV is responsible for most
morbidity and mortality in immunocompromised hosts
[3]. CMV is also a serious pathogen in patients who have
received an organ or bone marrow transplant. Estimates
indicate that one third of transplant recipients experience
CMV disease, and up to half will have lung infections,
resulting in a patient mortality rate of up to 50% [4].
Laboratory methods for the diagnosis of CMV are
evolving, and considerable progress has been made over
the past 10 years in the design of improved diagnostic
techniques. Their use can clearly inform patient
management. Traditional methods for laboratory
diagnosis of CMV infection include serology, virus
culture, modified culture (shell vial assay), and antigen
detection (antigenemia and NAT). Since CMV can be
transmitted from mother to fetus or from organ donor to
recipient, automated serological assays have proven
useful in the prenatal diagnosis of congenital CMV
infection and the determination of donor/recipient
serostatus for CMV. CMV IgG avidity testing in
pregnant women, especially during the first trimester,
can be used to distinguish between a primary and
nonprimary CMV infection, thereby stratifying the risk
of transmission of maternal CMV infection to the fetus.
Serological assays have limited utility in
immunocompromised patients whose ability to mount an
immune response is by definition abnormal. Although
quantitative culture methods have been used to predict
disease or follow a patients response to therapy, they
are relatively insensitive. Commercially available
molecular methods have been introduced to complement
nonmolecular methods and include diagnostic CMV
methods such as real-time PCR [5]. Emphasis is on the
search for improved diagnostic testing for early detection
of the increased viral replicative activity that presumably
precedes the onset of CMV disease in infected
individuals.
CMV-IgM Test
CMV-IgM assays are currently based on ELISA
technology. A specific IgM response may indicate a
recent primary infection, or it may be associated with a
nonprimary CMV infection in the following cases: (1)
IgM levels may occasionally persist for up to several
years following primary infection, and (2) detection of
false-positive IgM antibodies may occur in patients with
autoimmune diseases such as systemic lupus
erythematosus (SLE) due to presence of rheumatoid
factor (RF), since CMV can establish latency in the host
[6]. Pregnant women with CMV IgG- and IgM-positive
results require further testing using a CMV IgG avidity
assay as described below. In the absence of a
commercially available gold standard CMV IgM assay,
comparisons between various commercially available
assays yield variable results, making it difficult to assess
differences in sensitivity and specificity observed
between different commercial assays [7]. The growth
and purification of the virus, as well as the source of the
viral antigen utilized in the CMV IgM assay, affects
assay sensitivity and specificity with state-of-the-art
assays utilizing recombinant antigens [8].
CMV-IgG Test
Semi-quantitative CMV-IgG assays are also based on
ELISA. This assay documents a past infection, as well as
indicating the potential for an active infection, since
reactivation of a latent infection or reinfection with
another strain of the virus may occur. An IgG assay may
also identify an active infection by demonstration of IgG
seroconversion or a significant IgG rise between paired
sera taken 2 to 3 weeks apart [9]. Because no
international standard exists for CMV IgG, definition of
either a protective antibody level or a CMV-IgG
international unit (IU) has not yet been defined.
Comparisons between commercial IgG assays utilize
arbitrary units and correlate well with each other.
CMV IgG Avidity Test
The IgG avidity assay measures the functional binding
affinity (Avidity Index) of the IgG class of antibody in
406
Cytomegalovirus (CMV)
response to infection. Avidity is a measure of the overall
strength of binding of an antigen with many antigenic
determinants and multivalent antibodies, whereas affinity
refers to the strength of binding between a single
antigenic determinant and an individual antibody
combining site. The IgG avidity assay has been
implemented in a diagnostic algorithm for pregnant
women [10]. This diagnostic algorithm is based on
screening pregnant women with a CMV IgG assay and a
sensitive IgM assay, followed by reflex testing of CMV
IgG and IgM-positive specimens with a CMV IgG
avidity assay. Interpretation of a positive IgM assay in
pregnant women is assisted by determination of the
avidity index of CMV-specific IgG, since low-avidity
IgG is produced during primary CMV infection. As the
infection progresses, the avidity of the IgG increases,
and therefore high-avidity IgG is a marker of
nonprimary CMV infection. Detection of low-avidity
CMV IgG in specimens from pregnant women indicates
that primary CMV infection has occurred within the past
18 to 20 weeks, whereas detection of high-avidity CMV
IgG excludes primary infection [11].
CMV Neutralization Test
The presence of neutralizing antibodies can be utilized to
distinguish primary from nonprimary infection. These
antibodies appear shortly after primary infection (13 to
15 weeks), and therefore the presence of high titer
neutralizing antibody during a CMV infection may be
used to rule out a primary infection [12]. Neutralization
assays have been applied to an ELISA format but have
not yet been commercialized because they are labor
intensive.
Virus Isolation in Tissue Culture
Fetal infection can be assessed by detection of CMV
virus in cultured amniotic fluid (AF). Virus isolation
from AF is considered the gold standard method for
detection of fetal infection, owing to the high sensitivity
and 100% specificity of the method. Inoculated cultures
are prepared using human diploid fibroblast cells [13].
The shell vial assay is the preferred method because of
its shortened incubation, providing results within 16 to
72 hours. The method utilizes immunofluorescence to
detect binding of monoclonal antibodies to viral proteins
synthesized shortly after infection. The method has been
described in numerous publications and has been studied
either exclusively or in combination with PCR methods
[14-16].
PCR Test
Real-time PCR combines PCR chemistry with
fluorescent probe detection of amplified product in the
same reaction vessel. In general, both PCR and
amplified product detection are completed in an hour or
less, which is considerably faster than conventional PCR
detection methods.
One of the earliest applications of real-time PCR for
testing in the clinical microbiology laboratory was for
detection of CMV, and an extensive literature exists
describing considerations and implementation of
applying real-time PCR to the routine laboratory testing
of CMV [17,18]. Quantitation of CMV DNA is
performed by dedicated instruments and involves DNA
extraction and analysis. Primers used in different assays
may be derived from different viral genomic sites, and
hence there may be variation between manufacturers.
Quantitative PCR is available as commercial kits using
various technologies. Most assays utilize the highly
conserved early or immediate-early genes. Real-time
PCR CMV assays provide sensitivity and specificity
equivalent to that of conventional PCR combined with
Southern blot analysis, and since amplification and
detection steps are performed in the same closed vessel,
these assays do not suffer from false-positive results due
to contamination. The presence of CMV DNA in plasma
may be associated with active viral replication and
disease development [19]. Uniform reporting of results
between laboratories is currently lacking and will benefit
from assay standardization and uniform guidelines.
The detection of CMV viral transcript mRNA is now
performed using nucleic acid sequencebased
amplification that allows specific amplification of
unspliced viral mRNAs in a DNA background. This is a
promising technique for monitoring CMV infection in
transplant recipients [19].
Reference and Preferred Methods
Since internationally recognized reference methods or
standards do not yet exist for various CMV diagnostic
methods, it is important for users to identify the specific
method utilized with each test result.
Virological and molecular detection of CMV and
serological demonstration of a specific immune response
are used for diagnosis. An acute infection is usually
diagnosed through detection of the virus in body fluids
or deduced by the presence of a specific IgM antibody
immune response along with the seroconversion from
the IgG antibody negative to IgG antibody positive.
Serological tests are highly specific and sensitive, and
although antibody can decline with age and severe
immunosuppression, IgG seropositivity is usually
lifelong. Techniques for detecting the virus itself are
improving rapidly, and nucleic acid tests such as
polymerase chain reaction are now available at large
centers. The accepted gold standard for the diagnosis of
congenital CMV infection is the detection of virus in
urine or saliva within 2 weeks of birth.
Specimen
CMV is detected and quantified most commonly using
plasma, but other specimens include amniotic fluid,
cerebrospinal fluid, urine, throat washing, semen, and
tissue samples, particularly with the use of PCR. In
detecting congenital CMV, isolation of CMV utilizes
saliva or urine within 2 weeks of birth. Neonatal blood
dried on paper has also been utilized for viral DNA
testing [20]. CMV virus is very labile, and tissue culture
samples should be kept at 4C to 8C and tested within
48 hours. Freezing of amniotic fluid specimens destroys
CMV infectivity [13].
Interferences
407
Cytomegalovirus (CMV)
CMV-IgM Assay
Detection of false-positive IgM antibodies may occur in
patients with autoimmune diseases such as systemic
lupus erythematosus (SLE) and due to the presence of
rheumatoid factor (RF). Cross-reactivity with Epstein-
Barr virus (EBV) and polyclonal stimulation by EBV
can also result in false-positive results. Conventional
PCR may exhibit false positives due to possible
contamination by amplification products.
Interpretation
Diagnosis of primary infection may be achieved in the
majority of cases through concurrent analysis of serum
antibodies, virus detection, and clinical signs.
Assessment of CMV infection in pregnancy (Figures 1a
and 1b) begins with maternal serology, which should
establish recent primary, latent, or nonprimary infection.
Primary CMV infection occurs when the CMV virus
infects a previously uninfected, seronegative individual;
primary infection is related to worse fetal outcomes.
Seroconversion can be demonstrated by the production
of CMV IgM and IgG antibodies (low avidity) in a
seronegative individual. Documentation of CMV
seroconversion is rare in pregnant women, since they are
not routinely screened for CMV antibodies prior to
gestation. In these cases, the detection of CMV IgM has
been used as a marker of active or recent CMV infection
[21]. Since CMV-specific IgM can be produced during
nonprimary CMV infections, a CMV IgM-positive result
by itself does not permit an accurate assessment of the
risk of CMV infection to the fetus. Nonprimary infection
occurs when a previously infected, seropositive
individual experiences (1) reactivation of a previous
CMV infection from latency or (2) reinfection with a
new strain of CMV. Nonprimary CMV infection is
usually characterized by the presence of high avidity
CMV IgG antibodies. The IgG avidity assay is a
powerful tool, but it should be used and interpreted
properly. Interpretable results can be achieved mainly
for sera obtained within the first trimester of pregnancy.
CMV Performance Goals
The 2007 College of American Pathologists (CAP)
Quality Assurance Program (QAP) listed the frequency
of the following anti-CMV (total) methodologies among
171 participants: enzyme immunoassay (43%),
fluorescent EIA (9%), particle agglutination (18%), and
solid phase red cell ADH (25%). Satisfactory qualitative
evaluation criteria (90% participant consensus of
results against target) was achieved within each method
group for all samples, save for particle agglutination,
which only achieved 36% consensus.
References
1 de Jong, MD, Galasso GJ, Gazzard B, Griffiths
PD, Jabs DA, Kern ER et al. Summary of the II
International Symposium on Cytomegalovirus,
Antiviral Res 1998; 39:141-162.
2 Stagno S. Cytomegalovirus. In: Infectious
Diseases of the Fetus and Newborn Infant (4th
Edition). Remington JS, Klein JO, (Editors).
Philadelphia, USA, 1995, W.B.Saunders,
pp.312-353.
3 Rubin RH, Cosimi A, Tulkoff-Rubin NE,
Russell PS, Hirsch MS. Infectious disease
syndromes attributable to cytomegalovirus
recipients and their significance among renal
transplant recipients, Transplantation 1977; 24:
458-464.
4 Schmidt GM, Horak DA, Niland JC, Duncan
SR, Forman SJ, Zaia JA. A randomized,
controlled trial of prophylactic ganciclovir for
cytomegalovirus pulmonary infection in
recipients of allogenic bone marrow transplants,
N Engl J Med 1991; 324: 1005-1011.
5 Szczepura A, Westmoreland D, Vinogradova Y,
Fox J, Clark M. Evaluation of molecular
techniques in prediction and diagnosis of
cytomegalovirus disease in
immunocompromised patients, Health
Technology Assessment 2006; 10: 1-176.
6 Krast YJ, Stals FS, Christiaans MH, Lazzarotto
T, Landini MP, Bruggeman, CA. IgM antibody
detection of ppUL80a and ppUL32 by
immunoblotting: an early parameter for
recurrent cytomegalovirus infection in renal
transplant recipients. J Med Virol 1996; 48:
289-294.
7 Lazzarotto T, Brojanac S, Maine GT, Landini
MP. Search for cytomegalovirus-specific
immunoglobulin M: Comparison between a
new Western blot, conventional Western blot
and nine commercially available assays. Clin
Diagn Lab Immunol 1997; 4: 483-486.
8 Maine GT, Lazzarotto T, Landini M. New
Developments in the diagnosis of maternal and
congenital CMV infection, Expert Rev Diagn
2001; 1: 19-29.
9 Rawlinson WD. Broadsheet Number 50:
Diagnosis of human cytomegalovirus infection
and disease, Pathology 1999; 31: 109-115.
10 Munro SC, Hall B, Whybin LR, Leader L,
Robertson P, Maine G, Rawlinson WD.
Diagnosis and Screening for Cytomegalovirus
in Pregnant Women. J Clin Microbiol 2005; 43:
4713-4718.
11 Lazzarotto T, Spezzacatena P, Pradelli P, Abate
D, Varani S, Landini MP. Avidity of
immunoglobulin G directed against human
cytomegalovirus during primary and secondary
infection in immunocompetent and
immunocompromised subjects, Clin Diag Lab
Immunol 1997; 4: 469-473.
12 Eggers M, Metzger C, Enders G. Differentiation
between acute primary and recurrent human
cytomegalovirus infection in pregnancy, using a
microneutralization assay, J Med Virol 1998;
56: 351-358.
13 Hodinka RL, Human cytomegalovirus. In:
Murphy PR, Baron EJ, Pfaller MA, Tenover
FC, Yolken RH, editors. Manual of Clinical
microbiology. 1999;p.889-991.
14 Mendelson E, Aboudy Y, Smetana Z,
Tepperberg M, Grossman Z. Laboratory
408
Cytomegalovirus (CMV)

assessment and diagnosis of congenital viral Microbiology: Applications for Routine

infections: Rubella, cytomegalovirus (CMV),
varicella-zoster virus (VZV), herpes simplex
virus (HSV), parovirus B19 and human
immunodeficiency virus (HIV), Reproductive
Toxicology 2006; 21: 350-382.
15 Weiner CP, Grose C. Prenatal diagnosis of
congenital cytomegalovirus infection by virus
isolation from amniotic fluid. Am J Obstet
Gynecol 1990; 163: 1253-1255.
16 Nicolini U, Kustermann A, Tassis B, Fogliani
R, Galimberti A, Percivalle E, et al. Prenatal
diagnosis of congenital cytomegalovirus
infection. Prenat Diagn 1994; 14: 903-906.
17 Boeckh M, Huang M, Ferrenberg J, Stevens-
Ayers T, Stensland L, Nichols WG, et al.
Optimization of quantitative detection of
cytomegalovirus DNA in plasma by real-time
PCR. J Clin Microbiol 2004; 42: 1142-1148.
18 Espy M, Uhl J, Sloan L, Buckwalter S, Jones
M, Vetter E et al. Real-Time PCR in Clinical
Laboratory Testing, Clin Micro Rev 2006; 19:
165-256.
19 Gerna G, Baldanti F, Lilleri D, Parea M,
Alessandrino E, Pagani A et al. Human
cytomegalovirus immediate-early mRNA
detection by nucleic acid sequence based
amplification as a new parameter for pre-
emptive therapy in bone marrow transplant
recipients, J Clin Microbiol 2000; 38: 1845-
1853.
20 Barbi M, Binda S, Caroppo S. Diagnosis of
congenital CMV infection via dried blood spots,
Rev Med Virol 2006; 16: 385-392.
21 Nielsen SL, Sorensen I, Andersen HK. Kinetics
of specific immunoglobulins M, E, A and G in
congenital, primary and secondary
cytomegalovirus infection studied by antibody-
capture enzyme-linked immunosorbent assay, J
Clin Microbiol 1988; 26: 654-661.

Table 1: Summary of CMV Methods

Method 1. CMV IgG

Principle: ELISA methodology
Usage: Marker of past CMV infection
Comments: Limitation of result: Not conclusive of latent or active infection. Comparisons between
commercial IgG assays correlate well with each other, but no international standard yet exists.
Method 2. CMV IgM
Principle: ELISA methodology
Usage: Marker of active or recent CMV infection
Comments: Improved CMV IgM assays employing recombinant antigens or peptides are a significant
advance over conventional viral lysate-based immunoassays. IgM antibodies may persist for months or years.
Method 3. CMV IgG Avidity
Principle: Two CMV IgG ELISA tests are performed on the same sample. After primary antibody incubation,
the solid phase of each assay is washed with either buffer (control) or a chaotropic reagent (urea, diethylamine) to
remove low-avidity antibodies. After another wash, the conjugate is added and the signal measured. The avidity
index is the ratio of (CMV IgG titer with chaotropic wash step/CMV IgG titer with buffer wash step) 100, that
is, the ratio of CMV high-avidity IgG over the total CMV IgG titer.
Usage: Differentiate between primary infection (low avidity IgG) and recurrent or persistent infection
(high avidity IgG).
Comments: Avidity testing should be performed early in gestation (within the first trimester). Fully
automated high throughput analytical systems available with integrated avidity assays.
Method 4. Fetus Culture (Shell Vial Assay)
Principle: Gold standard for detection of fetal infection, owing to 100% specificity. Immunofluorescence
can confirm presence of CMV virus.
Usage: Virus isolation from amnionic fluid remains key method for demonstration of fetal infection and
is used either exclusively or in conjunction with molecular methods.
Comments: Strict handling of sample due to labile virus. CMV identified after 16 to 72 hours in culture
Method 5. Real-Time PCR
Principle: DNA extraction, amplification of DNA, detection using fluorescent probes.
Usage: Detection of viral DNA in clinical samples
Comments: General agreement that PCR is more sensitive than culture. Samples can be frozen for
subsequent analysis by other labs.

Figure 1
409
Cytomegalovirus (CMV)

Figure 2. Proposed diagnostic algorithm for CMV serology screening in pregnant women.
 410
Copper
Copper
K. Raja, R. Swaminathan
Name: Copper
Clinical significance: Refer to Chapter 42, Trace Metals, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Principles of Analysisi
Copper, an essential yet potentially toxic element, is a
transition group IB metal in period 4 of the periodic table.
Copper forms two series of inorganic salts: cuprous and
cupric salts. The cuprous salts are unstable in solution,
converting readily into the corresponding cupric salts.
Analytical techniques applied to the measurement of
copper in biological fluids and tissues include
spectrophotometry (Table 1, Method 1) [1-8], flame (Table
1, Method 2) [9-12], and flameless (Table 1, Method 3)
[13-17] atomic absorption spectrometry, inductively
coupled plasma mass spectrometry (ICP-MS)(Table 1,
Method 4) [18], and inductively coupled plasma optical
emission spectrometry (ICP-OES) (Table 1, Method 5)
[19]. Other methods using emission spectrography [20]
neutron activation analysis [21], anodic stripping
voltammetry [22] and total reflection fluorescence analysis
[23] have also been described in the literature. However,
they are not always practical to be used in busy clinical
laboratories. Flame atomic absorption spectrophotometry
remains by far the most widely used method, followed by
ICP-OES and ICP-MS. A few laboratories, however, still
use colorimetric methods.
Many spectrophotometric methods that utilize the
formation of colored copper complexes have been
published. Chromogens used in the analysis of copper
include the following: 4-(3,5-dibromo-2-pyridylazo)-N-
ethyl-N-sulfopropylaniline (3,5,Di-Br-PAESA) [1], 2-
(5bromo-2-pyridylazo(-5-(n-propyl-N-sulfopropylamino)
aniline [2],
2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)
phenol (nitro-PAPS) [3], ,,,-tetrakis(4-N-
trimehylaminophenyl)porphine [4], 2,5-dimercato-1,3,4-
thiadiazole(DMTD) [5], di-2-pyridyl ketone
benzoylhydrazone (dPKBH) [6], 2,2-bicinchoninic acid
i
Copper
Previous and current authors of this method:
First edition: Not done
Methods edition: Nancy W. Alcock
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: K. Raja, R. Swaminathan
(BCA) [7] and 3-[2-[2-(2-hydroxyimino-1-methyl-
propylideneamino)-(ethylamino]-ethyl-imino]butan-2-
one oxime [8]. Some of these methods have been adapted
for use in clinical laboratories with commercial reagent
kits being available for the determination of serum copper.
In the method using 3,5,Di-Br-PAESA, copper bound to
ceruloplasmin is released by a reducing agent at pH 4.7;
the released copper forms a stable complex with the
chromogen [1].
In flame atomic absorption spectrophotometry (Table 1,
Method 2), monochromatic light from a hollow-cathode
copper lamp is absorbed by ground-state atoms in a lean
air-acetylene flame. The amount of light absorbed at 324.7
nm is directly proportional to the concentration of atoms in
the gaseous state in the light path and hence to the
concentration of copper in the solution. Sensitivity will
vary depending on the kind of burner used and on burner
slit length.
A number of publications have described methods for the
measurement of serum and urine copper by flameless
atomic absorption spectrophotometry (Table 1, Method 3)
[13,14,17]. The sensitivity of this method is sufficient for
measurements to be made directly on urine or on a 10- to
50-fold dilution of serum. Although acidification of urine
has been recommended by some authors, its necessity
remains questionable.
Inductively coupled plasma optical emission/mass
spectrometry (ICP-OES/MS) are relatively new techniques
in clinical laboratories and have the potential to measure
multiple elements within a single sample [18,19,24]. This
capability saves time and volume of sample required for
analysis. The instruments are relatively expensive to
purchase and operate. The methods can, however, detect
many elements besides copper at very low concentrations
in various sample types and over a wide dynamic range. In
these techniques, the sample is introduced into the
inductively coupled plasma (ICP) as an aerosol. The larger
droplets are excluded from entry via the spray chamber,
but the smaller droplets get swept into the ICP, which is a
hard but rapid atomization/ionization technique. The
very high temperature of the plasma leads to the aerosol
droplets being desolvated, vaporized, atomized, and/or
ionized. The ionization efficiency is a function of the
elemental ionization potential, as well as plasma
temperature and sample matrix. In ICP-OES, the
characteristic radiation emitted by the excited atoms/ions is
 411
Copper
detected and electronically converted to elemental
concentrations. In ICP-MS, the ions are extracted through
a series of cones prior to reaching the analyzer vacuum
compartment. The mass spectrometer separates the ions
according to their mass-to-charge (m/z) ratio. As the ions
produced within the plasma are virtually singly-charged
ions, the m/z ratio equates to the mass of the ion. The
signal measured by the detector (usually an electron
multiplier) is thus proportional to the concentration of the
element(s) in the sample. The most common type of mass
analyzer used in ICP-MS instruments is a quadrupole.
However, time of flight and high-resolution magnetic
sector analyzers are also used in some instruments, where
very accurate mass resolution of complex mixtures is vital.
The process of ICPMS can also be applied to ascertain the
analyte concentration in a sample via isotope dilution
analysis, provided the isotopic composition is known. With
this method, a known quantity of the analyte enriched in
one isotope is added to the sample, and the resulting
change in isotope ratio is used to calculate the
concentration of the element in the sample.
The process of ICP-MS does, however, suffer from
spectral, matrix, and physical interferences, with spectral
interferences being probably the most serious. For
example, two or more atomic ions arising from the
matrix/solvent/plasma can combine to form a molecular
(polyatomic) species of equivalent mass to the
element/isotope of interest. The analysis of 63Cu can be
affected by the interaction between 40Ar and 23Na [24].
Interferences can be reduced by the use of a reactant gas in
a collision or reaction cell placed between the extraction
optics and mass analyzer. The interference is diminished
by the reactant gas (e.g., ammonia), either converting the
interfering polyatomic to a different species or reducing its
kinetic energy.
Currently, the most widely used method for the
measurement of copper in body fluids or tissues is either
flame or flameless atomic absorption spectroscopy. Only
6% of laboratories participating in the UK National
External Quality Assurance Service use a colorimetric
method for copper estimation.
Reference and Preferred Methods
A reference method for copper has not been published. In
the absence of a reference method, flame atomic
absorption spectrophotometry (FAAS) is the method of
choice when the concentration is 1.5 mol/L (96 g/L) or
greater. Flameless or electrothermal atomic absorption
spectrophotometry (EAAS) is more sensitive than FAAS;
the detection limit is around 30 nmol/L (2 g/L). However,
EAAS is also more sensitive to low levels of
contamination. The EAAS method may be preferable for
untreated urine. The assay time is, however, longer for
EAAS typically minutes as compared to seconds per
specimen when using the flame instrument. This is the
main disadvantage of the flameless technique. In addition,
extreme care is required to avoid contamination, especially
at the low concentrations when using EAAS.
When atomic absorption/ICP spectrometry is not available,
a colorimetric method based on 3,5-Di-Br-PAESA can be
used either manually or on an automated instrument.
Reagents for this method can be obtained form commercial
sources in the form of a kit (e.g., Randox copper assay). In
this method, the sample or standard is mixed with reagent
containing ascorbic acid and allowed to stand for 60
seconds before taking an absorbance reading. The
chromogen is added, and the final absorbance is taken 5
minutes later.
Interferences and Contaminants
As methods have progressively become more sensitive,
thereby allowing measurements to be made on small
volumes and down to trace (ppm/ppb)/ultratrace (ppb/ppt)
levels, the significance of contamination as a serious
source of error during the analytical process has become
even more apparent. An assessment of the potential source
of contamination and the need to take steps to prevent it
are therefore important when contemplating analytical
work from sample collection to the final analysis.
Contamination can arise from the environment, the
reagents, apparatus, or even the operator. Environmental
contamination is the presence of particulate/gaseous matter
in the air, which can arise from laboratory vents and/or the
air-conditioning unit(s) (especially when associated with
inefficient filtration), the slow deterioration of solid
objects, paints, cements or other construction materials,
plastics, or from chemical processes upon materials. Any
ongoing building works or high volume of traffic in the
vicinity of the laboratory can also contribute markedly to
the degree of contamination. It has been estimated that air
within an analytical lab can contain as much as 200 g/m3
of particulate matter, including copper [25]. Whereas the
heavier particles tend to settle, the lighter particles can stay
airborne and can, depending on the ventilation system and
air currents within a laboratory, be carried appreciable
distances. There are a number of ways of avoiding or
minimizing particulate/environmental contamination. The
more sophisticated and expensive approaches include the
construction of a clean room. The air entering the room
gets filtered using HEPA (high-efficiency particulate air)
filtrationwhich has the capacity to remove the vast
majority of particulate material (>99.9% of particles that
are 0.3 m or larger in size). In addition, the room could
also be maintained at positive pressure so that the flow of
the air is in one direction only (i.e., from laboratory to
outside). Very specialized centers may have, in addition,
an antechamber area that separates the main laboratory
from the clean room and provides an area where the
analyst(s) can put on clean apparel.
More practical approaches, however, include use of
laminar flow hoods. These self-contained units provide
 412
Copper
HEPA-filtered areas for the safe handling of samples,
reagents, standards, and so forth. To avoid the possibility
of any hazardous materials being blown at the analyst,
exhausted laminar flow systems are recommended.
However, correct operation of the unit (e.g., the
appropriate positioning of front shielding panel) is
necessary for optimal performance.
In laboratories, where the likelihood of a clean-
room/laminar-flow hood is not possible, owing to the cost
and/or the availability of space, the contamination risk can
be minimized by ensuring good laboratory working
practices. Usage of an ordinary enclosed room combined
with decent filtration of air vents/air-conditioning unit and
regular cleaning of walls, equipment, and floors with
deionized water/detergent (and lint-free towels or dust-
adsorbing cleaning tools) may be adequate to ensure the
production of reliable results within an analytical setting.
Sensible approaches to the working practices, such as
avoiding unnecessary shelving/cluttering/storage (which
can accumulate dust particles), working on easily cleaned
plastic sheets/trays, minimizing the number of
transfers/preparative steps and storage of
reagents/consumables in clean, closed cupboards/snap-top
plastic boxes helps to further reduce contamination.
The analysis of copper involves some initial preparative
step(s) of the sample, so that the resulting solution is as
homogenous/particle free as possible. Reagents, if not of
suitable high quality, can thus introduce contaminants
within the assay, especially when the analyst is dealing
with low concentrations. This factor becomes even more
important when considering the fact that the volume of
reagents used is often in excess of the volume of sample.
High-quality reagents should also be dedicated solely for
trace-metal work and must not be used for general
laboratory work. Moreover, pipetting should never be
carried out from the original reagent stock bottle. Always
transfer a small volume of the reagent to a suitable
container prior to use. Most preparations/dilutions within
the analytical laboratory involved in elemental analysis
also require the use of deionized/double distilled water as
the primary reagent. A large proportion of laboratories use
water that is produced by commercially available reverse
osmosis (i.e., passage of water through a membrane
against osmotic pressure) and/or ion-exchange (i.e.,
passage through a resin) purification systems. Many
commercial companies (e.g., Elga) make use of both
technologies within the same unit.
Another potential source of contamination is the apparatus
(e.g., containers, sample vials) that comes into contact with
the sample. Glass, a material that is extensively used
within a laboratory, is a potential source of elemental
contamination and should, wherever possible, be replaced
with plastic. The analyst should in fact attempt to use
plastics for every step of the protocol, namely sample
collection, preparation, and storage of reagents/standards.
The most popular plastics for trace analysis are
Perfluoroalkoxy (PFA) and high-/low-density polyethylene
(HDPE/LDPE). PFA is not only resistant to inorganic
acids (HF, HNO3, HCl)/bases and oxidizing agents, but
can also be microwaved, thus providing an ideal material
for microwave digestion vessels. However, the cost for this
type of plastic is high. LDPE or HDPE bottles are also
resistant to inorganic acids/bases and are typically used for
storing diluted samples/sample digests or working
standards. These bottles can withstand solutions of 10%
v/v HNO3 over extended periods of time.
Contamination from glassware can be reduced by soaking
it overnight in a dilute detergent solution (e.g., Decon at
5%), rinsing with deionized water, followed by soaking in
10% HNO3 for at least 24 hours and rinsing thoroughly
(3) with good quality deionized water. Alternatively, high
quality quartz (type III) may be used, as this form of glass
tends to have a lower level of elemental contamination.
Some workers also recommend soaking or rinsing of
plastic ware in dilute acid prior to rinsing with deionized
water. All lab ware should be air or oven dried before use.
It is important to note that often it is the contaminants from
the atmosphere and/or lab ware that is the cause of
interference rather than the impurities from the water.
The analyst should also take every precaution in
preventing contamination. Careless manipulation of
equipment or lab ware (e.g., by using bare hands) can
cause contamination arising from dried or dead skin,
sweat, and so forth. Use of hand lotions or creams can also
be a potential contaminant. Touching a dirty or metallic
surface, with or without gloves, before proceeding with a
protocol can also be a further source of contamination.
Wearing of jewelry should also be avoided in the
laboratory, since it can contribute to contamination if it
comes into contact with lab ware or a sample. The analyst
should also wear a fastened, lint-free lab coat and powder-
free gloves when carrying out all analytical procedures.
Specimen
Blood
Venous blood should be collected into a suitable
disposable vacutainer, using either a venipuncture vacuum
system or an indwelling butterfly cannula (attached to a
plastic syringe). If possible, the first 5 mL of blood should
be discarded or used for tests other than elemental analysis
(e.g., for general biochemistry). Subsequent collections
should then be made in suitable trace metalfree
vacutainers and used for determination of copper and other
elements. There are currently three suppliers of suitable
blood collection tubes: Beckton Dickinson, Greiner, and
Sarstedt. Although plasma can be used for analysis,
heparin, which is used as anticoagulant, may be
contaminated with copper. Trace metalfree tubes for
pediatric samples are currently unavailable. To avoid the
possibility of contamination, pediatric samples should be
 413
Copper

collected in anticoagulant-free tubes and centrifuged fairly post/van/courier. If there is to be a delay in transportation,
soon after clotting to obtain the serum fraction. the samples can be kept refrigerated or in a freezer. To
The transfer of serum or plasma to secondary tubes standardize calculations and interpretation of results, and
constitutes a further possible source of contamination. to correct for differing fluid content, tissues need to be
Trace metalfree tubes (e.g., Teklab) or polystyrene tubes completely dried prior to analysis.
(Sarstedt) or Hitachi cups (Clinicon) may be suitable. It is
preferable not to use colored caps or caps with o-rings to Hair
avoid the possibility of contamination. It is advisable to Though easy to obtain, this sample may suffer from
check each new batch of primary/secondary collection contamination from sweat, the environment (dust), water,
tubes for contamination by using water/dilute and previous beauty formulas (e.g., shampoos, hair dyes,
acid/serum/plasma as a matrix [26]. and other treatments) [28]. Diet has also been reported to
have an influence [29]. Moreover, because concentrations
Capillary blood sampling is more prone to contamination are affected by distance from the scalp, age, and rate of
and should be best avoided. hair growth, the use of hair copper concentration reflecting
nutritional status remains questionable [30].
Urine
A timed collection (24-hour) is the preferred specimen Reference Intervals
when assessing copper output, especially in the diagnosis Table 2 gives the reference ranges for serum and urine
of Wilsons disease [27]. It is necessary for collections to copper. Serum copper concentrations in females are
be made into acid-washed or copper-free plastic ware slightly higher than in males. Concentrations in children
containers. Metal containers should not be used. Subjects younger than 1 year are lower than in adults.
should also refrain from collecting urine in another
container and then transferring it to the allocated container. Table 2. Reference Values for Serum and Urine
Acid washing is accomplished by swirling a 10% to 20% Copper
HNO3 solution within a clean/new plastic container. After Serum/Plasma Age mol/L mg/L
disposing of the acid, the container (including the lid) must 0-4 months 1.4-7.2 0.09-
be rinsed out three times with deionized or double distilled 0.46
water then dried at room temperature. Containers need to 4-6 months 3.9-17.3 0.25-1.1
be kept away from possible sources of contamination 7-12 months 7.9-20.5 0.5-1.3
during these steps. It is not necessary to acidify urine after Over 1 year 12-25 0.76-1.6
collection for the measurement of copper. After Adult males 11.0-22.0 0.7-1.4
ascertaining the weight/volume of the collected urine, an Adult females 12.6-24.3 0.8-1.55
aliquot could be prepared (i.e., transferred to a sterile Pregnancy: 16 27-40 1.7-2.5
plastic universal container) and kept refrigerated if there is weeks to term
to be a delay in the analysis. Wilsons <4.0 <0.25
disease
If refrigerated, samples should be left to attain room
Urine mol/24hrs g/24hrs
temperature before analysis. Furthermore, it is better to
Adults <0.95 <60
mix urine samples 25 to 30 minutes before analysis and
Wilsons >1.57 >100
then to leave them standing. This period allows the
disease
particulate matter (e.g., urate and phosphates) to settle.
Thus the sample that is subsequently aspirated is usually Post <12 <760
free from particulate interferences. Pilot studies using penicillamine
urine samples and a known copper standard solution have challenge
revealed that the risk of copper co-precipitating or being normal adults
adsorbed onto the precipitates is negligible. Post >25 >1590
penicillamine
Tissue, Including Liver Biopsies challenge
Samples should be obtained with care. Use clean and Wilsons
metal-free instruments, if possible, to obtain and disease
manipulate soft-tissue specimens. Moreover, use clean
tools for each specimen to minimize carry-over Interpretation
contamination. Once obtained, the samples should be Copper is an essential, yet potentially toxic element. It is
stored and transported in suitable containers. Freshly involved in various cellular processes, primarily as a
obtained liver biopsies should be placed on a clean piece cofactor of enzyme/protein systems involved in
of filter paper moistened with deionized/double distilled catecholamine metabolism, oxidative metabolism,
water, and then placed into a plastic sterile universal neurotransmitter synthesis, iron absorption, free radical
container. The sample could then be sent by scavenging (SOD) and collagen cross-linking (lysyl
oxidase) [31-33]. Body concentrations of copper are
 414
Copper
usually low (100 to 150 mg), with the highest
concentrations being in the liver (10 to 20 mg) and brain.
Copper homeostasis is controlled mainly at the level of
excretion [34,35]. The average safe daily intake of dietary
copper in adults is 1.5 to 3.0 mg (24 to 47 mol) [36].
Useful dietary sources include beans, peas, whole grain
products, liver, seafood (oysters, crab, lobster), meats, and
nuts (almonds, walnuts). The intestine normally absorbs
about 30% to 50% of ingested copper via both passive and
active (energy-dependent) mechanisms [37,38]. It is then
transported to the liver, bound mainly to albumin, and to a
lesser extent amino acids. The liver plays a key role in
maintaining a physiological concentration of plasma
copper and in controlling copper homeostasis. It takes up
the copper from the circulation via a carrier-mediated
process and incorporates it into ceruloplasmin (the blue
glycoprotein), metallothionein, or glutathione.
In plasma, 90% of copper is bound to ceruloplasmin, with
about 10% loosely bound or associated with albumin. A
small fraction is complexed with low molecular weight
compounds such as amino acids.
Frank clinical copper deficiency is unusual. It has
however, been reported in malnourished children, in
infants on low-protein-diet, cows-milk formulas, after
long-term parenteral or enteral [39,40] nutrition, after
severe and persistent diarrheal illness, or when intake of
zinc has been excessive. Copper deficiency presents as a
microcytic, hypochromic anemia with marked neutropenia
and is resistant to iron therapy. Children and neonates may
also have defective collagen and elastin synthesis and may
develop bone disease (osteoporosis).
Subclinical copper deficiency may be more widespread
then previously thought and has been suggested as a risk
factor for cardiovascular disease [41].
The rare X-linked recessive neurodegenerative disorder
Menkes disease (prevalence 1 in 250,000 births), is
characterized by a failure of copper transport (efflux)
across the intestinal mucosa due to a defect in the MNK
gene located on long arm of chromosome 13, which codes
for a copper transporter [42,43]. Although copper
concentrations are low in plasma, brain, and liver, copper
paradoxically accumulates in many other tissues of the
body (i.e., kidney, spleen, and skeletal muscle) due to
abnormal copper transport. The intake into cells appears to
be normal, but there is defective utilization intracellularly.
The condition is therefore one of functional copper
deficiency as a result of a deficiency of copper-dependent
enzymes. As there are many variations of Menkes disease,
symptoms range from mild to severe, with onset occurring
in infancy (classical form) or childhood/adulthood (milder
variants such as occipital horn syndrome). Patients may
thus present with progressive cerebral degeneration,
mental retardation, poor immune function, poor thyroid
function, depigmentation, severe anemia, hypotonia,
skeletal abnormalities, and the strikingly peculiar steely
hair (possibly due to defective formation of disulphide
bonds in keratin). Affected infants tend to be identified
soon after birth, and most infants with the untreated, severe
(classic) form of Menkes die before their fourth birthday.
Diagnosis can be confirmed by low serum
copper/ceruloplasmin levels, abnormal catecholamine
concentrations in blood and CSF, microscopic examination
of hair, accumulation of copper in cultured fibroblasts
(skin biopsy) or placenta, or prenatally by measuring the
copper content of chorionic villi in the first trimester.
Toxicity of copper may arise from either a genetic disorder
or from acute or chronic copper ingestion. Acute ingestion
of copper salts produces nausea, vomiting, diarrhea,
circulatory collapse, intravascular hemolysis and in some
cases death. Acute toxicity has been reported in subjects
ingesting copper-containing solutions (either deliberately
or by accident), in patients given excessive
supplementation or low levels of other essential nutrients,
and in those on maintenance hemodialysis (leaching of the
element from copper-containing dialysis membranes
[Cuprophan]). In situations of acute toxicity, serum copper
concentration will be high, but the ceruloplasmin
concentration will be normal.
Chronic poisoning with copper leads to gross hepatic
copper overload with severe liver disease (cirrhosis) in
young children. Indian childhood cirrhosis has been
ascribed to boiling or storage of milk in corroded copper or
brass vessels. There have also been reports of poisoning in
young children as a result of high copper content in well
water. Liver copper content in such cases can exceed that
found in overload due to Wilsons disease.
Wilsons disease (hepatolenticular degeneration) is an
autosomal recessive disorder caused by a defect in the
gene encoding for the P type ATP7B transporter, mapping
to chromosome 13 [43]. Well over 100 mutations have
been identified so far, thus making routine DNA screening
an unlikely proposition. The condition is associated with
impaired incorporation of copper into ceruloplasmin, and
its subsequent excretion via the bile. Copper consequently
accumulates within the liver, where it can induce free-
radical formation. With progressive hepatic loading,
copper diffuses into the blood and accumulates in other
tissues, with damaging effects particularly on the brain.
Consequently, the subject presents with progressive
neurological symptoms (characterized by tremor,
dysphagia, dystonia, incoordination, and unusual
behavior), low serum concentrations of copper and
ceruloplasmin, raised urinary copper excretion, and
characteristic copper deposits in the Descemets membrane
in the limbus of the cornea (Kayser-Fleischer rings) [44].
Copper Performance Goals
The current target for acceptable performance criteria is
7.7% [45]. It is evident from the 2007 College of
 415
Copper
American Pathologists (CAP) survey that many methods
do not achieve this target. Atomic absorption with
deuterium background correction is the best of the current
methods used in the United States.
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Copper

Table 1. Methods for Copper Analysis in Biological Materials

Method Principle Usage Comments

1. Spectrophotometric Copper complexes with S

2. Flame atomic absorption
spectrometry
3. Flameless atomic
absorption spectrometry
4. Inductively coupled plasma
mass spectrometry (ICP-
MS)
5. Inductively coupled
plasma optical emission
spectrometry(ICP-OES)
chelator to form a strongly
chromogenic complex
Amount of light from copper S, U, tissue 1, 2
hollow cathode lamp
absorbed by ground state
atoms
As for Method 2, but electro S, U, tissue 1, 2
thermal heating used to
produce atoms
Atomization followed by S, U, tissue 1, 2, 3
ionization. Detection by
mass/charge ratio
Atomization followed by S, U, tissue 1, 2, 3
ionization. Emitted light by
excited atoms/ions is
detected

1 Digestion required for tissues.

2 Deproteinization or dilution required for serum.
3 Multi-element analysis.
S, Serum; U, urine.

Analysis of Copper centrifuge (3000 g for 10 to 15 min). The presence of an

Less than 10% of the analytical laboratories still use
colorimetric methods for the analysis of copper. Flame
atomic absorption spectrometry remains by far the most
common analytical method used in the analysis of copper
in biological specimens. It is thus imperative that the
presenting solution is as homogenous and particle-free as
possible to avoid blockage of the aspiration tube and/or
interference of the absorption signal. To aid in correcting
for background contamination, it is advisable to include a
blank which has been prepared in a similar fashion to the
samples and using the same reagents.
Analysis of Serum Copper
The preparative step(s) for producing a same dispenser/pipette(s) be used for the preparation of
clear/homogenous/particle-free solution varies from one
laboratory to another, but the principle of the actual
analytical process thereafter is very similar. The method
described below is one example of a method used in the
analysis of Cu in serum/plasma samples.
Prepare a 20%w/vTCA solution (100 g in 500 mL of
deionized water), and dilute this 1:1 to get a 10% solution.
To a plastic (polystyrene/polypropylene) tube containing
1.5 mL of 10% TCA, add a small volume of serum/plasma
(100 to 250 l), mix well (manual/automated vortexer), and
acidic milieu not only induces the proteins to precipitate
but also liberates the copper bound to the proteins.
Analyze the clear supernatant, ensuring that the aspiration
tube is directed away from any deposits precipitated on the
side of the tube. By comparing the signal to a calibration
curve that has been established by similarly preparing and
running a set of working aqueous standards (0 to 50
mol/L), the concentration of copper can be determined.
Note that blanking is carried out using a solution
containing water (instead of serum) and 10%TCA at the
same volumes as for the standards/serum samples. The
working standards are normally prepared from a
commercially available, high-quality stock copper
solution. To avoid pipetting errors, it is essential that the
both the standards and the samples.
There are many models of flame atomic absorption
currently in use todayof which Perkin Elmer Model
3100 is an example. Each atomic absorption instrument,
however, needs to be optimally set up prior to analysis.
The hollow cathode lamp must be run at a suitable current
(usually 10 to 15 mA), and the wavelength must be set at
324.7 nm. In instruments where the hollow cathode lamp is
not automatically adjusted, manual alterations are required.
Adjust the lamp gently (in/out and
clockwise/anticlockwise), and adjust the screws on the
 418
Copper
lamp housing to get the best possible intensity reading.
Because only a small proportion of sample that is aspirated
actually reaches the flame, and the sample is within the
light path very transiently, the burner-nebulization system
is very inefficient (only around 5% efficient). The
sensitivity of the analysis can be improved by using a
burner head having a longer slit (i.e., 10 cm). Moreover,
pre-mix burners are more popular, since they experience
less interference and have better signal/noise ratio than
total consumption burners. The burner head, however,
must be correctly aligned so that the maximal signal can be
detected. For checking the burner alignment, two steps are
necessary:
1. Rest a piece of fairly stiff paper/card in the middle of
the burner head and perpendicular to the slit. Turn
the horizontal/vertical adjustment screws/knobs in
turn, so that the burner head slot is centered
approximately below the light beam. Lower the
height of the burner head so that it is well below the
beam. Whilst monitoring the absorption signal on
continuous mode, auto-zero the reading and raise the
burner head until a small change in absorbance
becomes apparent (i.e. burner head interfering with
beam). Lower the burner head gently until the
absorbance reads zero again. A further marginal
lowering of the burner head ( to turn of the
screw) will suffice for not only copper but most
other analyses.
2. Once the flame is lit and warmed up, aspirate a dilute
standard solution of copper while continuously
monitoring the absorbance, make fine adjustments to
the burner by gently tweaking the horizontal and
vertical screws in turn until the maximum
absorbance is reached.
To obtain acceptable sensitivity, the oxidant/fuel
(air/acetylene) mixture for the flame should also be
optimized. Copper analysis usually performs better with a
fairly hot flame. Thus the analyst may need to make
adjustments to the fuel/oxidant knobs so that a lean, bluish
flame is evident. A 2:1 oxidant/fuel ratio usually suffices
in the case of copper. Optimization of the ratio can be
established by running a dilute copper standard solution
through the flame and under continuous reading mode,
making fine adjustments to the fuel/oxidant knobs to
acquire the maximal absorbance reading.
Analysis of Urine Copper
The instrument must be optimized as for the serum
measurements. In most cases, pretreatment of urine is not
necessary for copper measurement. Following blanking
with water, establish a calibration curve by aspirating a
number of aqueous copper standards over the
concentration range of interest. Ensure that the response is
linear over the range by comparing the absorbance at each
concentration. Run the urine samples against the
established calibration to ascertain the concentration
within samples. If the concentration within a sample
exceeds the concentration of the top-working standard,
dilute the sample with water and re-run, making sure to
correct for the dilution.
Analysis of Tissue Copper (e.g., Liver Biopsy)
The measurement of copper in a liver biopsy sample is
often the cornerstone in establishing the diagnosis of
Wilsons disease. As stated above, the fluid content of
tissue may vary from sample to sample, depending on
storage conditions and other factors. It is thus necessary to
totally dry the sample prior to digestion with concentrated
nitric acid. Transfer the tissue sample from the filter paper
to a clean, metal-free, plastic tube. If larger pieces are
available, they should be sliced into smaller pieces using a
clean scalpel. Dry the samples overnight with the lid of the
tube open but covered. In cases where larger pieces have
been provided, heating the samples to temperatures up to
100C on a hot plate or in an oven for a further 24 hours
helps with the drying process. The sample(s) are deemed
to be completely dry when there is no further change in
mass with time.
After drying, samples can be digested in a small volume of
concentrated nitric acid. Blank tubes (no tissue) and QC
samples (lyophilized liver sample of known concentration)
are also prepared at the same time to correct for
background noise (e.g., contamination) and to check for
accuracy and precision, respectively. Overnight exposure
to nitric acid is usually sufficient for complete digestion of
samples. In rare cases, heating at 70C to 80C for 2 hours
(hot plate) may be required to ensure complete digestion.
If available, the technique of high-pressure closed vessel
microwave digestion can be used to efficiently convert the
samples to homogenous solutions. The digested sample is
then diluted with deionized water. In cases where fat-
containing samples have been provided, a white insoluble
precipitate may be evident. In such cases, briefly
centrifuge the digest (10,000 rpm for 2 min) prior to
analysis.
Samples can then be analyzed as for the urine samples and
using the same set of aqueous standards. Where tissue
levels exceed the top aqueous standard concentration,
dilute the digest and re-run, and correct for dilution.
The Cu content (g/g) in the sample/digest, can be
calculated from the following equation:
[Cu]M total volume of digested sample (in mL)
63.54(atomic wt of copper)
Divided by 1000x dry wt (g)
The above-mentioned procedures and processes
mainly refer to the technique of flame AAS.
Electrothermal/Graphite furnace AA is equally
suitable for the analysis of copper in biological
419
Copper
samples. However, despite the increased sensitivity
(100 to 1000 times) and the ability to utilize small
volumes (e.g., pediatric samples), the instrument takes
much longer to analyze each sample (minutes as
opposed to seconds). Moreover, the set-up is more
complex than the flame system. The furnace-
programming steps (for drying, charring and
atomization) varies from matrix to matrix, thereby
requiring a greater degree of analyst expertise. Over
the last decade or two, the use of ICP (ICP-OES and
ICP-MS) has grown in popularity as an analytical tool.
The key advantage of these instruments is the ability
to perform multi-element analysis on a single sample.
Moreover, as a result of enhanced
sensitivity/specificity, a sample can be analyzed by
simply diluting it many-fold. This procedure
minimizes not only the preparative steps required but
also the matrix effects. Major drawbacks for the use of
ICP-MS are the high running costs and the
requirement for a greater degree of expertise.
420

Cortisol

Cortisol
R. Swaminathan
Name: Cortisol, hydrocortisone, compound F, glucocorticoid
Clinical significance: Refer to Chapter 51, Adrenal Hormones and Hypertension, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C21H30O5
Molecular mass: 362.47 D
Merck Index: 4689
Chemical class: Steroid
Structure:

soluble metabolites and glucuronide conjugates. The

Principles of Analysis and Current Usage i
Cortisol, also known as compound F and
hydrocortisone, is the most important glucocorticoid
secreted by the zona fasciculata and zona reticularis
of the adrenal gland. Under basal conditions, only
about 5% to 10% of circulating cortisol is free. The
rest is bound to proteins: 85% to transcortin (cortisol-
binding globulin) and 5% to 10% to albumin. It is the
free fraction which is physiologically active, although
the albumin-bound fraction is also available because
the binding affinity of albumin for cortisol is low.
Unbound cortisol is filtered at the glomerulus and
excreted. This fraction constitutes less than 1% of the
total cortisol synthesized daily; the rest is excreted as
i where cortisol is extracted and then converted by
Cortisol
Previous authors of this method:
First edition: Suman Patel, John A. Lott
Methods edition: Suman Patel, John A. Lott
Second edition: Not updated
Third edition: Suman Patel, John A. Lott
Fourth edition: Suman Patel, John A. Lott
Fifth edition: R. Swaminathan
excretory products of cortisol that contain the
dihydroxyacetone group are known as 17-
hydroxycorticosteroids (17-OH-CS). The
measurement of urinary 17-OH-CS has been used in
the past as an indirect measure of cortisol secretion
rate. Cortisol in urine is a reflection of free,
circulating cortisol in blood.
Many different types of methods have been used for
the determination of cortisol in biological fluids
(Table 1). Before the availability of immunoassay
methods for serum and urine cortisol, urinary 17-OH-
CS estimations by colorimetric or fluorometric
methods were used for the assessment of adrenal
function [1]. With the development of
immunoassays, these methods have become obsolete.
A fluorometric method developed by Mattingley,
ethanolic sulfuric acid to a fluorogenic chromogen,
has been used to measure plasma cortisol (Table 1,
Method 1) [2]. This method is also obsolete. These
methods lack specificity, require a large volume of
sample, and are labor intensive. Several competitive
protein binding assays were then used (Table 1,
Method 2) [3]. With the development of more
specific assays these methods are also no longer used.
421
Cortisol
Immunoassays are the main methods for estimation
of cortisol in clinical laboratories. A large number of
isotopic (Table 1, Method 3[i]) and non-isotopic
(Table 1, Method 3[ii]) immunoassays for the
measurement of cortisol have been described [4].
These immunoassays are competitive immunoassays,
where cortisol in the sample competes with labeled
cortisol for a limited number of anti-cortisol
antibodies. Almost all immunoassays used in clinical
laboratories do not require an extraction step [5].
Initially, labels used in these assays were radioactive.
Later, others were used, including enzymes,
luminescent, chemiluminescent, and fluorescent
labels. Many commercial kits are available for the
determination of plasma and urinary cortisol. Many
of the immunoassays are now automated and are
available on semi-automated or automated analyzer
platforms. These immunoassays can be
heterogeneous assays, where a separation step is
required to partition the bound from unbound
fractions, or homogenous assays, where no separation
is required. The specificity of immunoassays depends
largely on the properties of the antibody.
Chromatographic methods are an alternative to
immunoassay for the determination of cortisol, and
they have the advantage that there is less interference,
but these methods are time consuming and not
suitable for routine analysis. Of the chromatographic
methods, high-performance liquid chromatography
(HPLC) is often used in clinical laboratories [6]. All
HPLC methods require extraction of cortisol from the
sample, by either a liquid-liquid extraction or by use
of solid-phase extraction columns. Both reversed-
phase and normal-phase columns are used for the
chromatographic analyses, and the column effluents
are monitored by either ultraviolet (254 nm) or
fluorescence spectroscopy [6,7].
Other chromatography methods include gas
chromatography [8] and mass spectrometry
interfaced to gas chromatography (GC-MS) [9] or
liquid chromatography (LCMS) [10]. Methods using
capillary electrophoresis have also been described
[11].
In methods using gas-liquid chromatography, cortisol
is extracted, derivatized, and detected by flame
ionization [6]. In GC-MS methods, quantitation is by
mass spectrometry. LC-MS methods are similar to
HPLC methods, except the detection is by MS [9].
Reference and Preferred Methods
The definitive method for measurement of plasma
cortisol is isotope dilution mass spectrometry using
gas chromatography [12]. In this method [12],
labeled cortisol as internal standard is added to
plasma and the plasma extracted with
dichloromethane, purified using Sephadex LH-20,
followed by derivatization and detection by gas-
liquid chromatography.
The reference method for plasma cortisol utilizes
liquid chromatographymass spectrometry (LC-MS)
[13]. In this method, an internal standard is added to
the sample, followed by extraction with ethyl acetate.
Liquid chromatographymass spectrometry (LC-MS)
interfaced with electrospray ionization is used, and
selective monitoring of the [M+H]+ ions of cortisol
and isotopically labeled cortisol are used for
quantitation. A similar method but using
dichloromethane to extract cortisol has also been
described [14]. Guo et al. have described a much
simpler sample preparation. In this method, the
sample after addition of internal standard is mixed
with acetonitrile, centrifuged, and the supernatant is
injected into the LC-MS [15]. A recent study
suggests that care is necessary in using labeled
internal standard, since naturally occurring isotopes
of cortisol can cause interference [16].
A preferred method which gives good accuracy and
precision is the HPLC method. However, this method
is labor intensive, requires expertise, and is slow.
Direct (without extraction) immunoassay methods are
the most frequently used methods in laboratories, and
many are widely available on automated platforms
[17]. More than 90% of cortisol is bound to proteins,
so the bound fraction needs to be quantitatively
displaced before analysis in these direct assays.
Methods used for the displacement include use of
agents such as 8-anilino-1-naphthalene sulfonic acid
(ANS), danazol, and salicylate. Heat treatment or
lowering the pH are also effective in releasing bound
cortisol. It is important to note that the efficiency of
displacement by agents such as danazol may depend
upon the concentration of corticosteroid-binding
globulin (CBG). In situations of high CBG
concentration such as pregnancy, the displacement of
cortisol may not be complete. Furthermore, high
concentration of the releasing agent may affect the
binding of cortisol to the antibody.
Antibodies used in immunoassays are both
polyclonal and monoclonal. Common derivates used
in the preparation of antibodies are cortisol-21-
hemisuccinate and cortisol-3-carboxy methyloxime
conjugate. Measurement principles used in the non-
isotopic assays include photometry, fluorescence,
chemiluminescence, electro-chemiluminescence, and
422
Cortisol
time-resolved fluorescence. Table 2 gives details of
some of the automated immunoassays in common
use. Assay formats used include both heterogeneous
(separation of bound and free hormone required) and
homogenous (no separation required). Assays used in
the Roche system and Advia Centaur are examples of
heterogeneous assays. The most common methods
used in United States and the United Kingdom,
according to data from CAP and the United Kingdom
National External Quality Assessment Service
(UKNEQAS), are given in Table 3. The Bayer Advia
Centaur cortisol assay is a competitive immunoassay,
where cortisol in the sample and acridinium ester
labeled cortisol competes for a polyclonal rabbit anti-
cortisol antibody. Salicylate is added to release the
bound cortisol. The Roche Elecys assay is a
competitive immunoassay with competition between
the cortisol in the sample and ruthenium complex
labeled cortisol for a polyclonal ovine anticortisol
antibody. Treatment with danazol releases the bound
cortisol. The Abbott fluorescent polarization assay is
an example of a homogenous assay. Most of the
common automated immunoassays give satisfactory
performance. Assays that give relatively poor
performance are the Abbott AxSYM and DPC
IMMULITE 2000 methods.
Although cortisol immunoassays are automated, easy
to perform, and widely used, several problems have
been identified with these assays.
1. Commercial immunoassays for cortisol
show a positive bias when compared to
either isotope-dilution mass spectrometry or
gas chromatography mass
spectrophotometric methods [18]. Murphy et
al. [19] reported that without
chromatographic purification, all methods
grossly overestimated the amount of cortisol
present in urine.
2. Immunoassay methods work reasonably
well in patients with normal serum protein
levels. However, in patients with low protein
levels, such as patients in intensive care
units, these assays can give misleading
results. In a recent study, it was reported that
the Bayer Advia Centaur cortisol assay gave
consistently lower results when the albumin
concentration was lower than 24 g/L [20].
3. Specificity. Antibodies used in these
immunoassays show varying degrees of
cross-reactivity to related steroids (see
above), such as prednisolone, 11-
deoxycortisol, and 21-deoxycortisol. This
can be a problem in patients taking
prednisone/prednisolone, patients
undergoing metyrapone testing, and patients
with 21-hydroxylase deficiency. In addition,
immunoassays may give discrepant results
in patients whose hypothalamic-pituitary
axis is activated, such as in critical illness. In
these patients, stimulation of the
hypothalamic-pituitary-adrenal axis causes
increase in precursors of cortisol, which can
lead to varying degrees of cross-reactivity.
This was shown in a recent study of
critically ill patients [21], where cortisol
response to ACTH (Synacthen test) was
compared between 3 common
immunoassays and an HPLC method. This
study found that the Abbott TDX method
and IMMULITE 2000 method gave higher
results than HPLC by 95% and 79%,
respectively. Limits of agreement between
HPLC and the three methods were: 62% to
480%, 53% to 590% and 54% to 770%,
respectively, for the Centaur, TDX, and
IMMULITE methods.
4. Urine cortisol methods: Many
immunoassays are used for the
determination of urinary free cortisol, some
after extraction of urine with
dichloromethane and others without prior
extraction. In methods without prior
extraction, the measured concentrations are
nearly twofold higher than those determined
by HPLC methods [22,23].
Nevertheless, immunoassay techniques are the
current method of choice and most widely used
because of their overall acceptable specificity,
sensitivity, and precision and because results are
available fairly rapidly.
Specimen
Both serum and heparinized plasma are suitable
specimens for the measurement of cortisol in blood.
Specimens can be stored at 2C to 8C for 2 days.
For longer storage, specimens must be frozen [5].
Freeze/thaw cycles have not been found to
significantly alter cortisol concentrations.
Salivary cortisol has been found to be an excellent
indicator of serum free cortisol concentrations
[24,25]. A specimen is typically collected using a
special device containing a cotton swab, with or
without prior stimulation by chemicals such as citric
acid. Specimens are stable in the presence of citric
acid at a concentration of 10 g/L. In a citric-acid-
treated cotton swab, cortisol is stable for as long as 6
weeks at room temperature [26]. Presence of citric
acid (or lemon juice) may cause apparent under-
423
Cortisol
recovery in extraction-based assays [27]. Several
commercial collecting devices are available for
saliva, and of these the PE Salinette was found to be
the best [28].
Free cortisol in urine can be determined by
immunoassay or HPLC methods. These procedures
require an extraction step using methylene chloride,
and it is customary to monitor the efficiency of the
extraction technique by extraction of a urine control
with known cortisol concentration. A 24-hour urine
sample is preferred. Urine collected for a 24-hour
sample should be refrigerated during the collection
period. Some laboratories use boric acid
(approximately 10 g of boric acid per liter) or acetic
acid (approximately 7 mL of glacial acetic acid per
liter), placed in the collection container at the start of
the collection, to preserve the specimen. If the sample
cannot be analyzed within a day of collection, it
should be frozen at 20C.
Interferences
All immunoassays are prone to interference. Presence
of heterophilic antibodies in patient serum can give
false results. Although specific antibodies are used,
almost all immunoassays for cortisol used in clinical
laboratories show cross-reactivity with other related
steroids such as prednisolone. Cross-reactivity
against prednisolone varies from 27% for the Advia
Centaur to 171% for the Roche system [29]. Cross-
reactivity against prednisone is lower (0.3% to 6.6%).
However, prednisone is converted to prednisolone in
the body, and therefore these assays are not suitable
for measurement of cortisol in patients taking
prednisone or prednisolone. Cross-reactivity to 11-
deoxycortisol varies from 4.15% in the Roche system
to 21.6% in the Beckman Access analyzer. In patients
undergoing metyrapone testing, 11-deoxycortisol will
be high, and these assays will therefore give falsely
high values for cortisol. Some of the assays also
show cross-reactivity against 21-deoxycortisol, which
is elevated in patients with 21-hydroxylase
deficiency. Some of the interferences (e.g., due to 11-
deoxycortisol) could be removed by prior extraction
of samples with carbon tetrachloride.
Interpretation
Cortisol secretion is pulsatile, so isolated serum
cortisol values are of limited value. Stimulation and
suppression tests are required to diagnose
adrenocortical disorders.
Twenty-four-hour urine cortisol excretion reflects the
overall cortisol secretion rate and is not influenced by
the circadian rhythm. Only the free fraction in plasma
is filtered at the glomerulus, and this is excreted
unchanged, with very little tubular reabsorption.
Although less than 1% of the daily cortisol secretion
is excreted in the urine, urine cortisol measurements
reflect cortisol secretion rate. The reference range for
urinary free cortisol varies with the method used.
Typically, methods without extraction give much
higher values (e.g., up to 325 g/d (900 nmol/d)).
When an extraction method is coupled to an HPLC
analysis, the reference range is 10 to 54 g/d (27 to
150 nmol/d), and this is similar to that obtained using
an LC-MS/MS method [30]. Several studies have
shown a difference in cortisol excretion rate between
males and females.
Problems with urine free cortisol measurements
include incomplete urine collection and cross-
reactivity (especially in methods without prior
extraction). Sensitivity and specificity of urine
cortisol measurement for the diagnosis of Cushings
syndrome were reported to be 45% to 71% and
100%, respectively [31]. Owing to the difficulty in
collecting complete 24-hour urine samples, some
authors recommend the use of urine
cortisol/creatinine ratios in early morning urine as an
alternative. This is especially useful in children [32].
In addition, variation in the activity of 11-
hydroxysteroid dehydrogenase type 2, which
inactivates cortisol to cortisone, can significantly
affect the amount of cortisol excreted by the kidney.
It has also been suggested that measurement of both
urinary cortisol and urinary cortisone can provide
more meaningful information [33].
Serum free cortisol can be measured by equilibrium
dialysis method, steady-state gel filtration, or by
ultra-filtration methods [34-36]. All these procedures
are time consuming and labor intensive, and some
authors have suggested that calculation of a free
cortisol index from serum total cortisol and CBG
concentrations may be more useful [37]. An
alternative approach is the measurement of cortisol in
saliva. Salivary cortisol is a good index of free serum
cortisol [24]. The concentration of cortisol in saliva is
independent of salivary flow rate and reflects acute
changes in serum cortisol [25].
Cortisol Reference Interval
Reference values vary to some extent, depending on
the methods used for cortisol measurement. Other
factors affecting the reference interval are age,
gender, and diurnal variations. Values presented in
Table 4 are compiled from the literature. Because of
decreased cortisol production, children have lower
plasma cortisol levels and urinary excretion rates than
adults do. Plasma cortisol concentrations exhibit a
circadian rhythm, reaching a peak between 4 and 6
424
Cortisol
am; lowest values are seen by midnight. Total and
free plasma cortisol and cortisol-binding globulin
levels are elevated during pregnancy. Table 4 also
shows cortisol concentrations in saliva and urine
reported in the literature.
Cortisol Performance Goals
The current target for acceptable performance criteria
as mandated by the Clinical Laboratory Improvement
Amendments 88 is 25% of the peer-group mean
[38]. Precision goals suggested by Fraser et al. are a
maximum standard deviation of 23 g/L (63 nmol/L)
at a cortisol concentration of 300 g/L (828 nmol/L)
(a coefficient of variation [CV] of 7.6%) [39]. It is
evident from a recent study that intralaboratory
imprecision (%CV) of serum/plasma cortisol assays
by different vendors varies from 5% to 12% for
cortisol concentrations ranging from 100 g/L to 300
g/L (275 nmol/L to 828 nmol/L)[40]. Data from the
2007 CAP survey shows that the most widely used
method in the United States, the Bayer Advia Centaur
method, achieves this analytical goal. Cortisol assays
by the Abbott AxSYM and DPC IMMULITE 1000
methods show precision of 12.4% and 12.6%,
respectively. The newer DPC IMMULITE 2000 and
2500 assays show better performance [41].
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43 Burtis CA, Ashwood ER, Bruns DE. Tietz
Textbook of Clinical Chemistry and
Molecular Diagnostics. 4th ed. Philadelphia:
Saunders; 2006.
44 Heckmann M, Wudy SA, Haack D, Pohlandt
F. Reference range for serum cortisol in well
preterm infants. Arch Dis Child Fetal
Neonatal Ed. 1999;81:F171-4.
45 MacMahon W, Thompson J, Bowers W,
Sgoutas D. A simplified ultrafiltration
method for determination of serum free
cortisol. Clin Chim Acta 1983;131:171-84.
427
Cortisol

Table 1: Methods of Cortisol Analysis

Method Type of Analysis Principle Usage Comments

1.Fluorometry Chemical After extraction, ethanolic S, P Historical interest

derivatization sulfuric acid converts
cortisol to fluorogenic
chromogen
2. Competitive protein Radioassay After extraction, cortisol in S Historical interest
binding sample competes with
125
I-labeled cortisol for a
cortisol-binding protein
Heterogeneous assay
3. Immunoassay
(i) Radioimmunoassay Competitive radio As above, but antibody S, U Mainly of
assay specific for cortisol is the historical
binding agent interest

(ii) Non-isotopic Labeled with a non-isotopic

immunoassay label such as enzyme S, U

Separation of bound and free

label required
(a) Heterogeneous
No separation required Commonly used
method; no
extraction
(b) Homogeneous required

Commonly used
method; no
extraction
required
4. High-performance liquid Chromatographic Cortisol separated from other S, P, U Extraction required
chromatography Separation compounds by Not suitable for
(HPLC) chromatography and large number of
detected by native routine samples
fluorescence or UV Less interference
absorption
5. Gas chromatography As above Cortisol derivatized and S, P, U As above
separated by
chromatography and
detected by flame
ionization
6. Gas chromatography-mass As above; detection by MS S, P, U As above
spectrometry
7. Liquid chromatography- As above As for GC-MS S, P, U Non-extraction
mass spectrometry methods
available
Candidate
reference
method
428
Cortisol

Table 2: Commonly Used Automated Systems for the Measurement of Cortisol

Instrument Principle End point Manufacturer

Roche Elecys /E170 Paramagnetic beads Electro-chemiluminescence Roche
modular
ACS180and Centaur Paramagnetic particles Chemiluminescence Bayer/Siemens
IMMULITE 2000/2500 Coated beads Chemiluminescence DPC/Siemens
Access/DXI Paramagnetic particles Alkaline phosphatase Beckmann
Architect Paramagnetic particles Chemiluminescence Abbott

TDX Fluorescence Fluorescence Abbott

polarization
VITROS ECi Coated beads Enhance Ortho
chemiluminescence
TOSOH AIa Magnetic microbeads Kinetic fluorescence Tosoh
DELFIA Coated microtiter plate Time-resolved fluorescence Wallac

Table 3: Cortisol assays commonly used in the USA and UK

USA UK
(%) (%)
Bayer Advia Centaur ` 29.3 27.4
Beckman Access/DXI 24.3 11.1
DPC Immulite 2000/2500 11.9 16.7
RocheElcys/E170 modular 11.1 32.1
Abbott Architect 0.7 6.0
Abbott Axsym 4.2 2.0
Others 1.7 2.8

Data from CAP and UK NEQAS

429
Cortisol

Table 4: Cortisol Concentration in Serum, Saliva and Urine

Reference Ranges

Serum Total Cortisol

Adults am 51-230 g/L (140 635 nmol/L) [42]
4 pm 31-100 g/L ( 85 275 nmol/L)
midnight <51 g/L ( <140 nmol/L)

Children am 30-210 g/L ( 83 580 nmol/L) [43]

Infants 20-110 g/L ( 55 304 nmol/L) [43]
Preterm infants 26-203 g/L ( 73 562 nmol/L) [44]

Urine Free Cortisol

Adults unextracted <326 g/d (<900 nmol/d) [23]
extracted 20-90 g/d (55 248 nmol/d) [43]
HPLC <62 g/d (<170 nmol/d) [23]
LCMS <60 g/d (<165 nmol/d) [30]

Serum Free Cortisol

Adults ultra filtrate 2.0-22 g/L ( 6 - 61 nmol/L) [45]

Saliva
Adults 8 am 1.3-9.8 g/L (3.5 27 nmol/L) [34]
10 pm <2.0 nmol/L (<6 nmol/L)
430

Creatine Kinase

Creatine Kinase
Mauro Panteghini
Name: Creatine kinase (CK, adenosine triphosphate: creatine N-phosphotransferase)
Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Enzyme number: EC 2.7.3.2
Molecular mass: Varies from 78,500 to 85,100 D; molecular mass of individual
subunits is half that of intact molecule
Chemical class: Enzyme, protein
Known isoenzymes: CK1 (CK-BB), CK2 (CK-MB), CK3 (CK-MM), CKmt (mitochondrial)

i active site of the enzyme. Activity can be partially

Principles of Analysis and Current Usage
Creatine kinase (CK) is a dimer composed of two
subunits, each with a molecular weight of about 40,000
D. Three different pairs of subunits can exist: BB (or
CK1), MB (or CK2), and MM (or CK3). All three of these
isoenzyme species are found in the cytosol of the cell.
However, there exists a fourth isoenzyme (CKmt)
located between the inner and outer membranes of
mitochondria, and it constitutes, in the heart for example,
up to 15% of the total CK activity. A summary of
enzyme activity distribution is given in Table 1 [1].
CK catalyzes the formation of ATP, a high-energy
substance that is required for contractile or transport
systems. The enzyme also catalyzes the reversible
phosphorylation of creatine (Cr) with ATP as the donor
of the phosphate group. The reaction to form creatine
phosphate (CrP) (the forward reaction) is as follows:
Creatine + ATP pH 6.7 CK pH 9.0
creatine phosphate + ADP
At neutral pH, the formation of ATP is favored; a pH of
9.0 is optimal for the formation of CrP, another high-
energy compound. Mg2+ is an obligatory activating ion
that forms complexes with ATP and ADP. The optimal
concentration range for Mg2+ is quite narrow, and excess
Mg2+ is inhibitory. Many metal ions, such as Mn2+, Ca2+,
Zn2+, and Cu2+, inhibit enzyme activity, as do
iodoacetate and other sulfhydryl-binding reagents.
Activity is inhibited by excess ADP and by citrate,
fluoride, nitrate, acetate, iodide, bromide, malonate, and
L-thyroxine [2].
The enzyme is relatively unstable in serum, activity
being lost as a result of sulfhydryl group oxidation at the
i
Creatine Kinase
Previous and current authors of this method:
First edition: Lenox B. Abbott, John A. Lott
Methods edition: Kory M. Ward, John A. Lott
Second edition: Kory M. Ward, John A. Lott
Third edition: Kory M. Ward, John A. Lott
Fourth edition: Kory M. Ward, John A. Lott
Fifth edition: Mauro Panteghini
restored by incubating the enzyme preparation with
sulfhydryl compounds, such as N-acetylcysteine,
dithiothreitol (Cleland reagent), and glutathione. The
current agent of choice is N-acetylcysteine, which has
the advantage of being a very soluble substance used at a
final concentration of 20 mmol/L in the assay reagent.
Numerous photometric, fluorometric, and coupled-
enzyme methods have been developed for the assay of
CK activity, using either the forward (Cr CrP) or the
reverse (Cr CrP) reaction. Currently, all commercial
assays for total CK are based on the reverse reaction that
proceeds about six times faster than the forward reaction.
The ATP produced is measured by hexokinase
(HK)/glucose-6-phosphate dehydrogenase (G6PD)
coupled reactions that ultimately convert NADP+ to
NADPH, which is monitored spectrophotometrically at
340 nm. The rate of increase in absorbance is a measure
of CK activity present in the specimen.
2+
Creatine phosphate + ADP CK, Mg , pH 6.7
creatine + ATP
ATP + glucose HK glucose-6-phosphate + ADP
Glucose-6-phosphate + NADP+ G6PD 6-
phosphogluconate + NADPH + H+
Oliver first reported this method [3] that Rosalki
subsequently modified and improved by adding AMP to
inhibit adenylate kinase (AK) and cysteine to activate
CK [4]. Subsequently, Szasz and colleagues optimized
the assay by adding N-acetylcysteine to activate CK,
EDTA to bind Ca2+ and increase the stability of the
reaction mixture, and adenosine pentaphosphate (Ap5A)
in addition to AMP to inhibit AK [5]. A reference
method based on this previous experience was developed
by the International Federation of Clinical Chemistry
and Laboratory Medicine (IFCC) for the measurement of
CK at 37C [6].
Reference and Preferred Methods
The above reported Oliver-Rosalki reaction scheme is
the basis for the reference method [6]. The reaction and
measurement conditions of the reference method are
431
Creatine Kinase
listed in Table 2, and the procedure is described on page
11.
The major differences between the commercially
available optimized assays include the choice of buffer
and source of G6PD, the latter determining the type of
pyridine nucleotide that must be used. Imidazole is the
best buffer system, but Tris can also be used. The G6PD
is usually obtained from yeast or from the bacterium
Leuconostoc mesenteroides; the former requires NADP,
whereas the latter can use either NADP or the less
expensive NAD.
Specimen
Specimens for CK analysis include serum and plasma
heparin. Anticoagulants, other than heparin, should not
be used in collection tubes because they inhibit CK
activity. Collection of specimen in gel separator tubes
does not appear to affect CK activity when compared
with tubes not containing gel [7].
CK activity in serum is relatively unstable and rapidly
lost during storage. Average stabilities are less than 8
hours at room temperature, 48 hours at 4C, and 1 month
at 20C. Therefore, the serum specimen should be
stored at 80C if analysis is delayed for more than 30
days.
Interferences
A moderate degree of hemolysis (up to 0.32 g/dL of
hemoglobin) does not significantly influence the
measured CK activity because erythrocytes contain no
CK activity [8]. However, severely hemolyzed
specimens are unsatisfactory because enzymes and
intermediates (AK, ATP, and glucose-6-phosphate)
liberated from the erythrocytes may affect the lag phase
and the side reactions occurring in the assay system.
Turbid and icteric samples can be analyzed; appropriate
values are obtained if the starting absorbance is not too
high.
CK Reference Intervals
Serum CK activity is subject to a number of
physiological variations. Sex, age, muscle mass, physical
activity, and race all interact to affect measured serum
activity [9]. Males generally have a larger muscle mass,
which results in higher serum CK activities than those in
females. Race also has an effect on CK activities, and
the mean activity in whites is 66% of the mean activity
in blacks. In Caucasian subjects, the reference interval
was found to be 46 to 171 U/L for males and 34 to 145
U/L for females when measured with an assay traceable
to the IFCC 37C reference procedure [10].
Newborns generally have higher CK activities, resulting
from skeletal muscle trauma during birth. Serum CK in
infants decreases to the adult reference interval by 6 to
10 weeks. Table 3 contains details of CK activities in
apparently healthy populations.
The suggested upper reference limits when standardized
methods for CK determination are employed are:
Males up to 170 U/L
Females up to 145 U/L
Interpretation
Serum CK is increased in nearly all patients when there
is injury, inflammation, or necrosis of skeletal or heart
muscle.
Serum CK activity is greatly elevated in all types of
muscular dystrophy. In progressive muscular dystrophy
(particularly Duchenne sex-linked muscular dystrophy),
enzyme activity in serum is highest in infancy and
childhood (7 to 10 years of age) and may be increased
long before the disease is clinically apparent. Serum CK
activity characteristically falls as patients get older and
as the mass of functioning muscle diminishes with the
progression of the disease. About 50% to 80% of the
asymptomatic female carriers of Duchenne dystrophy
show threefold to sixfold increases of CK activity. Quite
high values of CK are noted in viral myositis,
polymyositis, and similar muscle diseases. However, in
neurogenic muscle diseases, such as myasthenia gravis,
multiple sclerosis, poliomyelitis, and parkinsonism,
serum enzyme activity is not increased [11,12].
In acute rhabdomyolysis due to crush injuries, with
severe muscle destruction, serum CK activities
exceeding 200 times the upper reference limit may be
found. If the CK remains below 5000 U/L (about 30
times the upper reference limit) during the first 3 days
after the insult, the probability of developing acute renal
failure appears to be low [13]. Very high activity is also
encountered in malignant hyperthermia, a familial
disease characterized by high fever and brought on by
administration of inhalation anesthesia (usually
halothane) to the affected individual. Serum CK can also
be increased by direct trauma to muscle, including
intramuscular injections and surgical interventions.
The changes of serum CK and its MB isoenzyme
following an acute myocardial infarction have been for
many years the mainstay for the diagnosis [14]. It is
now, however, more advantageous to use more cardiac-
specific nonenzymatic markers, such as cardiac troponin
I or T [15]. Other cardiac conditions have been reported
to increase serum CK and CK-MB in serum (e.g.,
coronary artery bypass surgery, cardiac transplantation,
myocarditis, and pulmonary embolism).
Serum CK activity demonstrates an inverse relationship
with thyroid activity. About 60% of hypothyroid
subjects show an average elevation of CK activity
fivefold more than the upper reference limit. The major
isoenzyme present is CK-MM, suggesting muscular
involvement.
Total CK activity may be significantly increased after
exercise. Strenuous, prolonged exercise will result in
large increases of serum CK activities [16]. In untrained
persons, serum CK appears to increase proportionately
to the duration and intensity of the exercise; however,
432
Creatine Kinase
conditioned persons often show smaller changes in
serum CK activity. Sustained exercise, such as in well
trained, long-distance runners, increases the CK-MB
content of skeletal muscle, owing to the phenomenon of
fetal reversion, in which fetal patterns of protein
synthesis reappear. Thus serum CK-MB isoenzyme may
increase in such circumstances. This explanation may
also account for the elevated CK-MB values sometimes
observed in chronic renal failure (uremic myopathy).
During normal childbirth, there is a sixfold elevation in
maternal total serum CK activity. Surgical intervention
during labor further increases the activity of CK in
serum.
A number of drugs can increase serum CK activities.
Release of CK from skeletal muscle may occur after
pharmacological doses of drugs listed in Table 4.
Decreased serum CK activity is observed in immobilized
persons (e.g., patients with spinal cord injury) and in
patients with cancer cachexia.
CK Performance Goals
For a single laboratory, the analytical goals for desirable
performance of CK assays, derived from biological
variation of the enzyme, are as follows [17]:
Imprecision (CV): 11.4%
Bias: 11.5%
Total error: 30.3%
The current interlaboratory coefficient of variation (CV)
on proficiency testing surveys at a CK activity of
approximately two times the upper reference limit ranges
from approximately 1.5% to 5% in different peer groups,
implying that current analytical techniques are adequate
in terms of imprecision [18]. In an international study
assessing the accuracy of CK results from six diagnostic
companies, all the evaluated assays showed significant
bias when compared to the IFCC reference procedure
[19]. Given the low assay imprecision, > 95% of
participating laboratories applying Abbott, Beckman,
and Roche analytical systems were, however, expected
to comply traceability within the biologically derived
total error budget [19].
References
1 Tsung SW. Creatine kinase isoenzyme patterns
in human tissue obtained at surgery. Clin Chem
1976; 22: 173-5.
2 Bais R, Edwards JB. Creatine kinase. CRC Crit
Rev Clin Lab Sci 1982; 16: 291-335.
3 Oliver IT. A spectrophotometric method for the
determination of creatine phosphokinase and
myokinase. Biochem J 1955; 61: 115-22.
4 Rosalki SB. An improved procedure for serum
creatine phosphokinase determination. J Lab
Clin Med 1967; 69: 696-705.
5 Szasz G, Gerhardt W, Gruber W. Creatine
kinase in serum. 3. Further study of adenylate
kinase inhibitors. Clin Chem 1977; 23: 1888-
92.
6 Schumann G, Bonora R, Ceriotti F, Clerc-
Renaud P, Ferrero CA, Ferard G, et al. IFCC
primary reference procedures for the
measurement of catalytic activity
concentrations of enzymes at 37 C. Part 2.
Reference procedure for the measurement of
catalytic concentration of creatine kinase. Clin
Chem Lab Med 2002; 40: 635-42.
7 Koumantakis G, Smith C, Wyndham L. More
on the PST blood collection tubes. Ann Clin
Biochem 1991; 28: 423-4.
8 Frank JJ, Bermes EW, Bickel MJ, Watkins BF.
Effect of in vitro hemolysis on chemical values
for serum. Clin Chem 1978; 24: 1966-70.
9 Meltzer HY. Factors affecting serum creatine
phosphokinase levels in the general population:
the role of race, activity, and age. Clin Chim
Acta 1971; 33: 165-72.
10 Schumann G, Klauke R. New IFCC reference
procedures for the determination of catalytic
activity concentrations of five enzymes in
serum: preliminary upper reference limits
obtained in hospitalized subjects. Clin Chim
Acta 2003; 327: 69-79.
11 Hood D, Van Lente F, Estes M. Serum enzyme
alterations in chronic muscle disease. A biopsy-
based diagnostic assessment. Am J Clin Pathol
1991; 95: 402-7.
12 Panteghini M. Enzyme and muscle diseases.
Curr Opin Rheumathol 1995; 7: 469-74.
13 Beetham R. Biochemical investigation of
suspected rhabdomyolysis. Ann Clin Biochem
2000; 37: 581-7.
14 Dolci A, Panteghini M. The exciting story of
cardiac biomarkers: from retrospective
detection to gold diagnostic standard for acute
myocardial infarction and more. Clin Chim
Acta 2006; 369: 179-87.
15 Panteghini M. The new definition of
myocardial infarction and the impact of
troponin determination on clinical practice. Int J
Cardiol 2006; 106: 298-306.
16 Lott JA, Landesman P. The enzymology of
skeletal muscle disorders. CRC Crit Rev Clin
Lab Sci 1984; 20: 153-90.
17 Ricos C, Garcia-Lario JV, Alvarez V, Caval F,
Domenech M, Hernandez A, et al. Biological
variation database, and quality specifications
for imprecision, bias and total error (desirable
and minimum). The 2004 update.
www.westgard.com/guest26.htm
18 College of American Pathologists. Survey
2007. CARB-B Cardiac MarkersParticipant
summary. Rev 4/2007.
19 Jansen R, Schumann G, Baadenhuijsen H,
Franck P, Franzini C, Kruse R, et al. Trueness
verification and traceability assessment of
results from commercial systems for
measurement of six enzyme activities in serum.
An international study in the EC4 framework of
433

Creatine Kinase

the Calibration 2000 project. Clin Chim Acta
2006; 368: 160-7.
20 Cherian GA, Hill JG. Age dependence of serum
enzymatic activities (alkaline phosphatase,
aspartate aminotransferase, and creatine kinase)
in healthy children and adolescents. Am Soc
Clin Pathol 1978; 70: 783-9.
21 Miller GW, Chinchilli VM, Gruemer H, Nance
WE. Sampling from a skewed population
distribution as exemplified by estimation of the
creatine kinase upper reference limit. Clin
Chem 1984; 30: 18-23.
22 Lott JA, Wolf PL. Creatine kinase. In: Clinical
enzymology: a case-oriented approach.
Chicago: Year-Book Medical Publishers, 1985;
pp. 163-97.
23 Young DS, Pestaner LC, Gibberman V. Effects
of drugs on laboratory tests. Clin Chem 1975;
21: 286D.
24 Thompson PD, Clarkson P, Karas RH. Statin-
associated myopathy. JAMA 2003; 289: 1681-
90.
25 Guthrie RM, Lott JA. Abnormal serum CK-MB
following an amitriptyline overdose: a case
report and review of the literature. J Family
Pract 1986; 22: 550-5.

Table 1: CK Activities and Cytoplasmic Isoenzyme Patterns in Human Tissues*

Tissue Relative CK Activity CK-MM% CK-MB% CK-BB%

(expressed as multiple
of serum activity)
Skeletal muscle
(type I, slow twitch) 50,000 97 3 <1
Skeletal muscle
(type II, fast twitch) 50,000 99 1 <1
Myocardium 10,000 78 22 <1
Brain 5000 0 0 100
Gastrointestinal tract 5000 3 1 96
Urinary bladder 4000 2 6 92
*Adapted from Panteghini 1995 [12]
434

Creatine Kinase

Table 2: Measurement Conditions for IFCC Reference Method

Temperature 37C
Wavelength 340 nm
Buffer, concentration Imidazole, 50 mmol/L
pH (at temperature) 6.5 (37C)
Volume fraction of sample 0.0435 (1:23)
Concentration of reagents in the final complete reaction mixture:
Creatine phosphate 30 mmol/L
ADP 2 mmol/L
EDTA 2 mmol/L
Mg acetate 10 mmol/L
N-acetylcysteine 20 mmol/L
AMP 5 mmol/L
Diadenosine-5-pentaphosphate 0.01 mmol/L
D-glucose 20 mmol/L
Pyridine nucleotide (NADP) 2 mmol/L
Hexokinase (37C) 4000 U/L
G6PD (37C) 2800 U/L

Table 3: Serum CK Activities in Apparently Healthy Populations

Population: Newborns
Subpopulation: Males and females with physiological vaginal deliveries
Enzyme activity: Up to 10 times the upper reference limits in adults

Population: Infants
Subpopulation: Males and females
Enzyme activity: Up to 3 times the upper reference limits in adults [16]

Population: Children and adolescents [9,20]

Subpopulation:
a. Males
b. Females
c. Males and females
Enzyme activity:
a. CK remains within the adult range from 5 to 12 years of age; thereafter, it rises maximally to 1.5 to 2.5
times the upper reference limit in male adults
b. CK remains fairly constant from 5 to 12 years and then gradually declines
with age up to 20 years
c. Male CK activities are 2 times those for females of the same age

Population: Adults [9,21,22]

Subpopulation:
a. Males and females
b. Whites and blacks
c. Ambulatory, hospitalized
d. Hospitalized, bedridden
Enzyme activity:
a. Mean CK activity for men is 1.5-fold that of women
b. Blacks have 1.2- to 1.3-fold higher CK activities than whites of the
same sex
c. Ambulatory patients show a trend toward higher CK activities
d. CK activities can be very low during inactivity
435

Creatine Kinase

Table 4: Drugs That Can Increase Serum CK Activities

Condition Relative Increase Comments

Drug-induced changes at pharmacological doses:
Danazol Slight [16] Some patients
Halofenate Slight [23] Mild, transient effect
Pindolol Slight [16] Some patients
Stanozolol Slight [16] Some patients
Succinylcholine Slight [16] Some patients; dramatic increases
if hyperpyrexia occurs
Statins Slight/Moderate [24] Myopathy observed in some patients;
may produce rhabdomyolysis (0.02%)
Aminocaproic acid Moderate [16] Myopathy observed in some patients
Amphotericin B Moderate [16] Proximal myopathy observed
Carbenoxolone Moderate [16,23] May cause hypokalemic myopathy
Haloperidol Moderate [16] Dystonia produced in some patients
Labetalol Moderate [16]
Lidocaine Moderate [16]
D-Penicillamine Moderate [16] Produced polymyositis
Perphenazine Moderate [16] Produced dystonia
Prochlorperazine Moderate [16] Produced dystonia
Quinidine Moderate [16]
Cyclopropane Pronounced [16] Dramatic increases if hyperpyrexia occurs
Diethyl ether Pronounced [16] Dramatic increases if hyperpyrexia occurs
Halothane Pronounced [16,23] Pronounced effect if causes malignant hyperpyrexia
Drug-induced changes in case of abuse or poisoning:
Amphetamine Pronounced [16] Huge increases
Barbiturates Pronounced [16] Huge increase in comatose patients
Ethanol Pronounced [16] Usually a result of alcoholic myopathy
Heroin Pronounced [16] Huge increases; may produce rhabdomyolysis
Amitriptyline Moderate [25] In suicide cases

Procedure Calculations
Average the change of absorbance (A) per min
Principle values. If the A values are decreasing or appear
CK is measured by a coupled reaction sequence inconsistent (e.g., not linear), repeat the assay with a
described earlier (Oliver-Rosalki method). After a 5-min diluted specimen.
incubation to allow the lag phase to go to completion,
the change in absorbance at 340 nm is monitored at 37C CK activity (U/L) = A/min ___1__ total vol_
(measurement interval, 2 min). The CK activity is molar absorptivity sample vol
directly related to the increase in absorbance of NADPH,

obtained from the zero-order portion of the curve of
= A/min _1_ _2.30_ 1000
absorbance versus time. The results are expressed in
U/L. 6.30 0.10

Reagents = A/min 3651

Reagents may be purchased from several
manufacturers.
Note: The molar absorptivity of NADPH is 630 m2/mol
Assay at 339 nm. The method should be linear to 1500 U/L.
Follow instructions of the manufacturers
analytical system (i.e., instrument, reagents, and
calibrators/controls).
436

Creatine Kinase Isoenzymes

Creatine Kinase Isoenzymes

Mauro Panteghini

Name: Creatine kinase (CK, adenosine triphosphate: creatine N-phosphotransferase)

Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation
Enzyme number: EC 2.7.3.2
Molecular mass: Varies from 78,500 to 85,100 D; molecular mass of individual
subunits is half that of intact molecule
Chemical class: Enzyme, protein
Known isoenzymes: CK1 (CK-BB), CK2 (CK-MB), CK3 (CK-MM), CKmt (mitochondrial)
Biochemical reaction: (See below.)

Principles of Analysis and Current Usage

i membrane of mitochondria. CK-MM is found
Creatine kinase (CK) is a dimeric enzyme that catalyzes predominantly in skeletal muscle; the richest source of
the reversible phosphorylation of creatine by ATP. CK CK-MB is myocardial muscle, and CK-BB is found
comprises two subunits which are designated M and mainly in brain and intestine. For details of relative CK
B; hence the protein may exist in three forms, CK- activities and isoenzyme composition of different tissues
MM, CK-MB, and CK-BB, respectively. All three of refer to Table 1. The CK activity in serum of healthy
these isoenzymes are found in the cytosol of the cell. people is due almost exclusively to MM activity (though
There is also a fourth isoenzyme (CKmt) located in the small amounts of CK-MB may be present) and is the
result of physiological turnover of muscle tissue.

i
Creatinine Kinase isoenzymes When electrophoresed, CK-MM runs closest to the
Previous and current authors of this method: cathode, CK-MB has intermediate mobility, and CK-BB
First edition: John F. Chapman, Linda L. Woodard, moves farthest from the point of application toward the
Lawrence M. Silverman anode. CKmt, which runs more cathodally than the MM
Methods edition: John F. Chapman, Linda L. Woodard, fraction, is usually associated with tissue necrosis that
Lawrence M. Silverman accompanies severe anoxic shock and severe liver
Second edition: John F. Chapman, Linda L. Woodard, disease. CK activity may also be found in a
Lawrence M. Silverman macromolecular formthe so-called macro-CK. Macro-
Third edition: Monica Payne, Peter E. Hickman CK is found, often transiently, in sera of up to 6% of
Fourth edition: Not updated hospitalized patients, but only a small proportion of
Fifth edition: Mauro Panteghini these have increased CK activities in serum. It exists in
437
2
two forms, types 1 and 2. Macro-CK type 1 is a complex
of CK, typically CK-BB, and an immunoglobulin, often
IgG. It often occurs in women older than 50. Macro-CK
type 2 is oligomeric CKmt found predominantly in
adults who are severely ill with malignancies or in
children who have notable tissue distress [1].
Both M and B subunits have a C-terminal lysine residue,
but only the former is hydrolyzed by the action of
carboxypeptidases present in blood. Carboxypeptidases
B (EC 3.4.17.2) or N (arginine carboxypeptidase; EC
3.4.17.3) sequentially hydrolyze the lysine residues from
CK-MM to produce two CK-MM isoformsCK-MM2
(one lysine residue removed) and CK-MM1 (both lysine
residues removed). The loss of the positively charged
lysine produces a more negatively charged CK molecule
with greater anodic mobility at electrophoresis. Because
CK-MB has only one M subunit, the dimer coded by the
M and B genes is named CK-MB2 and the lysine-
hydrolyzed dimer is named CK-MB1. The assay of the
CK isoforms requires special techniques, such as high-
voltage electrophoresis (with gel cooling), HPLC,
chromatofocusing, or immunoassay [2].
For many years, CK isoenzyme analysis has been used
to confirm or refute a diagnosis of acute myocardial
infarction. CK-MB content in myocardium is
approximately 20% of total myocardial CK, so damage
to myocardium is accompanied by release of CK-MB
into the circulating blood in higher proportion. It is now,
however, more advantageous to use more cardiac-
specific nonenzymatic markers, such as cardiac troponin
I or T [3].
Historically, CK-MB has been measured by determining
the catalytic activity after separation of the isoenzymes
by electrophoresis or ion-exchange chromatography.
Subsequently, immunological techniques measuring
catalytic activity were introduced. These methods were
limited in their use, since interferences with macro-CK
or CKmt gave low specificity. Currently, the most
common approach is to measure concentrations of the
CK-MB protein (mass) by using immunoassays with
monoclonal antibodies for the MB dimer [4].
Reference and Preferred Methods
At the present time, there is no reference method for CK
isoenzyme analysis. The techniques most commonly
used are electrophoresis and various immunological
methods.
Electrophoretic methods are useful for separation of all
of the CK isoenzymes. The isoenzyme bands are
visualized by incubating the support (e.g., agarose or
cellulose acetate) with a concentrated CK assay mixture
using the reverse reaction. The NADPH formed in this
reaction is then detected by observing the bluish-white
fluorescence after excitation by long-wave (360 nm)
ultraviolet light. NADPH may be quantified by
fluorescence densitometry, which is capable of detecting
bands of 2 to 5 U/L. The mobility of CK isoenzymes at
pH 8.6 toward the anode is BB > MB > MM, with the
MM remaining cathodic to the point of application. The
discriminating power of electrophoresis also allows the
detection of abnormal CK bands (e.g., macro-CK). The
disadvantages of electrophoresis include that the
turnaround time is relatively long, the procedure is
highly labor intensive and not adaptable to clinical
chemistry analyzers in emergency situations, and
interpretative skills are required.
Immunochemical methods are applicable to the direct
measurement of CK-MB. In the immunoinhibition
technique, an anti-CK-M subunit antiserum is used to
inhibit both M subunits of CK-MM and the single M
subunit of CK-MB and thus allow determination of the
enzyme activity of the B subunit of CK-MB, the B
subunits of CK-BB, and macro-CKs. To determine CK-
MB, this technique assumes the absence of CK-BB (and
of the other sources of interference such as macro-CKs)
from the tested serum. Because the CK-B subunit
accounts for half of the CK-MB activity, the change in
absorbance should be doubled to obtain CK-MB activity.
This results in a significant decrease of the analytical
sensitivity of the method. If present, atypical macro-CK
may result in falsely elevated CK-MB results. Owing to
its low sensitivity and specificity, the immunoinhibition
technique has been largely supplanted by mass assays of
CK-MB.
In contrast with immunoinhibition, which measures the
CK-MB isoenzyme by determination of its catalytic
activity, mass immunoassays measure CK-MB protein
concentrations. A number of mass assays using various
labels are now commercially available and are used for
routine determination of CK-MB. Measurements use the
sandwich technique, in which one antibody
specifically recognizes only the MB dimer. The
sandwich technique ensures that only CK-MB is
estimated, because neither CK-MM nor CK-BB reacts
with both antibodies. Mass assays are more sensitive
than activity-based methods, with a limit of detection for
CK-MB usually <1 g/L [4]. Other advantages include
sample stability, noninterference with hemolysis, drugs,
or other catalytic activity inhibitors, full automation, and
fast turnaround time.
Specimen
Fresh serum free from hemolysis is the specimen of
choice for analysis of the CK isoenzyme pattern. Of the
three commonly seen isoenzymes, CK-BB activity is the
least stable. Adding a thiol such as 2-mercaptoethanol to
the serum improves its stability [5]. CK-MB activity is
not significantly reduced when the separated serum is
stored for up to 48 hours at 4C or 1 month at 20C.
Since mass measurement is not subject to the loss of
enzyme activity, CK-MB protein concentration in serum
is stable for weeks, whether the specimen is stored under
refrigeration, and for several years if stored at 20C.
Interferences
EDTA, oxalate/fluoride, and citrated plasma must not be
used, since these inhibit CK activity. Because CK-MB
438
3
mass assays are not subject to the interferences of
activity assays, hemolysis and various anticoagulants
generally do not interfere.
CK Isoenzymes Reference Intervals
With the CK-MB mass assay, the upper reference limit
for males is 5.0 g/L, with values for females being less
than for males, although many laboratories use a single
reference interval (male). Sustained exercise, such as in
well trained, long-distance runners, increases the CK-
MB content of skeletal muscle, which may produce
abnormal serum CK-MB concentrations.
To better separate non-myocardial infarction from
myocardial infarction patients, the use of a relative
index (RI) is necessary. The RI relates CK-MB mass
concentration in g/L to measured total CK activity in
U/L. Results are expressed as a percentage:
Physiological: 3%
Equivocal: 3 to 5%
Consistent with myocardial necrosis: >5%
To appropriately use the RI, blood sampling between 8
and 36 hours from symptom onset is necessary.
CK-BB may be elevated in neonates, particularly in
brain-damaged or very low birth weight newborns. The
presence of CK-BB in blood, usually at low
concentrations, may however represent a physiological
finding in the first days of life.
Interpretation
Detection of CK-MB in increased amounts has been the
hallmark for the biochemical diagnosis of acute
myocardial infarction for the past several decades [6].
However, the interpretation of results in the presence of
other illness, particularly skeletal muscle injury, is
difficult [7]. It is important to remember that CK-MB
concentrations in serum may not always accurately
reflect myocardial damage. There have been numerous
reports of CK-MB elevation associated with non-cardiac
conditions, including polymyositis, malignancy, and
hypothyroidism. Serial sampling after the onset of chest
pain, showing the typical rise and fall of CK-MB values,
significantly increases the diagnostic accuracy for
myocardial infarction.
CK-MB determination can be used with some success to
estimate the extent of myocardial necrosis to assist with
assessment of infarct prognosis. When comparing peak
CK-MB with estimates of infarct size, good correlations
can be obtained (Table 2). A problem with using CK-
MB for this purpose is the requirement for frequent
sampling to ensure that peak CK-MB values are
correctly identified. It appears in some way provocative,
but it is quite possible that within a few years, CK-MB
determination will no longer be offered, because cardiac
troponin measurement is clearly superior in this clinical
setting [8].
CK-MB Performance Goals
For a single laboratory, the analytical goals for desirable
performance of CK-MB mass assays, derived from
biological variation of the protein, are as follows [9,10]:
Imprecision (CV): 9.2%
Bias: 16.0%
Total error: 31.3%
The current interlaboratory coefficient of variation (CV)
on proficiency testing surveys at a CK-MB mass
concentration around the upper reference limit ranges
from 3.7% to approximately 9% in different peer groups
[11]. At higher CM-MB concentrations (70 g/L), data
from the College of American Pathologists survey show
CVs between 2.5% and 6% [12].
References
1 Panteghini M, Bais R, van Solinge WW.
Enzymes. In: Burtis CA, Ashwood ER, Bruns
DE, eds. Tietz Textbook of Clinical Chemistry
and Molecular Diagnostics. 4th ed.
Philadelphia, PA: Elsevier Saunders, 2006:597-
643
2 Panteghini M. Serum isoforms of creatine
kinase isoenzymes. Clin Biochem 1988; 21:
211-8.
3 Panteghini M. The new definition of
myocardial infarction and the impact of
troponin determination on clinical practice. Int J
Cardiol.. 2006; 106: 298-306.
4 Panteghini M. Diagnostic application of CK-
MB mass determination. Clin Chim Acta 1998;
272: 23-31.
5 Abbott LB, Lott JA. Reactivation of serum
creatine kinase isoenzyme BB in patients with
malignancies. Clin Chem 1984; 30: 1861-3.
6 Adams JE III, Abendschein DR, Jaffe AS.
Biochemical markers of myocardial injury. Is
MB creatine kinase the choice for the 1990s?
Circulation 1993; 88: 750-63.
7 Adams JE 3rd, Sicard GA, Allen BT, Bridwell
KH, Lenke LG, Davila-Roman VG, et al.
Diagnosis of perioperative myocardial
infarction with measurement of cardiac
troponin I. N Engl J Med 1994; 330: 670-4.
8 Jaffe AS, Babuin L, Apple FS. Biomarkers in
acute cardiac disease. The present and the
future. Circulation 2006; 48: 1-11.
9 Ricos C, Garcia-Lario JV, Alvarez V, Caval F,
Domenech M, Hernandez A, et al. Biological
variation database, and quality specifications
for imprecision, bias and total error (desirable
and minimum). The 2004 update.
www.westgard.com/guest26.htm.
10 Ross SM, Fraser CG. Biological variation of
cardiac markers: analytical and clinical
considerations. Ann Clin Biochem 1998; 35:
80-4.
11 Reference Centre for Quality Assurance
External Quality Assessment Scheme (Regione
439
4

Toscana, Italy). Comprehensive Cardiac 13 Panteghini M. Enzyme and muscle diseases.

Markers Survey Report 2006. Curr Opin Rheumathol 1995; 7: 469-74.
12 College of American Pathologists. Survey
2007. CARB-B Cardiac Markers Participant
Summary. Rev 4/2007.
Tables
Table 1: CK Activities and Cytoplasmic Isoenzyme Patterns in Human Tissues*

Tissue Relative CK activity CK-MM% CK-MB% CK-BB%

(expressed as multiple
of serum activity)
Skeletal muscle
(type I, slow twitch) 50,000 97 3 <1
Skeletal muscle
(type II, fast twitch) 50,000 99 1 <1
Myocardium 10,000 78 22 <1
Brain 5000 0 0 100
Gastrointestinal tract 5000 3 1 96
Urinary bladder 4000 2 6 92
*Adapted from Panteghini 1995 [13]

Table 2: Decision Limits of CK-MB Mass Peak for Infarct Size Definition

Microscopic myocardial infarction (MI) (focal necrosis) <10 g/L

Small MI (<10% left ventricle) 10-60 g/L
Medium MI (10-30% left ventricle) 60-225 g/L
Large MI (>30% left ventricle) >225 g/L
440
Creatinine
Creatinine
Edmund J. Lamb
Name: Creatinine
Clinical significance: Increased concentrations in blood indicate renal impairment Refer to Chapter
30, Renal Function, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C4H7N3O
Molecular weight: 113.12 D
Merck Index: 2552
Chemical class: Creatine end-product metabolite
Structure:
Reference method: GC-IDMS
i was then commonly adsorbed onto aluminum
Principles of Analysis and Current Usage
Creatinine is a widely used test of kidney function. Most
chemical methods for measuring creatinine are primarily
based on the reaction with alkaline picrate (Table 1,
Method 1). In this reaction, first described by Jaffe in
1886 [1], creatinine reacts with picrate ion in an alkaline
medium to yield an equimolar orange-red Janovski
complex. Despite considerable literature on the subject,
the reaction mechanism and the structure of the product
remain unclear [2]. Use of this reaction to measure
creatinine in urine was first described by Folin in 1904
[3]. He later extended this principle to creatinine
measurement in deproteinized blood [4]. The reaction is
nonspecific for creatinine, and a variety of modifications
have been introduced in an attempt to remove
interference from noncreatinine chromogens (see
below). Measurements of creatinine in serum and plasma
are generally considered equivalent, and the term serum
will be used throughout this chapter.
End-Point Assays
-Ketomethylene groups found in proteins react with
alkaline picrate to form highly colored complexes. Early
end-point methods for the measurement of creatinine in
serum therefore required the use of a protein-free filtrate,
generated for example using tungstic acid. Creatinine
i differential rate of color development, thus allowing a
Creatinine
Previous and current authors of this method:
First edition: Robert L. Murray
Methods edition: Robert L. Murray
Second edition: Robert L. Murray
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Edmund J. Lamb
magnesium silicate clays, including Fullers earth
(Floridin) and Lloyds reagent, theoretically isolating it
from potential interferents (Table 1, Method 2) prior to
reaction with alkaline picrate. Such techniques were in
widespread use in the 1970s, and a variation of this
procedure was originally proposed as a reference method
[5]. However, it became apparent that such methods did
not eliminate interference from noncreatinine
chromogens. Further, they were imprecise and
unsuitable for automation, and by the 1980s they were
not widely used [6]. Other approaches that have been
used in an attempt to improve the general specificity of
the Jaffe reaction have included acid blanking, the use of
cation exchange resins [7,8], solvent extraction, and
oxidation of interferents with compounds such as cerium
sulfate; generally these modifications have not proved
practical. The use of dialysis membranes in continuous
flow automation in the 1960s reduced interference from
protein, although other noncreatinine chromogens
remained dialyzable.
Kinetic Assays
The Jaffe reaction is first order with respect to
creatinine, picrate, and hydroxide concentrations.
Kinetic alkaline picrate methods for the determination of
creatinine (Table 1, Method 3) take advantage of the fact
that creatinine and noncreatinine chromogens have a
rate-dependent separation of creatinine from interfering
substances. Deproteinization and/or adsorption of
interferents is not necessary, since the reaction between
alkaline picrate and noncreatinine chromogens is slow
and does not significantly occur during the usual kinetic
reaction interval. This approach significantly reduces but
does not eliminate interferences from noncreatinine
441
Creatinine
chromogens. Such methods gained in popularity with the
advent of instruments capable of making accurate
absorbance readings at precise, highly reproducible
intervals [6,9]. Kinetic Jaffe assays have been
implemented on various automated microprocessor-
controlled instruments and are currently the most widely
used approach to creatinine measurement. Extensive
literature exists on the choice of reactant concentrations
and reading interval, as well as on the choice of
wavelength and reaction temperature [6]. Brief
comments include:
Reading interval. Some noncreatinine chromogens have
a rapid reaction with alkaline picrate (e.g., acetoacetate),
whereas others show a slower reaction (e.g., protein). To
avoid the period of maximal reaction with these
interferents, the majority of kinetic methods read over
the approximate time interval 10 to 80 seconds after
mixing of the reagents and sample.
Picrate concentration. With respect to picrate, the Jaffe
reaction is pseudofirst order up to 30 mmol/L picrate,
with the majority of methods employing a concentration
between 3 and 16 mmol/L. At concentrations above 6
mmol/L, the rate of color development becomes
nonlinear, so a two-point fixed interval rather than
multiple-data-point approach is required.
Wavelength. While the absorbance maximum of the
Jaffe reaction is approximately 490 nm, improved
method linearity and reduced blank values have been
reported at other wavelengths, the choice varying with
hydroxide concentration. Generally, the absorbance of
the complex is measured between 510 and 520 nm.
Picrate ion, present in excess in the reaction solution,
absorbs significantly at wavelengths below 500 nm.
Temperature. The rate of Jaffe complex formation and
the absorptivity of the complex are temperature-
dependent, measurable differences being observed even
between 25C and 37C. The absorbance maximum of
the colored product is also temperature sensitive.
Consequently, temperature control is an important
component of assay reproducibility.
Enzymatic Assays
Several creatinine-degrading enzymes have been
investigated for use in creatinine analysis. This approach
was first attempted in 1937 by Miller and Dubos, using
crude extracts of aerobic bacteria capable of forming
creatinine-decomposing enzymes [10]. However, owing
to the requirement for large quantities of pure enzyme,
these approaches have only recently become
commercially feasible. There are primarily three
approaches, described below.
Creatininase. The enzyme creatininase (EC 3.5.2.10;
creatinine amidohydrolase) catalyzes the conversion of
creatinine to creatine. The creatine is then detected with
a series of enzyme-mediated reactions involving creatine
kinase, pyruvate kinase, and lactate dehydrogenase, with
monitoring of the decrease in absorbance at 340 nm due
to NADH disappearance (Table 1, Method 4) [11].
Initiating the reaction with creatininase allows for the
removal of endogenous creatine and pyruvate in a
preincubation reaction. The kinetics of the reaction are
poor, and a 30-min incubation is required to allow the
reaction to reach equilibrium. This shortcoming can be
overcome by a kinetic approach, but with a further
reduction in sensitivity. The approach has not been
popular, partly due to poor sensitivity, poor precision,
and the relatively high cost of reagents [6].
Creatininase and creatinase. An alternative, more
popular, approach has utilized the enzyme creatinase
(EC 3.5.3.3; creatine amidinohydrolase), which yields
sarcosine and urea, the former being measured with
further enzyme-mediated steps using sarcosine oxidase
(EC 1.5.3.1; yielding glycine, formaldehyde, and
hydrogen peroxide) and peroxidase (Table 1, Method 5)
[12]. The hydrogen peroxide can be detected with a
variety of methods. The potential interference due to
ascorbic acid can be overcome by the inclusion of
ascorbate oxidase. The influence of endogenous
intermediate creatine and urea can be overcome by a
preincubation step, initiating the reaction with
creatininase. This system has been incorporated in a
point-of-care testing device using polarographic
detection [13]. An alternative detection system involves
measurement of the reduction of NAD by formaldehyde
in the presence of formaldehyde dehydrogenase [14].
Creatinine deaminase. The third enzyme system used for
creatinine quantitation is creatinine deaminase (EC
3.5.4.21; creatinine iminohydrolase), which catalyzes the
conversion of creatinine to N-methylhydantoin and
ammonia (Table 1, Method 6) [15]. Early methods
concentrated on the detection of ammonia using either
glutamate dehydrogenase [16], an ammonia electrode
[17], or the Berthelot reaction. An alternative approach
involves the enzyme N-methylhydantoin amidohydrolase
[18]. In all of these procedures, the reaction mixture
must be free of ammonia-producing and ammonia-
consuming materials. In addition, a correction for
endogenous ammonia must be made. Although the
concentration of physiologically produced ammonia is
relatively low, substantial amounts of ammonia can be
formed by protein deamination if blood is left at room
temperature.
Dry Chemistry Systems
A number of multilayer dry reagent methods have been
described for the measurement of creatinine using
enzyme-mediated reactions. An early two-slide
approach employed creatinine deaminase, with the
ammonia diffusing through a semipermeable and
optically opaque layer to react with bromophenol blue to
give an increase in absorbance at 600 nm. A second
multilayer film lacking the creatinine deaminase enzyme
was used to quantitate endogenous ammonia, enabling
blank correction [19]. A later, more precise, single slide
method utilized the creatininase/creatinase reaction
sequence [20]. Lidocaine metabolites have been reported
to interfere with this method [21]. The creatinine
deaminase system described above has also been utilized
and adapted for use as a point-of-care testing device
[22]. In all cases, the color produced in the film is
quantitated by reflectance.
Other Chemical Methods
442
Creatinine
Other chemical approaches to the measurement of
creatinine have been tried. These include (1) reaction
with 1,4-naphthoquinone-2-sulphonate [23]; (2) use of o-
nitrobenzaldehyde to convert creatinine to
methylguanidine and its reaction with -naphthol and
sodium hypochlorite under alkaline conditions [24]; and
(3) reaction with 3,5-dinitrobenzoic acid (DNBA)
[25,26] or its derivatives [27,28]. The DNBA procedure
has been adapted for use with the dry-strip technology
[29]. In this procedure, creatinine percolates through the
initial layers of the strip to react with DNBA to form a
reddish chromogen. The reaction is monitored by
reflectance spectrophotometry at 560 nm. A reagent-free
mid-infrared method for urinary creatinine measurement
has been described [30]. None of these reactions are
widely used in clinical laboratories.
High-Performance Liquid Chromatography
Many authors have reported separation and quantitation
of creatinine with high-performance liquid
chromatography (HPLC) (Table 1, Method 7), using
both cation-exchange [31-33] and reversed-phase
techniques [34,35]. Separation is followed by
quantitation using either an on-stream Jaffe reaction
[31], use of creatinines native absorbance at
approximately 230 nm [32-37] or enzymatic detection
[38]. All HPLC approaches are reported to be rapid,
specific, and precise; several have been proposed as
candidate reference methods [33,38,39]. Thienpont et al.
reported between-day and within-run imprecision of
<1% with deviation from gas chromatography isotope
dilution-mass spectrometry (GC-IDMS) target values of
+0.1% [39].
Isotope-Dilution Mass Spectrometry
The use of mass fragmentography as a reference method
for creatinine was first proposed by Bjorkhem et al. in
1977 [40]. GC-IDMS is considered the method of choice
for establishing the true concentration of creatinine in
serum because of its excellent specificity and precision.
Definitive methods have been described by several
authors [41-43], and the Joint Committee on Traceability
in Laboratory Medicine (JCTLM) nominated three GC-
IDMS methods as reference measurement procedures
[44]. However, creatinine requires prior derivatization
before GC analysis and a cation-exchange clean-up step
is also necessary because creatine is derivatized into the
same chemical species as creatinine [44]. This limits the
throughput of analysis and hence the general
applicability of the technique. Recently, direct methods
in which HPLC is coupled with IDMS have been
reported, offering the potential for quicker turnaround
[45,46].
Reference and Preferred Methods
As described above, GC-IDMS has been proposed by the
JCTLM as the definitive reference method for serum
creatinine measurement. HPLC methods have often been
shown to give comparable performance. Although these
methods are generally used by commercial
manufacturers to establish traceability of their assays,
they are presently unlikely to find their way into more
general use. Measurement of serum and urine creatinine
is a core test for most diagnostic laboratories.
Laboratories must often provide results in a STAT mode
on a 24-hour-a-day basis, frequently on samples of small
volume (i.e., from pediatric patients) that may be
compromised by interferences (e.g., bilirubin in
jaundiced infants or adults). These factors will influence
a laboratorys choice of methodology. The methodology
for the measurement of creatinine is complex by virtue
of the number of variants of the Jaffe reaction and the
innovations attempted using enzymatic procedures to
overcome the limitations of the former. Kinetic Jaffe
approaches predominate in wet chemistry analytical
systems and are relatively inexpensive. Enzymatic
methods tend to be more expensive than Jaffe assays and
are the methods used in dry chemistry systems, including
some point-of-care testing devices. Any laboratorian
assessing a new creatinine method (e.g., as part of an
analyzer purchase) should review the data for that
method on interference due to bilirubin, protein, glucose,
and ketones/ketoacids. Bilirubin will also be an
important consideration in enzymatic procedures that
generate hydrogen peroxide. Despite criticism of the
Jaffe methods, there is invariably a good correlation
between them and enzymatic procedures, with the
differences likely to be due as much to calibration as to
interference. However, there is general agreement that
with appropriate standardization, commercial enzymatic
assays can, on most systems and in most samples,
provide results comparable to those of HPLC or IDMS.
Provided the increased costs can be justified, it seems
possible and probably desirable that enzymatic assays
will gradually replace the kinetic Jaffe assay currently in
widespread use.
Of 722 participants in the United Kingdom National
External Quality Assessment Scheme for serum
creatinine in May 2007, 73% used the kinetic Jaffe
reaction, 7% used an end-point Jaffe, 4% used an
OLeary modification of a kinetic Jaffe assay, 3% used
an enzymatic assay, and 13% used a dry-slide enzymatic
method. Of the kinetic Jaffe participants, the major
reagent manufacturers were Abbott Diagnostics (16%),
Beckman Coulter Ltd (13%), Roche Diagnostics Ltd
(36%, Modular and Integra platforms), and Olympus Ltd
(23%). A similar pattern of usage is seen in the Unites
States. Of 2931 participants in the 2007 College of
American Pathologists (CAP) program for serum
creatinine, 74% used the kinetic Jaffe reaction, 4% used
an end-point Jaffe, 3% used an enzymatic assay, and
18% used a dry-slide enzymatic method. Of the kinetic
Jaffe participants, the major reagent manufacturers were
Abbott Diagnostics (6%), Beckman Coulter Ltd (42%),
Dade Behring (37%), Bayer Diagnostics (2%), Roche
Diagnostics (5%), and Olympus Ltd (6%).
Specimen
The methods described are suitable for the measurement
of creatinine in serum, plasma, or diluted urine. Such
specimens are stable for at least 7 days at 4C. Serum
creatinine is also stable during long-term frozen storage
and after repeated thawing and refreezing [47].
443
Creatinine
However, it should be noted that delayed separation
(beyond 14 h) of serum from erythrocytes leads to a
significant increase in apparent serum creatinine
concentration using some kinetic Jaffe (but not
enzymatic) assays, possibly due to release of
noncreatinine chromogens from the red cells [48].
Bacterial contamination, which can occur in samples
stored for long periods of time, has been reported to
falsely lower creatinine values measured using the Jaffe
reaction, purportedly due to bacterial production of a
substance that retards the reaction [49]. Creatinine
concentration increases in blood after meals containing
cooked meat: ideally blood for serum creatinine
measurement should be obtained in the fasting state [50].
Interferences
The Jaffe reaction is not specific for creatinine. Many
compounds have been reported to produce a Jaffe-like
chromogen, including protein, glucose, ascorbic acid,
levulose, ketone bodies [51,52], pyruvate, guanidine,
aminohippurate, uric acid, blood-substitute products
[53], and cephalosporins [54]; the reader is referred to
several comprehensive reviews [6,55,56]. Ascorbic acid,
levulose, glucose, and uric acid are capable of reducing
picrate to picramate, and this product, with its
absorbance maximum of 485 nm, causes overestimation
of creatinine. Pyruvate, acetone, acetoacetic acid, amino
acids, and proteinor any compound with an active
methyl or methylene groupintroduce error by coupling
with picrate by a mechanism analogous to that of
creatinine. All these compounds can produce colored
compounds, which then increase the absorbance in the
region of the creatinine-picrate complex. The effect of
ketones and ketoacids is probably of the greatest
significance clinically, although the effect is very
method dependent. Thus reports on acetoacetate
interference vary from a negligible increase to an
increase of 310 mol/L in the apparent creatinine
concentration at an acetoacetate concentration of 8
mmol/L. Kinetic Jaffe approaches have significantly
reduced, but not eliminated, interferences from
noncreatinine chromogens. The degree of interference
from these compounds is dependent on the precise
reaction conditions chosen and the concentration of the
interferent present in the patients sample. It is generally
considered that noncreatinine chromogens contribute
approximately 20% of the apparent, Jaffe-measured,
creatinine concentration in normal serum samples.
Noncreatinine chromogens do not generally contribute to
measured urinary creatinine concentration.
Bilirubin or other hemoglobin degradation products are
negative interferents in the Jaffe reaction, probably by
their oxidation in strong base to colorless compounds
[57]. The resulting decrease in background decreases the
measured absorbance and is interpreted as a lower
creatinine concentration. The addition of buffering ions,
such as borate and phosphate, together with surfactant,
have been used to minimize the effects of this
interference. A popular maneuver in this context has
been the addition of potassium ferricyanide before the
alkaline picrate (OLeary method) which oxidizes
bilirubin to biliverdin, hence reducing its interference
[58,59]. Enzymatic (creatinine deaminase/creatininase)
assays are also not immune to negative interference by
bilirubin, which reacts with the peroxidase detection
system [57]. This problem has been approached with the
addition of potassium ferricyanide (with limited success)
or bilirubin oxidase.
Reference Interval and Interpretation
Given the foregoing discussion, reference intervals for
serum creatinine are clearly method dependent. A
selection of the reported reference values for different
methods are listed in the table below. Creatinine is
formed by the condensation of creatine into a ring
structure, with the concomitant loss of a water molecule.
The reaction occurs spontaneously in the body. Because
the bulk of creatine resides in muscle, the amount of
creatinine present in a person reflects muscle mass.
Therefore, women have lower serum creatinine
concentrations than men, and children and infants have
lower serum creatinine concentrations than adults.
Reference ranges in older people are similar to those in
younger adults, despite the decline in kidney function
that occurs on average with ageing. Typically, reference
intervals for serum creatinine measured by Jaffe methods
are 80 to 115 mol/L (0.9 to 1.3 mg/dL) in men and 53
to 97 mol/L (0.6 to 1.1 mg/dL) in women [60].
However, as can be seen from the table, lower reference
ranges are generally reported with enzymatic or
compensated assays. Serum creatinine concentration
in patients with untreated end-stage renal disease
(ESRD) may exceed 1000 mol/L (11 mg/dL).
Urinary creatinine excretion is higher in men (124 to 230
mol/kg/d [14 to 26 mg/kg/d]) than in women (97 to 177
mol/kg/d [11 to 20 mg/kg/d]). Creatinine excretion
decreases with age: typically, for a 70 kg man, creatinine
excretion will decline from approximately 14.5 to 9.1
mmol/d with advancing age from 30 to 80 years [61].
Measurement of urinary creatinine excretion can be a
useful indication of the completeness of a timed urine
collection. Creatinine excretion is often used as a method
of normalizing the urinary excretion of analytes. That is,
the excretion of the test analyte (in mmol or grams) is
divided by the total amount of creatinine (in mmol or
grams) excreted in the same urine specimen. This
method is a rough correction for volume differences
between patient specimens. Similarly, expressing the
concentration of a substance as a ratio to the creatinine
concentration is a useful method of adjusting for urinary
concentration differences in random (spot) urine
samples.
444
Creatinine

Serum Creatinine Reference Intervals

Study Method Age Serum Creatinine (mol/L)
Male n Female n
Gardner and Technicon 20-29 y 60-110 321 50-110 384
Scott (1980) [62] Autoanalyzer 30-39 y 60-120 537 50-110 698
method N11b 40-49 y 50-110 476 50-110 560
50-59 y 60-140 294 50-120 279
60-69 y 70-140 116 50-120 142
>69 y 50-160 38 60-140 50
Soldin and Hicks Kodak Ektachem 0-1 week 53-97 >100 53-97 >100
(1995) [63] (enzymatic) 1 week-1 month 27-62 >100 27-62 >100
1 month-1 y
1-18 y 18-35 >100 18-35 >100
18-62 >100 18-62 >100
Mazzachi et al. Hitachi 917 (rate- adults 62-106 293 44-80 269
(2000) [64] blanked,
compensated Jaffe)
Mazzachi et al. Hitachi 917 adults 59-104 293 45-84 269
(2000) [64] (creatininase/
creatinase)
Sugita et al. Hitachi 736 0-1 y 4-29* 18
(1995) [14] (creatinine 2-5 y 4-40* 16
amidohydrolase/sar 6-9 y 18-46* 41
cosine oxidase) 10 y 19-52* 15
adult 55-96 235 40-66 355
All values have been rounded up or down to the nearest whole number.
* males and females combined
To convert plasma creatinine in mol/L to mg/dL, multiply by 0.011
Creatinine Performance Goals GFR, and vice versa. Miller et al. have calculated that
Different methods for assaying serum creatinine have allowing for a total error in eGFR of 15%, the maximal
varying degrees of imprecision and accuracy. Targets for allowable bias of a creatinine assay at a concentration of
desirable imprecision are generally based on knowledge 88 mol/L (1.00 mg/dL) would be 3 mol/L (0.034
of the biological variation of an analyte [65]. Mean intra- mg/dL), a target achieved by very few laboratories or
individual biological variation (CVi) for serum methods [70]. As a result of reaction with noncreatinine
creatinine has been reported as 4.1% to 4.3% [66,67], chromogens, end-point Jaffe methods were typically
indicating a desirable analytical performance goal (based judged to overestimate true serum creatinine
on 50% of the upper estimate of the CVi) of 2.2% and a concentration by approximately 20% at physiological
desirable total error goal of 7.6% [44]. With the advent concentrations [44], and most commercially available
of automated kinetic analysis, within-laboratory, creatinine methods continue to demonstrate significant
between-day imprecision of approximately 3.0% can be positive bias compared to HPLC or IDMS methods,
expected at pathological concentrations, with slightly particularly at concentrations within the reference range
inferior performance within the reference interval [68]. [71,72]. A recent College of American Pathologists
A recent report suggests intra-laboratory imprecision at a (CAP) survey confirmed that most routine methods,
concentration of 88.4 mol/L (1.00 mg/dL) varying including some enzymatic assays, had significant
between 2.0% and 8.4% [44]. Clearly, many laboratories positive bias compared to a result obtained using an
do not perform to desirable performance standards IDMS reference procedure [70]. For example, at a true
defined in terms of biological variation [69], although concentration of 80 mol/L (0.90 mg/dL), kinetic
reference HPLC and IDMS methods do appear capable alkaline picrate assays on the Abbott Aeroset, Bayer
of achieving these performance goals. Advia 1650, Beckman LX20, Olympus AU5200, and
Vitros 950 systems had mean positive biases of 12
Additionally, accuracy remains a major issue and mol/L (0.14 mg/dL), 18 mol/L (0.20 mg/dL), 5
impinges on achievable between-laboratory agreement. mol/L (0.06 mg/dL), 11 mol/L (0.12 mg/dL), and 9
Acceptable performance criteria (CLIA-88) for mol/L (0.10 mg/dL), respectively. Variation in bias was
measurement of creatinine required laboratories to be related more to instrument manufacturer than method
accurate to within 27 mol/L (0.3 mg/dL) or 15% of principle, suggesting a strong influence of calibration
the peer group mean, whichever is greater. Given recent differences on bias. Indeed, one manufacturer (Roche)
emphasis on the use of estimated glomerular filtration had achieved almost zero bias across a range of assay
rate (eGFR) based upon serum creatinine data, this target principles and analytical platforms. This echoes earlier
is too liberal. The more a method overestimates true CAP data demonstrating that systematic differences in
creatinine, the greater will be the underestimation of the calibration of serum creatinine assays account for
445
Creatinine
85% of the observed differences between laboratories
[73]. In 2003, the International Measurement Evaluation
Programme (IMEP)-17 survey of approximately 1000
laboratories in 35 countries demonstrated almost
universal overestimation of serum creatinine in a pool
with IDMS certified concentration of 75 mol/L (0.83
mg/dL). Of the 14 method groups, 13 demonstrated
mean positive bias compared to the reference value,
typically varying between 10% and 15%. (The exception
was the Roche enzymatic method.) A pool at
pathological concentration (169 mol/L, [1.86 mg/dL])
showed an approximately equivalent number of
laboratories with positive and negative bias (see
www.imep.ws for further information). Calibration
differences contribute not only to bias but also to poor
interlaboratory agreement. Proficiency studies
demonstrate that whilst between laboratory CVs of <3%
are achievable within method groups, overall between-
laboratory agreement across methods is much poorer.
Systematic variation between laboratories of
approximately 18 mol/L (0.2 mg/dL) is common
[44,70].
Clearly, worldwide standardization of creatinine
measurement is a crucial aim. Recently, standardized
serum matrix reference materials (SRM 967) with
known creatinine concentrations (71 mol/L [0.80
mg/dL] and 354 mol/L [4.00 mg/dL]) have been
prepared by the National Institute of Standards of
Technology (NIST) and submitted to the JCTLM for
inclusion in a list of standardized reference materials
[44]. It is anticipated that the use of this material, in
combination with GC-IDMS reference methodology,
will assist reagent manufacturers in achieving better
consensus between methods [74]. Although undoubtedly
desirable, it must be recognized that standardization is
only one arm of the problem. Standardization will not
solve the problem of different reactivity with
noncreatinine chromogens across different patient
samples, which can only be resolved by the use of highly
specific creatinine methods.
Acknowledgments
Data on current method usage was provided by Finlay
Mackenzie from the United Kingdom National External
Quality Assessment Scheme, Birmingham, UK.
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449
Creatinine

Table 1: Creatinine Methods Summary Table

Method 1: Jaffe; spectrophotometric, end point, quantitative

Principle of analysis: Creatinine + alkaline picrate Janovski complex (red)
Comments: Serum, plasma, diluted urine; described by Jaffe, 1886. Significant interference from noncreatinine
chromogens.
Method 2: Jaffe/Fullers earth; same as Method 1; creatinine isolated before analysis; can be removed with buffer or
picrate reagent added directly to creatinine adsorbent suspension
Principle of analysis: Same as Method 1
Comments: Serum, plasma, diluted urine; alternatively cation exchangers used as adsorbent. Interference from
noncreatinine chromogens reduced. No longer in routine use.
Method 3: Jaffe, kinetic; spectrophotometric, quantitative, kinetic analysis during early color formation
Principle of analysis: Same as Method 1
Comments: Serum, plasma, diluted urine; requires automated equipment for accurately timed and precise
absorbance measurements. Precise conditions vary between instruments. Interference from noncreatinine
chromogens reduced. This is currently the most popular method in diagnostic laboratories.
Method 4: Creatininase (creatinine amidohydrolase); enzymatic hydrolysis to creatine, which reacts in indicator reactions
monitored spectrophotometrically at 340 nm
Principle of analysis:
_
Creatinine + H2O creatinine amidohydrolase creatine
_
Creatine + ATP CK creatine phosphate + ADP
_
ADP + phosphoenolpyruvate pyruvate kinase ATP + pyruvate
_
Pyruvate + NADH + H+ LD NAD+ + lactate
(CK, creatine kinase; LD, lactate dehydrogenase)
Comments: Poor sensitivity and precision, not widely used.
Method 5: Creatininase (creatinine amidohydrolase) is coupled with creatinase, generating creatine and sarcosine
sequentially. A further reaction with sarcosine oxidase leads to the production of hydrogen peroxide, which can be
detected by several indicator reactions.
Principle of analysis:
_
Creatinine + H2O creatinine amidohydrolase creatine
_
Creatine + H2O creatinase sarcosine + urea
_
Sarcosine + O2 + H2O sarcosine oxidase formaldehyde + glycine + H2O2
_
Indicator (reduced) + H2O2 peroxidase indicator (oxidized) + 2H2O
Comments: Widely used specific method. Bilirubin interference remains a problem.
Method 6: Creatinine iminohydrolase; enzymatic hydrolysis of creatinine with formation of ammonia, which can be
quantitated spectrophotometrically or electrometrically
Principle of analysis:
_
Creatinine creatinine iminohydrolase N-methylhydantoin + NH3
NH3 measured by: GLDH reaction, ammonia electrode, or colorimetrically
Comments: Requires high enzyme purity, used in some dry-slide techniques.
Method 7: High-performance liquid chromatography (HPLC); cation-exchange or reversed-phase chromatographic
separation of creatinine from other compounds
Principle of analysis: Creatinine quantitated by Method 1 or absorption at 230 nm or by enzymatic assay
Comments: Highly specific and precise, not used in routine practice, possible reference method.
Method 8: Isotope-dilution mass spectrometry (IDMS)
Principle of analysis: Measurement by IDMS following either initial GC or HPLC separation. GC approaches
require prior clean-up step
Comments: Highly specific and precise measurement. Reference method proposed by JCTLM. HPLC-IDMS
approaches may be applicable to routine use in the future.
450
Creatinine
Appendix: Glomerular Filtration Rate, Creatinine
Clearance, and Estimated GFR
The GFR is widely accepted as the best overall measure
of kidney function, enabling a statement of the complex
functions of the kidney in a single numerical expression.
A decrease in GFR precedes kidney failure in all forms
of progressive disease. A number of methods are used to
measure the GFR; most involve the kidneys ability to
clear either an exogenous or endogenous marker. The
renal clearance of a substance is defined as the volume
of plasma from which the substance is completely
cleared by the kidneys per unit time.
Provided a substance S is in stable concentration in the
plasma, physiologically inert, freely filtered at the
glomerulus, and neither secreted, reabsorbed,
synthesized, nor metabolized by the kidney, then the
amount of that substance filtered at the glomerulus is
equal to the amount excreted in the urine (i.e., the
amount of S entering the kidney must exactly equal the
amount leaving it). The amount of S filtered at the
glomerulus = GFR multiplied by plasma S concentration:
GFR PS. The amount of S excreted equals the urine S
concentration (US) multiplied by the urinary flow rate
(V, volume excreted per unit time).
Since filtered S = excreted S
GFR PS = US V
GFR = (US V)/PS
where: GFR = clearance in units of milliliters of plasma
cleared of a substance per minute
US = urinary concentration of the substance
V = volumetric flow rate of urine in milliliters
per minute
PS = plasma concentration of the substance
The term (US V)/PS is defined as the clearance of
substance S and is an accurate estimate of GFR,
providing the aforementioned criteria are satisfied. Inulin
satisfies these criteria and has long been regarded as the
most accurate (gold standard) estimate of GFR. A
variety of other exogenous (radioisotopic and non-
radioisotopic) and endogenous markers have been used
to estimate clearance. Commonly, clearance estimations
are adjusted to an average body surface area (BSA) of
1.73 m2.
Creatinine Clearance
Because creatinine is endogenously produced, released
into body fluids at a constant rate, and freely filtered at
the glomerulus, its clearance can be measured as an
indicator of GFR. Creatinine clearance has in the past
been seen as more sensitive for detection of renal
dysfunction than serum creatinine measurement.
However, it requires a timed urine collection, which
introduces its own inaccuracies, and is inconvenient and
unpleasant. To do the test, one needs a precisely timed
urine collection and a blood sample taken during the
collection period. The test is initiated by having patients
empty the bladder at the beginning of the timed period.
Urine is collected throughout the period, the bladder is
again emptied at the end of the time period, and a blood
sample is obtained. Creatinine determinations are
performed on both samples, the urine volume measured
and the clearance calculated using the above formula.
In adults, the intra-individual day-to-day coefficient of
variation for repeated measures of creatinine clearance
exceeds 25% [75]. Although tubular secretion
undermines the theoretical value of creatinine as a
marker of GFR, in the context of creatinine clearance,
this has previously been offset to some extent by the use
of nonspecific Jaffe methods to measure serum
creatinine, which leads to an overestimation of serum
concentration (see above). Nevertheless, creatinine
clearance usually equals or exceeds inulin GFR in adults
by a factor of 10% to 40% at clearances above 80
mL/min. However, as GFR falls, tubular secretion of
creatinine rises proportionately, and the creatinine
clearance can reach nearly twice that of inulin [76].
Hence at best, creatinine clearance can only provide a
crude index of GFR.
Estimated GFR
The mathematical relationship between serum creatinine
and GFR can be improved by correcting for confounding
variables. Many equations have been derived which
estimate GFR using serum creatinine corrected for some
or all of gender, body size, race, and age. These may
produce a better estimate of GFR than serum creatinine
alone. The Cockcroft and Gault and Modification of Diet
in Renal Disease (MDRD) Study equations have been
widely used in adults. Two further equations, Schwartz
and Counahan-Barratt, are recommended for use in
children.
The Cockcroft and Gault equation, published in 1976, is
one of the earliest and most widely used of these
equations [77].
Creatinine clearance (mL/min) = [140 age (years)]
weight (kg)/(0.814 serum creatinine (mol/L)
(0.85 if female)
The observed correlation coefficient between their
predicted creatinine clearance and measured creatinine
clearance was 0.83 (R2 0.69), with predicted and mean
measured clearance values differing by 20% or less in
67% of patients.
The 6-variable (6-v) MDRD formula was published in
1999 following a retrospective re-analysis of the MDRD
Study [78]. GFR was determined in 1628 predominantly
middle-aged patients with known kidney disease, using
125
I-iothalamate and expressed per 1.73 m2 BSA.
Creatinine was measured in a central laboratory using a
kinetic Jaffe assay on a Beckman Astra CX3 analyzer.
Stepwise multiple regression modeling on log-
transformed data was used to determine the set of
451
Creatinine

variables that best predicted GFR. The inclusion of
serum urea and albumin concentrations was found to
improve the predictive strength of the model (R2 0.903
against 125I-iothalamate GFR).
GFR (mL/min/1.73 m2) = 170 [serum creatinine
(mol/L) 0.011312]-0.999 [age]-0.176 [0.762 if
patient is female] [1.180 if patient is black]
[serum urea (mmol/L) 2.801]-0.170 [serum albumin
(g/L) 0.1]+0.318
The lack of requirement of knowledge of body weight
was a practical advantage compared to the Cockcroft and
Gault equation, which also gave poorer accuracy in their
hands. The requirement for measurement of albumin and
urea in addition to creatinine was clearly a limitation of
the 6-v MDRD equation, increasing both analytical costs
and the contribution of analytical variation. Recognizing
this, these workers subsequently published (in 2000) a
simplified 4-variable (4-v) version of the formula, which
does not require these measurements and did not result
in appreciable loss of accuracy (R2 0.892 against 125I-
iothalamate GFR), with 90% of subjects having a GFR
estimate within 30% of the measured value [79].
GFR (mL/min/1.73 m2) = 186 [serum creatinine
(mol/L) 0.011312]-1.154 [age]-0.203 [1.212 if black]
[0.742 if female]
MDRD equation, the constant factor 186 is replaced with
the constant 175.
There are several clinical situations in which precise
knowledge of GFR is important, and where reliance on
equation-based estimates of GFR should be avoided.
These include cancer chemotherapy or the use of any
other drug that is renally excreted and has a narrow
therapeutic margin; the assessment of potential living,
related kidney donors; and the assessment of GFR in
patients with muscle-wasting disorders, including spina
bifida and paraplegia. When accurate GFR information
is required, reference methods should be used.
Serum creatinine is an imperfect marker of GFR, and
therefore it is not altogether surprising that formulae
based predominantly upon it are imperfect. Their use
cannot circumvent the very significant spectral
interferences affecting serum creatinine measurement,
and the formulae are critically susceptible to variations
in creatinine assay calibration and specificity. However,
despite these major limitations, the use of estimates of
GFR has been shown to improve the recognition of
patients with chronic kidney disease, compared to use of
serum creatinine alone. Internationally,
recommendations now generally favor use of the 4-v
MDRD formula for estimating GFR in adults.

The 4-v MDRD equation has several advantages

compared to the Cockcroft and Gault equation: It was
developed and validated in a large population; there is no
requirement for patient weight; the validation was
against an iothalamate clearance estimate of GFR, and
reported GFR is corrected for BSA. Unlike the
Cockcroft and Gault equation, the model also takes into
account the fact that the relationship between serum
creatinine and GFR differs between Caucasians and
African Americans. Since its publication, a series of
studies have compared the applicability of the Cockcroft
and Gault and 4-v MDRD equations in a variety of
clinical settings [80]. From these, a general conclusion
can be drawn: For the detection of patients with GFR
<60 mL/min/.73 m2, the MDRD equation provides a
more accurate and clinically acceptable assessment of
GFR compared to the Cockcroft and Gault equation.
Both equations demonstrate deteriorating performance as
GFR increases, and neither adequately estimates GFR in
patients with physiological-range GFRs.

Interlaboratory differences in creatinine measurement

have been discussed previously. These differences,
compounded by poor precision around the upper limit of
the reference interval, can have a significant impact on
the estimation of GFR using these equation-based
approaches. Further, the MDRD equation was anchored
to a nonspecific kinetic rate assay for creatinine on an
analytical platform that will not be available in
perpetuity. Recognizing this, the authors of the MDRD
study have published a re-analysis of their data, enabling
the MDRD formula to be anchored to an ID-MS
creatinine assay [81,82]. In this version of the 4-v
 452

CICLOSPORIN (CYCLOSPORINE A)
David W. Holt, Denise A. McKeown and Atholl Johnston

Name: rINN Ciclosporin, USAN Cyclosporine

Also known as: cyclosporin A, Neoral and Sandimmune
Clinical significance: Refer to Chapter 54, Laboratory Evaluation of the Transplant Recipient, in the
5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C62H111N11O12
Molecular mass: 1201.84 D
Merck Index: 2748
Chemical class: Cyclic peptide
CH3
|
CH
||
CH
|
CH3 CH2
| |
CH --- CH3 CH3 CH --- CH3 CH3
| | | |
CH2 CH3 CH --- CH3 CH3 CH --- HO CH2 CH3
| | | | | | |
CH3 --- N --- CH --- CO --- N ---------- CH --- C --- N --- CH --- CO --- N --- CH --- C --- N --- CH2
| L L || L | L || |
CH3 CO O H O CO
| | |
CH3 --- CH --- CH2 --- CH N --- CH3
|
CH3 --- N H O H
| D L | L || L |
OC --- CH ---------- N --- CO --- CH --- N --- CO --- CH --- N --- C --- CH --- N --- CO --- CH L
| | | | | | |
CH2 H CH3 CH3 --- CH2 CH3 CH --- CH3 CH2
| | | |
O CH3 CH3 CH --- CH3
| |
CH2 CH3
|
CH2 --- OH
Structure:

Principles of Analysis and Current Usage prescribed, including psoriasis, atopic dermatitis,
rheumatoid arthritis, nephrotic syndrome and Behets
Ciclosporin is a unique immunosuppressive agent disease.
isolated from Tolypocladium inflatum Gams (NRRL
8044). It is approved for the prevention of graft rejection Ciclosporin is metabolized extensively to more than 30
following kidney, liver, heart, combined heart-lung, metabolites [1,2], and the ratio of metabolite to parent
lung, or pancreas transplantation. It can also be used for drug in the circulation is often greater than 3:1. There is
the treatment of transplant rejection in patients who have little evidence that the metabolites possess any
previously received other immunosuppressive agents. significant pharmacological activity, so assay
Following bone marrow transplantation, it can be used to development has focused on the measurement of the
prevent graft rejection and for the prophylaxis of graft- parent compound. The choice of sample matrix has been
versus-host disease (GVHD). There are several determined by the partitioning of ciclosporin between
autoimmune indications for which ciclosporin can be plasma and red cells. The drug distributes extensively
into red cells such that plasma/serum concentrations are
only about one third of those in whole blood [3]. In
addition, the distribution into the red cell is temperature
i
Ciclosporin (Cyclosporine A) dependent [4]. Thus the combination of much lower drug
Previous and current authors of this method: concentrations in plasma/serum, and the need to separate
First edition: Not done samples at a fixed temperature to maintain consistency,
Methods edition: Larry D. Bowers has resulted in the use of whole blood as the matrix for
Second edition: Not updated therapeutic drug monitoring (TDM) of the drug [5].
Third edition: Leslie M. Shaw
Fourth edition: Leslie M. Shaw, Larry D. Bowers Essentially, two analytical techniques have been used to
Fifth edition: David W. Holt, Denise A. McKeown, measure the drug: immunoassays and high-performance
Atholl Johnston liquid chromatography (HPLC). During the early clinical
use of ciclosporin, TDM was performed using a
 453
polyclonal antibody-based radioimmunoassay kit
produced by the manufacturer of the drug (Sandoz
Pharma, Basel, Switzerland, now Novartis Pharma)[6].
This kit was withdrawn some time ago, as were its
successor monoclonal antibodybased
radioimmunoassays, one relatively specific for the parent
compound, the other with a broad range of cross-
reactivity for the parent compound and its metabolites
[7]. A number of commercial immunoassays have been
developed over the intervening 2 decades [8,9].
Immunoassays are very widely used to measure the drug,
partly because of their relative ease of use [10]. Over the
years, there has been some interest in the use of
immunoassays that cross-react with both ciclosporin and
its major metabolites. This is because some centers
wanted a measure of the high concentrations of
ciclosporin metabolites that can accumulate in some
clinical settings [11]. However, interest in nonspecific
measurements has declined substantially.
A popular assay for such nonspecific measurements has
been the polyclonal antibody fluorescence polarization
immunoassay produced by Abbott Diagnostics.
Immunoassays in current use are listed in Table 1. They
all have a relatively high specificity for the parent
compound, but their lack of total specificity for the
parent drug is well documented [12,13].
Until comparatively recently, the only chromatographic
technique available for the measurement of ciclosporin
was HPLC with ultraviolet (UV) detection. The
molecule contains no selective chromophores, but low
wavelengths can be used for detection. Although a wide
variety of reversed-phase HPLC-UV methods have been
developed [14-18], they have been largely superseded by
the introduction of assays based on mass-spectrometric
detection. This has brought both selectivity and
sensitivity to the measurement of the drug, and for the
first time, the possibility of attaining a gold standard
reference technique. These single (HPLC-MS) [19-26]
and tandem (HPLC-MS/MS) [27-38] mass-spectrometric
assays have been developed for the analysis of
ciclosporin alone, for the simultaneous, multi-analyte
analysis of the drug and its metabolites, and for
ciclosporin together with other immunosuppressive
drugs. Typical chromatographic assays currently in use
are summarized in Table 1.
Reference and Preferred Methods
There is no defined reference method for ciclosporin.
Consensus documents on ciclosporin monitoring have
advocated that a well-validated HPLC method should be
used for the selective measurement of ciclosporin ]5,39-
43]. The immunoassays in current use have all been
shown to correlate with HPLC results but almost
invariably produce a higher result. The bias associated
with immunoassays is very dependent on the specificity
of the antibody for the parent molecule. Within-subject
and between-subject, the proportion of cross-reacting
metabolites can vary, depending on such factors as
hepatic function and concomitant drug therapy.
Although it is possible to say that a particular
immunoassay has an average bias of, say, 10%
compared with HPLC, no universal correction factor can
be applied to an immunoassay result to take into account
interference due to ciclosporin metabolites. For clinical
samples from kidney transplant patients, immunoassay
results can have a bias of approximately 10% to 40%,
compared with a chromatographic assay. This bias can
be substantially higher for samples from patients with
hepatic dysfunction, since cross-reacting metabolites
tend to accumulate in the blood [7]. However, it must be
remembered that both immunoassay and
chromatographic techniques are prone to bias from
calibrator inaccuracy, whether the calibrators are
prepared in-house or supplied with commercial kits
[44,45]. Between-center differences might be minimized
if common calibrators or control material were used
more widely [46]. Calibrator and control material
carrying a CE mark (the symbol indicating that the
product conforms to a number of European Community
Directives on manufacturing standards) and U.S. Food
and Drug Administration (FDA) approval for tacrolimus
is already available, and similar material for ciclosporin
should be available in the near future [47]. A certified
reference material would be helpful, since it would allow
both those preparing in-house calibrators and diagnostics
companies preparing kit calibrators to have access to a
common material with a defined value.
Despite the lack of a selective chromophore, HPLC-UV
has the required sensitivity for the analysis of ciclosporin
[48,49]. However, HPLC-UV is susceptible to
interferences from the sample matrix, co-elution from
ciclosporin metabolites, and co-administered drugs and
their metabolites, resulting in laborious extraction
procedures and lengthy chromatographic run times [48-
50].
As a result there has been an increasing trend towards
the use of HPLC-MS and HPLC-MS/MS for the
measurement of ciclosporin and other
immunosuppressive drugs, largely because of the
availability of bench-top apparatus at competitive prices.
The increased specificity of these techniques has allowed
the use of less tedious extraction procedures and shorter
chromatographic run times [48,49]. There have also been
advances towards the automation of these techniques.
The trend away from the use of HPLC-UV towards the
use of mass-spectrometric techniques is reflected by
recent returns to the International Proficiency Testing
Scheme (www.bioanalytics.co.uk, accessed February 15,
2008), with only 4 centers reporting use of HPLC-UV,
but 66 centers reporting use of HPLC-MS(/MS).
However, immunoassay is still the predominant assay
technique, with 470 centers (87%) reporting results with
this methodology. Immunoassays require less technical
input to operate, can be automated, and provide fast
turnaround times. However, the advantages of
automation and rapid analysis times are now being seen
for HPLC-MS(/MS). There are recent reviews on the use
of HPLC-MS(/MS) for the analysis of
immunosuppressive drugs by McKeown et al. [50],
Taylor et al. [49], and Deters et al.[48].
 454
Specimen
The debate over which sample matrix to use for the
TDM of ciclosporin continued for some years after the
introduction of the drug. For the practical reasons
mentioned above, whole blood was adopted as the
matrix of choice. The introduction of the more sensitive
and selective mass-spectrometric assays has enabled
researchers to investigate alternative samples for
ciclosporin measurement. Assays validated for one
matrix should not be used for other matrices without at
least partial revalidation.
Keevil et al. [32] and Yonan et al. [51] have investigated
the use of fingerprick (capillary blood spot) samples
using sample volumes as low as 10 L. Mendonza et al.
[33] have investigated the use of saliva as the sample
matrix as a measure of unbound drug in the circulation.
The separation of bound and unbound fractions of
ciclosporin in plasma is complex. However, saliva
analysis avoids the need for this step; it is only the
unbound portion that diffuses into the salivary glands.
These alternative matrices are less invasive to collect
than venous blood and lend themselves to allowing
patients to collect their own samples outside the hospital
environment. The enhanced sensitivity associated with
mass-spectrometric techniques also allows the
measurement of the drug in individual subsets of cells
[52-54] to study the pharmacokinetic/pharmacodynamic
relationships of the drug at its target level.
Ciclosporin is relatively stable in whole blood, allowing
the shipment of samples at ambient temperatures. If
samples are to be analyzed within a week of receipt, they
should be kept refrigerated at approximately 4C. If they
are to be stored for longer periods before
analysis/reanalysis, they should be deep frozen at
approximately 20C. Deep frozen, the drug is stable for
at least 1 year and through 3 freeze/thaw cycles.
Interferences
A challenging problem associated with ciclosporin
measurement is interference from metabolites of the
drug, either the variable cross-reactivity with antibodies
used in immunoassays or interference in
chromatographic assays.
Some users tend to consider HPLC-MS(/MS) to have
absolute specificity for the analyte, which has given rise
to a dilute (protein precipitation) and shoot (limited
chromatography) approach to the analysis. However,
HPLC-MS(/MS) is prone to interferences from co-
eluting compounds, the sample matrix, and detector
cross-talk [55,56]. (With tandem mass spectrometers,
cross-talk occurs as a result of ions from the previous
scan events not being cleared from the collision cell
before the next scan event occurs. This is more of a
problem in multi-analyte methods in which ions of
similar masses are formed. Many modern mass
spectrometers are designed to reduce cross-talk, but it
may present problems in the use of the emerging ultra-
performance liquid chromatography [UPLC] systems,
which enable the analysis of many compounds in a
shorter time frame than traditional HPLC.) Vogeser et al.
[57] highlighted the interference that isobaric ions of
ciclosporin metabolites can have on the HPLC-MS/MS
analysis of the drug, utilizing the analogue ciclosporin D
as the internal standard. Analytical data from one patient
sample investigated in detail gave results of 277, 254,
and 65 g/L for analyses made by immunoassay and
HPLC-MS/MS with extended or limited
chromatography, respectively. The inaccurate result
obtained using a short chromatographic run time was
probably due to the co-elution of a ciclosporin
metabolite in the internal standard channel.
Co-eluting sample matrix components can affect the
efficiency of ionization of the analyte of interest, and
this can result in ion suppression or ion enhancement of
the signal [55,56,58]. These interferences can arise from
the sample of interest or a previous sample injection, or
from buildup of interfering compounds on the analytical
column. Ion suppression can also occur as a result of
additives in the mobile phase. Electrospray ionization
(ESI) is more prone to ion suppression than atmospheric
pressure chemical ionization (APCI).
Although the majority of matrix effects are observed in
the void volume of the chromatographic run, explaining
why problems can arise using the dilute-and-shoot
approach, they can also occur later in the
chromatographic run [56]. Matrix effects can be
minimized or eliminated by improving extraction
techniques and/or chromatographic conditions [55].
Taylor et al. [58] and McKeown et al. [50] have
demonstrated how ion suppression can be minimized if
drug-free whole blood is extracted using solid-phase
extraction (SPE) rather than by a simple protein
precipitation (PP) method. Matuszewski et al. [55]
provide a detailed account on how to assess for the
presence of matrix effects.
The use of internal standards that are analogs of the
analyte of interest labeled with stable isotopes can aid in
eliminating the problems of matrix effects, since both
compounds should be affected to the same extent [56].
Until recently, a deuterated analogue of ciclosporin was
not readily available. Salm et al. [36] described the
HPLC-MS/MS analysis of ciclosporin using ciclosporin-
D12 as the internal standard. As alternatives, authors
have used structural analogs of ciclosporin (e.g., D, B, or
G) and nonstructurally related compounds (e.g.,
ascomycin) as internal standards. Recently, Taylor et al.
[59] compared the use of the deuterated and D analogues
and ascomycin as internal standards for the analysis of
ciclosporin. The results were as expected; the deuterated
analog gave superior resultsas judged by the
comparison of linearity, between-day precision, and the
results from 104 whole-blood samples from solid organ
transplant recipients receiving ciclosporin therapy
followed by the D analogue and then ascomycin. Taylor
et al. also hypothesized that the negative bias reported
for the patient sample results, when using the D analog
as the internal standard, may have been due to
 455
interfering ciclosporin metabolite ions in the ciclosporin
D mass channel.
In an effort to promote consistency in the performance of
HPLC-MS(/MS) assays for the measurement of
ciclosporin, the manufacturer of the drug, Novartis
Pharma, has donated stocks of the deuterated analogue to
the International Association of Therapeutic Drug
Monitoring and Clinical Toxicology for distribution
amongst its members. Details on how to obtain the
standard can be found on the Associations website,
www.iatdmct.org.
Although HPLC-MS(/MS) methods are very sensitive
and have the potential to be highly selective, they still
need to be well validated and any pitfalls addressed
before they can be used reliably for patient management.
A useful scheme to follow for assay validation is that
described in the FDA bioanalytical methods validation
document
(http://www.fda.gov/cder/guidance/4252fnl.pdf,
accessed 2008-02-15). Viswanathan et al. [60], Peters et
al. [61], and Shah et al. [62,63] also provide good
guidance on the validation of bioanalytical methods.
Ciclosporin Therapeutic Range
Therapeutic drug monitoring is recommended for
ciclosporin because the drug has erratic
pharmacokinetics, it is difficult to distinguish its
therapeutic efficacy from symptoms and signs of
toxicity, there is the potential for pharmacokinetic drug
interactions with other prescription or nonprescription
drugs, and long-term, there are problems related to
patient compliance with the prescribed dose.
Most centers monitor the drug using a pre-dose, or
trough (C0), blood sample. For this approach to
monitoring, ciclosporin concentrations are of the order
100 to 400 g/L, depending on the indication for which
the drug is being used. However, it has been known for
some time that ciclosporin efficacy is related to exposure
during a dosing interval, based on the area under the
time-concentration curve (AUC), and that the trough
sample is not a good reflection of exposure [64]. For
logistical reasons, measurement of AUC is difficult,
even if based on a limited sampling approach rather than
on several samples collected across a dose interval [65].
For this reason, the measurement of AUC has seen
negligible uptake. Following the introduction of the
Neoral oral formulation, designed to reduce some of the
pharmacokinetic variability associated with absorption
[66], there was considerable interest in monitoring,
based on a sample collected 2 hours post-dose (C2)[67].
The reason for using this time point is that it is a good
approximation for AUC0-4, when most of the variability
in the 12-hour AUC occurs [68]. Although the uptake of
this monitoring strategy has not been widespread, there
is a substantial body of literature, including a meta-
analysis of results and consensus guidelines [69,70].
Measurement of ciclosporin in C2 samples has
methodological implications [71]. Concentrations are of
the order 500 to 1800 g/L, outside the calibration range
for most immunoassays, although two kit manufacturers
have produced an extended calibration range for their
assays. It is also worth noting that the relative
differences between several of the immunoassays are
smaller when C2 sample results are compared with C0
results, probably because the proportion of metabolites
in the 2-hour sample is smaller [72].
There are few reasons to measure ciclosporin in patients
receiving the drug for autoimmune indications. Doses
are generally lower than those used for transplant
indications, and there is often an easily observable
pharmacodynamic marker of efficacy [73-75]. In some
instances, it is necessary to ensure that the drug has been
absorbed and to rule out noncompliance if there is
therapeutic failure.
For compliance testing, it is important to have
established the lowest concentration that can be
measured reliably by an assay. The lower limit of
quantification (LLOQ) is the concentration of the lowest
calibrator, and concentrations measured below that
should be reported as less than the LLOQ value. Ideally,
a concentration of about 25 g/L should be measurable,
but this is not always possible with immunoassays or
HPLC-UV assays. It is prudent to assay a ciclosporin-
free blood sample periodically as a check on assay
sensitivity.
Most centers report results in g/L or ng/mL, since the
literature uses these units almost exclusively. For those
who wish to report in mol/L, then 1 g/L is equivalent
to 0.8315 mol/L.
The inherent differences between the results obtained by
the immunoassays (related to their cross-reactivity with
ciclosporin metabolites) and the generally lower values
obtained by chromatographic assays impacts on the
target concentration ranges for this drug. These
methodological differences have given rise to method-
related target ranges. Such differences must be kept in
mind if a center changes methodology or if clinicians
move between centers at which different methods are in
use.
Interpretation
The object of TDM is to ensure that there is sufficient
exposure to the drug to achieve therapeutic efficacy
while avoiding excessive exposure that might lead to
acute and chronic nephrotoxicity or other unwanted
effects. There are many publications in which
therapeutic concentration ranges have been suggested,
but by and large, the approach to establishing them has
been empirical rather than systematic [5,40]. There are
no randomized trials in which TDM is compared with
empirical dosing. The concept of a generally applicable
therapeutic range for this drug cannot be justified. There
are several factors that affect the interpretation of the
measured ciclosporin concentration, including the
transplant type, the time since transplantation, other drug
 456
therapy, the time of blood sampling relative to the last
dose, and the assay method employed [76,77].
Applying data on therapeutic concentration ranges that
have been generated over the almost 30-year history of
ciclosporin TDM is complicated by a number of changes
that have taken place over that period: concomitant drug
therapy, changes in the selection of patients and
transplanted organs, and advances in surgical techniques.
To avoid long-term toxicity, principally chronic allograft
nephropathy, there is a trend towards the use of lower
doses of the calcineurin inhibitors, both ciclosporin and
tacrolimus, by combining them with mycophenolic acid
and/or an mTOR inhibitorsirolimus or everolimus.
The introduction of induction therapy using monoclonal
anti-IL2 antibodies has also had an impact on ciclosporin
dosing and its target concentrations [78,79]. As a result,
lower target concentrations of ciclosporin have been
proposed, based either on empirical observation or by
analysis of data from concentration-controlled clinical
studies.
Given the caveats that can be placed on the interpretation
of any given measured concentration, it is not surprising
that centers often adopt an in-house set of ranges that are
based on their own experience, patient demographics,
combination of drug therapy, and assay methodology. As
was noted in a previous review [77], the last set of
guidelines, based on data from 21 centers monitoring the
drug in kidney transplant patients [40], there was a
sixfold range in the concentrations considered effective
and a threefold range in the concentrations considered
toxic. In general, higher concentrations are aimed for
early after transplantation, when the immunological risk
is highest. After a period of 3 to 6 months, there is
usually a gradual tapering of the dose, together with
more reliance on adjunctive therapy. It is also common
for method-specific concentration ranges to be used,
with allowance being made for the higher results
produced by methods with least specificity for the parent
compound.
For those centers new to this field, it is always worth
consulting recent literature on the interpretation of
ciclosporin concentrations, but one must always take
into consideration any trends in prescription or clinical
practice subsequent to these publications. Consulting
those already in the field, who are engaged in a similar
transplant setting, is probably of more value. It must be
remembered that the measured concentration of
ciclosporin is only one part of the dosing algorithm,
which must also take into account clinical and other
laboratory data.
Ciclosporin Performance Goals
Guidelines that have been developed for the monitoring
of therapeutic drugs indicate that inaccuracy and
imprecision of the analytical method used should not
exceed 10% [80]. For the assays currently available
(see below), this is not likely to be the case. For
immunoassays, it is not possible to achieve so close an
agreement with a chromatographic method for many
samples from transplant patients because of antibody
cross-reactivity with ciclosporin metabolites. Added to
this is the variability in calibration accuracy [45].
Bioanalysts should be aware of these assay differences
and strive to minimize them, but it is fair to say that
clinicians are often more concerned about the
consistency of the result rather than the absolute number.
This is because they may be looking at trends in
concentration within fairly broad limits and are anxious
to know that an assay is performing in the same manner
over a period of time. Between-method differences may
assume more importance if the assay method is changed,
if a patient visits more than one center at which different
assays are performed, or if different assays are used in
multicenter clinical studies.
Consistency of assay performance and comparability
with peers using the same method is best addressed by
participation in an external proficiency testing scheme
[81]. Data from these schemes can also be used to assess
assay performance, especially if pooled samples from
patients receiving ciclosporin are circulated. Choosing
which assay to use in a clinical setting will be heavily
influenced by the apparatus available, the technical
expertise available, and the sample throughput. When
reviewing published data on assay kits, remember that
changes in kit configuration may have occurred since
publication of the data. When comparing absolute
differences between assays, it is useful to check whether
common control material has been analyzed by each
assay. In addition, the analytical platform on which an
immunoassay was tested may differ from that in current
use, and the names of many diagnostics companies have
changed owing to mergers and acquisitions. In some
locations, diagnostic kits may only be available through
a local agent and may be rebadged under another name.
The Tables below show the immunoassays and
chromatographic assays currently in use to measure
ciclosporin. Typical data for the measurement of
ciclosporin in a blood sample spiked with the drug and
in a pooled sample from patients receiving the drug are
shown in Figure 1, using data available online from the
International Proficiency Testing Scheme
(www.bioanalytics.co.uk). It is clear that the between-
method differences are influenced by both calibration
and antibody specificity and that HPLC assays are
subject to some wide variations in performance. The
package inserts for each kit give details of assay
performance and cross-reactivity with ciclosporin
metabolites. However, bear in mind that in vitro tests of
cross-reactivity do not set out to investigate the various
combinations of ciclosporin metabolites that might be in
any sample and cannot reproduce every clinical situation
that could arise. All immunoassays have the potential to
produce higher-than-true values, especially in clinical
situations in which ciclosporin metabolites may be
relatively high, such as following liver and heart
transplantation, during hepatic dysfunction, and when
the kinetics of ciclosporin have been altered by
pharmacokinetic drug interactions.
 457
There are clear differences between the various methods
related to calibration and selectivity which must be taken
into consideration when a result is interpreted. In
general, the most selective assays are to be preferred, but
the final choice is likely to be governed by operational
constraints such as access to appropriate apparatus. All
the methods can be used to investigate the
pharmacokinetics of ciclosporin following chronic
doses, but preference should be given to the most
selective assays. For single-dose studies and for the
investigation of pharmacokinetic drug interactions, a
chromatographic assay with mass-spectrometric
detection is the method of choice.
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 461

Table 1: Methods for Ciclosporin (CSP) Measurement

IMMUNOASSAYS
1. Radioimmunoassay
CYCLO-Trac SP-Whole Blood - DiaSorin Inc.
Principle of analysis:
Competitive binding of 125I-CSP and nonlabeled CSP to a limited amount of monoclonal specific antibody, using
a methanolic extract of a whole blood sample. Double antibody phase separation technique. Sample size: 100 L
of whole blood collected into EDTA anticoagulant. Calibration range 0 to 1200 g/L.
Comments:
Problems related to the use and disposal of radioactive materials; some cross-reactivity with CSP metabolites.
2. Fluorescence Polarization Immunoassay (FPIA)
TDx Cyclosporine Monoclonal Whole Blood Abbott Laboratories
Principle of analysis:
A competitive immunoassay performed on the TDx platform, involving competitive binding of fluorescein-
labeled CSP and nonlabeled CSP to a limited amount of monoclonal specific antibody, using a whole blood
sample pretreated with a solubilization reagent and precipitation reagent. The polarization of fluorescent light is
inversely proportional to the concentration of CSP in the sample. Sample size: 150 L of whole blood
(anticoagulant not specified). Calibration range 0 to 1500 g/L
Comments:
Relatively high cross-reactivity with CSP metabolites.
3. Fluorescence Polarization Immunoassay (FPIA)
Cyclosporine AXSYM Abbott Laboratories
Principle of analysis:
A competitive immunoassay performed on the AXSYM platform, involving competitive binding of fluorescein-
labeled CSP and nonlabeled CSP to a limited amount of monoclonal specific antibody, using a whole blood
sample pre-treated with a solubilization reagent and precipitation reagent. The polarization of fluorescent light is
inversely proportional to the concentration of CSP in the sample. Sample size: 150 L of whole blood collected
into EDTA or heparin anticoagulant. Calibration range 0 to 800 g/L
Comments:
Some cross-reactivity with CSP metabolites.
4. Chemiluminescent Microparticle Immunoassay (CMIA)
ARCHITECT Cyclosporine Abbott Laboratories
(Available in Europe from March 2008)
Principle of analysis:
A two-step immunoassay performed on the Architect platform. Samples are solubilized and extracted with a
precipitation reagent. The sample and monoclonal antibodycoated paramagnetic microparticles are combined.
CSP binds to the antibody on the beads, which are washed, and a CSP acridinium conjugate is added to occupy
vacant CSP antibody sites. A trigger solution then initiates chemiluminescence. Sample size: 200 L of whole
blood collected into EDTA anticoagulant. Calibration range 0 to 1500 g/L
Comments:
Initial reports suggest relatively little cross-reactivity with CSP metabolites.
5. Enzyme Multiplied Immunoassay Technique (EMIT)
Emit 2000 Cyclosporine Specific Assay Dade Behring
Principle of analysis:
A homogeneous competitive immunoassay in which the drug competes with enzyme-labeled CSP for antibody
sites. Unbound enzyme reacts with nicotinamide adenine dinucleotide (NAD), converting it to NADH, which can
be measured spectrophotometrically. The sample requires pretreatment to solubilize the drug and precipitate
protein components before analysis. Sample size: 100 L of whole blood collected into EDTA anticoagulant. Two
calibration ranges available 0 to 500 g/L and 0 to 2000 g/L.
Comments:
Some cross-reactivity with metabolites. Several analytical platforms can be used.
6. Affinity Chrome-Mediated Immunoassay (ACMIA)
Cyclosporine Felx Reagent Cartridge CSA and CSAE
Dimension Clinical Chemistry System Dade Behring
Principle of analysis:
An automated immunoassay technique with no pretreatment step, performed with a method specific reagent
cartridge on the Dimension platform. The sample is mixed and lysed on the analytical platform then mixed with a
-galactosidase-CSP conjugate. Magnetic particles coated with CSP then bind unbound antibody-enzyme
conjugate, which are separated magnetically. -galactosidase is then used to catalyze the hydrolysis of a
chromophore, and the change in absorption measured. Samples must remain on the analytical platform for 30
minutes prior to analysis. Sample size: 200 L of whole blood collected into EDTA anticoagulant. Calibration
range 25 to 500 g/L (CSA), 350 to 2000 g/L (CSAE).
 462
CICLOSPORIN (CYCLOSPORINE A)

Comments:
Some cross-reactivity with metabolites. Performed on a random-access analyzer.
7. Cloned Enzyme Donor Immunoassay (CEDIA)
CEDIA Cyclosporine Plus Microgenics
Principle of analysis:
A homogeneous enzyme immunoassay which uses genetically modified -galactosidase as two inactive
fragments. Samples are lysed off the analytical platform, but no other pretreatment is required. CSP in the sample
competes for antibody with CSP conjugated to one enzyme fragment. Higher concentrations of CSP allow the
enzyme fragments to reassociate and form active enzyme, which cleaves a colored substrate. Sample size: 100 L
of whole blood collected into EDTA anticoagulant. Calibration range 25 to 450 g/L (CSA), 450 to 2000 g/L
(CSAE).
Comments:
Some cross-reactivity with metabolites. Several analytical platforms can be used.
8. Direct Chemiluminescence
Cyclosporine (CsA) Siemens Healthcare Diagnostics
Principle of analysis:
A competitive immunoassay using direct chemiluminescent technology, performed on the ADVIA Centaur and
ADVIA Centaur XP systems. Samples must be manually pretreated to lyse the cells. The labeled antibody binds
to streptavidin coupled to paramagnetic particles on a solid phase. Sample size: 100 L of whole blood collected
into EDTA anticoagulant. Calibration range 0 to 2000 g/L.
Comments:
Preliminary data suggest relatively low cross-reactivity with metabolites. Performed on a random access analyzer.

CHROMATOGRAPHIC ASSAYS
High-Performance Liquid Chromatography Techniques
a. Ultraviolet (HPLC-UV) detection at low wavelengths, 205 to 227nm.
b. Mass spectrometric (HPLC-MS) detection, typically by monitoring the stable sodium adduct [M+Na]+.
c. Tandem mass-spectrometric (HPLC-MS/MS) detection, typically by multiple reaction monitoring of the less
stable ammonium adduct [M+NH4]+ precursor ion to the [M+H]+ product ion.
Principle of analysis: Extraction of CSP from whole blood, using protein precipitation followed by a variety of
off-line and semi-automated on-line clean up techniques; chromatography typically on reversed-phase HPLC
systems; then detection by one of the above methods (a-c)
Comments: Well validated HPLC based techniques provide a selective measurement of CSP and its metabolites.
 463

Figures

Figure 1A

Figure 1B
Figure 1: Blood ciclosporin concentrations in A, a ciclosporin-free whole blood sample spiked to a nominal concentration
of 150 g/L and B, in a pooled whole blood sample from transplant patients receiving ciclosporin following bone marrow
transplantation. The data are presented as box and whisker plots, by analytical technique.
ACMIA, Affinity Chrome-Mediated Immunoassay; CEDIA, cloned-enzyme donor immunoassay; EMIT, enzyme-
multiplied immunoassay technique; FPIA TDx, fluorescence polarization immunoassay performed on the TDx platform;
FPIA AxSYM, fluorescence polarization immunoassay performed on the AxSYM platform; RIA CYCLO-Trac, radio-
immunoassay using CYCLO-Trac SP kit; HPLC/MS, high-performance liquid chromatography with MS or MS/MS
detection; HPLC/UV, high-performance liquid chromatography with UV detection. (Data from International Ciclosporin
Proficiency Testing Scheme [unpublished].)
 464

Figure 2: Typical HPLC-UV chromatograms of CSP. A, 250 g/L CSP standard (4.3 min) and 750 g/L CSP-D (5.6 min).
B & C, Chromatograms of whole blood extracts from transplant patients receiving the drug; whole blood CSP
concentrations were 79 g/L (B) and 298 g/L (C).
 465

Good chromatographic conditions

e.g. extended chromatography or rapid
No Further Extraction chromatography utilising ballistic gradients and
divertor valves

Manual LLE
e.g. in house, utilising 0.1M sodium hydroxide and
Liquid-Liquid methyl-tert-butyl ether or chlorobutane
Extraction
(LLE) Semi-automated 96-well plate LLE
e.g. utilising the Tomtec Quadra 96 workstation

Manual SPE
Protein
Precipitation
Semi-automated off-line SPE
e.g. utlising the Gilson ASPEC XL4

Solid Phase Extraction Semi-auotmated 96-well plate SPE

(SPE) e.g. utilising the Tomtec Quadra 96 workstation

On-line SPE (or LC/LC) utilising an additional

extraction column, additional HPLC pumps, and a
6-port column switching valve, prior to the analytical
column

Turbo Flow On-line TFC utilising a specially designed TFC

extraction column, additional HPLC pumps, and two
Chromatography 6-port column switching valve, prior to the analytical
(TFC) column

Figure 3: Schematic diagram of several sample preparation methods that have been used to extract ciclosporin from whole
blood.
 466

Figure 4: HPLC-MS/MS chromatograms of ciclosporin extracted from a whole-blood sample spiked at a concentration of
25 g/L (left-hand panels). The MRM transitions monitored were: A, m/z: 1203/100 for the [M+H]+ precursor
ion; B, m/z: 1201/1089 for the [M-H]- precursor ion, and C, m/z: 1220/1203 for the [M+NH4]+ precursor ion. Note
the increase in sensitivity when monitoring the [M+NH4]+ precursor ion compared with the [M+H]+ in positive
ionization mode. The comparable transitions for the ciclosporin-D12 internal standard are shown in the right-hand
panels.
 467
CICLOSPORIN (CYCLOSPORINE A)

Figure 5: Typical calibration curve used for the analysis of pre-dose (trough, C0) samples from patients receiving
ciclosporin therapy. Whole-blood calibrators over the concentration range 25 to 500 g/L were extracted, and the
concentration was plotted against the ratio of peak area for the drug and the internal standard (ciclosporin-D12).

Procedure: High-Performance Liquid Internal Standards: Methods have used structural

Chromatography
HPLC-UV
Sample Preparation
Whole blood samples are typically protein precipitated
using reagent mixtures such as aqueous 10% zinc
sulphate solution (ZnSO4):acetonitrile:methanol
(50:20:30, v/v/v),[15] or 5% ZnSO4 and acetone [18].
The supernatant is then liquid-liquid extracted (e.g.,
diethyl ether then partitioned twice with hexane [15] or
undergoes SPE (e.g., Bond-Elut cartridge, C18, 200 mg,
3 mL). Sample volumes up to 1 mL can be required to
achieve lower limits of quantitation (LLOQ) of 20 g/L
[18].
Chromatography and Detector Conditions
Mobile Phase: Typically, mixtures of acetonitrile-
deionized water, acetonitrile-methanol-deionized water,
or acetonitrile-methanol-phosphate buffer [15,16,18].
Analytical Column: Beckman Ultrasphere C8 (75
4.6mm, 3 m) or a Hypersil ODS C18 (100 4.6mm, 5
m) are examples of the types of reverse-phase columns
that have been utilized for ciclosporin analysis [17,18].
Column Oven: Elevated temperatures of 60C [18] or
70C [15] tend to be used to obtain acceptable peak
shapes. Investigations have shown that the peak
broadening observed during the chromatographic
analysis of ciclosporin is attributable to the presence of
rapidly interconverting conformers of the ciclosporin
molecule [82].
Run Times: Chromatographic analysis can take up to 18
min for ciclosporin [16], and 36 min [18] and 40 min
[15] for the multi-analyte analysis of ciclosporin and
everolimus, or ciclosporin and two of its metabolites,
respectively.
analogues such as ciclosporin D and various structurally
unrelated compounds [15,18].
UV Detector Wavelength: Low wavelengths ranging
from 205 nm to 227 nm [15,17].
Figure 2 shows an HPLC-UV chromatogram for
ciclosporin, using the D analogue as the internal
standard.
HPLC-MS(/MS)
The following section summarizes HPLC-MS and
HPLC-MS/MS method conditions that can be used for
the analysis of ciclosporin and for the simultaneous
multi-analyte analysis of ciclosporin with other
immunosuppressive drugssirolimus (SIR), everolimus
(EVR), tacrolimus (TAC) and mycophenolic acid
(MPA).
Sample Preparation
Whole blood can be protein precipitated using the
successive addition of reagents or the addition of
premixed reagents (83). Methanol:0.4M ZnSO4 (4:1,
v/v) [19] and acetonitrile:0.1M ZnSO4 (70:30, v/v) [36]
are examples of the premixed reagents utilized.
Annesley et al. [83] reported cleaner extracts with the
successive addition of deionized water (water
hemolysis), followed by 0.1 M ZnSO4, then methanol, in
comparison to premixed reagents.
Following protein precipitation, the extracts are either
analyzed by HPLC-MS(/MS), or further off-line or on-
line sample cleanup procedures can be utilized prior to
HPLC-MS(/MS) analysis (see Figure 3).
Sample volumes as low as 100 L have been used to
achieve a LLOQ of 7.5 g/L for HPLC-MS [19]. Keevil
et al. [31] reported a HPLC-MS/MS method for
 468
ciclosporin that was linear over a wide analytical range
(1 to 5000 g/L), using only 100 L of sample.
Chromatography Conditions
Mobile Phase:
HPLC-MS: Typically methanol-deionized water [21] or
acetonitrile-deionized water [25] systems, sometimes
with the addition of formic acid [19] or acetic acid [24].
Both isocratic and gradient elution systems are utilized.
HPLC-MS/MS: Typically methanol-deionized water or
acetonitrile-deionized water and may be supplemented
with ammonium acetate [29,36] or ammonium formate
[38] (important for tandem mass spectrometric analysis,
see below). Both isocratic and gradient elution systems
are utilized.
Analytical Column: Various reverse-phase HPLC
columns have been used, including C8, C18, phenyl-
hexyl [19,34,36].
Column Oven: Elevated temperatures of 65C [38] or
70C [36] are often still used. However, the specificity
of mass spectrometry allows rapid isocratic and gradient
elutions compared to HPLC-UV, which aids in
improving the broad ciclosporin peak shape.
Run Times: Simultaneous HPLC-MS and HPLC-MS/MS
analysis of ciclosporin, SIR, EVR and TAC can be
achieved in less than 10 min [19] and 3 min [35],
respectively. Recently, Salm et al. [36] reported a total
analysis time of 2 min for the HPLC-MS/MS analysis of
ciclosporin.
Internal Standards: The stable isotope ciclosporin-D12
[20,36], structural analogues such as ciclosporin D
[19,35], and various structurally unrelated compounds,
such as ascomycin.
Mass Spectrometry
Ion Source: Electrospray (ESI) or atmospheric pressure
chemical ionization (APCI), operated in positive or
negative ionization mode.
Principle of HPLC-MS: Following chromatography, the
molecules enter the mass spectrometer, where they are
ionized and detected according to their mass-to-charge
ratio (m/z). Various ions are formed; [M-H]- m/z: 1201,
[M+H]+ m/z: 1203, [M+Na]+ m/z: 1225 and [M+K]+ m/z:
1241. However, ciclosporin (m/z: 1225) and the D
analogue (m/z: 1239) are typically detected by the single
ion monitoring (SIM) of their stable sodium adducts
[M+Na]+ [48].
Principle of HPLC-MS/MS: Following chromatography,
the molecules enter the mass spectrometer, where they
are ionized (precursor ion), filtered according to their
m/z, and undergo fragmentation using a collision gas
(e.g., Ar, N2 or He) to produce product ions, which are
again filtered according to their m/z. Analytes are
detected by monitoring the precursor-to-product-ion
transitions in multiple reaction monitoring (MRM) mode
[48].
Ciclosporin can gain [M+H]+ or lose [M-H]- a proton
when analyzed in positive or negative ionization modes,
respectively. MRM transitions monitored in positive
ionization mode are m/z: 1203/425,100 (ciclosporin),
m/z: 1217/425 (ciclosporin D) and m/z: 1215/437,100
(ciclosporin-D12), for ciclosporin and its internal
standards. In negative ionization mode, the following
MRM transitions are typically monitored for ciclosporin
m/z: 1201/1089, ciclosporin D m/z: 1215/907, and
ciclosporin-D12 m/z: 1213/1101. Typical
chromatograms for the analysis of an extracted
ciclosporin calibrator together with ciclosporin-D12 are
shown in Figure 4.
For reasons related to assay sensitivity (see Figure 4),
the less stable ammonium adduct precursor ion
[M+NH4]+, which fragments back to the [M+H]+ ion, is
typically monitored for ciclosporin (m/z: 1220/1203),
ciclosporin D (1234/1217), and ciclosporin-D12
(1232/1215) [48]. The mobile phase must be
supplemented with ammonium ions to form this adduct.
Analyzers: Typically, single quadrupole and triple
quadrupole analyzers are used for the measurement of
ciclosporin in clinical samples. However, any suitable
analyzer can be used.
Ciclosporin Calibrator and Quality-Control Sample
Preparation
In order to determine the concentration of whole blood
samples from patients receiving ciclosporin therapy, it is
necessary to construct a calibration curve. A sufficient
number of whole blood calibrators (Cals) (n 6),
analyzed in duplicate, must be used to cover the
expected concentration range. For the TDM of
ciclosporin C0 and C2 samples, a range of 25 to 2000
g/L should be suitable. Two ciclosporin-free samples
should be analyzed, one with internal standard (zero
sample) and one without internal standard (blank
sample); neither should be included when fitting the
calibration curve.
(http://www.fda.gov/cder/guidance/4252fnl.pdf,
accessed February 15, 2008)
Quality-control (QC) samples (n 3) that cover the
entire calibration range should be run in duplicate, with a
specified frequency, depending on the size of the
analytical run, to determine accuracy and precision. The
low QC should be 3 times the concentration of the
LLOQ, the mid QC approximately midway between the
low and high QC, and the high-range QC near the upper
end of the concentration range.
(http://www.fda.gov/cder/guidance/4252fnl.pdf,
accessed February 15, 2008)
The purity, storage, and expiry/retest dates of the
ciclosporin reference material used to prepare the Cals
and QCs should be known. If possible, all critical
analytical measurements should be made from
equipment which is traceable to a national/international
standard (e.g., glassware, calibration check weights for
balances, etc.).
Preparation of Ciclosporin Calibrator and Control
Stock Solutions:
Prepare two separate stock solutions for the Cals and
QCs by dissolving 10 mg ciclosporin in 10 mL of
methanol. Remember to correct for purity.
Preparation of Ciclosporin Whole-Blood Calibrators:
Pipette 100 L of the Cal stock solution into a 10-mL
volumetric flask, and make up to the mark with
ciclosporin-free human blood to produce a 10 g/mL Cal
sub-stock (CSS). The percentage solvent content should
always be 5% when spiking biological matrices.
Prepare working calibrators by diluting the CSS with
human blood as follows:
 469
CICLOSPORIN (CYCLOSPORINE A)

Cal Concentration
Calculations
Vol of CSS (mL) Total Vol Blood (mL) (g/L) Calibrators:
0.000 10 0 The peak area ratio (PAR) for ciclosporin is
0.025 10 25 calculated as follows:
0.050 10 50 Peak area of calibrator
0.100 10 100 Peak area of internal standard
A calibration curve is generated by using the simplest
0.250 10 250
regression model that adequately describes the
0.500 10 500 concentration-response (PAR) relationship.
1.000 10 1000 (http://www.fda.gov/cder/guidance/4252fnl.pdf,
1.500 10 1500 accessed February 15, 2008) For a calibration curve to
2.000 10 2000 be considered acceptable, the back-calculated
concentrations should be within 15% of the expected
Preparation of Ciclosporin Whole-Blood Quality value for all non-zero calibrators, except the LLOQ
Controls: (20%). At least 75% of the calibrators must fall within
Pipette 100 L of the QC stock solution into a 10-mL these criteria, including the LLOQ and the upper limit of
volumetric flask, and make up to the mark with quantification (ULOQ). Figure 5 shows a typical
ciclosporin-free human blood to produce a 10 g/mL QC calibration curve for ciclosporin.
sub-stock (QCSS). The percentage solvent content Quality Controls and Patient Samples:
should always be 5% when spiking biological The concentrations for the QCs and patient samples are
matrices. calculated using the calibration regression model. For an
Prepare working control samples by diluting the QCSS analytical run to be considered acceptable, at least 67%
with human blood as follows: of the QCs must be within 15% of their expected
concentrations
Vol of QCSS (mL) Total Vol Blood (mL) QC Concentration (g/L)
0.075 10 75 (http://www.fda.gov/cder/guidance/4252fnl.pdf,
0.750 10 750 accessed February 15, 2008).
1.750 10 1750
470

D-Xylose

D-Xylose
Michael D.D. McNeely

Name: D-Xylose
Clinical significance: Refer to Chapter 34, The Pancreas: Function and Chemical Pathology, in the
5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C5H10O5
Molecular mass: 150.13
Merck Index: 9897
Chemical class: Carbohydrate
Structure: click here

Other methods include the measurement of serum-

Principles of Analysis and Current Usage
Three general analytical approaches have been used for
the measurement of d-xylose: reaction with ortho-
toluidine,[1] para-bromoaniline,[2] and
phloroglucinol.[3]
In hot (100 C) glacial acetic acid, o-toluidine reacts
with xylose and other 5- and 6-carbon sugars to form
colored aromatic complexes with different maximal
absorbances (method 1, D-Xylose Methods Summary
Table).
Using a two-wavelength technique, one can subtract the
absorbance resulting from the reaction of other sugars to
assess the amount of color produced by xylose.
Measurements are made at 480 and 630 nm (D-Xylose
Figure 1). Borate is added to form a complex with
hydroxyl groups and allow greater reactivity and
absorbance. The method of Goodwin is the preferred
version.[4] The advantage of this approach is that it can
be performed directly on serum, plasma, and urine. The
reaction time is only 10 min.
The phloroglucinol method, as described by Eberts,[3] is
based upon Tollens test[5] for pentoses. It relies on the
reaction of furfural, which is produced by the acid-
catalyzed dehydration of xylose, with the phloroglucinol
to produce a colored complex (method 2, D-Xylose
Methods Summary Table). The reaction is monitored at
554 nm. This method is less susceptible to glucose
interference than the other two, requires a single reagent
without protein precipitation, requires a 4-min
incubation step, and does not require dual-wavelength
measurements.
The most commonly used method to measure d-xylose is
the p-bromoaniline procedure (method 3, D-Xylose
Methods Summary Table). The basis of the method is
dehydration of d-xylose (a pentose) to furfural, which
then combines with p-bromoaniline to produce a pink
chromogen in the presence of a thiourea antioxidant.[2]
The reaction conditions are mild in order to prevent the
formation of furfurals from other carbohydrates,
glucuronic acid, and ascorbic acid. The reaction is
monitored at 520 nm.
reducing substances with alkaline ferricyanide,[6] breath
testing with 14C-labeled xylose,[7] an enzyme method
using d-xylose isomerase and d-glucitol
dehydrogenase,[8] and various procedures that use acids
to dehydrate pentoses to furfurals, which are determined
by reaction with reagents such as aniline,[9] orcinol
ferric chloride (Bials reagents),[10] benzidine,[11] or
xylidine.[12] These approaches have no advantage over
the three described above, and they are rarely used.
Reference and Preferred Methods
The o-toluidine method was very convenient when
ortho-toluidine was a common manual reagent for
glucose. On the other hand, o-toluidine reagent is a
noxious, possibly carcinogenic, and corrosive material
that is difficult to pipet accurately. When plasma glucose
concentrations are greater than 3 g/L, the absorbance of
the reaction solution becomes impossible to read
accurately. The method is susceptible to interference
from other pentoses. Hexoses other than glucose may
inaccurately elevate or depress the results by altering the
A480/A630 ratio. Fortunately, the plasma and urine
concentrations of sugars other than glucose is very low
and quite stable under conditions of the clinical test
protocol.
The p-bromoaniline analytical approach is
recommended. In our experience, the p-bromoaniline
method is the most consistent assay, particularly if
performed only occasionally. Glucose and other sugars
do not interfere, except at very high levels. It is
necessary to perform a protein precipitation on serum
and plasma. Nevertheless, d-xylose analyses do not
require a rapid turnaround time, and the relatively long
incubation time (70 C and room temperature, for a total
of 80 min) is seldom a constraining feature. A definitely
negative feature of this method is that it uses a reagent
(thiourea) that is considered to be a carcinogen in
animals.[13]
The phloroglucinol method should also be considered as
a recommended alternative method. Its short reaction
time and use of nontoxic reagents are attractive features.
A comparison of this procedure to a highly specific gas-
chromatographic (GC) procedure[14] showed an
471
D-Xylose
excellent correlation for both serum (r = 0.98) and urine
(r = 0.99). The slopes of the regression lines comparing
the results of the phloroglucinol assay to the results of
the GC procedure were both 1. The phloroglucinol
method did have a slight positive bias compared to the
GC method at lower concentrations of d-xylose. A
comparison of the reactions listed in d-Xylose Methods
Summary Table is found in d-Xylose Table: Comparison
of reaction conditions.
Specimen and Patient Protocol
The patient is instructed to fast overnight but is
encouraged to drink an ample amount of water. At the
start of the test, the patients bladder is emptied, and then
a blood specimen is drawn into a fluoridated or
iodoacetate phlebotomy tube. Twenty-five grams of d-
xylose, dissolved in 150 to 200 mL of hot tap water and
cooled with 300 to 350 mL cold tap water, is ingested by
the patient (0.5 g per kilogram of body weight to a
maximum of 25 g is given to smaller subjects). All urine
passed over the next 5 h is collected in a single
container. Urine specimens are stable for 48 h at 4 C
and for many weeks if frozen. Blood specimens may be
drawn at 1 and 2 h.
Fluoridated plasma samples are preferred over serum,
but both can be used. The samples, if separated within 2
h, are stable at least 2 weeks in the refrigerator and for
many months when frozen. Heparin anticoagulant and
iodoacetate preservative are also acceptable.
d-Xylose Reference Interval
Adults given the full 25 g dose will excrete at least 25%
over the next 5 h if renal function is normal. One-hour
and 2-h plasma levels usually rise above 300 mg/L.
Children should excrete 25% of the dose into the urine,
but the plasma levels may be slightly lower (80 to 280
mg/L).
With a 5 g dose, the 1-h plasma concentration should be
above 200 mg/L in young children. In adults and
cooperative children, a 5-h urine collection is best. An
excretion of less than 30% of the oral dose is considered
abnormal.
Interpretation
The assessment of d-xylose absorption is used as a
general test of the small intestines ability to absorb
nutrients. d-Xylose is a dextrorotatory aldopentose,
which is known to be absorbed at a much slower rate
than glucose.[15] Helmer and Fouts in 1937[16]
observed that two patients with gastrointestinal disease
absorbed xylose at a slower than expected rate. This
started the investigations that ultimately led to the
verification of the test as an indicator of gastrointestinal
disease.
We now know that d-xylose is absorbed most efficiently
in the jejunum. Since there is no active renal tubular
reabsorption, the sugar is excreted rapidly into the urine.
It has been demonstrated that d- and l-xylose are
absorbed at different rates. Thus some factor other than
passive diffusion must play a part. A weak carrier
mechanism has been postulated.
Of an ingested dose, some 30% to 40% is excreted in the
urine. After an intravenous dose of d-xylose,
approximately 40% appears in the urine. Up to 20%
undergoes cellular metabolism, since radioactive
remnants can be detected in the expired breath as CO2.
This leaves about 40%, which either is not absorbed or is
metabolized by some unknown pathway. Studies have
demonstrated that it is not stored as glycogen.
Presumably, the remaining amount is metabolized
(possibly in the liver).[17]
Whatever its fate, the essential fact is that d-xylose is
primarily absorbed by a passive mechanism that is
largely dependent on the general integrity of the
gastrointestinal mucosa. Once absorbed, a large fraction
of it is excreted rapidly into the urine.
This phenomenon is the basis of the d-xylose absorption
test in which the urinary excretion of d-xylose after an
oral load is directly correlated with the passive
absorptive capacity of the small intestine.
Variations in the clinical test have centered on the dose
of d-xylose, the timing of specimen collection, the
choice of blood or urine, and the analytical technique for
d-xylose quantitation.
Dose
The standard dose of d-xylose has been 25 g.
This amount is considered too large by some workers
who believe that it causes an osmotic assault on the
small intestinal lumen, which then reduces sugar
absorption. To counter this criticism, a 5 g dose has been
recommended. However, the smaller doses have also
been criticized because of the observation that up to 10 g
of d-xylose can be metabolized by intestinal bacteria.
This claim has been countered by Kendall, who
demonstrated that there is little effect on D-xylose from
Klebsiella, Enterobacter aerogenes, Proteus mirabilis,
and Streptococcus fecalis.[18]
d-Xylose is relatively expensive ($0.40/g), and
therefore the use of a 5 g rather than a 25 g dose will
result in a saving of $8.00 per test.
Prolonged administration of d-xylose to rats has
been shown to be carcinogenic. However, specially
formulated d-xylose has recently been approved for use
in this test (Xylo-Pfan, Adria Laboratories, Inc., P.O.
Box 16529, Columbus, OH 43215).
Timing
After its absorption, the blood concentration of
d-xylose peaks at 2 h in adults and 1 h in children.[19]
However, this is a highly variable phenomenon, and
therefore the collection of a 5-h, postingestion urine is
advocated. The administration of a 5 g dose permits the
use of a 2-h urine collection.
A test relying solely on a 1-h blood sample was
first reported by Rolles in 1973 and confirmed by
others.[20,21]
472
D-Xylose
The 1-h approach has been criticized by
Lamabadusuriya,[22] Christie,[23] and Liebman,[24]
who cited a sensitivity of 39% to 95% and a specificity
of 24% to 78%. Kraut and Lloyd-Still[25] used a 5 g
dose and a 60-min collection and compared their results
with those of intestinal biopsy. They obtained a
sensitivity of 68% to 96%, depending on the category of
the disease, and a specificity of 100%.
Santini and Sheehy[26] used a 5 g dose with a
5-h urine collection in a study of 125 normal subjects.
All the subjects excreted more than 30% of the ingested
dose, whereas 60 patients with untreated tropical sprue
excreted 5% to 15%, and 36 patients with tropical sprue
excreted 14% to 30% of the ingested dose.
Hill and Cherian showed that the specificity of
the test was 95% and the sensitivity was 70% for celiac
disease and 25% for nonceliac gastrointestinal disorders.
All factors considered, the use of a 25 g dose in
combination with a 5-h urine collection is recommended.
For routine use, a blood sample is not necessary.
Low serum levels of d-xylose or low excretion
rates are seen in a wide variety of conditions affecting
the jejunum. Impaired absorption indicates decreased
mucosal absorption ability. In addition, low levels are
associated with ascites (where much of the dose may be
retained in the abdominal cavity), vomiting, delayed
gastric emptying, improper urine collection, high-dose
aspirin therapy, neomycin, colchicine, indomethacin, and
atropine. Impaired renal function will cause a dramatic
reduction in xylose excretion.
References
1 Goodwin JF. Method for simultaneous direct
estimation of glucose and xylose in serum. Clin
Chem 1970;16:85-91.
2 Roe JH, Rice EW Photometric method for
determination of free pentoses in animal tissues.
J Biol Chem 1948;173:507-12.
3 Eberts TJ, Sample RHB, Glick MR, Ellis GH.
A simplified colorimetric micromethod for
xylose in serum or urine with phloroglucinol.
Clin Chem 1979;25:1440-3.
4 Goodwin JF. Method for simultaneous direct
estimation of glucose and xylose in serum. Clin
Chem 1970;16:85-91.
5 Oshima K, Tollens B. Ueber Spectral-
reactionen des Methylfurfurols. Ber Dtsch
Chem Ges 1901;34:1425.
6 Haeney MR, Culank LS, Montgomery RD,
Sammons HG. Evaluation of xylose absorption
as measured in blood and urine: a one-hour
blood xylose screening test in malabsorption.
Gastroenterology 1978;75: 393-400.
7 Roberts RK, Campbell CB, Bryant SJ, Adames
L. Xylose-1-14C absorption test: the use of
urine, serum and breath analysis, and
comparison with a colorimetric assay. Aust NZ
J Med 1976;6:532-6.
8 Kersters-Hilderson H, Van Doorslaer E, De
Bruyne CK, Yamanaka K. Quantitative
determination of d-xylose by a coupled reaction
of d-xylose isomerase with d-glucitol
dehydrogenase. Anal Biochem 1977;80:41-50.
9 Goodwin JF. Micromethod for measuring
pentoses by use of an aniline reagent. Clin
Chem 1971;17:397-9.
10 Rozental M, Tomaszewski L. A new simple
ultramicromethod for the determination of d-
xylose in blood and urine. Clin Chem Acta
1974;50:311-7.
11 Tauber H. Color test for pentoses. Proc Soc Exp
Med Biol 1937;37:600-1.
12 Suminokura K. Colorimetric method for
determination of pentoses. J Biochem
1931;14:343-59.
13 Department of Health and Human Services,
USPHS, Centers for Disease Control, National
Institute for Occupational Safety and Health.
Registry of toxic effects of chemical substances.
Vol. 11 981-1982.
14 Johnson SL, Bliss M, Mayersohn M, Conrad
KA. Phloroglucinol-based colorimetry of xylose
in plasma and urine compared with a specific
gas-chromatographic procedure. Clin Chem
1984;30:1571-4.
15 Cori CF. Fate of sugar in animal body; rate of
absorption of hexoses and pentoses from
intestinal tract. J Biol Chem 1925;66:691-715.
16 Helmer OM., Fouts PJ. Gastrointestinal studies:
excretion of xylose in pernicious anemia. J Clin
Invest 1937;16:343-9.
17 Hiszczynskyj R, Keitges PW. D-Xylose
absorption test. Advanced Clinical Chemistry
No. ACC-3 (1972), American Society of
Clinical Pathologists, Commission on
Continuing Education, Check sample program,
1973.
18 Kendall MJ, Nutter S, Hawkins CF. Bacteria
and the xylose test. Lancet 1972;1:1017-8.
19 Rolles CJ, Nutter S, Kendall MJ, Anderson CM.
One-hour blood-xylose screening-test for
coeliac disease in infants and young children.
Lancet 1973;2:1043-5.
20 Schaad U, Gaze H, Hadorn B. Value of 1-hour
blood-xylose test in diagnosis of childhood
coeliac disease. Arch Dis Child 1978;53:420-2.
21 Buts JP, Morin CL, Roy CC, Weber A, Bonin
A. One-hour blood xylose test: a reliable index
of small bowel function. J Pediatr 1978;92:729-
33.
22 Lamabadusuriya SP, Packer S, Harries JT.
Limitations of xylose tolerance test as a
screening procedure in childhood coeliac
disease. Arch Dis Child 1975;50:34-9.
23 Christie DL. Use of the one-hour blood xylose
test as an indicator of small bowel mucosal
disease. J Pediatr 1978;92:725-8.
24 Liebman WM. Xylose test in malabsorption. J
Pediatr 1979;94:508-9
25 Kraut JR, Lloyd-Still JD. The 1-hour xylose test
in the evaluation of malabsorption in infants
and children. Am J Clin Nutr 1980;33:2328-33.
473

D-Xylose

26 Santini R, Sheehy TW, Martnez-de Jess J. Gastroenterology 1961;40:772-4.

The xylose tolerance test with a five-gram dose.
474

D-Xylose

Tables

D-Xylose Methods Summary Table

Method 1: o-Toluidine
Principle of analysis: o-Toluidine reacts with pentoses to form products with different absorbance maxima;
differential spectrophotometry is carried out
Comments: Serum, plasma, urine; 10-min incubation at 100 C, no precipitation step; glucose may interfere at
high concentration
Method 2: Phloroglucinol
Principle of analysis: Acetic and HCl acid produce furfural, which reacts with phloroglucinol to produce a
colored compound with absorbance at 554 nm
Comments: Serum, plasma, urine; 10-min incubation; single reagent; no protein precipitation step
Method 3: p-Bromoaniline
Principle of analysis: p-Bromoaniline acetate and thiourea antioxidant react with furfural to develop a pink
color, monitored at 520 nm
Comments: Serum, plasma, urine; standard method; protein precipitation step and 70-min incubation

Comparison of Reaction Conditions for d-Xylose

Condition p-Bromoaniline o-Toluidine Phloroglucinol
Temperature 70 C; 100 C 100 C
ambient temperature
Sample volume Plasma, 1 mL Serum, 0.1 mL Serum, 50 L
Urine, 1 mL Urine, 5 L
Sample pretreatment Deproteinization None None
of serum
Fraction of sample 0.167 0.0196 Serum, 0.0099
volume Urine, 0.001
Reagent p-Bromoaniline o-Toluidine Phloroglucinol
Reaction time 80 min 13 min 4 min
Linearity 13.32 mmol/L 13.2 mmol/L
Precision 4.4% urine Coefficient of variation
7.4% blood 3.0% to 5.2%
Interferences Substances other Bilirubin Glucose
than xylose that Hemolyzed serum Ribose
produce furfurals Glucose Arabinose
Methylxanthines
475

D-Xylose

Figures

D-Xylose Figure 1

Absorbance spectra of ortho-toluidine reaction with D-xylose, dashed curve, and D-glucose, solid curve (each at 1 g/L).
Absorbance versus reagent blank.

D-Xylose Figure 2

Absorbance spectra of p-bromoaniline reaction with D-xylose (100 mg/L). Reagent blank versus water, dashed curve; D-
xylose versus reagent blank, solid curve.
476

D-Xylose

D-Xylose Figure 3

Absorbance spectra of phloroglucinol reaction with D-xylose (5 mg/L). Reagent blank versus water, dashes; D-xylose
versus water, dashes with dots; D-xylose versus reagent blank, solid curve.

undissolved excess solid. Stable for 2 months at room

Procedure: p-Bromoaniline Reaction for d-Xylose
Principle
Urine or protein-free supernatant of plasma is
added to a reagent consisting of p-bromoaniline in
glacial acetic acid to produce a pink chromogen. The
absorbance spectrum for the chromogen is shown in D-
Xylose Figure 2.
Reagents
1. Zinc sulfate, ZnSO4 7H2O, 50 g/L (0.174
mol/L). Place 50 g of ZnSO4 7H2O in a 1 L flask.
Dissolve in approximately 900 mL of distilled water.
Bring to volume, and mix. Store at room temperature for
up to 1 year.
2. Barium hydroxide, Ba(OH)2 8H2O (0.158
mol/L). Dissolve 25 g in 500 mL of distilled water; then
boil, cool, and filter. Stable at room temperature for up
to 1 year.[27]
3. p-Bromoaniline reagent (116 mmol/L) in
saturated thiourea. To prepare saturated thiourea, add
7.5 g of thiourea to 150 mL of glacial acetic acid, and
allow to mix overnight. (Caution: Believed to be an
animal carcinogen.) Place 2.0 g of p-bromoaniline in a
100 mL volumetric flask, and bring to mark with the
saturated solution of thiourea, which is decanted off the
temperature.
4. Saturated benzoic acid. Add 15 g of benzoic
acid to 500 mL of distilled water. Allow to mix for 48 h
at room temperature. Use if solid benzoic acid is still
visible.
5. Stock Place 200 mg of d(+)-xylose in a 100
mL volumetric flask and dissolve in and bring to mark
with saturated benzoic acid. Stable for 12 months at
refrigerated temperature.
6. Working Dilute 5 mL of stock standard to 100
mL with saturated benzoic acid. Stable for 3 months at
refrigerated temperature.
7. Working standard, 200 mg/L (1.33 mmol/L).
Dilute 10 mL of stock standard to 100 mL with saturated
benzoic acid. Stable for 3 months at refrigerated
temperature.
Assay
Equipment: Heating bath set at 70 2 C;
spectrophotometer with a band pass less than 10 nm at
520 nm.
1. Prepare protein-free supernatant of plasma by
mixing 1.0 mL of plasma, 5.0 mL of distilled
water, and 2.0 mL of 0.158 M barium
hydroxide. Then add 2.0 mL of 0.174 M zinc
sulfate, mix, and centrifuge at 500 g for 5 min.
477
D-Xylose
2. Prepare 1:50 and 1:100 aqueous dilutions of
urine (stable 48 h in refrigerator, longer if
frozen).
3. Pipet 1.00 mL of distilled water, standards,
plasma protein-free supernatant, and urine
dilutions into a series of labeled, duplicate glass
tubes.
4. Add 5.00 mL of p-bromoaniline reagent to all
tubes.
5. Incubate one set of tubes in a heating bath at 70
2 C for 10 min. Cool under running water.
The second set will serve as blanks.
6. Place all tubes in the dark at room temperature
for 70 min.
7. Read absorbance of each tube within 30 min at
520 nm. Unheated tube serves as blank. If
absorbance is too high, dilute both tubes with p-
bromoaniline reagent, reread, and make
appropriate correction.
Calculation
Plasma:
Urine:
D-Xylose Reference Interval
click here
Procedure: Phloroglucinol Reaction for d-Xylose
Principle
Simplified colorimetric procedure using
phloroglucinol (Trinder and Tollen application) wherein
d-xylose is heated in strong acid to form furfural.
Furfural reacts with phloroglucinol to produce a pink-
colored compound, with a high molar absorptivity at 554
nm (d-Xylose Figure 3).
Reagents
1. Glacial acetic acid
2. Concentrated HCl
3. Working phloroglucinol reagent (36
mmol/L). Place 0.5 g of phloroglucinol (Sigma
Chemical Co., St. Louis, P-3502) in an Erlenmeyer flask,
and dissolve in 100 mL of glacial acetic acid and 10 mL
of concentrated HCl. Keep in a dark bottle. Stable only
for 4 days at room temperature. Use gloves in handling
reagent.
4. Stock xylose Place 200 mg of d(+)-xylose in a
100 mL volumetric flask and dissolve in and bring to
mark with saturated benzoic acid. Stable for 12 months
at refrigerated temperature.
6. Working xylose Dilute 5 mL of stock standard
to 100 mL with saturated benzoic acid. Stable for 3
months at refrigerated temperature.
7. Working standard, 200 mg/L (1.33 mmol/L).
Dilute 10 mL of stock standard to 100 mL with saturated
benzoic acid. Stable for 3 months at refrigerated
temperature.
Assay
Equipment: Heating block (105 C);
spectrophotometer (band pass <10 nm) capable of
reading at 554 nm.
1. Set heating block at 105 C (under fume hood).
2. Run standards and patient samples in duplicate.
Label 13 100 mm disposable tubes as follows:
1 for each serum blank, 1 for each standard
blank, 2 for each standard, 2 for each patient.
3. Pipet 5 mL of working reagent into each tube.
4. Add 50 L of distilled H2O to standard blank.
Add 50 L of any normal (xylose-free) serum to
serum blank. Add 50 L of plasma, serum, or
standard to respective tubes or 5 L of urine (if
analyzing a urine). Vortex mix well.
5. Simultaneously transfer all tubes to heating
block, and set clock for exactly 4 minutes.
NOTE: Do not add tubes at intervals because
temperature will drop during incubation, and different
tubes will be affected differently.
6. After incubation, immediately cool tubes in an
ice-water bath. Mix again.
7. Read absorbance of each tube at 554 nm, using
the standard blank to set the spectrophotometer
to 100%T (zero absorbance).
Calculation
Serum:
A554 patient A554 serum blank mmol/L standard
A554 standard
= mmol/L
Use the standard whose absorbance is closest
to that of the sample.
Urine:
1. Calculate as for serum, but multiply urine
results by 10 (5 L of urine and 50 L of
standard were used) to find mmol/L.
2. Convert mmol/L to mg/L: mmol/L 150
mg/mmol = mg/L.
3. Calculate for 5 h = mg/L Total volume (in
liters).
4. Divide by 1000 mg/g, and report as g/5-h urine.
Notes
1. Perform a glucose analysis on the patients
serum specimen. Record on slip. Slight
interference is seen in samples with high level
of glucose (>5000 mg/L).
478

D-Xylose

2. Color reaction is linear to 10.0 mmol/L. on the requisition. The total volume of the urine must be
known to calculate results and interpret the test. If this
information is unavailable, send out measured value
D-Xylose Reference Interval (mm/L) and free text comment that no reference
click here intervals are available for uncalculated results.
3. Both urine and serum samples are stable at 28
d-Xylose in Serum/Plasma and Urine C for 1 week.
I. Principle of Test and Clinical Significance
III. Reagents
A. Principle: Simplified colorimetric procedure
using phloroglucinol (Trinder and Tollen application). d- A. Phloroglucinol (Sigma P-3502)
Xylose is heated in strong acid to form furfural. Furfural B. Glacial acetic acid (Fischer A38-212)
reacts with phloroglucinol to produce a pink colored C. Hydrochloric acid (Fischer A144st-212)
compound with high molar absorptivity at 550 nm. This D. d-Xylose (Sigma X-1500)
test can be used to differentiate between intestinal E. Saturated benzoic acidadd benzoic acid to
malabsorption and pancreatic insufficiency. 500 mL distilled water until it settles out.
F. Working reagentWeigh out 0.5 gm
B. Clinical Significance: Xylose is not normally phloroglucinol and place in a 200 mL dark brown bottle.
present in significant amounts in the blood. When given Carefully add 100 mL glacial acetic acid and 10 mL
orally, it is passively absorbed in the proximal small concentrated HCl to bottle. Mix together under fume
(duodenojejunal) intestine (to about 60%) and most is hood. Stable for 4 days at room temperature. This
excreted by the kidneys. The amount of xylose reagent is very caustic. Wear gloves and eye shields
recovered in the urine or blood in a specified time when handling and preparing reagent and perform test
interval after administration is used to evaluate intestinal under the hood.
absorption. Low absorption of xylose is observed in
intestinal malabsorption. In malabsorption due to IV. Equipment
pancreatic insufficiency, the absorption of xylose will be
essentially normal. Therefore, this test is of some help A. Heating block (Thermolyne Dri-Bath Model
to distinguish between these two types of malabsorption. #DB-17615)
B. Thermometer that reads up to 105 C
NOTE: The accuracy of the procedure depends C. Spectrophotometer (PerkinElmer Jr. model 35)
not only on the rate of absorption but also on the rate of D. 13 x100 disposable test tubes
excretion of xylose by the kidneys. In order to eliminate E. 5 mL serological pipet
misinterpretations as a result of renal retention, a blood F. 50 L pipettor (MLA)
determination of xylose is carried out along with the G. MLA adjustable volume pipettor (adjusted to
determination of xylose in urine. A normal blood xylose 450 l)
level in the presence of decreased urine xylose would H. 12 x 75 mm glass cuvettes
suggest renal retention.
V. Calibration
Low values for d-xylose absorption are also
observed in a number of other conditions, including A. Stock d-xylose standard (50 mmol/L). Weigh
celiac disease, tropical sprue, Crohns disease, out 0.3755 g d-xylose. Place in 50 mL volumetric flask
immunoglobulin deficiency, pellagra, ascariasis, and QS to line with saturated benzoic acid.
vomiting, delayed gastric emptying, inadequate
hydration, decreased circulation, intrinsic renal disease, B. Working Standards
thyroid disease, sequestration in body fluids as occurs in
pregnancy and ascites, and incomplete urine collection. 1. 5.0 mmol/Lusing volumetric pipet add 1 mL
of the 50 mmole/L stock d-xylose standard and 9 mL of
II. Sample (Specimen) saturated benzoic acid to a 16 x 100 mm test tube. Mix
thoroughly.
A. Patient Preparation Instructions: A 25 g oral 2. 1.0 mmol/Ladd volumetrically 1 mL of the 5
dose of xylose in water is given to a fasting patient. mmol/L standard and 4 mL of saturated benzoic acid in a
Blood should be drawn at 1 and 2 h intervals after the d- 16 x 100 mm test tube. Mix thoroughly.
xylose is administered. All urine should be collected for
5 h following the dose. All the above standards are stable for 3 months at room
temperature.
B. Sample
Routinely run the 5 and 1 mmol/L standards every time
1. A minimum of 150 l serum or plasma. the assay is run and select the one with O.D. closest to
2. A minimum of 50 l urine is required. Urine the O.D. of patient sample for calculations. Standard
should be mixed and measured. Send a 10 ml aliquot of duplicates should have <5% variation between tubes.
the 5 h collection for analysis. Record the total volume
479

D-Xylose

VI. Quality Control F. After incubation, immediately cool tubes (use

ice water). Mix again.
A. Run any patient with a normal glucose as a
negative OC (serum) with each run. G. Read on PerkinxElmer Jr.:
B. Run an aliquot of the spiked pool as positive
QC (found in -70 C freezer) with each run. 1. Pour solutions to be read into clean 12 x 75
C. The QC should be within + 2SD of the mean spectrophotometer cuvettes.
from several interassay determinations (see overlap 2. Set to 550 filter.
policy). See range in QC notebook. Record QC on run 3. Set to absorbance.
sheet and in QC notebook. 4. Zero against proper blank (standard or serum).
D. If QC is out of range, repeat the assay with 5. Read absorbance of patients and QC.
fresh standards and reagent. 6. Write down absorbance values of QC # and
E. If QC is still out of range, consult supervisor. patients on worksheet.
To make spiked pool: Add 1 mL of 50 mm/L standard 7. Calculate results.
to 24 mL of normal serum, not normal control (there is
color interference). (Glucose should be normal.) The H. After incubation, solutions to be read are stable
value will be approximately 2 mm/L. Mix well, aliquot, for 1 h. Keep on ice until they are read.
and store in -70C freezer. Run several times to
accumulate data and calculate range. I. Solutions may be discarded down drain under
hood but must be followed by running water for 12
VII. Procedure min.

WEAR GLOVES AND PROTECTIVE J. Clean 12 x 75 cuvettes by washing in solution

EYEWEAR DURING COMPLETE PROCEDURE. of Acationox (C6330 American Scientific), 1 capful to 2
L water. Rinse well with distilled water. Place upside-
A. Set Dow heat block to 105 C (under hood). down in rack and dry in oven.
Acceptable temperature 105 C + 3 C. Write down
temperature on run sheet at beginning of incubation. VIII. Calculation
B. Run standards and patients in duplicate. Label
13 x 100 disposable tubes: O.D. patient x mmol/L std. = mmol/L
O.D. standard
1 serum blank
1 standard blank Urine:
2 for each standard
2 for each patient 1. Multiply urine results by 10 (dilution factor) =
2 for each QC mmol/L.
2. Convert mmol/L to mg/dL: mmol/L x 15 =
C. Pipet 5 ml of working reagent into each tube mg/dL.
(volumetrically). 3. Calculate for 5 h = mg/dL x TV/100.
4. Divide by 1000 and report as grams/5 h urine.
D. Pipet QC, standards and patients into
appropriate tube as follows (using calibrated pipets): Expected Results:

1. Standard blank: 50 L H2O to standard blank Blood:

(zero standards against this)
2. Serum blank: 50 L of any normal (xylose-
free) serum-to-serum blank (zero all patients against
this).
3. 50 l of plasma, serum or standard to respective
tubes.
4. 50 l urine (make 10 dilution of urine before
pipetting into tube with working reagent). Mix well
(vortex) and cap with Multifit stoppers (Kew Scientific).
E. Simultaneously add all tubes to heat block and
set clock for 4 minutes. NOTE: Do not add tubes at
intervals because temperature will drop during
incubation and different tubes will be affected
differently.
Adult (after 25 g dose) > 25 mg/dL(> 1.66 mm/l)
Child (after 25 g dose) > 30 mg/dL (> 2.0 mm/l)
Urine:
Adult (after 25 g dose) > 4 g/5 h urine
Child (1633% of given dose)
X. Procedural Notes
A. This procedure is sensitive to pipetting
technique. Be sure to use a volumetric pipet when
dispensing the working regent and to use good pipetting
technique when adding the 50 L of sample to the
reagent.
B. This procedure may be run in triplicate instead
of duplicate to ensure that absorbances agree within 5%.
When running in triplicate, the best 2 out of 3
absorbances may be used in calculating the average.
480

D-Xylose

C. When cooling the tubes after inoculation, make

sure to immerse the tubes at least halfway in the ice-
water bath and to leave them in a sufficient amount of
time to allow complete cooling (about 5 min).
XI. Procedure Performance

A. Color reaction is linear to 10.0 mmol/L.

Samples exceeding this amount should be diluted 10x
with distilled water and repeated.
B. Patients with high glucose (>500 mg/dL) may
have false positive results. Run and record on the
worksheet a glucose on each patients specimen to check
that it is normal. If it is >500 mg/dL, attach a comment
to the patients result, noting that there will be a slight
positive interference. False negative results may occur if
patient is on high-dose aspirin therapy or taking
neomycin, calcine, indomethacin, or atropine.
C. Day-to-day precision at a level of 1.9, SD =
0.12, CV = 6.3%

XII. References
Eberts, T.J., et al.: A simplified colorimetric
micromethod for xylose in serum or urine, with
phloroglucinol, Clin. Chem. 25(8):1440-1443, 1979.
481
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
Gus Koerbin
Name: Dehydroepiandrosterone, DHEA, Prasterone,
3-hydroxyandrost-5-en-17-one
Clinical Significance: Refer to Chapter 48, General Endocrinology, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular formula: DHEA, C19H28O2; DHEA-S, C19H28SO5
Molecular mass: DHEA, 288.41; DHEA-S, 368.51
Merck Index: DHEA, 7606 (Prasterone)
Chemical class: Steroid
Structure:
Principles of Analysis and Current Usage
The measurement of dehydroepiandrosterone (DHEA)
and dehydroepiandrosterone sulfate (DHEA-S) is
important for the assessment of adrenal androgen
production [1]. Gas-liquid chromatography (GLC) was
first used in a number of procedures (Table 1, Method 1)
[2]. In each procedure, direct measurement was not
possible, since DHEA and its sulfate are thermo-labile
and required derivatization.
An exhaustive double-isotope derivative method was
suggested by Gandy (Table 1, Method 2) [3]. This
procedure required five chromatographic steps and
quantitation by liquid scintillation.
A competitive protein-binding assay [4] for total DHEA
and DHEA-S in plasma actually measured the derivative
5-androstenediol (5-A-diol) (Table 1, Method 3).
Early radioligand assays for DHEA and its sulfate
generally required chromatographic separation before
RIA because of cross-reactivity of the antisera with these
i
Dehydroepiandrosterone and Its Sulfate (DHEA and
DHEA-S)
Previous and current authors of this method:
First edition: Not done
Methods edition: Karen L. Nickel
Second edition: Not updated
Third edition: Not updated
Fourth edition: Karen L. Nickel
Fifth edition: Gus Koerbin
closely related steroids (Table 1, Method 4). Nieschlag
[5] described an RIA procedure for both steroids. For
DHEA, plasma was extracted with ether and then
purified by TLC before RIA. DHEA-S was extracted
from plasma into ethyl acetate, hydrolyzed in acetic acid
at 75C to form free DHEA, and then purified by TLC.
Separation of bound from free labeled DHEA was
achieved with globulin-dextran-coated charcoal.
Direct RIA measurement of DHEA-S in plasma was
described by Buster and Abraham [6] and Cattaneo [7].
Because of the relatively high concentration of DHEA-S
in plasma, simple dilution of plasma reduced
interferences, making extraction and chromatography
unnecessary. Haning [8] improved the direct assay for
DHEA-S by using an antiserum that reduced previously
observed cross-reactivity with other steroid sulfates. An
RIA from blood spotted on filter paper was developed by
Grueters [9]. Determination of urinary DHEA-S was
reported by Causon, using high-performance liquid
chromatography and RIA [10]. It was generally found
that analysis of DHEA by RIA required extensive
treatment of plasma before RIA. This was necessary
because of the relatively low concentration of DHEA
compared with DHEA-S and other interfering steroids.
Assay of DHEA-S using solid-phase enzyme-linked
immunosorbent assay (ELISA) based on the principle of
competitive binding is now the most commonly
performed assay (Table 1, Method 5). Microtiter wells or
micro-particles (beads) are coated with a polyclonal
antibody directed towards a unique antigenic site of the
DHEA-S molecule. Endogenous DHEA-S of a patient
sample competes with a DHEA-S horseradish
482
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
peroxidase conjugate for binding to the coated antibody.
After incubation, the unbound conjugate is removed. The
amount of bound peroxidase conjugate is inversely
proportional to the concentration of DHEA-S in the
sample.
Reference and Preferred Methods
There is no reference method for either DHEA or
DHEA-S. Determination of DHEA and DHEA-S in
serum or plasma has generally replaced urinary analysis.
However, total 17-ketosteroid assay in urine is used by
some clinicians as a screen for hypersecretion of adrenal
androgens.
Analysis of DHEA-S in plasma is routinely performed
by solid-phase competitive chemiluminescent enzyme
immunoassay.
With direct RIA, neither extraction nor chromatography
is required in most methods. These procedures use a
tritiated isotope. Commercial availability of 125I-(3)-
radioiodinated DHEA-S and antiserum raised to DHEA-
S-3-betamonohemisuccinate-human serum albumin
allow the use of methods with reduced counting time and
greater ease of handling.
Specimen
Serum, heparinized plasma, or EDTA plasma may be
used for analysis of DHEA or DHEA-S. Patients should
not be given any steroids or gonadotropin medications
for at least 48 hours prior to sample collection [11]. The
specimen is stable for 2 days at 4C and for many
months if frozen.
Interferences
Heterophilic antibodies are the most common cause of
interference when using ELISA techniques. The use of
separator gel tubes (SST) may affect results. No
significant differences have been noted between
concentrations in specimens collected in plastic and
glass SST tubes [12]. If using RIA, impure absolute
ethanol may leave a residue on drying that interferes
with binding. Care must be taken to avoid contamination
of the alcohol.
DHEA and DHEA-S Reference Interval
Normal values should be established by each individual
laboratory for each particular assay. Normal adult males
have typical values of 80 to 550 g/dL, (2.2 to 15.0
mol/L). A typical adult female reference interval is 37
to 430 g/dL (1.0 to 11.6 mol/L) and for
postmenopausal women < 190 g/dL (<5.1mol/L).
DHEA-S levels in women also vary according to the
menstrual cycle, with the follicular and luteal phases
showing lower levels than midcycle. DHEA-S
concentrations in infants decrease between 1 and 5
months after birth but are generally less than 150 g/dL
(4 mol/L). Between 6 months and 7 years of age,
DHEA-S is normally less than 18 g/dL (0.05 mol/L)
in boys and less than 37 g/dL (1.0 mol/L) in girls
[13]. In teenage children, indicatively, values are 3.7 to
170 g/dL (0.1 to 4.6 mol/L).
Interpretation
DHEA is produced in substantial quantity in the adrenal
gland. It is converted to its sulfated form in either the
adrenal gland or the liver and can be desulfated in
peripheral tissues. The circulating concentration of
DHEA-S is many times higher than DHEA.
DHEA has weak androgenic actions and may be
considered a prohormone, with conversion to
testosterone and/or estrogen occurring in peripheral and
genital tissue [14]. Adrenal production of DHEA is an
important contributor to androgen production and effects
in women but not in men.
The steady decrease in serum levels of these steroids
from early adulthood onwards suggests that they may be
markers of aging and that high levels may protect against
the pathological consequences of becoming old. Studies
on humans demonstrate associations of DHEA and
DHEA-S with survival and various measures of health
status, including diabetes, cardiovascular disease,
obesity, cognitive impairment, physical limitations, and
depressive symptoms [15,16].
Despite extensive research on these steroids, little is
known about their specific functions or the mechanisms
by which they affect health [17]. Research suggests that
DHEA may counterbalance the immunosuppressive
effects of glucocorticoids, although the receptor for
DHEA has not been fully characterized [18]. DHEA has
been shown to have biological actions on hemostasis,
cell proliferation, lipid metabolism, stress response, and
immune function [15]. Knowledge about causal effects
of DHEA-S for health in humans is limited and often
inconclusive because many of the experimental studies
have been performed on nonprimates, which have far
lower concentrations of these steroids than humans [19].
DHEA-S is important in the investigation of hirsutism
and alopecia in women. It is also useful in the
assessment of delayed puberty and adrenal disorders.
Plasma levels increase steadily from about 7 years of
age, declining after about 30 years of age. Diurnal
variation is not observed.
DHEA-S is frequently analyzed in conjunction with free
testosterone in the investigations of hirsutism and
hyperandrogenism. One of these two hormones is likely
to be elevated in these conditions. High levels are also
seen in polycystic ovary syndrome. Extremely high
levels (>700 g/dL) in women are suggestive of
hormone-secreting adrenal tumors.
DHEA and DHEA-S Performance Goals
Survey data from the 2007 College of American
Pathologists (CAP) Participant Summary Report show
imprecision values (% coefficient of variation) for
measurement of DHEA-S range from 4.2% to 14.3% at
concentrations of approximately 40 g/dL (1.0 mol/L)
and 4.5% to 12.6% at 140 g/dL (3.8 mol/L) for assays
with > 10 participants. Less than 5% of all assays are
performed using RIA, and these methods have higher
483
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
imprecision, 5.4% to 14.3%, when compared with
ELISA, 4.2% to 11.8%.
DHEA-S is not regulated under the Clinical Laboratory
Improvement Amendments (CLIA 88) for proficiency
testing. Analytical performance goals set by the Royal
College of Pathologists of Australasia (RCPA) are 37
g/dL, up to 370 g/dL and 10% > 370 g/dL (1.0
mol/L up to 10 mol/L and 10% > 10 mol/L.
The 2007 CAP data show that there is a lack of
harmonization between commonly used DHEA-S
ELISA assays. There are differences in concentration
between the Roche assays (16% of all assays) and other
assays, with the Roche assays showing values up to 60%
higher at 100 g/dL (2.7 mol/L). The RCPA 2007 data
also show a lack of harmonization with the Roche
assays. In this program, differences in concentrations are
up to 30%. Within-individual biological variation has
been found to be about 4% and between-individual
variation approximately 29% [20].
References
1 Nelson DH. Adrenal androgens. In: The
Adrenal Cortex: Physiological Function and
Disease. Vol 18. Major Problems in Internal
Medicine. Philadelphia: Saunders; 1980.
2 Kumari GL, Collins WP, Sommerville IF.
Further studies on the gas-liquid
chromatographic determination of C19-steroids
in human plasma using nickel-63 electron
capture detection. J Chromatogr 1969;41:22-36.
3 Gandy HM, Peterson RE. Measurement of
testosterone and 17-ketosteroids in plasma by
the double isotope dilution derivative
technique. J Clin Endocrinol 1975;4:505-512.
4 Rosenfield RL. A competitive protein binding
method for the measurement of unconjugated
and sulfate-conjugated dehydroepiandrosterone
in peripheral plasma. Steroids 1971;17:689-
696.
5 Nieschlag E, Loviaux DL, Lipsett MB.
Radioligand assay for delta-5-3-beta-
hydroxysteroids. I. 3-Beta-hydroxy-5-
androstene-17-one and its sulphate. Steroids
1972;19:669-679.
6 Buster JE, Abraham GE. Radioimmunoassay of
plasma dehydroepiandrosterone sulphate. Anal
Lett 1972;5:543-551.
7 Cattaneo S, Forti G, Fiorelli G, Barbieri U,
Serio M. A rapid radioimmunoassay for
determination of dehydroepiandrosterone
sulphate in human plasma. Clin Endocrinol
1972;4:505-512.
8 Haning RV, Carlson IH, Shapiro SS, Nolten
WE. Testosterone free index correlates best
with dehydroepiandrosterone sulphate. Fertil
Steril 1981;36:757-765.
9 Grueters A, Korth-Schutz S. Longitudinal study
of plasma dehydroepiandrosterone sulfate in
preterm and full-term infants. J Clin Endocrinol
Metab 1982;55:314-320.
10 Causon RC, Collins SL, Fry DE. Determination
of urinary dehydroepiandrosterone sulphate by
combined high-performance liquid
chromatography and radioimmunoassay. J
Chromatogr 1982;227:485-491.
11 Inter Science Institute. Current Unique and
Rare Endocrine Assays. Inglewood, CA:
InterScience Institute; 1997.
12 Reinartz JJ, Ramey ML, Fowler MC et al.
Plastic vs glass SST evacuated serum-separator
blood-drawing tubes for endocrinologic
analytes. Clin Chem 1993;39:2535-2536.
13 Babalola AA, Ellis G. Serum
dehydroepiandrosterone sulfate in a normal
pediatric population. Clin Biochem
1985;18:184-189.
14 Labrie F, Luu-The V, Belanger A et al. Is
dehydroepiandrosterone a hormone? J
Endocrinol 2005;187:169-96.
15 Feldman HA, Johannes CB, Araujo AB, Mohr
BA, Longcope C, McKinlay JB. Low
dehydroepiandrosterone and ischemic heart
disease in middle-aged men: prospective results
from the Massachusetts Male Aging Study. Am
J Epidemiol 2001;153:79-89.
16 Trivedi DP, Khaw KT.
Dehydroepiandrosterone sulfate and mortality
in elderly men and women. J Clin Endocrinol
Metab 2001;86:4171-7.
17 Celec P, Starka L. Dehydroepiandrosterone: is
the fountain of youth drying out? Physiol Res
2003;52:397-407.
18 Butcher SK, Lord JM. Stress responses and
innate immunity: aging as a contributory factor.
Aging Cell 2004;3:151-60.
19 Dhatariya K, Bigelow ML, Nair KS. Effect of
dehydroepiandrosterone replacement on insulin
sensitivity and lipids in hypoadrenal women.
Diabetes 2005;54:765-9.
20 Westgard QC. Desirable Specifications for
Total Error, Imprecision, and Bias, Derived
from Biologic Variation. Available at
<http://www.westgard.com/biodatabase1.htm>
Accessed 2008.10.18.
21 Anderson DC, Hopper BR, Lesley BL, Yen SC.
A simple method for the assay of eight steroids
in small volumes of plasma. Steroids
1976;28:179-196.
22 Smith MR, Rudd BT, Shirley A, Rayner PH,
Williams JW, Dugnan ND, Bertrand PV. A
radioimmunoassay for the estimation of serum
dehydroepiandrosterone sulphate in normal and
pathological sera. Clin Chim Acta 1975;65:5-
13.
484

Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)

Table 1: Methods of DHEA and DHEA-S Analysis

Method 1: Gas-liquid chromatography (GLC)
Principle of analysis: Extraction; analyte partially purified by chromatography, derivatized, analyzed by GLC,
detected to electron capture
Comments: Plasma; rarely used
Method 2: Double-isotope derivatization
Principle of analysis: Radioactive derivatives are formed, purified by chromatography, and quantitated by
radioisotopic counting
Comments: Plasma; very rarely used
Method 3: Competitive protein binding (CPB)
Principle of analysis: Extraction, purification by thin-layer chromatography; enzymatic conversion to 5-
androstenediol; CPB using testosterone-binding globulin
Comments: Plasma; very rarely used
Method 4: Radioimmunoassay (RIA)
Principle of analysis:
a. DHEA-S: Dilution of plasma and direct RIA using iodinated or tritiated tracer and antiserum
b. DHEA: Extraction, chromatography, RIA using tritiated tracer and antiserum
Comments:
a. Plasma; in common use
b. Plasma; in common use
Method 5: Enzyme-linked immunosorbent assay (ELISA)
Principle of analysis: Solid-phase ELISA based on the principle of competitive binding.
Comments: Serum, plasma; most widely used assay.
485

Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)

Table 2: Comparison of Some Radioimmunoassay Methods for DHEA-S

Buster [6] Cattaneo [7] Smith [22] Haning [8]
1972 1972 1975 1981
Extraction No No Yes No
Recovery correction No No Yes No
Antiserum DHEA-3- DHEA-3-BSA DHEA- DHEA-BSA
hemisuccinate hemisuccinate
Isotope 3H-DHEA 3H-DHEA-S 3H-DHEA-S 3H-DHEA-S
(not S)
Incubation Overnight, 4C 2 hr, 4C 2 hr, room Overnight, 4C
4C temperature
Separation Charcoal-dextran Charcoal-dextran Ammonium Charcoal-dextran
sulfate
Cross-reactivity DHEA 100% A-S 50%; DHEA 129% EpiS 33%
A-dione 13% DHEA 25%; A-dione 30% A-S 20%
Andro 11%; A-diol 3% Epi 8%
EtioS 10%
Recovery Not stated Satisfactory 87% 95%
Interassay precision 18.8% 7.0% 12.3% 11.2%
Sensitivity 10 pg 50 pg 800 pg 35 pg
Normal range (g/dL): M, 199334 M, 169315 M and F, M, 230470
F, 82338 F, 150328 98456 F, 130350

A-diol, 5-Androstenediol; A-dione, 5-androstenedione; A-S, conjugated androstene; BSA, bovine serum albumin; DHEA,
dehydroepiandrosterone; Epi, epiandrosterone; EpiS, epiandrosterone sulfate; EtioS, etiocholanolone sulfate; F, female; M,
male; S, sulfate (conjugated).
Standard
Volume (mL) reaction
Procedure: DHEA-S by Radioimmunoassay
Standard of inter-of absolute tube
Principle (pg/mL) mediate B ethanol (pg/tube) (pmol/tube)
The plasma sample is diluted 300- to 2000-fold in 10,000 10.0 0 1000 2.71
buffer. For routine analysis, a dilution of 1 to 1000 is 5,000 5.0 5.0 500 1.36
2,500 2.5 7.5 250 0.68
convenient. The radioimmunoassay is then performed by 1,000 1.0 9.0 100 0.27
use of an antiserum to DHEA-3-hemisuccinate-BSA and 500 0.5 9.5 50 0.14
tritiated DHEA-S. Incubation is conducted at 4C for 2
hr. Separation of bound and free is achieved using a Pipet intermediate B according to the scheme above. For
charcoal-dextran mixture. One obtains a concentration of convenience, use 10.0 mL volumetric flasks. DHEA-S
DHEA-S in the specimen by comparing its observed standard solutions should be stored at 2C to 8C.
binding with that of standard DHEA-S. This procedure is Always allow ethanolic solutions to come to ambient
based on the method of Cattaneo.[7] temperature, and mix well before opening and use. Care
Reagents must be taken to avoid evaporation (and concentration)
1. Tris buffer, 0.1 mol/L, pH 7.4. Dissolve 12.1 of standard solutions. Properly stored, standard solutions
g of Tris (tris[hydroxymethyl]aminomethane) in 950 mL are stable for 6 months.
of distilled deionized water. Adjust pH to 7.4 with 6 M 5. DHEA-S antiserum. An antiserum is raised
HCl or 6 M NaOH, as needed. Adjust volume to 1000 using either DHEA-3-hemisuccinate or DHEA-3-BSA.
mL and mix. Stable for 2 months at 4C to 8C. This material is also commercially available from
2. DHEA-S standards. Weigh 20 mg of DHEA- Radioassay Systems Laboratory, Carson, CA. The
S (Sigma Chemical, St. Louis, or equivalent) and antiserum is diluted appropriately to achieve proper
dissolve in 150 mL of absolute ethanol. Swirl to binding characteristics so that maximum binding is 40%
dissolve, and bring to 200 mL. Label Stock DHEA-S to 50%, and the 50% intercept is about 300 pg per tube.
Standard. This solution contains 100 g/mL (0.27 This usually requires about a 1:1000 to 1:2000 dilution.
mmol/L). Aliquot and store at 20C.
3. Intermediate A. Dilute 1.0 mL of stock 6. Tritiated DHEA-S. Stock isotope can be
standard to 100 mL in absolute ethanol. This solution obtained from New England Nuclear (0.25 mCi, 0.0012
contains 1 g/mL (2.7 mol/L). mg in 0.25 mL of benzene).
4. Intermediate B. Dilute 1.0 mL of intermediate 7. Intermediate trace. Dilute stock isotope 1:10
A to 100 mL in absolute ethanol. This solution contains in absolute ethanol. This solution contains 96 ng/mL
10 ng/mL (or 10,000 pg/mL, or 27 nmol/L). Use this (0.26 nmol/L).
solution for preparing working standards. 8. Working trace. Dilute intermediate trace 1:50
Working standards in absolute ethanol. This solution contains 1.9 ng/mL (52
486
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
pmol/mL) and is equivalent to 96 pg/tube (0.26
pmol/tube). Store all isotope solutions at 2C to 8C.
9. Charcoal-dextran solution. Weigh 250 mg of
washed Norite A (Amend Drug Co., Philadelphia, PA)
and 25 mg of Dextran T-70 (Pharmacia Fine Chemicals,
Piscataway, NJ). Add 100 mL of Tris buffer. Mix and
store at 2C to 8C. Suspend well before use.
10. Scintillation fluid. Mix 64 mL of Spectrofluor
PPOPOPOP (Aldrich Chemicals, Milwaukee, WI) and
25 mL of methanol with 1 L of toluene.
Assay
Equipment: Automatic liquid scintillation spectrometer
and vortex mixing apparatus.
1. Dilution of plasma samples and controls: Dilute
0.1 mL of well-mixed plasma with 2.0 mL of
Tris buffer. Vortex mix. Then continue dilution
by pipetting 0.1 mL of diluted plasma and
adding 5.0 mL of Tris buffer. Vortex mix and
use this for the assay. The overall dilution is
1:1071.
2. Remove working antiserum, isotope, and
standards from storage, and allow to come to
ambient temperature. Mix gently.
3. Label duplicate 10 75 mm glass tubes for
zero, TC (total counts), NSB (nonspecific
binding), standards, and for each specimen and
control.
4. Pipet 0.1 mL of absolute ethanol into the tubes
labeled zero and 0.1 mL of each working
standard into the appropriately labeled tubes.
Dry under nitrogen, taking care not to overdry.
5. Pipet 0.05 mL of working isotope into all tubes.
Dry under nitrogen, taking care not to overdry.
6. Pipet 0.1 mL of Tris buffer into the zero and all
standard tubes. Pipet 0.3 mL of Tris buffer into
TC and NSB tubes. Pipet 0.1 mL of diluted
plasma samples and controls into appropriately
labeled tubes. Vortex mix gently.
7. Add 0.2 mL of diluted working antiserum to
each tube except the NSB tubes. Vortex mix
gently.
8. Incubate all tubes at 4C for 2 hr. After
incubation, place tubes into an ice bath for 10
min.
9. Add 0.2 mL of charcoal-dextran solution to all
tubes except TC. Maintain charcoal slurry using
an ice bath above a magnetic stirrer. Add slurry
rapidly, using automatic pipettes. Vortex mix
tubes immediately after addition of charcoal,
and continue incubation at 4C for 10 min.
10. Centrifuge all tubes at 3000 g for 15 min.
11. Transfer 0.2 mL of each tube supernatant into
labeled counting vials, using an automatic
pipet. Add 10 mL of scintillation fluid, mix, and
count.
Calculations
1. Determine the average TC, NSB, and zero
standard counts; the last should be 40% to 50%
of TC.
2. Calculate %B/B0 binding for each standard,
unknown, and control, according to the
following formula:
% B/B0 = Counts (standard, unknown) NSB 100%
Counts (zero standard) NSB
3. Plot a smooth curve through the standard points
using logit-log graph paper, placing %B/B0 on
the ordinate and DHEA-S concentration on the
abscissa.
4. Calculate the final DHEA-S concentration
using the following formula:
mg/L = [pg/tube from graph][107][1000 mL/L]__
[0.1 mL of plasma/tube][1,000,000 pg/mg]
The factor 1071 is the usual dilution factor. If a different
dilution protocol is used, use the appropriate factor.
1 mg/L = 2.71 nmol/L
Procedure: DHEA by Radioimmunoassay
Principle
An aliquot of plasma is mixed with 3H-DHEA,
incubated for 30 min, and then extracted with diethyl
ether. After drying, the extract is dissolved in isooctane
for chromatography. The DHEA is separated from other
steroids by Celite partition chromatography. One then
performs radioimmunoassay using an antiserum to
DHEA-17-o-carboxymethyl-HSA and tritiated DHEA.
Incubation proceeds overnight at 4C. Separation of
bound and free forms is achieved with charcoal-dextran.
One can obtain the concentration of DHEA in the
specimen by comparing its observed binding with that of
the DHEA standards. Correction is made on each
specimen for individual recovery losses. This procedure
is based on the methods of Anderson [21].
Reagents
1. Solvents. Spectroquality solvents (Merck,
Rahway, NJ), diethyl ether, isooctane, ethyl acetate,
propylene glycol, and ethanol, are used without further
purification.
2. Chromatographic solutions:
1. 10% ethyl acetate Add 10 mL
of ethyl acetate to 90 mL of isooctane.
2. 15% ethyl acetate Add 15 mL
of ethyl acetate to 85 mL of isooctane.
Solutions should be prepared fresh
before each chromatography
procedure.
3. Celite 545, reagent grade. Obtained from
Alltech Associates, State College, PA, washed in
benzene, dried at 800C for 16 to 18 h, and kept dry at
100C until use.
4. Phosphate-gelatin buffer (P-G buffer) (0.4
mol/L, pH 7.4, 0.1% gelatin). Weigh 1 g of gelatin
(Knox unflavored) and add to 500 mL of deionized or
distilled water. Heat to 60C to dissolve, and then cool.
Add the following to the gelatin solution: 5.38 g of
NaH2PO4, 8.80 g of Na2HPO4, 9.00 g of NaCl, and
1.00 g of NaN3 (all reagent grade). Stir to dissolve.
Adjust pH to 7.4 if necessary, using 1 M NaOH or 1 M
487
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
HCl. Adjust volume to 1 L and mix. Store at 2C to 8C.
Stable for 1 month after preparation.
5. DHEA standards. Weigh 24 mg of DHEA
(Sigma Chemical, St. Louis) and add to 150 mL of
absolute ethanol. Swirl to dissolve, and adjust volume to
200 mL with ethanol. Label stock DHEA standard.
This solution contains 120 g/mL. Store at 2C to 8C
for 6 months.
6. Intermediate A. Dilute 10.0 mL of stock
standard to 100 mL in absolute ethanol. This solution
contains 12 g/mL. Store at 2C to 8C for 6 months.
7. Intermediate B. Dilute 1.0 mL of intermediate
A to 100 mL in absolute ethanol. This solution contains
120 ng/mL (120,000 pg/mL or 416 pmol/mL). Store at
2C to 8C for 6 months.
Preparation of Working Standards
Standard in
Volume (mL) reaction
Standard P-G tube
(pg/mL) intermediate buffer (pg/tube) (pmol/tube)
C 6000 0.5 mL of B 9.5 600 2.08
D 3000 5.0 mL of C 5.0 300 1.04
E 1500 5.0 mL of D 5.0 150 0.52
F 750 5.0 mL of E 5.0 75 0.26
G 375 5.0 mL of F 5.0 38 0.13
For convenience in preparing working
standards, use 10-mL volumetric flasks. Prepare working
standards fresh before use and discard unused volumes.
Care must be taken in handling of ethanolic
standard solutions. Always allow ethanol to come to
ambient temperature, and mix well before opening and
use. Care must be taken to avoid evaporation (and
concentration) of standard solutions.
DHEA antiserum. An antiserum is raised
using DHEA-17-o-
carboxymethyl-HSA. This antiserum is commercially
available from Radioassay Systems Laboratory, Carson,
CA. The antiserum is diluted appropriately to achieve
proper binding characteristics with a maximum binding
of 40% to 50% of total counts and a %B/B0 equaling
50% at about 200 pg/tube. The dilution is typically
1:2500. Aliquot and store at 20C.
8. Tritiated DHEA. Stock isotope can be
obtained from New England Nuclear. Stock solutions (1
mCi/mL) are stable at 4C for 2 months. Intermediate
aliquots are diluted in absolute ethanol to achieve 12.5
Ci/mL and are stored at 4C. Prepare working trace
solutions just before use by diluting intermediate trace in
buffer to a level of 5000 disintegrations per minute
(dpm) per 0.1 mL. Discard unused working trace after
each preparation.
9. Celite/propylene glycol mixture. Add 200 g
of Celite to 100 mL of propylene glycol. Slurry well
before use.
10. Charcoal-dextran solution. Weigh 250 mg of
washed Norite A (Amend Drug Co. Irvington, NJ) and
25 mg of Dextran T-70 (Pharmacia Fine Chemicals,
Piscataway, NJ). Add 100 mL of P-G buffer. Mix and
store at 2C to 8C. Slurry well before dispensing.
11. Scintillation fluid. Mix 64 mL of Spectrofluor
PPOPOPOP and 25 mL of methanol with 1 L of toluene.
Assay
Equipment: Automatic liquid scintillation spectrometer.
Extraction of Plasma
1. Add 0.1 mL of 3H-DHEA solution (spiking
isotope) having about 5000 dpm to 0.5 mL of
plasma. Vortex mix.
2. Incubate spiked plasma for 30 min at ambient
temperature.
3. Into separate liquid scintillation vials, pipet 0.1
mL of spiking isotope (in duplicate). Add 0.4
mL of P-G buffer, and save for later counting.
These are total recovery tubes.
4. Add 10 mL of diethyl ether to each plasma
sample, and vortex for 1 min.
5. Freeze extract on dry ice, and pour off ether.
6. Dry extract, and redissolve in 1.5 mL of
isooctane.
Preparation of Columns
1. Plug 5-mL siliconized pipets (Kimble) with a
small ball of glass wool at the bottom, and
mount on a supporting rack. Use separate
columns for each specimen and control.
2. Add a slurry of Celitepropylene glycol
mixture equivalent to 0.75 g (about 0.4 mL) to
each pipet. Allow to drain.
3. Add 5 mL of isooctane to each column, and
allow to drain.
Sample Chromatography
1. The extract from ether extraction is layered on
top of the Celite column and allowed to drain.
2. The column is washed with 5 mL of isooctane.
3. Add 5 mL of 10% ethyl acetateisooctane to
each column, allow to drain, and discard eluate.
4. Add 5 mL of 15% ethyl acetateisooctane to
each column, allow to drain, and collect and dry
the eluate.
Radioimmunoassay
1. Redissolve the dried chromatography eluates in
4 mL of P-G buffer, vortex, and heat at 50C
for 5 min. Vortex again after letting it stand for
10 min at ambient temperature.
2. Pipet duplicate 0.5-mL aliquots of each
reconstituted eluate into liquid scintillation vials
for recovery determination. These are sample
recovery counts tubes.
3. Label 10 75 mm glass tubes in duplicate for
TC, NSB, standards, samples, and controls.
4. Pipet two 0.5-mL and two 0.2-mL aliquots of
P-G buffer eluates in P-G buffer into labeled 10
75 mm tubes.
5. Pipet 0.1 mL of working standards into labeled
standard tubes.
6. Add 1.1 mL of P-G buffer to TC tubes, 0.6 mL
to NSB tubes, 0.4 mL to standard tubes, and 0.3
mL to sample tubes that contain only 0.2 mL of
eluate. Vortex gently.
7. Add 0.1 mL of working tracer to each tube.
8. Add 0.1 mL of working antiserum to each tube
except TC and NSB. Vortex tubes gently.
9. Incubate tubes at 37C for 30 min and then
overnight at 4C.
488
Dehydroepiandrosterone and Its Sulfate (DHEA and DHEA-S)
10. Add 0.5 mL of charcoal-dextran solution to all
tubes except TC. Maintain charcoal slurry using
an ice bath above a magnetic stirrer. Add slurry
rapidly using an automatic pipettor. Vortex mix
tubes immediately after addition of charcoal,
and continue incubation for 10 minutes at 4C.
11. Centrifuge all tubes at 3000 g for 15 min.
12. Transfer 0.5 mL of each tube supernate into
labeled counting vials using an automatic pipet.
Add 10 mL of scintillation fluid, mix, and
count. Also at this time, count the total recovery
and sample recovery vials for each sample.
Calculations
1. Determine the average TC, NSB, zero standard
counts, total recovery, and sample recovery.
2. Calculate B/B0 binding for each standard,
unknown, and control, according to the
following formula:
% B/B0 = Counts (standard, unknown) NSB 100%
Counts (zero standard) NSB
3. Plot a smooth curve through the standard points
using logit-log graph paper, placing %B/B0 on
the ordinate and DHEA concentration on the
abscissa.
4. Calculate the final DHEA concentration using
the following formula:
ng/mL = [pg/tube from graph][Dilution factor]______
[0.5 mL of plasma][Fract. rec.][1000 pg/ng]
where the dilution factor is 4.0/0.5 = 8, or 4.0/0.2 = 20,
depending on volume of redissolved eluate used
Fract. rec. is fractional recovery, defined as
Average sample recovery counts
Average total recovery counts
and 0.5 mL is volume of plasma extracted:
DHEA 1 ng/mL = 3.47 nmol/L
Notes
1. Any residue left from a contaminated solvent
will potentially interfere with binding in this
procedure. Recoveries less than 70% indicate
deficiencies in the extraction-chromatography
procedure.
2. Each Celitepropylene glycol system should be
calibrated with 15% ethyl acetateisooctane
and radiolabeled DHEA to ensure proper
elution of peak.
3. Care must be taken in handling ether so that no
peroxides are present.
4. Cross-reactivity of the antiserum should be
characterized, especially toward other
conjugates of DHEA. Chromatography,
however, eliminates most of any cross-reacting
steroid present.
Procedure: DHEA-S by ELISA
1. Secure the desired number of microtiter wells in the
holder.
2. Dispense 25 L of each standard, controls, and
samples with new disposable tips into appropriate wells.
3. Dispense 200 L enzyme conjugate into each well.
4. Thoroughly mix for 10 seconds. It is important to
have a complete mixing in this step.
5. Incubate for 60 minutes at room temperature without
covering the plate.
6. Briskly shake out the contents of the wells.
Rinse the wells 3 times with diluted sash Solution (400
L per well). Strike the wells sharply on absorbent paper
to remove residual droplets.
IMPORTANT NOTE:
The sensitivity and precision of this assay is markedly
influenced by the correct performance of the washing
procedure!
7. Add 100 L of substrate solution to each well.
8. Incubate for 15 minutes at room temperature.
9. Stop the enzymatic reaction by adding 50 L of stop
solution to each well.
10. Read the absorbance at 450 10 nm with a
microtiter-plate reader within 10 minutes after adding
the stop solution.
Calculation of Results
1. Calculate the average absorbance values for each set
of standards, controls, and patient samples.
2. Construct a standard curve by plotting the mean
absorbance obtained from each standard against its
concentration with absorbance value on the vertical (Y)
axis and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample,
determine the corresponding concentration from the
standard curve.
4. Automated method: Computer programs using cubic
spline, 4 PL (4 Parameter Logistics) or Logit-Log can
generally give a good fit.
5. The concentration of the samples can be read directly
from this standard curve. Samples with concentrations
higher than that of the highest standard have to be
further diluted. For the calculation of the concentrations,
this dilution factor has to be taken into account.
A typical example of a standard curve for the DHEA-S
ELISA:
Standard Optical Units (450 nm)
Standard 0 (0 g/dL)
Standard 1 (10 g/dL)
Standard 2 (50 g/dL)
Standard 3 (100 g/dL)
Standard 4 (250 g/dL)
Standard 5 (500 g/dL)
Standard 5 (1000 g/dL)
489

Digoxin and Digitoxin

Digoxin and Digitoxin

Randal J. Schneider
Name: Digoxin and digitoxin
Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C41H64O14 (- and -acetyldigitoxin, digoxin)
C41H64O13 (digitoxin)
Molecular mass: 780.97 D and 764.92 D, respectively
Merck Index: 3148, 3143
Chemical class: Cardiac glycoside
O
Digoxin
O Lactone Ring

OH CH3
H

CH3 H
Steroid Nucleus
H
H OH

O
CH3 H

Sugar Residues

3
O OH

Structure: H

Principles of Analysis and Current Usage
The term digitalis is used to represent all steroid
glycoside compounds sharing common features of
chemical structure (a steroid nucleus with an unsaturated
lactone ring at the C17 position and one or more sugar
residues at C3) and common effects on cardiac activity.
Although digoxin and digitoxin are the two digitalis
preparations used in the United States, digoxin is more
frequently prescribed.
Digoxin and digitoxin are obtained in the crystalline
form from the leaves of Digitalis lantana and Digitalis
purpurea, respectively. Digoxin is sparingly soluble in
water, chloroform, ether, ethyl acetate, and acetone, but
soluble in dilute (50%) alcohol or pyridine, whereas
digitoxin, a nonpolar molecule (structure is that of
digoxin without the C12-OH group), is relatively more
soluble in organic solvents. Digoxin and digitoxin are
i
Digoxin and Digitoxin
Previous and current authors of this method:
First edition: I-Wen Chen, Linda A. Heminger
Methods edition: I-Wen Chen, Linda A. Heminger
Second edition: I-Wen Chen, Linda A. Heminger
Third edition: I-Wen Chen, Linda A. Heminger
Fourth edition: I-Wen Chen, Linda A. Heminger
Fifth edition: Randal J. Schneider
used to control cardiac arrhythmias through
improvement of the strength of myocardial contraction.
However, intoxication is a frequent complication of
digitalis therapy and can be life threatening, frequently
requiring stat analysis for digitalis levels and in severe
cases, administration of the digoxin-specific antibody
fragments (Digibind, DigiTAb). Determination of
digitalis concentrations in blood has been helpful in the
diagnosis of toxicity and the establishment of an optimal
dosage, because a reasonably close correlation exists
between blood and tissue concentrations of digitalis.
Many techniques have been used to determine blood
levels of digoxin. They include gas chromatography [1]
(Table 1, Method 1), methods based on inhibition by
digoxin of sodium-potassium-activated adenosine
triphosphatase (ATPase) in red blood cells [2] or in
microsomes [3] (Table 1, Method 2), and an isotopic
displacement method using a sodium/potassium-
activated ATPase (Table 1, Method 3) [4]. However,
these methods are relatively tedious and have been
largely replaced by more practical and sensitive
immunoassay techniques.
Digoxin was one of the first hapten-like small molecules
to be analyzed by the RIA technique (Table 1, Method 4)
[5]. Digoxin antisera were successfully raised in animals
immunized with digoxin coupled to a variety of
490
Digoxin and Digitoxin
macromolecules, including poly-l-lysine, human serum
albumin, and bovine serum albumin. Unlike thyroid
hormones, a digoxin molecule contains neither iodine
nor any functional groups that can be iodinated. Tritiated
digoxin preparations were initially the only radioligands
available for digoxin RIA. Tritiated radioligands are
beta-particle emitters and must be counted by means of
liquid scintillation counters. This is expensive and
inconvenient in comparison to the counting of gamma-
ray emitters such as iodine (125I) by a crystal
scintillation well counter. This problem was overcome
by the development of techniques that attach a functional
group that can be easily iodinated to a digoxin molecule.
Tyrosine, histamine, and tyramine have been coupled to
a digoxin or digoxigenin molecule for radioiodination.
An example of a derivative formed in this way is 3-O-
succinyl digoxigenin tyrosine. When this compound is
radioiodinated, a monoiodinated and di-iodinated
derivative can be obtained. It is important that the
radioiodinated 3-O-succinyl digoxigenin tyrosine used in
a digoxin RIA be free of the di-iodinated derivative,
since the di-iodinated derivative is known to bind to
thyroxine-binding globulins [6]. Nearly every separation
technique used in RIAs has been applied successfully to
the RIA of digoxin.
Other types of labels have been developed as alternatives
to radionuclides. In the homogeneous enzyme-multiplied
immunoassay technique (EMIT) (Table 1, Method 5),
the enzyme glucose-6-phosphate dehydrogenase is
chemically coupled to digoxin and used as a label. When
the enzymedigoxin conjugate is bound to the digoxin
antibody, the enzyme is inactivated. In the presence of
digoxin in a sample, some of the enzymedigoxin
conjugate does not bind to the digoxin antibody, and the
enzyme is active. Therefore, one can monitor the extent
of digoxin-antibody bindingand thus the digoxin
concentrationby measuring the enzyme activity
spectrophotometrically without physically separating the
bound and free fractions. Although the EMIT was
developed originally as a manual assay system, it has
been successfully adapted to automated chemistry
analyzers.
A sensitive affinity columnmediated immunometric
assay (Table 1, Method 6) has also been developed [7].
In this assay, the F(ab)2 fragment of the digoxin
antibody is labeled with -galactosidase. An excess
amount of this enzymeantibody conjugate is incubated
with the sample so that digoxin contained in the sample
can be rapidly and quantitatively bound to the antibody.
To accomplish this, equal volumes of serum sample and
reagent containing -galactosidaseantibody conjugate
are mixed and incubated manually for 10 minutes to 2
hours at room temperature. The sample cup is then
introduced into the instrument along with a digoxin test
pack containing ouabain (digoxin analog)-bovine serum
albuminSephadex G-10 affinity column positioned in
the pack head and a substrate tablet. The system then
automatically pumps the sample through the column,
allowing only the enzymeantibody conjugate with the
bound digoxin to be eluted from the column. The
column eluate is mixed with the substrate tablet
(containing o-nitrophenyl--d-galactopyranoside), and
the enzyme activity is quantified by measurement of the
rate of change of absorbance at 405 nm. The -
galactosidase activity present in this fraction is
proportional to the digoxin concentration in the serum.
A fluorescent probe has also been used as an alternative
to radioactive labels in the determination of digoxin. In
the homogeneous fluorescence polarization
immunoassay developed for digoxin, the label is a
fluorescent dye (fluorescein) (Table 1, Method 7). When
the digoxin-fluorescein complex is excited by a
polarized beam of light, it emits polarized fluorescent
light. The degree of polarization depends on the extent
of rotation of the molecule during the period between
excitation and emission (rotational relaxation time). The
rotational relaxation time is proportional to the size of
the molecule; the larger the molecule, the longer the
rotational relaxation time and the larger the degree of
fluorescence polarization. Therefore, in the fluorescence
polarization immunoassay, the quantity of antibody-
bound digoxin-fluorescein complex (a much larger
molecule than the unbound complex) present can be
determined, in the presence of the unbound complex, by
measurement of the increase in the degree of
polarization of the emitted fluorescence.
More recently, assays based on drug-coated
microparticles have been developed. The particle-
enhanced turbidimetric immunoassay method (PENTIA,
Table 1, Method 8) is based on competition between
exogenous drug in the specimen and digoxin-coated
microparticles for anti-digoxin antibody. The rate of
absorbance change is directly proportional to the rate at
which the microparticles agglutinate in the presence or
absence of digoxin in the specimen. Other microparticle-
based assays include latex (Table 1, Method 9),
magnetic enzyme immunoassay (Table 1, Method 10),
and magnetic microparticles (Table 1, Method 11). In
these assays, the microparticles serve as the solid phase
for capture of the antigen of interest, followed by a
detection method such as chemiluminescence or
fluorescence.
All methods described in Table 1 are also applicable to
(and for some, developed for) the measurement of
circulating digitoxin [3,8,9]. As with digoxin, digitoxin
is determined in the clinical laboratory almost
exclusively by immunoassay. However, only a few
commercial immunoassay kits are currently available
because digitoxin analyses are infrequently requested,
owing to the lower frequency of digitoxin prescribing as
compared to digoxin.
A review of the laboratory methods reported in the
College of American Pathologists (CAP) Proficiency
Summary Report (2007 CAP Z-B) indicates more than
half of the methodologies include microparticle
technologies. The majority of the remaining methods are
491
Digoxin and Digitoxin
based on enzymes, fluorescence, and
chemiluminescence.
Gas chromatography, ATPase inhibition, and isotope-
displacement methods are described briefly in Table 1
for historic interest or for specialized use.
Reference and Preferred Methods
Recently a candidate reference method for digoxin and
digitoxin was developed and implemented by the
Institute for Standardization and Documentation in the
Medical Laboratory (INSTAND). The method employs
high-pressure liquid chromatography with isotope-
dilution tandem mass spectroscopy (LC-IDMS/MS)
[10]. The method is used to set target values for
materials used in external quality-assessment surveys.
Prior to the development of the IDMS/MS method, an
interlaboratory transferability study for digoxin assays
sponsored by the Centers for Disease Control, the
National Committee on Clinical Laboratory Standards,
and the American Association for Clinical Chemistry
developed a candidate reference method. The RIA
method was based on tritium-labeled digoxin as the
radioligand and charcoal as the separation agent [11].
Although most commercial kits are well-characterized
systems, their performance characteristics should be
evaluated carefully in each laboratory before they are
selected for routine use. Immunoassay methods for
digoxin have been fully automated. The sensitivity of the
EMIT assay is relatively low, primarily because of the
steric hindrance introduced into the antigen-antibody
reaction by the presence of the enzyme macromolecule
and also because of the need to differentiate the
antibody-enzyme complex from the unbound enzyme-
substrate complex (bound from unbound) and the
competitive mode of the assay (competition between
labeled and unlabeled antigens for the limited number of
antibody-binding sites). Some of the limitations have
been circumvented by the affinity columnmediated
immunometric assay and by the homogeneous
fluorescence polarization immunoassay. Non-isotopic
immunoassays have been semiautomated. Pretreatment
steps may be required, because endogenous fluorescent
compounds bound to serum proteins interfere with the
assay. It has been shown that digoxin binds to the protein
pellet during the trichloroacetic acid precipitation step,
and thus falsely high or low values may be expected
with patient specimens having abnormally low or high
protein contents [12]. Although the non-isotopic reagents
are considerably more expensive than those of RIA, they
are attractive alternatives to RIA in laboratories that
have such units already in use. The assay precision of
these two systems compared favorably with that of RIA.
Specimen
In most instances, either serum or plasma may be used
for the assay, though some commercial manufacturers
specifically indicate that plasma is not to be used with
their assay kits. Digoxin in serum is quite stable, with no
appreciable loss of digoxin observed in serum stored at
room temperature for up to 2 weeks. However, if the
specimen is not to be tested within 24 hours, it should
preferably be stored frozen.
Time of blood collection is an important factor in
determining digoxin toxicity. Serum digoxin levels will
rise sharply during the first hour after an oral dose,
indicating post-absorption uptake. This is followed by a
sharp decrease as digoxin is taken up by the myocardium
and other tissues. Usually 4 to 6 hours are needed for
serum and tissue stores of digoxin to reach equilibrium.
Thereafter, serum digoxin levels tend to stabilize and
decrease very slowly over the next 24 to 40 hours.
Clinically, the time of peak tissue (not plasma)
concentrations is used to accurately correlate plasma and
tissue concentrations. One obtains the greatest diagnostic
accuracy by drawing the blood sample at a standard time
during the stable phase, when serum digoxin levels
reflect the average cardiac concentrations.
Recommendations are to collect specimens 8 hours or
more after the dose.
Digoxin concentrations cannot be accurately determined
in a patient who is being switched from digitoxin to
digoxin therapy, since both drugs may be present in the
serum. The digoxin antisera used in most commercial
digoxin immunoassay kits cross-react significantly with
digitoxin (2% to 6% cross-reactivity, with some as high
as 40%). The problem of cross-reaction is further
exacerbated by the fact that therapeutic levels of
digitoxin are about 10 times higher than those of
digoxin. The digitoxin antisera also cross-react with
digoxin (2% to 4% cross-reactivity), but because of the
higher therapeutic levels, the interference by digoxin is
less significant in the digitoxin immunoassay.
Interferences
The presence of digoxin-like immunoreactive substances
(DLIS) in human plasma and other body fluids can
cross-react with anti-digoxin antibodies, causing falsely
increased digoxin concentrations [13]. Increased DLIS
concentrations have been found in patients with
conditions typically characterized by volume expansion,
such as uremia, essential hypertension, liver disease, and
pregnancy. Exogenous DLIS have been found in serum
following ingestion of various Chinese medicines, and
following therapy with spironolactone. FPIA procedures
generally exhibit the greatest degree of interference.
Falsely decreased digoxin results due to the presence of
DLIS have been described. Negative interference in
digoxin measurements have been described in the MEIA
procedures. Positive and negative interference due to the
presence of DLIS can be overcome by measuring free
digoxin rather than total digoxin concentrations. The
presence of hemoglobin, lipemia, or icterus usually does
not interfere with the commonly used immunoassay
procedures unless these compounds are in extremely
increased amounts [14].
Digoxin and Digitoxin Therapeutic Range
As with all diagnostic tests, it is advisable for the
individual laboratory to accumulate and analyze its own
data to establish a range of toxic and nontoxic values;
492
Digoxin and Digitoxin
differences in assay techniques may affect assay results.
One study found that 90% of nontoxic patients had
serum digoxin levels below 2 ng/mL (2.56 nmol/L),
whereas 86% of the toxic patients had levels greater than
2 ng/mL [15].
Beller et al. [16] reported a mean serum digitoxin
concentration of 20 11 ng/mL (26.1 nmol/L), with a
range of 9 to 32 ng/mL (11.8 to 41.8 nmol/L), for
nontoxic patients on oral doses of 0.07 to 0.15 mg/day
(0.89 to 1.96 mol/day) and a mean concentration of 34
18 ng/mL (44.4 nmol/L), with a range of 16 to 52
ng/mL (20.9 to 67.9 nmol/L), for toxic patients on doses
of 0.10 mg/day (1.31 mol/day).
It is generally understood that infants are more resistant
to cardiac glycosides than adults are. OMalley et al.
found that a mean nontoxic digoxin level of 3.8 1.2
ng/mL (4.87 nmol/L) for eight infants younger than 1
month was significantly higher than 1.4 1.1 ng/mL
(1.79 nmol/L) for 16 adults, but the digoxin
concentration in infants older than 1 month (1.4 0.5
ng/mL, n = 5) was no higher on average than that of the
adult controls [17].
Interpretation
Digitalis is a frequently prescribed drug in clinical
practice in the United States. Because of its ability to
increase the strength of cardiac contraction, digitalis
preparations have been used medically for the control of
supraventricular tachycardias and the treatment of
congestive heart failure. Oral administration is the least
expensive and the safest route of digitalis administration.
The intravenous route is used only when conditions such
as coma and vomiting prevent oral administration or
when rapid onset of digitalis action is imperative, as in
patients with pulmonary edema from acute left-
ventricular failure. Digoxin, the most commonly used
digitalis preparation in the United States, has an average
excretory half-life of about 35 hours. Since digoxin is
eliminated primarily by renal excretion, patients with
reduced renal function show a longer plasma half-life
as long as 5 days in patients who are essentially
anephric. In contrast, digitoxin is more than 90% bound
to serum albumin, has a half-life averaging 5 to 9 days,
and is relatively uninfluenced by impaired renal
function.
Digitalis is given until a therapeutic effect is achieved
(such as relief of cardiac failure) or until the earliest
toxic effects (such as anorexia or arrhythmia) appear.
The typical initial oral dose of digoxin in adults is 0.75
to 1.5 mg. This is followed by the administration of 0.25
to 0.5 mg every 6 hours until adequate digitalization is
achieved. The oral maintenance dose ranges from 0.0625
to 0.75 mg daily. The initial oral dose of digitoxin of 1.0
to 2.5 mg is administered in several equal aliquots every
6 hours. The dose should be administered within 36 to
40 hours. The oral maintenance dose ranges from 0.005
to 0.2 mg daily in divided doses. Digitalis doses in
children are about 50% larger than those used in adults
when calculated on the basis of milligrams per kilogram
of body weight. The higher doses are necessary because
of the greater ratio of cardiac mass to total body mass in
children. These higher doses are reflected in higher
serum digitalis concentrations in children.
Digitalis toxicity occurs frequently and can be life
threatening. Common clinical symptoms of a digitalis
overdose are anorexia, nausea and vomiting, blurred
vision, flickering objects of yellow and green color, and
disorientation. The most common important myocardial
manifestation of digitalis toxicity is cardiac arrhythmia.
Clinical diagnosis of digitalis toxicity is often difficult in
patients with serious heart disease, because most of its
manifestations could be caused by either the disease or
the drug. Immunoassays of digitalis were initially
developed for diagnosis and avoidance of digitalis
toxicity. Mean serum digitalis concentrations in patients
with drug toxicity (arrhythmia) are about two to three
times higher than those in patients receiving
therapeutically appropriate doses. Although this
difference is statistically significant in most studies,
substantial overlap exists between groups with and
without toxicity. Therefore, knowledge of high serum
digitalis levels is helpful in the clinical recognition of
digitalis toxicity, but under no circumstances can it be
used as the sole basis for determining the presence of
drug toxicity in place of sound clinical judgment. Serum
digitalis levels are especially helpful in the interpretation
of arrhythmias. In the presence of high digitalis levels,
arrhythmias by digitalis toxicity are predicted, whereas
arrhythmias in patients with low serum digitalis levels
are probably related to the underlying disease.
Furthermore, hypokalemia can precipitate digitalis
toxicity because of the combined effects an elevated
digoxin with a low potassium will have on normal
cardiac rhythm. The digitalis assay can also be used to
detect noncompliant patients, especially when they are
known to have had adequate digitalis levels in the past.
Fab fragments of digoxin-specific antibodies have been
used in the management of advanced, life-threatening
digitalis toxicity unresponsive to conventional
therapeutic modalities [18]. The use of Fab fragments
permits glomerular filtration and relatively rapid renal
excretion of antibody-bound digoxin, resulting in
progressive removal of digoxin from receptor sites and
rapid reversal of advanced digoxin toxicity without signs
of adverse effects. Because of the presence of digoxin-
specific antibodies, serum samples obtained from
patients receiving antibody treatment cannot be assayed
directly. They must be either deproteinized or subjected
to equilibrium dialysis before the assay. Total and free
digoxin concentrations can be obtained from the
deproteinized and equilibrated dialysis samples,
respectively. It has been reported that free (active)
digoxin serum concentrations decreased to undetectable
levels within minutes after administration of the digoxin-
specific Fab fragments, whereas the total digoxin serum
concentrations decreased more gradually. Immunoassays
that measure total digoxin levels vary in their cross-
reactivities to digoxin-specific Fab fragments, and
493
Digoxin and Digitoxin
laboratories should be aware of this limitation for 8.
potentially overestimating digoxin levels generated after
the administration of the Fab antidote. Some laboratories
have validated ultra-filtration systems for separating free
from Fab-bound digoxin to provide healthcare 9.
professionals with a more accurate bioavailable serum
digoxin level.
Digoxin Performance Goals 10.
Survey data from the 2007 CAP Participant Summary
Report show imprecision values (% coefficient of
variation) for digoxin measurements range from 4.6% to
14.1% at a mean digoxin concentration of approximately
1.24 ng/mL. Acceptable performance criteria (Clinical 11.
Laboratory Improvement Amendments, 1988) for
measurement of digoxin and free digoxin require that
laboratories be accurate to within the greater of 20% or
0.2 ng/mL and 3S.D. or 10% of the peer-group 12.
mean, respectively. Desirable specifications for
analytical imprecision derived from studies of biological
variation indicate an assay SD of 0.03 and 0.08 ng/mL at
concentrations of 0.8 and 2.0 ng/mL [19]. 13.
References
1. Watson E, Kalman SM. Assay of digoxin in
plasma by gas chromatography. J Chromatogr
1971;56:209-18. 14.
2. Bertler A, Redfors A. An improved method of
estimating digoxin in human plasma. Clin
Pharmacol Ther 1970;11:665-73.
3. Burnett GH, Conklin RL. The enzymatic assay 15.
of plasma digoxin. J Lab Clin Med
1971;78:779-84.
4. Brooker G, Jelliffe RW. Serum cardiac
glycoside assay based upon displacement of 3 16.
H-ouabain from Na-K ATPase. Circulation
1972;45:20-36.
5. Butler VP Jr, Chen JP. Digoxin-specific
antibodies. Proc Natl Acad Sci U S A 17.
1967;57:71-8.
6. Painter K, Vader CR. Interference of iodine-
125 ligands in radioimmunoassay: evidence 18.
implicating thyroxine-binding globulin. Clin
Chem 1979;25:797-9.
7. Leflar CC, Freytag JW, Powell LM, Strahan JC,
Wadsley JJ, Tyler CA et al. An automated, 19.
affinity-column-mediated, enzyme-linked
immunometric assay for digoxin on the Du Pont
aca discrete clinical analyzer. Clin Chem
1984;30:1809-11.
Lukas DA, Peterson RE. Double isotope
dilution derivative assay of digitoxin in plasma,
urine, and stool of patients maintained on the
drug. J Clin Invest 1966;45:782-95.
Oliver GC Jr, Parker BM, Brasfield DL, Parker
CW. The measurement of digitoxin in human
serum by radioimmunoassay. J Clin Invest
1968;47:1035-42.
Kaiser P, Akerboom T, Wood WG, Reinauer H.
A novel LC-IDMS/MS method for the
determination of the cardiac glycosides digoxin
and digitoxin using caesium adducts. Clin Lab
2006;52:37-42.
Fast DM, Hannon WH, Burtis CA, Bayse DD.
An interlaboratory study of the determination of
digoxin by immunoassay. Clin Chem
1980;26:480-6.
Porter WH, Haver VM, Bush BA. Effect of
protein concentration on the determination of
digoxin in serum by fluorescence polarization
immunoassay. Clin Chem 1984;30:1826-9.
Dasgupta A. Endogenous and exogenous
digoxin-like immunoreactive substances:
impact on therapeutic drug monitoring of
digoxin. Am J Clin Pathol 2002;118:132-40.
Miller JJ, Straub RW Jr, Valdes R Jr. Analytical
performance of a monoclonal digoxin assay
with increased specificity on the ACS:180. Ther
Drug Monit 1996;18:65-72.
Park HM, Chen IW, Manitasas GT, Lowey A,
Saenger EL. Clinical evaluation of
radioimmunoassay of digoxin. J Nucl Med
1973;14:531-3.
Beller GA, Smith TW, Abelmann WH, Haber
E, Hood WB Jr. Digitalis intoxication. A
prospective clinical study with serum level
correlations. N Engl J Med 1971;284:989-97.
OMalley K, Coleman EN, Doig WB,
Stevenson IH. Plasma digoxin levels in infants.
Arch Dis Child 1973;48:55-7.
Wenger TL, Butler VP Jr, Haber E, Smith TW.
Treatment of 63 severely digitalis-toxic patients
with digoxin-specific antibody fragments. J Am
Coll Cardiol 1985;5(5 Suppl A):118A-123A.
Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biological variation: pros, cons
and progress. Scand J Clin Lab Invest
1999;59:491-500.
494

Digoxin and Digitoxin

Tables

Table 1: Methods of Digoxin Analysis

Method 1: Gas chromatography
Principle of analysis: Physicochemical separation with 3H-digoxin as internal standard
Comments: Plasma; may be applied to other biological specimen; requires prepurification by thin-layer
chromatography and derivatization; tedious and time consuming
Method 2: ATPase inhibition
Principle of analysis: Dose-dependent inhibition of Na- and K-activated ATPase
Comments: Plasma; may be applied to other biological specimens; requires extraction; tedious and time
consuming; less precise and less specific than RIA
Method 3: Isotope displacement
Principle of analysis: Displacement of 3H-ouabain from purified Na-K ATPase preparation by digoxin
Comments: Plasma; may be applied to other biological specimens; requires extraction and beta counting; tedious,
nonspecific
Method 4: Radioimmunoassay
Principle of analysis: Competitive binding of 3H- or 125I-labeled and unlabeled digoxin with digoxin antibody
Comments: Plasma or serum; requires phase separation and beta-particle or gamma-ray counting; fully
automated; most sensitive and precise
Method 5: Enzyme-multiplied immunoassay techniques
Principle of analysis: Competitive binding for antibody between endogenous digoxin and enzyme-labeled
digoxin. Antibody binding to enzyme-digoxin complex inhibits enzymatic activity.
Comments: Serum; lacks sensitivity; requires sample pretreatment
Method 6: Enzyme immunometric assay
Principle of analysis: Quantitative binding of digoxin by excess amount of enzyme-labeled antibody
Comments: Plasma or serum; requires no sample pretreatment but requires phase separation by affinity column;
system semiautomated
Method 7: Fluorescence polarization immunoassay
Principle of analysis: Competitive binding of fluorescein-labeled and unlabeled digoxin with digoxin antibody
Comments: Plasma or serum; requires sample pretreatment but requires no phase separation; quantitation
depends on degree of polarization of emitted fluorescence; system semiautomated
Method 8: Particle-enhanced turbidimetric immunoassay
Principle of analysis: Competitive binding for antibody between endogenous drug and drug coated onto
microparticles. Antibody binding of digoxin in the specimen to microparticles slows the agglutination process that
is measured photometrically.
Method 9: Latex microparticle agglutination
Principle of analysis: Competition between digoxin and digoxin-labeled latex microparticles for digoxin-
specific antibodies. The level of agglutination is measured turbidimetrically.
Method 10: Microparticle enzyme immunoassay
Principle of analysis: Digoxin in the specimen is sandwiched between bound antibodies to microparticles and
digoxin-specific, enzyme-labeled antibodies. Digoxin is proportional to the fluorescence generated from the
enzyme-substrate interaction.
Method 11: Magnetic particle immunoassay
Principle of analysis: Antibody conjugate reagent mixing with digoxin in the specimen. Bound digoxin is
separated from excess antibody conjugate by magnetic microparticles coated with the digoxin analog, ouabain.
The remaining supernatant containing the bound digoxin-antibody-enzyme complex is mixed with substrate.
Enzymatic conversion of the substrate is proportional to the amount of digoxin the specimen.
495

Drug Screens

Drug Screens
Christopher R. McCudden

Name: Drug screens

Clinical significance: Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.

to detect the drug. Screening methods typically use

Principles of Analysis and Current Usage
Drug screens are a general term for analysis of toxic
and controlled substances. These tests are known by a
variety of names such as coma panel, toxic screen,
complete drug screen, comprehensive screen, drug-
abuse screen, stimulant panel, or overdose panel.
Despite the fact that these test names suggest an
exhaustive list of compounds, drug screens are
individually defined by each laboratory and may only
contain a handful of commonly encountered drugs.
There are many different types of qualitative drug
screens, each developed with that particular laboratorys
interests, finances, and experience in mind. There are
various types of urine screening tests used which rely on
spot tests, immunoassays, or chromatography. Whereas
immunoassays are directed toward specific drugs or drug
groups, chromatographic systems such as TLC and GC-
MS can detect a much wider range of compounds and
metabolites.
To define a specific drug screen, one must consider the
reasons for screening. Areas in which drug screening is
often indicated include (1) clinical toxicology, (2)
forensic/medicolegal toxicology, (3) employee-screening
programs, (4) drug trials, and (5) drug-abuse programs.
Although screening for forensic purposes is still largely
the domain of the coroners laboratory, other drug-
screening programs have been adopted by the clinical
laboratories with special emphasis on clinical toxicology
(the overdose patient) and drug-abuse screening
programs.
Defining the Test
Qualitative analysis for a drug or a group of drugs can
generally be divided into three basic steps: (1) isolation
or separation of the compounds of interest from the
biological specimen, (2) analysis of the drug content of
the partially purified sample by a screening method, and
(3) if positive, employment of one or more confirmatory
methods that are different from the method used initially
i tests to employ on a limited budget.
Drug screen
Previous and current authors of this method:
First edition: F. Michael Hassan
Methods edition: F. Michael Hassan
Second edition: F. Michael Hassan
Third edition: F. Michael Hassan
Fourth edition: F. Michael Hassan
Fifth edition: Christopher R. McCudden
analytical techniques that give either a positive or
negative finding, whereas confirmation tests may be
either qualitative or quantitative. Numerous possible
analytical permutations exist for the screening-
confirmation format (see Table 1).
Most drug screens fall into one of two classes: (1) a
specific screen for a particular known or suspected drug
(or class of drug), or (2) a comprehensive screen, with
analysis of as many drugs as are technically and
economically feasible for the laboratory to measure.
Searching for one particular compound is much easier
than screening for a large number of possible
compounds. If a single known or suspected drug is of
interest, the screening test can be optimized to ensure
adequate specificity and sensitivity. If the screen is
broadened to include several compounds, a series of
specific analyses for each compound can be combined
together to form a limited or panel type of screen. In
this format, tests are performed on each compound using
a procedure that optimizes sensitivity. If the number of
compounds is increased further, the time required for
performing specific individual assays for a large group
of drugs will eventually limit the number of drugs that
can be included in the screen. When it is necessary to
expand the screen to include many substances, less-
specific assays that enable the analyst to look for a vast
array of compounds at a single glance may be employed.
The actual number of drugs included in this expanding
screening process varies from laboratory to laboratory
and depends heavily on the experience of the analyst
performing the assay.
When defining the screen in a particular laboratory, it is
important to consider the clinical need to detect a given
compound. In the case of drugs of abuse, certain regions
of the country are more or less likely to encounter a
particular drug. An example of this is shown in Figure
1A, where PCP is commonly encountered in parts of
Texas but is less frequently seen in the Southeast and
Northwest. In contrast (Figure 1B), cocaine is prevalent
in both Texas and throughout the Southeast. Historical
experience can be a valuable tool when deciding which
Once the laboratory has decided on the test menu, it
must gather the appropriate analytical data on these
drugs and their metabolites. The necessity of
understanding metabolic pathways of drugs of interest
cannot be overemphasized. As many drugs are present in
urine only as metabolites, it is essential to have standards
496
Drug Screens
of both known metabolites and the parent drugs for
accurate identification and confirmation.
General Analytical Procedures
A set of general references is provided below. These
detail not only general screening procedures but also
specific individual assay techniques and technologies
involved in toxicological analyses [1-6].
Extraction
With the exception of spot tests and immunoassays, most
procedures require significant preparation of the
specimen before actual analysis. For the drug screen,
isolation is usually accomplished by solvent extraction
procedures or by column chromatography, employing
exchange resins or solid, bonded-phase stationary
phases. Solvent extraction of urine samples involves a
two-phase partitioning system in which the drug is
separated from an aqueous phase and redistributed into a
second phase, which is usually organic. This removes
many of the interfering compounds present in the
specimen. The degree of complexity of the extraction
depends on the type of analytical method used and the
degree of freedom from interfering compounds required
of the processed specimen.
Since drugs are extracted from matrices according to
their ionization state, partitioning of the specimen
constituents into acidic, neutral, and basic fractions is
possible. Enhanced extraction efficiencies are obtained
by preparation of extractions from specimens made
acidic or alkaline to yield higher recoveries of drug.
When shortened turnaround times are a necessity, single
pH extractions can be performed. These shortened
extractions are essentially a compromise, since most
drug groups are extracted to some extent but with a
lower overall recovery.
For extraction of drugs, the most commonly used resin is
known as XAD-2 (non-ionic polystyrene exchange
resin). This resin is typically immobilized in a column
bed, and urine is applied. Drugs are adsorbed onto the
resin and then subsequently eluted with appropriate
solvents. As with conventional solvent extractions, the
proper selection of sample pH and solvents for elution
optimize the selection process. Column extraction is
amenable to batch extraction using semiautomated
procedures [7].
Drug extraction can also be achieved using other
compounds such as bonded-phase sorbents. Bonded-
phase sorbents are usually composed of silica gels and
enable convenient batch processing in commercially
available columns. As with XAD-2 resins, column-
bound drugs can be selectively eluted off with various
solvents.
Spot Tests
Spot tests are among the simplest and quickest methods
for initial drug screening or a confirmation test (Table
2). Usually an unprocessed specimen (urine or serum) is
added directly to test reagents to assay for the presence
of a specific drug (or class of drugs, see Table 3), which
is indicated by the formation of a colorimetric reaction
product [5,8-10]. Although spot tests offer inexpensive
testing with rapid turnaround times, they are relatively
nonspecific and often require subjective interpretation.
Ultraviolet Spectroscopy
As with the spot tests, ultraviolet analysis is best
employed to test for a specific drug or confirm the
presence of compounds detected by other techniques.
The ultraviolet scan has relatively poor specificity unless
the unknown drug is isolated by other test procedures,
such as thin-layer chromatography, or extracted by
procedures that minimize spectral interferences from
body fluids and from other drugs. As evident in Figure 2,
compounds may have overlapping or similar ultraviolet
spectra, necessitating further confirmatory techniques.
Sources have compiled spectra of most commonly
encountered drugs [5]. However, whereas ultraviolet
spectra are readily obtained, sample preparation time can
be lengthy (Table 2).
Immunochemical Methods
Immunochemical methods rely on antibodies and
various chemical, enzyme, and small-particle labels to
identify drugs. There are numerous different formats for
antibody-based drug detection that have become popular
because of their rapid turnaround times, accuracy, and
ease of use. Currently available formats include enzyme-
multiplied immunoassay technique (EMIT), cloned-
enzyme donor immunoassay (CEDIA), colloid metal
immunoassay (CMI), fluorescence immunoassay,
fluorescence polarization immunoassay (FPIA),
immunochromatography, lateral-flow immunoassay,
microparticle immunoassays (turbidimetric or
nephelometric), and radioimmunoassay (RIA). Because
these assays are antibody based, is important to consider
the drug target and related compounds that may be
detected. Commercial assay users should be familiar
with the metabolism of target drugs and consult the
manufacturers package inserts for compound cross-
reactivity. External proficiency tests also provide a
useful source for antibody specificity.
Enzyme Immunoassay
Commercially available EMIT kit assays for an array of
individual drugs or drug classes are offered by several
manufacturers (Table 2). The sample is added to a drug-
specific antibody mixture, and then an enzyme-labeled
drug reagent is introduced. Drug in the patient specimen
competes with conjugated drug for antibody. A
colorimetric reaction occurs between the enzyme and
substrate, where the amount of drug in the specimen is
proportional to the signal. These assays offer good
specificity and adequate sensitivities for detection of
drugs in both overdose and drug-abuse situations. The
EMIT test menu includes an array of both prescription
drugs and drugs of abuse (see Table 4).
As with other immunoassay, is important to consider the
drug target and related compounds that may cross-react
with the antibody. For example, the Emit II Plus opiate
497
Drug Screens
assay (Syva, Dade Behring, USA) is targeted towards
urinary morphine, morphine-3-glucuronide, and codeine
but can cross-react with high concentrations of synthetic
opiates such as oxycodone and hydromorphone.
Similarly, the Emit II plus assay for amphetamine
detects related compounds such as
methylenedioxymethamphetamine (MDMA or
Ecstasy), as well as unrelated drugs like bupropion and
propranolol.
Radioimmunoassay
Although infrequently used today, RIA procedures have
historically provided the analyst with commercially
available assays for a select number of individual drugs
[4]. As with EMIT assays, RIA specificity depends
completely on the antibody. RIAs have the highest
analytical sensitivities, enabling detection of drugs at
concentrations found in the majority of clinical
specimens. However, most assays require more than 30
min of incubation time, and few laboratories can offer
immediate results for more than one or two of these
assays. Radioactive-material handling and waste
disposal are also a significant drawback. As with EMIT
assays, users should be aware of antibody specificity.
Thin-Layer Chromatography (TLC)
Despite its technical demands, TLC serves as an
excellent screening or confirmation technique for both
large laboratories and those with more limited
capabilities. The method requires no instrumentation and
is relatively inexpensive (Table 2). Additional
advantages include the flexibility to perform
simultaneous analyses of a larger number of drugs on a
larger number of specimens. Sensitivities of most TLC
screening procedures are adequate for both overdose and
drug-abuse cases, and specificity is often acceptable. The
major limitation of this method is lengthy turnaround
time. Depending on the method utilized, the procedure
may take 2 to 4 hours [11]. Modified methods have been
introduced that are based on the same principles of TLC
but with accelerated development and detection steps
[12,13].
Gas Chromatography (GC) and Gas Chromatography
Mass Spectrometry (GC/MS)
GC offers one of the most powerful analytical tools for
the separation of complex mixtures in biological fluids.
As with thin-layer chromatography, GC enables the
evaluation of a large number of possible constituents
simultaneously (Table 2). Unlike thin-layer
chromatography, however, GC techniques cannot screen
a large number of patient specimens simultaneously;
therefore individual samples must be analyzed
consecutively, lowering the overall throughput.
For analysis by GC, specimens must generally be
extracted, and the drug must either be volatile or
derivatized to make it volatile [14-18]. For screening
purposes, samples can be separated on a single column
or, to enhance analytical specificity and sensitivity, can
be injected into various compound-selective columns.
Compounds are identified by their retention times
relative to an internal standard (Figure 3A), using
thermal conductivity detection. More compound-specific
detectors are available for nitrogen and phosphorus, or
halogens. For comparison of detection methods, contrast
Figure 3A with Figure 3B, which shows a sample
detected using nitrogen detection.
Alternatively, a gas chromatograph can be interfaced
with a mass spectrometer detector (GC/MS). This
combination provides the most objective analytical data
of existing methods for structural elucidation of an
unknown compound (Figure 4). The GC/MS system has
been utilized both as a confirmatory procedure and as a
primary screening method for drug screens when
improved turnaround times are necessitated (immediate
overdose screening) [19]. The drawbacks of
conventional GC and GC/MS are (1) the considerable
technical expertise required to develop and perform
testing and (2) high initial equipment costs.
High-Performance Liquid Chromatography (HPLC)
HPLC is another technique that can be used to separate
complex mixtures of drugs extracted from biological
fluids. Although specimens usually require extraction
before analysis, HPLC has an advantage over GC in that
the sample does not have to be volatile or derivatized.
However, HPLC has limited ability to separate many
different classes of drugs simultaneously using the same
column and buffer conditions. Therefore, several runs
may be required, using columns and conditions with
specificity for a given class of drugs (e.g.,
benzodiazepines). Like other forms of chromatography,
HPLC is also limited in the number of patient samples
that can be processed at a time, particularly when runs
are long (>60 min). It is noteworthy that application of
these traditional methods to ultra-pressure
chromatography (UPLC) will eventually shorten analysis
times. As with GC, it is possible to connect several
detectors, spectrophotometers, fluorometers,
electrochemical detectors, or mass spectrometers in
series to characterize the compounds partially as they are
eluted from the column.
Liquid Chromatography and Mass Spectrometry or
Tandem Mass Spectrometry
When coupled to mass spectrometry (MS) or tandem
mass spectrometry (MS/MS), liquid chromatography
(LC) has the potential to definitively identify a vast array
of compounds. Although it is not yet widely utilized,
LC-MS/MS has a few advantages over GC-MS,
including analysis of polar, nonvolatile, and heat-labile
drugs [20,21]. As with HPLC, some of the laborious
derivatization steps may be omitted. In addition, reagent
costs may be substantially reduced in comparison with
immunoassays. As with GC-MS, limitations of LC-MS
drug screening are the technical expertise required to
develop and validate the assays and the initial capital
costs [22,23].
Point-of-Care Testing
As with all critical analytes, it is attractive to consider
screening for drugs of abuse as point-of-care (POC) tests
498
Drug Screens
(Table 2). These potentially offer rapid turnaround times,
simplified work processes, and could alleviate the
relatively high cost of maintaining low-volume, 7-day,
24-hour service in the central testing laboratory [24,25].
For POC drug testing, urine is the preferred specimen,
since the more common drugs of abuse can be detected
for longer time periods in urine compared with serum.
The amount of sample required ranges from a few drops
to as much as 20 to 30 mL. Other samples have also
been considered for POC drug testing, including sweat,
breath, and saliva [25]. Of these, saliva is considered an
attractive, readily accessible and noninvasive alternative
to urine. However, because some urinary drug
metabolites differ from those found in saliva, traditional
cutoffs for urinethose specified by the U.S. Substance
Abuse and Mental Health Services Administration
(SAMSHA), for examplemay not be appropriate.
Advantages of POC testing include rapid turnaround
time and simplified chain-of-custody issues
(confirmation tests are still required for tests with legal
implications). The demands for proper specimen
identification, labeling, and transport can also be
simplified with the use of POC devices that are used at
the bedside [26].
Devices for POC testing have been designed for single-
or multiple-drug detection. Test devices include a range
of formats, including dipsticks, cards, and plastic
cassettes. The methods used in these devices are
typically immunochromatographic, analogous to home
pregnancy tests, which permit simple visual
interpretation of results. One example of a POC device is
based on competitive binding of antibody to drug present
in urine and drug conjugate bound to a porous
membrane. If the urine contains drug, dye-conjugated
antibodies bind to an immobilized antibody strip,
resulting in the formation of a colored line. Although the
performance of POC tests for drugs is improving [27],
they are not as accurate as immunoassays or GC-MS and
are more expensive [28]. In addition, it is essential that
users be appropriately trained with the testing device and
be aware of its limitations. The National Academy of
Clinical Biochemistry (NACB) has published detailed
guidelines on POC drugs-of-abuse screening [25].
Future Technologies
There are several developing technologies that may be
used for drug screening and confirmation in the future.
These newer technologies include infrared spectroscopy
[29], nuclear magnetic resonance spectroscopy [30], and
capillary zone electrophoresis [31,32]. CZE has been
used experimentally to screen opiates [31,32], but
because of the very small injection volumes, it is subject
to detection-limit problems. This requires derivatization,
which essentially nullifies the ease of use and analytical
time savings. The other methods are expensive and
largely experimental, so they will not be discussed here.
Reference and Preferred Methods
As is so often found with esoteric laboratory tests, drug-
screening procedures do not have a reference method.
The optimal reference method is difficult to establish,
since it would entail (1) defining which compounds
should be included in various screens, (2) establishing a
preferred separation or extraction scheme, (3) describing
the best screening method, and (4) suggesting the best
confirmation procedures. Given that the drugs of interest
invariably change over time, these criteria cannot readily
be achieved.
The type of screen required is dependent on both
economic and clinical needs. If there is good clinical-
laboratory interaction, it is possible to offer more
efficient specific testing. For example, in the acute care
setting, an agitated or hyperactive patient does not need
to be screened for morphine, tricyclic antidepressants,
and benzodiazepines, because drugs such as
amphetamines, phencyclidine, and the cocaine agents are
more probable causes of this altered mental status.
Therefore, the laboratory can offer faster, more accurate
service if the drug screen is limited to a few agents.
Similarly, most drug-abuse screening related to
employee testing or drug-rehabilitation programs
require only the testing for a limited number of drugs.
Examples of (1) comprehensive, (2) stimulant panel, and
(3) drug-abuse screening procedures are presented
below.
Specimen
The most commonly used samples for drug screens are
blood or urine (Table 5). However, the type of specimen
used for drug screens depends on many factors. Whole
blood or serum samples are useful for quantitative
analysis of circulating agents such as therapeutic drugs.
This is particularly true when drug levels correlate with
toxicity and there are available antidotes (e.g., N-
acetylcysteine for acetaminophen poisoning). For past
exposure and drugs-of-abuse screening, it is generally
accepted that the optimal specimen is urine. Immediate
and appropriate collection is critical for all drug screens
(see Table 6 for drug-detection windows).
For forensic drug analysis, a wide variety of specimens
may be employed, including vitreous fluid, hair, urine,
blood, and body-cavity fluid. For drugs-of-abuse testing
in the workplace, alternate specimens such as hair and
saliva are being researched. At this time, these
specimens are not used, owing to lack of standardization,
lack of reference ranges, and potential ethical bias, but it
is noteworthy that the U.S. Department of Health and
Human Services (DHHS) is actively exploring the use of
alternate specimens [33]. This may accelerate the
development of methods for such specimens, as well as
the use of point-of-collection testing.
Interpretation
Interpretation of results is largely method dependent.
When using tests that rely on antibodies, such as EMIT
assays, one should consider drug cross-reactivity. For
example, the EMIT amphetamine assay can cross-react
499
Drug Screens
with ephedrine and phenylpropanolamine, causing false-
positive results. Thus even though the theoretical
specificities of immunochemical assays are generally
excellent, all positive results encountered must be
confirmed by a secondary procedure. In fact, if potential
medicolegal specimens are routinely analyzed (that is,
employee, athletic, or forensic screening), a second
confirmatory analytical procedure must be employed.
A discussion of drug-abuse screening would not be
complete without some comment on the proper
processing of specimens, including collection,
transportation, storage, and analytical phases of the
process. Since the ramifications of this type of screening
are of concern, both analytical accuracy and specimen
integrity must be assured. Where chain-of-custody is
required, all urine collections should be witnessed,
followed by a foolproof sample-identification method.
Results from forensic and nonstandard specimens should
be interpreted with caution.
Medicolegal specimens should be properly stored
(refrigerated at 4C) and secured until time of
transportation. To assure their integrity, they must be
adequately sealed; this is best done with standard legal-
specimen tape. The number of people handling the
specimen during its transportation should be kept to a
minimum, and the sample should be delivered directly to
laboratory personnel. The assignment of an accession
number by the laboratory must be straightforward and
easy to follow through all phases of the testing procedure
(such as instrument logs, labeling of chromatogram data
sheets, and final reports). All steps must be thoroughly
documented when testing has legal ramifications.
Drug-Screening Performance Goals
Quality Control
As with all laboratory tests, the performance and
reliability of drug screens must be continuously
monitored. One can accomplish this by employing an
internal quality-control program and by participating in
external proficiency surveys, such as those conducted by
the College of American Pathologists (CAP).
Proficiency surveys enable labs to monitor screening
accuracy and allow evaluation of both the sensitivity and
specificity of the methods utilized. CAP proficiency
surveys from 2006-2007 are summarized in Table 7. The
data summary includes cannabinoids (THC-COOH),
cocaine (benzoylecgonine), amphetamine (amphetamine
and methamphetamine), opiates (heroin, opium, codeine,
morphine, and 6-acetyl morphine), and PCP
(phencyclidine). Additional information resources are
available on the CAP website, http://www.cap.org.
Labs are also required to perform internal quality control
(QC), where known control materials spiked with an
assortment of drugs are tested daily. Control materials
can be obtained commercially or can be prepared in-
house if the laboratory possesses the proper licenses for
handling controlled substances. Control samples should
be processed in the same manner as patient specimens
and should be submitted to the same rigorous analyses as
unknowns. Where appropriate, it is important to monitor
all three stages of the procedure: extraction or
preparation, screening, and confirmation. Screening test
performance can also be monitored by correlating data
with confirmation test results (e.g. GC-MS).
References
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2 Mikkelsen SL, Ash KO. Adulterants causing
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for Analytical Toxicology Programs. Cleveland:
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liquid-liquid extraction. J Anal Toxicol.
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8 Berry DJ, Grove J. Emergency toxicological
screening for drugs commonly taken in
overdose. J Chromatogr. 1973;80:205-220.
9 Faulkner WR, American Association for
Clinical Chemistry. Selected Methods of
Emergency Toxicology. Washington, DC:
AACC Press; 1986:x,102.
10 Fischer DS. A method for the rapid detection of
acute iron toxicity. Clin Chem. 1967;13:6-11.
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chromatographic screening procedure for
detecting drug abuse. Techn Bull Reg Med
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12 Blass KG, Thibert RJ, Draisey TF. A simple,
rapid thin-layer chromatographic drug
screening procedure. J Chromatogr.
1974;95:75-79.
13 Lillsunde P, Korte T. Comprehensive drug
screening in urine using solid-phase extraction
and combined TLC and GC/MS identification.
J Anal Toxicol. 1991;15:71-81.
14 Bastos ML, Jukofsky D, Mule SJ. Routine
identification of drugs of abuse in human urine.
3. Differential elution of the XAD-2 resin. J
Chromatogr. 1973;81:93-98.
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15 Mule SJ. Routine identification of drugs of
abuse in human urine. I. Application of
fluorometry, thin-layer and gas-liquid
chromatography. J Chromatogr.1971;55:255-
266.
16 Mule SJ, Bastos ML, Jukofsky D, Saffer E.
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human urine. II. Development and application
of the XAD-2 resin column method. J
Chromatogr. 1971;63:289-301.
17 Fujimoto JM, Wang RI. A method of
identifying narcotic analgesics in human urine
after therapeutic doses. Toxicol Appl
Pharmacol. 1970;16:186-193.
18 Breiter J, Helger R, Lang H. Evaluation of
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analysis of drugs in body fluids. Forensic Sci.
1976;7:131-140.
19 Deutsch DG, Bergert RJ. Evaluation of a
benchtop capillary gas chromatograph-mass
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20 Maurer HH. Screening procedures for
simultaneous detection of several drug classes
used for high throughput toxicological analyses
and doping control: a review. Comb Chem High
Throughput Screen. 2000;3:467-480.
21 Politi L, Groppi A, Polettini A. Applications of
liquid chromatography-mass spectrometry in
doping control. J Anal Toxicol. 2005;29:1-14.
22 Polettini A. Systematic toxicological analysis of
drugs and poisons in biosamples by hyphenated
chromatographic and spectroscopic techniques.
J Chromatogr B Biomed Sci Appl. 1999;733:47-
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23 Svensson JO, Andersson M, Gustavsson E,
Beck O. Electrospray LC-MS method with
solid-phase extraction for accurate
determination of morphine-, codeine-, and
ethylmorphine-glucuronides and 6-
acetylmorphine in urine. J Anal Toxicol.
2007;31:81-86.
24 Harvey MA. Point-of-care laboratory testing in
critical care. Am J Crit Care. 1999;8:72-83;
quiz 4-5.
25 Ian D, Watson RB, Hammett-Stabler C,
Nicholes B, Smith B, George S, Verstraete A,
Goldberger B. Drugs and alcohol. In: Nichols
JH, ed. NACB Laboratory Medicine Practice
Guidelines: Evidence-Based Practice for Point-
of-Care Testing. Washington, DC: National
Academy of Clinical Biochemistry; 2007.
26 Yang JM, Lewandrowski KB. Urine drugs of
abuse testing at the point-of-care: clinical
interpretation and programmatic considerations
with specific reference to the Syva Rapid Test
(SRT). Clin Chim Acta. 2001;307:27-32.
27 Moody DE, Fang WB, Andrenyak DM, Monti
KM, Jones C. A comparative evaluation of the
instant-view 5-panel test card with OnTrak
TesTcup Pro 5: comparison with gas
chromatography-mass spectrometry. J Anal
Toxicol. 2006;30:50-56.
28 George S, Braithwaite RA. Use of on-site
testing for drugs of abuse. Clin Chem.
2002;48:1639-1646.
29 Petibois C, Deleris G, Cazorla G. Perspectives
in the utilisation of Fourier-transform infrared
spectroscopy of serum in sports medicine:
health monitoring of athletes and prevention of
doping. Sports Med (Auckland, NZ).
2000;29:387-396.
30 Grootveld M, Algeo D, Silwood CJ, Blackburn
JC, Clark AD. Determination of the illicit drug
gamma-hydroxybutyrate (GHB) in human
saliva and beverages by 1H NMR analysis.
BioFactors (Oxford, England). 2006;27:121-
136.
31 Alnajjar A, Idris AM, Multzenberg M, McCord
B. Development of a capillary electrophoresis
method for the screening of human urine for
multiple drugs of abuse. J Chromatogr B Analyt
Technol Biomed Life Sci. 2007;856:62-67.
32 Wei F, Zhang M, Feng YQ. Application of
poly(methacrylic acid-ethylene glycol
dimethacrylate) monolith microextraction
coupled with capillary zone electrophoresis to
the determination of opiates in human urine.
Electrophoresis. 2006;27:1939-1948.
33 Bush DM. The U.S. Mandatory Guidelines for
Federal Workplace Drug Testing Programs:
current status and future considerations.
Forensic Sci Int. 2008;174:111-119.
34 Verstraete AG. Detection times of drugs of
abuse in blood, urine, and oral fluid. Ther Drug
Monit. 2004;26:200-205.
35 Figure adapted from:
http://www.questdiagnostics.com/employerso
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2009-03-03.
36 Moeller MR, Steinmeyer S, Kraemer T.
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37 Natelson S. Techniques in Clinical Chemistry.
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Thin-layer chromatographic screening and
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Drug Screens

Table 1. Suggested Confirmation Procedures for Screening Tests

Screen Confirmations
Spot test UV, TLC, or GC/MS
TLC UV or GC/MS
EMIT TLC, GC/MS, or UV
RIA TLC or GC/MS
GC GC/MS

Table 2. Types of Assays Used in Screening Procedures

Assay 1: Spot test

Principle: Drug or agent in specimen reacts chemically, giving specific color Advantages: Very rapid test;
indicates possible drug group present
Disadvantages: Not specific; different spot test required for each group of drugs; limited to a few drug groups
Assay 2: Ultraviolet spectroscopy
Principle: Extracted drug identified by its specific absorbance spectrum
Advantages: Inexpensive; spectrum often gives specific identification and quantitation of compound
Disadvantages: Not all drugs have suitable absorbance spectra; limited sensitivity; some degree of ambiguity
among drugs of same class; extensive sample preparation
Assay 3: Enzyme-multiplied immunoassay technique (EMIT)
Principle: Drug in the specimen competes with a conjugated drug-enzyme complex for a specific antibody;
unbound drug-enzyme conjugate is detected colorimetrically
Advantages: Rapid for single or several assays; most convenient method for detection of certain drugs
Disadvantages: Class specific; assays must be done separately for each class of drugs or individual drug; costly
Assay 4: Thin-layer chromatography
Principle: Extracted drug is separated by TLC and detected by specific chemical staining reaction or
physicochemical properties
Advantages: Can separate and detect more compounds than any other method; inexpensive
Disadvantages: Not possible to detect all drugs with one extraction or one chromatography system; detection is
subjective; long turnaround times
Assay 5: Gas chromatography (GC) and GC/mass spectrometry (MS)
Principle: Extracted drug is separated by GC and identified by retention time; with GC/MS, exact mass is used
to identify compounds.
Advantages: Can separate and detect large variety of drugs; GC/MS provides definitive identification of drugs
Disadvantages: Not possible to detect all drugs; extraction and confirmation necessary
Assay 6: Point-of-Care Tests
Principle: Variable, but typically drug in specimen interacts with conjugated antibody and is immobilized by
capture antibody for visual detection
Advantages: Rapid and easy to use
Disadvantages: Test devices are expensive; limited performance; only applicable to a subset of drugs
502

Drug Screens

Table 3. Spot Tests

Drug: Acetaminophen
Specimen required: Urine
_
Reaction: o-Cresol + acetaminophen NH4OH blue color
Test time: 15 min
Comments: Highly sensitive and relatively specific
Reference: [8]
Drug: Ethanol
Specimen required: Urine, serum
Reaction: Microdiffusion into dichromate
2K2Cr2O7 + 10H2SO4 + 3C2H5OH 2Cr2(SO4)4 + 2K2SO4 + 3CH3COOH +
11H2O + 4H+ (green to blue color)
Test time: 15 to 30 min
Comments: Good sensitivity; nonspecific for ethanol
Reference: [5]
Drug: Salicylate
Specimen required: Urine, serum
Reaction: Trinders solution: Salicylate + FeCl3 violet-colored complex
Test time: 2 min
Comments: Good specificity if serum used; good sensitivity
Reference: [37]
Drug: Carbamates (meprobamate)
Specimen required: Urine
Reaction: Furfurol + meprobamate + antimony trichloride black color on thin-layer chromatography plate
Test time: 5 minutes
Comments: Not specific for meprobamate; good sensitivity
Reference: [38]

Drug: Imipramine/desipramine
Specimen required: Urine
Reaction: Forrest reagent K2CrO3 (acidic) + imipramine green-colored complex
Test time: 2 min
Comments: Phenothiazines may interfere
Reference: [5]

Drug: Ethchlorvynol
Specimen required: Urine, serum
Reaction: Diphenylamine + ethchlorvynol in acid red color
Test time: 10 min
Comments: Good sensitivity and specificity
Reference: [9]

Drug: Phenothiazines
Specimen required: Urine
Reaction: FPN reagent (ferric chloride/perchloric acid/nitric acid) FeCl3 (acidic oxidizing agent) red- to violet-colored
complex
Test time: 2 min
Comments: Nonspecific; poor sensitivity for some phenothiazines
Reference: [5]

Drug: Iron
Specimen required: Serum
Reaction: Bathophenanthroline color change with iron (blue)
Test time: 15 minutes
Comments: Will not react at normal serum concentrations
Reference: [10]
503

Drug Screens

Table 4. EMIT Menu of Drug Tests

Urine Serum
Amphetamine Acetaminophen
Barbiturates Anticonvulsants:
Cannabinoids Carbamazepine, ethosuximide,
Cocaine phenobarbital, primidone, valproic Acid
Ethanol Antimicrobial drugs:
LSD Amikacin, gentamicin, tobramycin,
Methadone vancomycin
Methaqualone Caffeine
Opiates Cardioactive drugs:
Phencyclidine Digoxin, disopyramide, lidocaine, N-
Propoxyphene acetylprocainamide, procainamide,
Propoxyphene quinidine
Adulterates: Methotrexate
Bleach, chromate, pH, Salicylate
specific gravity, nitrates Theophylline

Table 5. Specimens of Choice in Drug Screen

Specimen: Urine
Volume required: 30 mL
Indication: Drug abuse screening; overdose screening; employment screening
Advantages: Generally easy to obtain in high volume; most drugs found in sufficient concentration to enable
identification
Disadvantages: Contains many metabolic products that may interfere with identification; parent drug may not be
present; quantitation offers little correlation with clinical effects
Specimen: Blood, serum, and plasma (tube type may depend on analyte)
Volume required: 10 mL
Indication: Overdose screening; therapeutic drug monitoring
Advantages: Parent drug present; quantitative level may assist with patient management (therapeutic and toxic
reference levels often known)
Disadvantages: Limitation of sample volume; concentration of select drugs often too low to enable detection
(especially in non-overdose situations)
Specimen: Gastric lavage or emesis
Volume required: 30 mL
Indication: Overdose screening
Advantages: Parent drug present
Disadvantages: Matrix problems (interference from foodstuffs); drugs quickly absorbed may be missed by
gastric screen; drugs not orally ingested will not be detected
504

Drug Screens

Table 6. Detection Window for Commonly Encountered Drugs of Abuse [34].

Drug/Metabolite Acute/Single Use Chronic/Heavy Use
Alcohol 0-4 hrs 6-12 hrs
Amphetamines 2-7 hrs 2-4 days
Anabolic Steroids 4-6 hrs 2-3 weeks (oral dose)
1-3 months injected dose
Barbiturates 2-4 hrs 1-4 days short-acting
(secobarbital, allobarbital)
2-3 weeks long-acting
(phenobarbital, barbital)
Benzodiazepines 2-7 hrs 4-6 weeks with long-acting
Cannabinoids 6-18 hrs Up to 30 days with chronic use
Cocaine metabolite 1-4 hrs 2-4 days
(benzoylecgonine)
Lysergic acid diethylamide (LSD) 2 hrs 1-4 days
Mescaline 1-2 hrs 2-4 days
Methadone 2 hrs 2-6 days
Methamphetamines 1-3 hrs 2-4 days
Methaqualone 3-8 hrs Up to 10 days
Methylenedioxymethamphetamine 1 hr 2-3 days
(MDMA)
Nicotine 4-6 hrs 7-14 days
Opiates (heroin, morphine, codeine) 2 hrs 2-3 days
Oxycodone 1 hr 1-2 days
Phencyclidine (PCP) 5-7 hrs 6-10 days
Propoxyphene 4-6 hrs 3-6 days
Psilocybin (mushrooms) 2 hrs 1-3 days
Rohypnol 1 hr 8 hrs
Gammahydroxybutyric acid (GHB) 1 hr 8 hrs
Tricyclic antidepressants (TCA) 8-12 hrs 2-7 days
*NOTE: Detection depends on dose, usage, physiological factors, and excretion rates. Data represent average rates.

Table 7. Summary of College of American Pathologists Proficiency Testing from 2006-2007.

DRUG CLASS
Cocaine
Amphetamine Metabolite Cannabinoid Opiate PCP

Cutoff (1000 ng/mL) (300 ng/mL) (20 ng/mL) (300 ng/mL) (25 ng/mL)
METHOD Sensitivity Sensitivity Sensitivity Sensitivity Sensitivity
CEDIA 98% 100% 100% 100% 100%
CMI 97% 99% 99% 100% 99%
EIA 98% 99% 99% 99% 100%
FIA 57%** 99% 94% 99% 100%
FPIA 99% 100% 99% 100% 99%
IC 63%** 99% 98% 99% 94%
LFI 55%** 96% 92% 98% 98%
MIA 63%** 99% 96% 100% 100%
Specificity of
All Assays 99% 100% 100% 100% 100%
Combined
*Data exclude PT samples below the cutoff and methods with different cutoff values than those listed above.
** No consensus was reached for some samples in these assays.CEDIA, cloned-enzyme donor immunoassay; CMI, colloid
metal immunoassay; EIA, enzyme immunoassay; FIA, fluorescence immunoassay; FPIA, fluorescence polarization
immunoassay; IC, immunochromatography; LFI, lateral-flow immunoassay; MIA, microparticle immunoassay.
505

Drug Screens

Figures
Drug Screen: Figure 1
A.B.

Prevalence of drug positivity in employer drug screens from January to June 2005 [35].
A, Phencyclidine-positive (PCP-positive) rate. B, Cocaine-positive rate. ND, Not determined.
506

Drug Screens

Drug Screen: Figure 2

Absorbance Spectra for A, Theophylline in 0.1 M NaOH. B, Sulfamethoxazole in 0.1 M NaOH.

Drug Screen: Figure 3

Gas chromatograms for A, Nonselective detector (flame ionization detector). B, Selective detector (nitrogen-phosphorus
detector).
507

Drug Screens

Drug Screen: Figure 4

GC-MS chromatogram of serum samples spiked with THC (2 ng/mL), OH-THC (5 ng/mL) and THC-COOH (20 ng/mL).
Section 1: m/z 313, 328, 316, 331; Section 2: m/z 313, 314, 358; Section 3: m/z 313, 357, 372, 375.

Drug Screen: Figure 5

Gas chromatographic pattern of standard solutions of amphetamines. 1,d-amphetamine; 2, phentermine; 3,

methamphetamine; 4, phenylpropanolamine; 5, pseudoephedrine; 6, ephedrine; 7, phenmetrazine. Note that nicotine has
the same retention time as phenylpropanolamine.
508
Drug Screens
Drug Screen: Figure 6
Gas chromatographic pattern of patient urine extract to confirm positive-EMIT cocaine. Note positive cocaine (peak 2) and
its methyl ester (peak 1).
Drug-Screening Methods and Procedures
Procedure: Comprehensive Drug Screen
Principle
Urine or gastric specimens are extracted with organic
solvents at a selected pH followed by analysis by thin-
layer chromatography. Selected ancillary spot tests and
enzyme immunoassays are simultaneously performed on
urine. Positive findings are confirmed by use of
appropriately chosen techniques.
Indications
The comprehensive drug screen is ordered to establish
information regarding drug usage as it may pertain to
cases of a toxicological nature, including accidental or
suicidal overdose with coma or for evaluation of
potential drug involvement in other medical
emergencies, such as seizures, jittery baby syndrome, or
drug interactions.
Reagents
All reagents should be of chromatographic
grade.
1. Trinders solution: Fe(NO3)3, 100 mmol/L;
HgCl2, 14.7 mmol/L; HCl, 120 mmol/L. Add 4 g of
ferric nitrate and 4 g of mercuric chloride to a 100 mL
volumetric flask. Dissolve salts in 12 mL of 1 M
hydrochloric acid. Dilute to mark with distilled water.
Store at room temperature. This is stable for 6 months.
2. Potassium dichromate (3.4 mmol/L). Add 1 g
of potassium dichromate to a 1 L volumetric flask.
Dissolve salt in 500 mL of distilled water. Add 0.1 g of
silver nitrate to the flask, and then slowly, with stirring,
add 500 mL of concentrated sulfuric acid. Store in a
brown bottle at room temperature. This is stable for 1
year at room temperature.
3. o-Cresol reagent (0.096 mol/L). Add 10 mL
of o-cresol to a 1 L volumetric flask, and dilute to mark
with distilled water. Store at room temperature. This is
stable for 1 year at room temperature.
4. Forrest reagent
Nitric acid (7.8 mol/L). Add 50 mL of
concentrated nitric acid to 50 mL of distilled water.
Perchloric acid (1.85 mol/L). Add 20 mL of 70%
perchloric acid to 50 mL of distilled water. Allow to
cool, and bring volume to 100 mL.
Sulfuric acid, 12 N (6 mol/L). Add 30 mL of
concentrated sulfuric acid slowly, with constant stirring,
to 50 mL of distilled water. Allow to cool, and bring
volume to 100 mL.
Potassium dichromate, 2 g/L (6.8 mmol/L). Add 200 mg
of potassium dichromate to 80 mL of distilled water.
Dissolve, and bring to a final volume of 100 mL.
Mix 100 mL of each of the above reagents together. This
solution is stable for 1 year at room temperature when
stored in an opaque (amber) glass container.
5. FPN reagent (ferric chloride/perchloric
acid/nitric acid)
Nitric acid (7.8 mol/L). See above, under Forrest
reagent.
Perchloric acid (1.85 mol/L). See above, under Forrest
reagent.
Ferric chloride (0.308 mol/L). Dissolve 5 g of ferric
chloride in 50 mL of distilled water in a volumetric
flask. Bring to final volume of 100 mL.
Mix these solutions in the following proportions: 1 part
ferric chloride, 9 parts perchloric acid, and 10 parts nitric
509
Drug Screens
acid. This is stable for 1 year at room temperature when
stored in an opaque (amber) glass container.
6. Concentrated hydrochloric acid.
7. Ammonium hydroxide, 4 mol/L. Dilute 284
mL of concentrated ammonium hydroxide to 1 L with
distilled water. This is stable for 2 years at room
temperature.
8. EMIT d.a.u. (drug-of-abuse urine) opiate
and benzodiazepine assay kits. These kits are usually
stable for 1 year before opening. Follow manufacturers
recommendations for storage and use.
9. Extraction buffer. Prepare a saturated
ammonium chloride solution by dissolving 600 g in 1 L
of hot, distilled water. Cool, and add concentrated
ammonium hydroxide to adjust the pH to 9.5. Store at
room temperature. This is stable for 6 months.
10. Extraction solvent. Prepare a mixture of
dichloromethane and isopropanol (90:10 by volume).
Store at room temperature. Prepare weekly.
11. Methanolic 0.1 M hydrochloric acid. Add 0.9
mL of concentrated hydrochloric acid to a 100 mL
volumetric flask. Dilute to mark with methanol. Store at
room temperature. This is stable for 1 year.
12. Reconstitution solvent. Prepare a 1:1 (v/v)
mixture of dichloromethane and methanol. Store at room
temperature. This is stable for 1 year.
13. Thin-layer chromatographic developing
solution. Mix 85 mL of ethyl acetate, 10 mL of
methanol, and 5 mL of ammonium hydroxide. Prepare
fresh for each development.
14. Thin-layer chromatographic sprays
Ninhydrin (5.61 mmol/L). In a 100-mL volumetric flask,
dissolve 100 mg of 1,2,3-indantrione in 5 mL of acetone,
and dilute to mark with acetone. Prepare fresh daily.
Diphenylcarbazone (2 mmol/L). In a 100-mL volumetric
flask, dissolve 10 mg of diphenylcarbazone in 5 mL of
acetone, and dilute to mark with a 1:1 acetone-water
solution. This is stable for 1 month at room temperature.
Mercuric sulfate (11.5 mmol/L). Add 0.5 g of mercuric
oxide to 20 mL of concentrated sulfuric acid. Add this
acid solution slowly to 150 mL of distilled water, and
bring to a final volume of 200 mL. This is stable for 6
months at room temperature.
15. Iodoplatinate
Stock solution (0.38 mol/L). Dissolve 10 g of platinum
chloride in 100 mL of distilled water. refrigerate at 4C.
This is stable for 1 year.
Working solution. Add 5 mL of stock solution to 3 g of
potassium iodide in 100 mL of distilled water. Dilute
this mixture to 125 mL with distilled water, and then
dilute this with an equal volume of methanol (final
volume 250 mL). Refrigerate at 4C. This is stable for 6
months.
Dragendorff reagent. Dissolve 1.3 g of bismuth
subnitrate in a solution composed of 60 mL of distilled
water and 15 mL of glacial acetic acid. Dissolve 12 g of
potassium iodide in 30 mL of distilled water. Combine
the two solutions. Dilute this mixture with 100 mL of
distilled water and 25 mL of glacial acetic acid.
Refrigerate at 4C. This is stable for 6 months.
16. Standards
Ethanol (789 mg/L, 17.15 mmol/L). Pipet 0.1 mL of
absolute ethanol into a 100-mL volumetric flask. Dilute
to mark with distilled water. Store at 4C. This is stable
for 2 months.
Salicylate standard (100 mg/L, 724 mol/L). Dissolve
10 mg of salicylic acid (USP grade) in 0.5 mL of
methanol in a 100-mL volumetric flask, and dilute to
100 mL with distilled water. Store at 4C. This is stable
for 6 months.
Acetaminophen urine control (100 g/mL, 0.66
mol/mL). Add 10 mg of acetaminophen to a 100-mL
volumetric flask. Dissolve in 1 mL of methanol. Dilute
to mark with urine previously noted to be free of the
drug. Refrigerate at 4C. This is stable for 6 months.
Urine drug control. Select a suitable commercially
available control or prepare one using the following
procedure:
1. Collect approximately 2 L of drug-free urine (urine
previously noted to be negative for all drugs through
testing). To clarify, filter through Whatman No. 1 filter
paper.
2. Prepare a stock drug standard by adding 4 mg each of
phenobarbital, secobarbital, morphine, codeine,
meperidine, methyprylon, and oxazepam and 10 mg each
of d-amphetamine and methamphetamine to a 10-mL
volumetric flask. Dissolve the drugs in methanol, and
dilute to mark with methanol.
3. Transfer the entire contents of the drug standard flask
to a 2-L volumetric flask. Rinse the flask several times
with distilled water, and add rinses to the 2 L flask.
Dilute to the 2-L mark with the urine pool. Mix well.
4. Divide into 20-mL aliquots, and store in plastic vials.
5. Store at 20C until used. This is stable for 1 year.
6. Control contains 2 g/mL of each drug except the
amphetamines, which have concentrations of 5 g/mL.
17. Thin-layer chromatographic reference
standard (1 g/mL). Add 10 mg each of
propoxyphene, methadone, chlorpromazine, meperidine,
quinine, phenobarbital, phenylpropanolamine, and
morphine to a 10-mL volumetric flask. Dissolve in
methanol, and dilute to mark with methanol. Refrigerate
at 4C. This is stable for 6 months.
18. Individual drug standards (1 g/mL).
Prepare in the same manner as previous standard with
drug of choice. This is stable for 6 months at 4C.
Assay
Equipment:
Porcelain spot-test plates
Conway microdiffusion dish
Teflon 125-mL volume separatory funnels (Fisher
Scientific)
60-mL glass centrifuge tubes
Sample concentrator/evaporator (Brinkmann, Buchii
Rotavap, or other convenient evaporation system)
Thin-layer chromatographic apparatus
Spotting platen
20 20-cm glass, silica-gel 60 F-254 plates
Developing tank to accommodate 20 20-cm plates
Chromatographic spray cans
Air blower
510
Drug Screens
Short (254 nm)/long (366 nm) combination
ultraviolet lamp
Procedure: Comprehensive Drug Screen
(Alternative)
Ancillary Tests
Salicylate spot test. Add 3 drops of salicylate control or
patient specimen to a porcelain dish. Serum is the first
specimen of choice, followed by urine. Gastric lavage or
emesis is unacceptable without special treatment. (If
urine is used, boil the specimen first to remove diacetic
acid, a potentially interfering substance.) Add 3 drops of
Trinders reagent. A violet color indicates the presence
of salicylate.
Alcohol spot test. To the center well of a
microdiffusion dish, add 0.5 mL of potassium
dichromate reagent. To the outer ring, add 0.5 mL of
alcohol control or patient specimen (urine or serum).
Lightly grease the outer ring of the microdiffusion dish
lid with vacuum grease. Cover and allow to stand at
room temperature. The dichromate solution will change
from a yellow to a light green or blue color in the
presence of methanol, ethanol, or isopropanol.
Acetaminophen spot test. To 1 mL of patient or
control urine, add 1 mL of concentrated hydrochloric
acid. Heat in a boiling water bath for 10 min. Dilute 0.1
mL of this sample with 0.9 mL of o-cresol reagent. Add
2 mL of 4 M ammonium hydroxide. Blue color indicates
presence of acetaminophen.
EMIT II Plus d.a.u. For amphetamines, barbiturates,
benzodiazepines, cannabinoids (THC), cocaine, opiates,
phencyclidine (PCP), methadone, methaqualone, and
propoxyphene assays. Use as described by
manufacturer. Cross-reactivity of some compounds is
indicated in package inserts.
Extraction of Urine
1. Process the drug-screen control along with
patient specimen.
2. To a 125-mL separatory funnel, add 20 mL of
urine and 2 mL of ammonium chlorideammonium
hydroxide buffer, and mix.
3. Add 30 mL of extraction solvent, and extract
for 10 min.
4. Allow layers to separate. Collect bottom solvent
layer in a 50-mL glass centrifuge tube, and centrifuge for
5 min at 500 g at room temperature.
5. With a glass stirring rod, break emulsions that
may have formed by aggregation, and re-centrifuge.
Aspirate off top aqueous layer and discard. Filter solvent
through filter paper (P5, Fisher Scientific, qualitative)
into evaporation flask.
6. Add 2 drops of methanolic 0.1 M hydrochloric
acid, and evaporate to dryness at 50C under nitrogen.
7. Using a Pasteur pipet, reconstitute with 10
drops (approximately 150 L) of methylene dichloride
methanol solution, being sure to rinse down the sides of
the evaporation flask.
Thin-Layer Chromatography (TLC)
TLC spotting. Use a conditioned (preheated for 1 hr at
70C) TLC plate. Using a spotting platen, mark the spot
origin at 2 cm from the bottom of the plate. From this
mark, measure 15 cm upward, and draw the end line.
Apply approximately 1 L of reference standard in the 3,
10, and 17 positions on the plate. Use positions 9, 11,
and 12 to spot drug standards of interest (drugs indicated
by history). Apply extracted control and patient
specimens in open positions. Apply approximately 3 L
at a time, and spot 10 times, allowing the applied spots
to dry between applications.
Plate development. Prepare the TLC development
solvent, and pour into tank lined with 20 20-cm filter
paper on both sides. Position the plate or plates in the
development tank, and allow time for migration to
proceed to the end line (approximately 40 min).
Detection. Air-dry the plate. Observe the plate under a
long-wave (366 nm) ultraviolet lamp. Mark any
fluorescing compound by lightly tracing the spot with a
pencil. Mark subsequent positive spots with a pencil.
Place under short (254 nm) light, and look for any
absorbing spots. Spray with Ninhydrin, irradiate with
long-wave ultraviolet radiation for 5 min, and then heat
plate at 70C for 5 min. Amines, such as
phenylpropanolamine and d-amphetamine, will stain
pink. Spray with diphenylcarbazone, followed by the
mercuric sulfate spray. Observe the blue-violet colors of
barbiturates, phenytoin, glutethimide, and ethchlorvynol.
Re-spray with diphenylcarbazone, and heat plate at 70C
for 10 min. The pink, red, and violet colors of
metabolites of phenothiazine drugs will appear. Observe
under long-wave ultraviolet lamp. Observe the yellow
fluorescence of the benzodiazepine drugs and their
metabolites. Spray with iodoplatinate and Dragendorff
reagents. Most nitrogenous basic drugs will stain dark.
Soak the finished plate in water for 30 seconds, remove,
and dry with a jet of hot air. Observe the chalk-white
colors of methyprylon and carbamates such as
meprobamate. Rf values (distance of spot migration
divided by distance of developing solvent migration),
spray reactions, and ultraviolet detections of unknowns
should be compared to known standards.
Confirmation
The presence of drugs indicated as being positive by the
thin-layer chromatographic or ancillary test screening
methods must be confirmed by a second analytical
procedure different from that used to screen for the
compound (see Table 1, suggested confirmation
procedures for screening tests). Occasionally, when
serum is provided and when indicated, as in overdose
cases, the quantitation of the suspected drug in serum is
the confirmatory step in the method. At other times,
additional tests must be performed on the urine to
confirm the presence of the drug.
511

Drug Screens

Drugs Which Can Be Identified by a Comprehensive Methyprylon (Noludar)*

ScreenListed Alphabetically Morphine
Nortriptyline (Aventyl)*
Orphenadrine
Acetaminophen (Tylenol)*
Oxycodone
Amitriptyline (Elavil)*
Pentazocine (Talwin)
Amobarbital*
Pentobarbital*
Amoxapine (Asendin)*
Phencyclidine (PCP)
Amphetamine
Pheniramine
Atropine
Phenmetrazine (Preludin)
Benzocaine
Phenobarbital*
Benzodiazepinesincludes metabolites of:
Phenothiazinesincludes metabolites of:
Chlordiazepoxide (Librium)*
Trifluoperazine (Stelazine)
Chlorazepate (Tranxene)*
Propiomazine (Largon)
Diazepam (Valium)*
Perphenazine (Trilafon)
Oxazepam (Serax)*
Prochlorperazine (Compazine)
Benzotropine (Cogentin)
Promazine (Sparine)
Benzphetamine (Didrex)
Chlorpromazine (Thorazine)
Brompheniramine
Thioridazine (Mellaril)
Butalbital*
Phentermine (Ionamin)
Caffeine
Phenylpropanolamine
Cannabinoids
Phenyltoloxamine
Carbamazepine (Tegretol)*
Phenytoin (Dilantin)*
Carbromal
Primidone (Mysoline)*
Carisoprodol (Soma)
Procainamide (Pronestyl)*
Chlorpheniramine
Procaine
Cimetidine/ranitidine
Promethazine (Phenergan)
Cocaine
Propranolol (Inderal)
Codeine
Propoxyphene (Darvon)
Cyclobenzaprine (Flexeril)
Pyrilamine
Desipramine (Norpramine)*
Quinidine/quinine*
Dimenhydrinate (Dramamine)
Quinine
Diphenhydramine (Benadryl)
Salicylate*
Disopyramide (Norpace)
Secobarbital*
Doxepine (Sinequan)*
Strychnine
Doxylamine
Theophylline (Aminophylline)
Ephedrine/pseudoephedrine
Trimethoprim
Ethanol*
Trimipramine
Ethchlorvynol (Placidyl)*
Trip-(Pyribenzamine)
Ethinamate (Valmid)
Tripelennamine
Fluoxetine (Prozac)
Triprolidine
Flurazepam (Dalmane)
Verapamil
Glutethimide (Doriden)*
Haloperidol (Haldol)
*Quantitative assays available.
Hydrocodone
Not available in stat screen.
Hydromorphone (Dilaudid)
Not routinely reported
Hydroxyzine (Vistaril)
Imipramine (Tofranil)*
Isopropanol*
Lidocaine (Xylocaine)*
Loxapine (Loxitane)
Maprotiline (Ludiomil)
Meperidine (Demerol)
Mepivacaine
Meprobamate (Miltown)*
Methadone
Methamphetamine
Methanol*
Methapyrilene
Methaqualone (Quaalude)*
Methocarbamol (Robaxin)
Methylphenidate (Ritalin)
512

Drug Screens

Drugs Which Can Be Identified by a Comprehensive NARCOTIC ANALGESICS

ScreenListed by Class Codeine
Hydrocodone
Hydromorphone (Dilaudid)
ALCOHOLS
Meperidine (Demerol)
Ethanol*
Methadone
Isopropanol*
Morphine
Methanol*
Oxycodone
Pentazocine (Talwin)
ANTICONVULSANTS
OTHER ANALGESICS
Carbamazepine (Tegretol)*
Acetaminophen (Tylenol)*
Phenobarbital*
Benzocaine
Phenytoin (Dilantin)*
Mepivacaine
Primidone (Mysoline)*
Procaine
ANTIDEPRESSANTS
Propoxyphene (Darvon)
Amitriptyline (Elavil)*
Salicylate*
Amoxapine (Asendin)*
STIMULANTS
Cyclobenzaprine (Flexeril)
Amphetamine
Desipramine (Norpramin)*
Benzphetamine (Didrex)
Doxepin (Sinequan)*
Caffeine
Fluoxetine (Prozac)
Cocaine
Imipramine (Tofranil)*
Ephedrine/pseudoephedrine
Loxapine (Loxitane)
Methamphetamine
Nortriptyline (Aventyl)*
Methylphenidate (Ritalin)
Maprotiline (Ludiomil)
Phencyclidine (PCP)
Trimipramine
Phenmetrazine (Preludin)
ANTIHISTAMINES
Phentermine (Ionamin)
Brompheniramine
Phenylpropanolamine
Chlorpheniramine
Strychnine
Dimenhydrinate (Dramamine)
TRANQUILIZERS
Diphenhydramine (Benadryl)
Benzodiazepinesincludes metabolites:
Doxylamine
Chlorazepate (Tranxene)*
Methapyrilene
Chlodiazepoxide (Librium)*
Orphenadrine
Diazepam (Valium)*
Pheniramine
Oxazepam (Serax)*
Promethazine (Phenergan)
Carisoprodol (Soma)
Phenyltoloxamine
Haloperidol (Haldol)
Pyrilamine
Hydroxyzine (Vistaril)
Triprolidine
Meprobamate (Miltown)*
Tripelennamine (Pyribenzamine)
Methocarbamol (Robaxin)
CARDIAC DEPRESSANTS AND BETA
Phenothiazinesincludes metabolites:
BLOCKERS
Chlorpromazine (Thorazine)
Disopyramide (Norpace)
Perphenazine (Trilafon)
Lidocaine (Xylocaine)*
Prochlorperazine (Compazine)
Procainamide (Pronestyl)*
Promazine (Sparine)
Propanolol (Inderal)
Propiomazine (Largon)
Quinidine/Quinine*
Thioridazine (Mellaril)
Verapamil
Trifluoperazine (Stelazine)
HYPNOTICSSEDATIVES
MISCELLANEOUS
Amobarbital*
Atropine
Butalbital*
Benztropine (Cogentin)
Pentobarbital*
Cannabinoids
Secobarbital*
Cimetidine/ranitidine
Carbromal
Quinine
Ethchlorvynol (Placidyl)*
Theophylline (Aminophylline)
Ethinamate (Valmid)
Trimethoprim
Flurazepam (Dalmane)
Glutethimide (Doriden)*
*Quantitative assays available.
Methaqualone (Quaalude)*
Not available in STAT screen.
Methyprylon (Noludar)*
Not routinely reported
513

Drug Screens

Procedure: Stimulant Panel (Urine) assay. Report in quotation marks those test samples
measuring less than the EMIT low calibrator for that
Principle assay. Proceed to the confirmatory techniques listed
Various drugs of interest are first screened using enzyme below if positive results are obtained from the EMIT
immunoassay, and presumptive-positive cases are assay.
confirmed by either gas or thin-layer chromatographic
methods. Amphetamine Confirmation by Gas Chromatography
Indications Equipment: Gas chromatograph with flame
The stimulant panel is used to rule out the following ionization detector, utilizing a 6-foot, 2-mm inner
drugs as the cause of hyperactivity, psychosis, or other diameter glass column packed with GP 10% Apiezon
stimulated states of consciousness: cocaine, L/2% KOH on 80/100 Chromosorb W AW column of 2-
phencyclidine (PCP), d-amphetamine, mm inner diameter (Supelco, Inc., Bellefonte, PA).
methamphetamine, phenmetrazine, phentermine, 1. Sample preparation by Toxi-Lab tube extraction
phenylpropanolamine, ephedrine. a. Shake Toxi-Lab tube A to mix
The schema of analysis is as follows: contents.
b. Add 5 mL of urine, and mix for 5 min.
PCP c. Centrifuge for 2 min at 1000 g.
Cocaine Amphetamine d. Transfer half of the supernatant to a
Screen EMIT EMIT EMIT Reacti-Vial, add 2 drops of 0.1 mol of HCl per
Confirmation 1 GLC (OV-17) GC (OV-17) liter of methanol, and evaporate to dryness.
GC (Apiezon)
Confirmation 2 Toxi-Lab or Toxi-Lab equivalent for e. Save the other half of the supernatant
each drug for further confirmation if necessary.
f. Reconstitute the dried residue from
EMIT is the enzyme immunoassay d.a.u. (drug of abuse step (d) in 20 L of methanol, and vortex mix
urine) kit sold by Dade-Behring (Syva), GLC is gas- the vial.
liquid chromatography utilizing 3% OV-17 and Apiezon g. Inject 1 L of the mixed standard into
column types, and Toxi-Lab is a drug detection system the gas chromatograph.
sold by the analytical systems division of Marion h. Allow the column to re-equilibrate
Laboratories, Inc., Laguna Hills, CA. after the run.
Reagents i. Inject 4 L of the reconstituted
1. EMIT II Plus (Syva, Dade Behring) reagents. specimen.
Reagents for cocaine, phencyclidine (PCP), and 2. Chromatographic parameters
amphetamine, including negative controls, and low a. Column temperature program, 130C
calibrator. to 210C, 10C per minute
2. Toxi-Lab reagents, including A tubes b. Injection port temperature, 250C
(Marion Labs). c. Detector temperature, 270C
3. Methanol. Chromatoquality (Fisher Scientific). d. Nitrogen flow, 30 mL/min
4. 0.1M HCl in methanol. Prepare by diluting e. Attenuation, 8
8.5 mL of concentrated HCl to 1 L with methanol. f. Range, 10-11 amp/mV
5. Gas chromatography amphetamine reference g. Chart speed, 2 cm/min
standards 1 mg/mL. Add 5 mg of each of the h. Sample run time, 8 min
following to a 5-mL volumetric flask: d-amphetamine, 3. Confirm by comparing relative retention times
methamphetamine, phentermine, phenmetrazine, of unknown peaks to peaks from standard run. Drug
ephedrine, phenylpropanolamine, and nicotine. Bring to Screen: Figure 5 shows a typical separation of
volume with methanol. Store at 4C. Stable for 3 amphetamine standards.
months. 4. Perform Toxi-Lab TLC plate development for
6. Gas chromatography PCP, cocaine, and stimulants as described by the manufacturer for
benzoylecgonine standards 1 mg/mL. Add 5 mg of secondary confirmation.
each of the following to a 5-mL volumetric flask:
cocaine, benzoylecgonine, and phencyclidine. Bring to Cocaine Confirmation by Gas Chromatography
volume with methanol. Stable for 1 month at 4C. Equipment: Same GC detector and column as
Note. A primary cocaine metabolite, the ecgonine above but column packing is 3% OV-17 on 100/120
methyl ester, is presently not routinely available. It is Supelcoport.
suggested that urines known to have tested positive for 1. Sample preparation by Toxi-Lab tube extraction
cocaine be used as a source of this metabolite. a. Shake Toxi-Lab tube A to mix
Assay contents.
EMIT Screening Procedure b. Add 5 mL of urine, and mix for 5 min.
Perform the EMIT PCP, cocaine, and c. Centrifuge for 2 min at 1000 g.
amphetamine screening procedures according to d. Transfer all the upper fraction to a
manufacturers directions. Phentermine, phenmetrazine, Reacti-Vial.
ephedrine, and phenylpropanolamine are detected, as are
other amphetamines with the amphetamine EMIT d.a.u.
514

Drug Screens

e. Extract the remaining bottom layer for l. Allow the column to re-equilibrate
benzoylecgonine by adding 2 mL of after the run.
dichloromethane. m. Inject 1 L of the unknown extract,
f. Mix for 2 min. and initiate run program.
g. Centrifuge for 2 min at 1000 g. 2. Chromatographic parameters
h. Remove the colored aqueous layer, a. Column temperature program, 150C
and transfer the dichloromethane to the same to 270C, 15C per minute
evaporation vial as in step (d). b. Injection port temperature, 250C
i. Evaporate to dryness at 50C under a c. Detector temperature, 250C
gentle stream of nitrogen. d. Nitrogen carrier flow, 20 mL/min
j. Reconstitute in 20 L of methanol, and e. Attentuation, 8
vortex mix. f. Chart speed, 1.2 cm/min
k. Inject 1 L of the GC cocaine standard g. Sample run time, 25 min
into the gas chromatograph, and follow run 3. Confirm by comparing retention times of
program. unknown peaks to peaks from standard run. Drug
l. Allow the column to re-equilibrate Screen: Figure 6 shows a patient urine positive for
after the run. cocaine.
m. Inject 1 L of the unknown extract, 4. Perform Toxi-Lab analysis for cocaine as
and initiate run program. described by the manufacturer for secondary
2. Chromatographic parameters confirmation.
a. Column temperature program, 150C
to 270C, 15C per min Procedure: Drug Abuse Screen
b. Injection port temperature, 250C Principle
c. Detector temperature, 250C Drugs of interest are first screened by enzyme
d. Nitrogen carrier flow, 20 mL/min immunoassay, with presumptive positive cases being
e. Attenuation, 8 confirmed by TLC.
f. Chart speed, 1.2 cm/min Indications and Comments
g. Sample run time, 25 min The screen is used to rule out the presence of select
3. Confirm by comparing relative retention times drugs of abuse as part of employee-screening programs,
of unknown peaks to peaks from standard run. Drug to monitor clients in drug-rehabilitation programs, or for
Screen: Figure 6 shows a positive urine cocaine any clinical situation requiring knowledge of drug usage.
chromatogram.
4. Perform Toxi-Lab analysis for cocaine as As with the comprehensive drug screen, the drugs
described by the manufacturer for secondary chosen to be included in this screen will be selected
confirmation. based upon the current prevalence of their abuse, along
with technical and economic considerations. The
PCP Confirmation by Gas Chromatography constituents of the screen will be continuously changing,
Equipment: as above for Cocaine just as drug-usage patterns are. When a limited approach
1. Sample preparation by Toxi-Lab extraction to drug-abuse screening is the goal, as presented here,
a. Shake Toxi-Lab tube A to mix the participation by commercial suppliers of drug assay
contents. kits is essential for a successful program.
b. Add 5 mL of urine, and mix for 5 min. Reagents and supplies
c. Centrifuge for 2 min at 1000 g. 1. EMIT d.a.u. assay kits. (Dade-Behring)
d. Transfer the entire upper fraction to a 2. Toxi-Lab A and B drug detection systems
Reacti-Vial. and cannabinoid (THC) assay kits. (Analytical
e. Extract the remaining bottom layer for Systems, Inc., Laguna Hills, CA)
benzoylecgonine by adding 2 mL of Assay
dichloromethane. 1. Perform the following EMIT d.a.u. enzyme
f. Mix for 2 min. immunoassays as described by the manufacturer:
g. Centrifuge for 2 min at 1000 g. amphetamines, barbiturates, benzodiazepines,
h. Remove the colored aqueous cannabinoids (THC), cocaine, opiates, phencyclidine
layer, and transfer the dichloromethane (PCP), methadone, methaqualone, and propoxyphene.
to the same evaporation vial as in step 2. If found to be presumptively positive by
(d). immunoassay, confirm with the appropriate Toxi-Lab
i. Evaporate to dryness at 50C under a procedure as described by the manufacturer.
gentle stream of nitrogen. a. Positive amphetamines. Confirm using
j. Reconstitute in 20 L of methanol, and the Toxi-Lab Sympathomimetic Amines
vortex mix. (Amphetamine) Differentiation special
k. Inject 1 L of the GC cocaine procedure using Toxi-Lab A system.
standard into the gas chromatograph, b. Positive barbiturates. Confirm using
and follow run program. the Toxi-Lab B system.
515

Drug Screens

c. Benzodiazepines. Confirm using the

Toxi-Lab Benzodiazepines: Hydrolysis
special procedure using Toxi-Tubes B and
Toxi-Lab A system.
d. Cocaine. Confirm using conventional
Toxi-Lab A system for basic drugs and the
Benzoylecgonine: Extraction and Detection
special procedure.
e. Phencyclidine. Confirm using the
Toxi-Lab Phencyclidine: Confirmation by
Remigration special procedure.
f. Methaqualone, methadone, and
propoxyphene. Confirm using the Toxi-Lab A
system. For methaqualone, also use the
Methaqualone: Confirmation with Sodium
Borohydride special procedure.
g. Opiates. Confirm using the
conventional Toxi-Lab A system and the
Morphine and Codeine: Confirmation using
Sodium Hydroxide special procedure. In
addition, the special procedure Morphine:
Hydrolysis of the Glucuronide Conjugate may
be required.
h. Cannabinoids. Confirm using the
Toxi-Lab Cannabinoid (THC) assay system.
516
Estradiol
Estradiol
Greg Ward
Name: Estradiol, E2 , estra-1,3,5(10)-triene-3,17-diol
Clinical significance:
Molecular weight: 272.39 D
Structure: C18H24O2
Chemical Class: Steroid
Refer to Chapter 44, Pregnancy, and Chapter 50, The Gonads, in the 5th edition of Clinical Chemistry: Theory, Analysis,
Correlation.
Principles of Analysis and Current Usage
Early methods of estradiol analysis used the competitive-
binding assay format with tritiated estradiol as the
radiolabel. Estradiol is bound to protein, and these
assays used organic extraction with diethyl ether prior to
assay of total estradiol levels. Early commercial
immunoassay kits achieved separation of free from
bound with dextran-coated charcoal [1]. Later
immunoassays used estradiol-6-(o-
carboxymethyl)oxime-[125I]-histamine as tracer. Organic
extraction was replaced by chemical agents such as
danazol, dihydrotestosterone, mesterolone, or
anilinonaphthalene sulfonic acid to displace estradiol
from its binding proteins [2]. These direct estradiol
immunoassays used either double-antibody techniques or
antibody bound to solid phase for separation of free
tracer from antibody-bound tracer. Further SHBG
interference [3] and binding of iodinated tracers to
SHBG [4] has been reported in direct assays, leading to
potentially discordant results.
Direct radioisotopic methods have been replaced with
more sensitive, direct, automated non-isotopic
proprietary assays for total estradiol. One example is the
Vitros immunodiagnostic method by the Ortho Clinical
Diagnostics Division of Johnson and Johnson
(Rochester, NY), which uses a competitive
immunoassay technique. The estradiol in the sample
competes with estradiol labeled with horseradish
peroxidase for biotinylated antibody to estradiol. The
antibody complex is captured by streptavidin bound to
plastic wells. The peroxidase is used to catalyze the
oxidation of a luminol derivative with the production of
light. The Siemens (Ciba Corning Bayer Corporation)
Advia Centaur or ACS estradiol procedure is a
competitive chemiluminescent immunoassay. Estradiol
in the patient serum binds to an acridinium ester (AE)-
labeled mouse monoclonal antibody (Lite Reagent).
i
Estradiol (E2)
Previous and current authors of this method:
First edition: Not done
Methods book: Not done
Second edition: Not done
Third edition: Not done
Fourth edition: Unidentified author
Fifth edition: Greg Ward
Unbound antibody binds to an estradiol derivative
coupled to paramagnetic particles (Solid Phase). The
acridinium ester remaining on the solid phase is reacted
chemically to release light. An inverse relationship exists
between the amount of estradiol in the patient sample
and the amount of relative light units (RLUs) detected by
the ACS:180 system.
Comparison of eight automated, non-isotopic estradiol
assays with an isotope-dilution gas chromatography
mass spectrometry (ID-GC-MS) reference method
showed variable agreement, with slopes of 0.87 to 1.20
observed. Further, the interassay precision at an estradiol
concentration of 18 pg/mL (66 pmol/L) varied from
6.9% to 42.6% [5]. Liquid chromatographytandem
mass spectrometry (LC-MS/MS) demonstrated poor
agreement with direct immunoassay and observed
interference in immunoassays at low estradiol
concentrations [6]. The functional sensitivity of this LC-
MS/MS method was 6.3 pg/mL (23.2 pmol/L). In
general, current direct estradiol immunoassays have a
wide measuring range, with the desired aim of a single
assay for clinical application in menopause (low levels)
and assisted reproduction (high levels). This analytical
tradeoff has not been successful, and as a result, the
functional sensitivity of these immunoassays is
inadequate for use at low concentrations. Future
estradiol immunoassay design may require two reagent
formulations with different antibody concentrations,
providing for precise measurement of estradiol at either
low- or high-concentration ranges. It has been suggested
that extraction of samples prior to immunoassay allows
for the measurement of low estradiol levels [7,8].
Overestimation of estradiol levels at concentrations less
than 30 ng/L (110 pmol/L) has been observed for all
types of immunoassay with indirect (extraction)
immunoassays, correlating better with ID-GC-MS than
direct immunoassays [9]. The need to measure estradiol
in clinical practice for men, children, and
postmenopausal women with either fracture risk or
breast cancer risk, together with the inaccuracy and
imprecision of immunoassays at low estradiol levels, has
led to the development of MS methods which can
measure low levels of estradiol using low sample
volumes [10,11,12,13,14]
517
Estradiol
Reference and Preferred Methods
There are currently three reference methods for estradiol
[15,16,17]. Of the 1255 laboratories reporting estradiol
levels in the 2007 College of American Pathologists
(CAP) Survey Participant Summary Report,
approximately 98.4% used direct non-isotopic
immunoassay. Two laboratories used mass spectrometry,
and 18 (1.5%) used radioimmunoassay.
The problems with estradiol assays are the same as those
described for testosterone assays, with repeated calls for
standardization [18,19]. The availability of reference
materials linked to reference methods will allow for
appropriate standardization of methods by manufacturers
and laboratories. The increasing clinical requirement for
measurement of low estradiol levels will see further
development and utilization of MS methods in routine
laboratories [20].
Specimen
The choice of serum or plasma is dependent in part on
the manufacturer of the test system. For example, for the
ACS 180 system, serum is the preferred specimen, but
for the Vitros system, serum, EDTA, or heparinized
plasma are all acceptable. The sample should be
refrigerated if not analyzed immediately. Exposure to air
should be minimized. Samples may be stored at 20C
but not frozen and thawed more than once [21]. It is
important that information relating to the clinical status
of the woman with respect to the luteal cycle accompany
the specimen.
Reference Intervals for Adults, Reported in pmol/L and pg/mL
Patient Vitros Vitros ACS 180
pmol/L pg/mL pg/mL
Female follicular 97-592 26-161 28-172
Pre-ovulary peak 685-1404 187-382
Female luteal 120-738 32-201 55-246
Postmenopausal 19.7-141 5.4-38 13-93
Males 19.7-242 5.4-66 21-76
Interpretation
The adrenals, testes, ovaries, and placenta all produce
estradiol. In serum or plasma, 1% to 3% is nonprotein
bound, 40% is bound to sex-hormone-binding globulin
(SHBG), and the remainder is bound to albumin [2]. The
principal and most potent estrogen secreted by the ovary
is estradiol-17. Estradiol promotes the development of
the female secondary sexual characteristics, uterine
growth, thickening of the vaginal mucosa, thinning of
the cervical mucus, and development of the ductal
system of the breast.
Measuring the circulating level of estradiol is important
for assessing ovarian function and monitoring follicular
development for assisted-reproduction protocols [27]. In
normal, nonpregnant females, estradiol is secreted
mainly by the combined function of the theca and
granulosa cells of the developing follicle and the corpus
It has been recommended that preanalytical factors and
procedures, both technical and biological, which
contribute to error in the measurement of estradiol be
addressed and standardized prior to method
harmonization and development of reference intervals
[22].
Interferences
Specimens collected in glass gel-separator tubes have
been shown to have significantly lower results compared
with specimens collected in plain evacuated tubes [23].
Direct assays have been reported to yield incorrect
results in neonates [24]. Heterophile antibodies such as
human anti-mouse antibody can interfere in many
immunoassays.
Samples from patients receiving estrogen replacement
therapy, including Premarin, may yield spurious
estradiol results because this replacement therapy can
suppress estradiol release. Further cross-reactivity of
exogenous estrogens in estradiol assays is variable.
Unconjugated estriol has been reported to interfere in
some estradiol immunoassays [25].
Estradiol Reference Interval
Early discussions of estradiol reference intervals
considered total estradiol composed of the conjugated
and unconjugated forms [26]. The reference interval is
dependent on the stage of the luteal cycle of the woman.
Univ. Virginia
pg/mL
30-65 early
30-200 mid
<30
30-56
luteum [28]. During pregnancy, the placenta is a source
of estradiol secretion. The primary function of estradiol
is to stimulate growth of the female sex organs and
development of secondary sexual characteristics.
Estradiol plays an essential role throughout the human
menstrual cycle [28]. For interpretation, it is important to
know the clinical status of the woman with respect to the
luteal cycle. During the early follicular phase, the
estradiol level is relatively constant and low. By day 7,
the dominant follicle is established, and the estradiol
level rises significantly. The elevated estradiol level
suppresses the FSH level by negative feedback on the
hypothalamus and pituitary gland and triggers a rapid
rise of LH. The estradiol level falls significantly as LH
reaches its peak. Normally, ovulation occurs 10 to 12
hours after the LH peak and 24 to 36 hours after the
estradiol peak. During the luteal phase, the estradiol
518
Estradiol
level increases again, reaching its peak luteal level about
8 days after ovulation. The elevated estradiol level is
involved in the regression of the corpus luteum. Unless
fertilization of the ovum takes place, the estradiol level
decreases, signaling the start of a new cycle.
Elevated estradiol levels in females may also result from
primary or secondary ovarian hyperfunction [29]. Very
high estradiol levels are found during the induction of
ovulation [27] for assisted reproduction therapy or in
pregnancy. Automated immunoassays have suitable
performance for these protocols [30,31]. Decreased
estradiol levels in females may result from either the
lack of ovarian synthesis (primary ovarian hypofunction
and menopause) or a lesion in the hypothalamus-
pituitary axis (secondary ovarian hypofunction).
Estradiol levels are normally low in males. Elevated
estradiol levels in males may be due to increased
aromatization of androgens, resulting in gynecomastia
[29,32].
Estrogen replacement therapy can effect estradiol
concentrations, complicating interpretation. Target
concentrations for therapeutic estradiol levels have been
published; however, use of these target values is not
valid, owing to some or all of assay variability, lack of
method documentation, lack of reference to timing of
sample in relation to patch change or gel application,
small number of women in studies, failure to specify
type of patch studied, and lack of standardization of gel
area [33].
Estradiol Performance Goals
Survey data from the CAP 2007 participant summary
report shows imprecision values (% coefficient of
variation [CV]) for estradiol ranging from approximately
3% to 18% at a mean concentration of 360 pg/mL and
from 3% to 23% at a mean concentration of 1608 pg/mL.
Acceptable Clinical Laboratory Improvement
Amendments performance criteria (CLIA 88) for
measurement of estradiol requires that laboratories be
accurate to within 3 SD of the peer-group mean.
Within-subject biological variation has been determined
to be approximately 11.3% [34]. Desirable specifications
for analytical imprecision derived from studies of
biological variation indicate that an assay imprecision of
no greater than 5.15% is required for an individual
laboratory.
Laboratories will meet CLIA 88 performance criteria,
since this only requires agreement with the same
method. Estradiol methods currently do not meet the
precision, specificity, and accuracy required for clinical
use [20]. Harmonization of immunoassay methods
against reference methods and the use of MS at low
estradiol levels is required to address these issues.
References
1 Toro G, Ackerman PG. Practical Clinical
Chemistry. Boston: Little Brown & Co;
1974:571-572.
2 Ratcliffe WA, Carter GD, Dowsett M et al.
Oestradiol assays: applications and guidelines
for the provision of a clinical biochemistry
service. Ann Clin Biochem 1988;25:466-83.
3 Masters AM, Hahnel R. Investigation of sex-
hormone binding globulin interference in direct
immunoassays for testosterone and estradiol.
Clin Chem 1989;35:979-984.
4 Micallef JV, Hayes MM, Latif A, Ahsan R,
Sufi SB. Serum binding of steroid tracers and
its possible effect on direct steroid
immunoassay. Ann Clin Biochem 1995;32:566-
574.
5 Yang DT, Owen WF, Ramsay CS, Xie H,
Roberts WL. Performance characteristics of
eight estradiol immunoassays. Am J Clin Pathol
2004;122:332-337.
6 Nelson RE, Grebe SK, OKane DJ, Singh RJ.
Liquid chromatography-tandem mass
spectrometry assay for simultaneous
measurement of estradiol and estrone in human
plasma. Clin Chem 2004;50:373-384.
7 Dighe AS, Sluss PM. Improved detection of
serum estradiol after sample extraction
procedure. Clin Chem 2004;50:764-766.
8 Ankarberg-Lindgren C, Norjavaara E. A
purification step prior to commercial sensitive
immunoassay is necessary to achieve clinical
usefulness when quantifying serum 17beta-
estradiol in prepubertal children. Eur J
Endocrinol 2008;158:117-124.
9 Lee JS, Ettinger B, Stanczyk FZ, Vittinghoff E,
Hanes V, Cauley JA, Chandler W et al.
Comparison of methods to measure low serum
estradiol levels in postmenopausal women. J
Clin Endocrinol Metab 2006;91:3791-3797.
10 Santen RJ, Demers L, Ohorodnik S, Settlage J,
Langecker P, Blanchett D, Goss PE, Wang S.
Superiority of gas chromatography/tandem
mass spectrometry assay (GC-MS/MS) for
estradiol for monitoring aromatase inhibitor
therapy. Steroids 2007;72:666-671.
11 Hsing AW, Stanczyk FZ, Blanger A,
Schroeder P, Chang L, Falk RT, Fears TR.
Reproducibility of serum sex steroid assays in
men by RIA and mass spectrometry. Cancer
Epidemiol Biomarkers Prev 2007;16:1004-
1008.
12 Kushnir MM, Rockwood AL, Bergquist J,
Varshavsky M, Roberts WL, Yue B, Bunker
AM, Meikle AW. High-sensitivity tandem mass
spectrometry assay for serum estrone and
estradiol. Am J Clin Pathol 2008;129:530-539.
13 Guo T, Gu J, Soldin Op, Singh RJ, Soldin SJ.
Rapid measurement of estrogens and their
metabolites in human serum by liquid
chromatographytandem mass spectrometry
without derivatization. Clin Biochem
2008;41:736-741.
14 Sluss PM, Hayes FJ, Adams JM, Barnes W,
Williams G, Frost S, Ramp J et al. Mass
spectrometric and physiological validation of a
sensitive, automated, direct immunoassay for
estradiol using the Architect. Clin Chim Acta
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Estradiol

2008;388:99-105. DL. Analytical performance of a new two-step

15 Siekmann L. Determination of oestradiol-17 ADVIA Centaur estradiol immunoassay during
beta in human serum by isotope dilution-mass ovarian stimulation. Clin Chem Lab Med
spectrometry. Definitive methods in clinical 2006;44:105-109.
chemistry. II. J Clin Chem Clin Biochem 31 Taieb J, Mendez Lozano DH, Benattar C,
1984;22:551-557. Messaoudi C, Pos C. Enlightenment about the
16 Thienpont LM, Verheghe PG, Van Brussel KA, new Architect-i2000 estradiol (Abbott
De Leenheer AP. Estradiol-17 beta quantified Laboratories) immunoassay during in-vitro
in serum by isotope dilution-gas fertilization. Clin Biochem 2007;40:1423-1426.
chromatography-mass spectrometry: reversed- 32 Martin RM, Lin CJ, Nishi MY, Billerbeck
phase C18 high-performance liquid AEC, Latronico AC, Russell DW, Mendonca
chromatography compared with immuno- BB. Familial hyperestrogenism in both sexes:
affinity chromatography for sample clinical, hormonal, and molecular studies of
pretreatment. Clin Chem 1988;34:2066-2069. two siblings. J Clin Endocrinol Metab
17 Tai SSC, Welch MJ. Development of a 2003;88:3027-3034.
reference measurement procedure for the 33 Armston A, Wood P. Hormone replacement
determination of estradiol-17 in human serum therapy (oestradiol-only preparations): can the
using isotope-dilution liquid chromatography laboratory recommend a concentration of
tandem mass spectrometry. Anal Chem plasma oestradiol to protect against
2005;77:6359-6363. osteoporosis? Ann Clin Biochem 2002;39:184-
18 Middle J. Standardization of steroid hormone 193.
assays. Ann Clin Biochem 1998;35:354-363. 34 Fraser CG. Biological Variation: From
19 Stanczyk FZ, Lee JS, Santen RJ. Principles to Practice. Washington, DC: AACC
Standardization of steroid hormone Press; 2001.
immunoassays: why, how, and when? Cancer
Epidemiol Biomarkers Prev 2007;16:1713-
1719.
20 Demers LM. Testosterone and estradiol assays:
current and future trends. Steroids 2008;May 15
Epub.
21 Diver MJ, Hughes JG, Hutton JL, West CR,
Hipkin LJ. The long-term stability in whole
blood of 14 commonly-requested hormone
analytes. Ann Clin Biochem 1994;31:561-5.
22 Raff H, Sluss PM. Pre-analytical issues for
testosterone and estradiol assays. Steroids
2008;May 21 Epub.
23 Reinartz JJ, Ramey ML, Fowler MC et al.
Plastic versus glass SST evacuated serum-
separator blood-drawing tubes for
endocrinologic analytes. Clin Chem
1993;39:2535-2536.
24 Diver MJ. Plasma estradiol concentrations in
neonates. Clin Chem 1987;33:1394.
25 Cao Z(T), Swift TA, West CA, Rosano TG, Rej
R. Immunoassay of estradiol: unanticipated
suppression by unconjugated estriol. Clin Chem
2004;50:160-165.
26 Tietz NW. Clinical Guide to Laboratory Tests.
Philadelphia: Saunders; 1983:180.
27 Muse K, Wilson EA. Monitoring ovulation
induction: use of biochemical and biophysical
parameters. Semin Reprod Endocrinol
1986;4:301-09.
28 Speroff L, Glass RH, Kase NG, eds. Clinical
Gynecologic Endocrinology and Infertility. 4th
ed. Baltimore: Williams & Wilkins; 1989:91-
119.
29 Kicklighter EJ, Norman RJ. The gonads. In:
Kaplan LA, Pesce AJ. Clinical Chemistry:
Theory, Analysis, Correlation. 2nd ed. St Louis:
Mosby; 1989:650-663.
30 Massart C, Gibassier J, Laurent MC, Le Lannou
520
Estriol
Estriol
Lawrence A. Kaplan
Name: Estriol, 1,3,5,(10)-estratriene-3,16, 17-triol, E3
Clinical significance: Refer to Chapter 44, Pregnancy, and Chapter 50, The Gonads, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation
Molecular formula: C18H24O3
Molecular mass: 288.37 D
Merck Index: 3654
Chemical class: Steroid
Structure:
Principles of Analysis and Current Usage
Estriol is the major estrogen steroid produced during
pregnancy. The concentration of plasma and urinary
estriol rises a thousandfold during pregnancy, and by the
third trimester estriol represents 85% to 90% of the C18
estrogenic steroids present in plasma and urine. The
serum and urine levels of the estriol derivatives are
compared in Estriol Table: Estriol metabolites present in
plasma and urine. Urinary estriol is present only in the
conjugated forms, since the protein-bound, unconjugated
estriol is not filtered at the glomerulus.
Estriol is most frequently measured as part of the
biochemical monitoring of high-risk pregnancies, and
thus the sample analysis time required before a physician
can obtain a result is an important consideration when
one is choosing a method. The type of method used will
depend most importantly on the decision to monitor
estriol in either serum or urine. In either case, the
monitoring is usually initiated in the beginning of the
third trimester, when estriol levels rise to measurable
amounts. The absolute measurement at any one time is
less important than changes over time.
The urinary procedures were traditionally performed on
a 24-h sample. This is a difficult specimen to collect
accurately and repeatedly. When reported as milligrams
of estrogen per 24 h, the collection error is added to the
i
Estriol
Previous and current authors of this method:
First edition: Lawrence A. Kaplan
Methods edition: Lawrence A. Kaplan
Second edition: Lawrence A. Kaplan
Third edition: Not updated
Fourth edition: Lawrence A. Kaplan
Fifth edition: Not updated
already significant biological and analytical errors. The
introduction of reporting estrogen output normalized to
creatinine output, the estrogen/creatinine (E/C) ratio[1,2]
improved the assay by eliminating the need for an
accurate, timed collection. The premise of the E/C ratio
is that the kidneys excrete both the conjugated estriol
and creatinine by similar mechanisms and that a random
urine measured for an E/C ratio yields results as valid as
results obtained from the longer collection.[3]
Urinary estriol output is dependent not only on fetal and
placental biochemical pathways that result in the
synthesis of estriol, but on maternal hepatic conjugation
and renal clearance of the estriol as well. It has therefore
been suggested that monitoring urinary estrogen is less
valid than measurement of serum estriol for the
assessment of the status of the fetoplacental unit (fetus
and placenta) because of the dependence of the urinary
measurement on both maternal and placental
function.[4,5] The measurement of plasma estriol has
been recommended, since it monitors only the
fetoplacental unit. Unconjugated estriol, with a half-life
of only 20 min, reflects most specifically the output of
the fetoplacental unit at the time of phlebotomy.[4,6]
Few studies have demonstrated an overwhelming utility
of one measurement (urine versus plasma) over the
other. Many studies have demonstrated a good
correlation between plasma estriol and urinary estriol
measurements; included are studies both on entire
populations and on individual, high-risk
pregnancies.[7,8] Estriol output by the fetoplacental unit
is variable, and circadian rhythms and hour-to-hour
fluctuations have both been noted.[9,10] These
variations appear to affect the concentration of plasma
estriol more than urinary estriol. Urinary estrogens, the
E/C ratio, and total serum estriol seem to have maximal
values at around midday with minimum concentrations
noted about midnight; the daytime concentrations for
521
Estriol
serum free estriol are greater than the nighttime
values.[9]
Urine
The estrogen composition of urine of a pregnant woman
in the third trimester is approximately 80% to 95%
estriol conjugates (sulfate and glucuronate) with the
remainder consisting of conjugates of estrone and
estridiol.[11,12] The oldest, and still most widely used,
procedure for the analysis of estrogen in urine is the
colorimetric reaction developed by Kober in 1931.[13]
This method is based on the reaction of all estrogens
with sulfuric acid to form a pinkish red Kober
chromogen of a yet unknown structure[14] (method 1,
Estriol Methods Summary Table). Oliver et al. suggested
that at least three chromogenic reaction products that
form a colloidal suspension of differing particle size are
produced by the Kober reaction.[15] Ferrous sulfate is
often added to the color reaction to serve as a catalyst
and to minimize certain interferences.[14-16] Since most
of the urinary estrogen in the third trimester is estriol,
this assay is effective in monitoring changes in excretion
of estriol during pregnancy though it is not specific for
this compound.
The Kober reaction is usually performed after a
hydrolysis step (acid or enzymatic) to convert
conjugated estriol into the free form. Performing the
hydrolysis step before extraction has been shown to
increase the final estrogen yield by about 20%, but for a
screening procedure this may.[17] Hydrolysis of the
estriols before extraction can also lead to interferences.
The Kober reaction can be performed directly on the
hydrolyzed urine or on unhydrolyzed or hydrolyzed
estrogens that have been first extracted into an organic
phase (such as ethyl acetate), which is removed by
evaporation. In the latter case, the Kober reaction is then
performed on the residue. A pinkish red color is formed
with estrogens, and a brownish color results from
nonestrogenic compounds that are also extracted. Since
the interfering compounds produce a linear background
absorbance over the spectral region about the absorbance
maximum of the Kober chromogen,[17] the Allen
correction has been used to correct the absorbance for
background interference. The Kober chromogen in
aqueous solutions has an absorbance maximum at 514
nm, and the Allen correction measurements are made at
472 and 556 nm.[17]
An important modification of the Kober reaction was the
extraction of the Kober chromophore into chloroform
(containing 2% p-nitrophenol plus 1% ethanol), the so-
called Ittrich extraction.[18] This extraction step was
designed to remove the estrogen reaction product from
other, more water-soluble, interfering chromogens. The
Ittrich chromogen can be measured colorimetrically or
fluorometrically. In chloroform, the chromophore has an
absorbance maximum between 530 and 540 nm[17,18]
(method 2, Estriol Methods Summary Table). The Allen
correction has also been applied to the chloroform
extract, with the secondary absorbance measurements
made at 505 and 565 nm.[19] When the extracted
chromophore is excited at 530 nm, an intense, yellow-
green fluorescence, which can be monitored at 550
nm,[18,20] is produced. The fluorescent reaction has
been adapted to continuous-flow, automated
analysis.[16,21,22] Ittrich also found that hydroquinone
can enhance the color formed in the Kober reaction.[23]
Trichloroacetic acid has been used in place of p-
nitrophenol.[24]
Brown and his co-workers employed several extraction
steps to separate estriol from estrone and
estradiol.[25,26] The estrogens were further purified
and separated on alumina columns, where the fractions
were monitored by the Kober reaction. This complex
procedure allows for quantitation of each individual
estrogen (estriol, estrone, and estradiol).
Urinary estriol can be specifically quantitated by a
variety of chromatographic techniques, all of which
require a hydrolysis step to convert estriol to the
unconjugated form. In addition, all chromatographic
assays require an extraction and partial purification of
the estrogens before analysis. Liquidliquid partitioning,
column extractions, and carbon black adsorption[27]
have been used successfully. Gasliquid
chromatographic techniques usually require an
extraction of the hydrolyzed estrogens before volatile
derivatives, such as acetate, methyl, and tetramethylsilyl
conjugates, are formed. Most separations have been
performed on 3% OV-1 columns, with nitrogen gas as
the mobile phase and flame ionization as the detection
method.[28,29] Some methods utilize temperature
programming (method 3, Estriol Methods Summary
Table). High-performance liquid chromatography
(HPLC) analyses of urinary estriol using reversed-phase
columns have been reported. These methods employ
several different types of detection methods (method 4,
Estriol Methods Summary Table) including
measurement of absorbance at 280 nm (see Estriol:
Figure 1 for absorbance spectrum),[30] measurement of
native fluorescence at 308 nm after excitation at 220 nm
(see Estriol: Figure 2 for the fluorescence spectrum),[31]
and derivatization with dansyl chloride and measurement
of the fluorescent derivative.[32]
Immunoassays developed for measurement of urine
estriol include radioimmunoassay (RIA) and enzyme
immunoassays (EIA) (methods 5 and 6, Estriol Methods
Summary Table). These assays require a hydrolysis step
to convert estriol conjugates to the free form. Several
RIAs have been reported using tritium (3H) or
radioactive iodine (125I) labeling and employing a
variety of techniques for separating bound from free
label, including dextran-coated charcoal,[33] double-
antibody precipitation,[34] and ammonium sulfate
precipitation.[35] The EIA is a heterogeneous assay
requiring polyethylene glycol to separate the antibody-
bound estriolenzyme conjugate from the free label.[36]
A unique luminescence immunoassay has been
described for the measurement of total urinary
estrogens.[37] After enzymatic treatment of the urine to
522
Estriol
hydrolyze the conjugates, the estrogens are allowed to
compete for antibody binding sites with 17-estradiol
conjugated to aminobutylated isoluminol. The amount of
light emitted in the subsequent luminescence assay is
inversely related to the concentration of total estrogens
in the sample.
Plasma Estriol
The thousandfold lower estriol concentration in serum as
compared to urine has, for the most part, precluded the
use of colorimetric or fluorometric assays[33] for
analysis of blood estriol. Most of the assays for serum
estriol, whether one is measuring total or only the
unconjugated fraction, have been RIAs (method 7,
Estriol Methods Summary Table). The RIAs use either
3H- or 125I-estriol as the tracer and employ a variety of
procedures to separate bound from free label,[38]
including polyethylene glycol,[39] ammonium
sulfate,[40] a second precipitating antibody,[10,41] and
solid-phase bound systems.[8,42] The use of the last
technique has led to the automation of the assay.[43]
Measurement of total serum estriol requires the
hydrolysis of estriol conjugates to the free form before
the analysis; either acid or enzyme hydrolysis is used.
Since there can be significant antibody cross-reactivity
between unconjugated estriol and its conjugated forms
(present in five to 10 times higher concentrations), the
analysis for free estriol often requires separation of the
free estriol from its conjugates. This is most frequently
accomplished by a liquidliquid extraction step, in
which the less water-soluble, unconjugated estriol is
extracted into the organic phase with high efficiency.
Alternatively, gel exclusion chromatography (such as
Sephadex) has been used for this purpose.[44] RIAs that
directly measure serum unconjugated estriol without a
prior extraction step are also available.[38,45,46] These
methods employ solid-phase procedures[38,46] and
double-antibody techniques to separate bound from free
label and can be automated as well.[46]
The measurement of total serum estrogens by EIA has
been reported by several groups.[47,48] These assays
are not specific for estriol but measure total serum
estrogens, including conjugates of estriol. These assays
utilize a horseradish peroxidase antibody label, and the
reaction is monitored at 463 nm (method 8, Estriol
Methods Summary Table). An EIA that allows direct
measurement of serum unconjugated (as well as
conjugated) estriol is also available.[49] This assay
employs a double-antibody precipitation technique to
separate bound from free tracer. A competitive
heterogeneous EIA has been developed for
determination of unconjugated estriol in serum.[50]
This assay consists of a fluoresceinated capture
antibody, an estriol labeled alkaline phosphatase
conjugate, and a magnetic particle solid phase. The assay
produces P-nitrophenoxide, which is measured at 405
nm. HPLC assays for unconjugated serum estriol have
also been reported.[51,52,53] The estriol is extracted by
liquidliquid partitioning or adsorption chromatography
and is separated from interfering compounds by
reversed-phase chromatography (method 9, Estriol
Methods Summary Table). Detection and quantitation of
the estriol peak is either by electrochemical analysis at
+0.75 volts using a glassy carbon electrode[51] or by
fluorescence at 308 nm after excitation at 280
nm.[52,53]
A fluorescence polarization assay (method 10, Estriol
Methods Summary Table) for the measurement of total
estriol in serum, plasma, or urine is also available.[54]
This assay, which is partially automated, requires no
extraction of the sample. Hydrolysis of total (conjugate
and free) estriol is achieved by the use of Escherichia
coli glucuronidase to hydrolyze the glucuronide
conjugate.
Saliva
Saliva has been investigated as a possible sample for the
analysis of estriol. Salivary unconjugated estriol has
been measured by radioimmunoassay (RIA), either as a
manual assay, employing 3H-estriol and a charcoal
adsorption step for separating bound from free
tracer,[55] or as an automated, continuous-flow RIA
using 125I-estriol as the tracer.[40]
Other Measurements
Although most analyses for estriol measure either the
unconjugated fraction or total estriol after a hydrolysis
step, assays have been developed that allow
measurement of the individual conjugates of estriol. This
includes a radioimmunoassay that specifically measures
estriol 3-sulfate 16-glucuronide[56] and an HPLC assay
that measures three other conjugates of estriol in
addition to the diconjugate.[57] There does not appear
to be widespread clinical use of this type of analysis at
this time.
Reference and Preferred Methods
There is no reference method for the measurement of
either urine or serum estriol. The lack of a reference
method may be partly ascribed to the lack of a consensus
as to how to best monitor estrogens during pregnancy.
Urine
The original colorimetric method for the measurement of
urinary estriol, the Kober reaction, has sufficient
sensitivity (0.2 g) to detect only total pregnancy
estrogens in urine. When the product of the Kober
reaction is measured by fluorometry, the sensitivity can
be increased to the low-nanogram range needed to
measure serum estriol.[33] However, the poor
specificity of the method and the variability of
endogenous fluorescence reduce the usefulness of this
assay unless the chromogen is further purified. Both the
colorimetric and fluorometric procedures have been
adapted to automated, continuous-flow
analysis[16,22,24,58] to increase the throughput and
precision. Manual assays have reported a between-day
percent coefficient of variation (%CV) of about
7.5%,[17] whereas the automated assays have reported
CVs of 4% to 5% (X, 12 to 29 mg/L).[22]
523
Estriol
The HPLC analysis of urinary estriol with fluorescence
detection may have the advantage of increased
sensitivity and fewer possible interfering
chromatographic peaks. Gas chromatographic analysis is
probably not suited for clinical use because of the labor
and time necessary for such assays. Measurement of
urinary estriol by RIA has good precision and
specificity.
Plasma
The antibody used to measure total estriol after a
hydrolysis step usually has a cross-reactivity of less than
1% to 2% with estradiol or estrone. Similarly, when one
is measuring free serum estriol, the antibody should have
low cross-reactivity with the conjugated forms of estriol,
unless an extraction step is used to separate the
conjugated from the unconjugated forms.
125I seems to be the label of choice in most RIAs. The
use of a second, precipitating antibody to separate bound
from free radioactivity may have a small advantage over
other techniques (polyethylene glycol, ammonium
sulfate) in terms of reproducibility of technique.[10] An
important advantage of solid-phase RIAs is that they
can be readily adapted to automated analysis with a
concomitant increase in precision.[40,46] Automated,
solid-phase free estriol assays are commercially
available.[46]
In addition to having the capability of being
semiautomated, the HPLC assays are more flexible in
terms of performing one or many analyses per analytical
run at a reasonable cost. The very quick start-up times
allow HPLC to be used to provide rapid stat analysis.
The EIAs are quite sensitive and are able to detect
approximately 0.2 nmol of estriol. However, many of
these assays suffer from a lack of specificity; they have
significant cross-reactivity with several estrogenic
compounds and are primarily designed to measure total
plasma estrogens.
The method of choice for the monitoring of pregnancy
estrogens will depend on factors to be evaluated by each
laboratory: cost, ease and reproducibility of analysis, and
the clinical value of the information obtained. The lower
biological day-to-day variability of the random urine
estrogen/creatinine (E/C) ratio and the low cost and
rapid turnaround time for results from the colorimetric
assay still make this test an important method for
consideration. The analysis time for a random urine E/C
analysis is approximately the same as for free serum
estriol by RIA. A rapid manual method for urinary
estrogens is presented below. For those laboratories with
large numbers of samples, an automated Kober method
on continuous-flow analysis would be appropriate.
For those laboratories asked to measure plasma estriol,
the free estriol analysis provides physicians with a
slightly more rapid turnaround time than total serum
estriol assays (because there is no hydrolysis step) and
gives results with less daily fluctuation. However,
because of the cost of reagents, most laboratories batch
their estriol analyses and cannot provide clinicians with
estriol measurements throughout the day. Low-cost,
rapid, automated HPLC analysis also can easily provide
an alternative if results are to be available 24 hours a
day. Estriol Table: Comparison of representative assays
compares the assay characteristics of several methods of
estriol analysis. Specimen
Either 24-h collections or single voidings are appropriate
urine specimens. Serum or plasma specimens are both
valid for blood analysis. When one is monitoring
patients with consecutive sample analyses, the random
urine or serum specimens should be obtained at
approximately the same time of the day to minimize any
error caused by biological variation. It has been shown
that bacterial contamination of urine left unpreserved
leads to an apparent increase in estrogens measured by
an automated fluorometric assay.[59] Preservation of
urine during collection with thimerosal (about 25 mg/L)
or storage at 4 C effectively prevents changes in
measured estrogen.
Certain antibiotics, most notably ampicillin, have been
shown to cause a decrease in the urinary excretion of
estriol conjugates by reducing the intestinal bacteria that
hydrolyze estriol conjugates.3 This reduces the
recirculation of estriol into serum and its eventual
urinary excretion.
Interferences
The direct Kober assay, performed on prehydrolyzed,
unextracted urine, is subject to interferences from a wide
number of compounds, especially those that form
acetaldehyde during the acid hydrolysis step. The
acetaldehyde reacts with the hydroquinone to give false-
negative results. One of the most important of these
interfering compounds is glucose, which, in the
monitoring of the high-risk pregnancies of diabetic
women, can cause significant negative interferences.
Glucose, at concentrations as low as 2.5 g/L, and
methenamine mandelate, which interfere with the acid
hydrolysis step, cause an overall low recovery of estriol
and false-negative results. In addition to glucose,
galactose, lactose, and mannose have also been found to
interfere.[60] Glucose can also reduce the fluorescence
produced by estrogens in fluorescence assays.[61] One
can reduce interference by glucose by performing the
acid hydrolysis on diluted urine or by extracting estriol
before performing the Kober reaction.[17] If an initial
chemical reduction step is performed with sodium
borohydride (NaBH4) to eliminate glucose interference
during a direct Kober reaction or automated fluorescence
assay, accurate results can be obtained without the
necessity of making multiple readings for an Allen
correction.[21,61] The use of enzymatic hydrolysis can
certainly prevent the interference from glucose but
requires a much longer time for the incubation step. A
general correction for nonspecific color can still be made
using the Allen correction.
524
Estriol
Estriol Reference Interval
The reference interval for both urinary and serum and
plasma estriol varies with gestational age. The biological
variability of the fetoplacental unit (as the 95th
percentile range) is very broad during the last month of
pregnancy. This is illustrated in Estriol: Figure 4 for
plasma estriol. The following reference intervals can
serve to illustrate expected estriol values in the last
trimester of pregnancy. Concentrations are also higher in
mothers with twin pregnancy than in those with
singleton pregnancies with concentrations just before
delivery significantly correlated with total birth
weight.[62] Each laboratory must determine its own
reference interval. The reference intervals are shown in
Estriol Table: Reported reference intervals.
The levels of salivary estriol (all unconjugated) are
approximately one-tenth the levels of serum
unconjugated estriol, with the plasma to salivary estriol
concentrations ranging from 6.1 to 14.0 ng/mL.[40,63]
The daily fluctuations in total serum estriol appear
greater than those of free serum estriol, though the
percentage of fluctuation for both appears to be
similar.[8] The individual diurnal variations of plasma
estriol can vary from 15% to 45%, with total urinary
estrogens varying from 30% to 40% during the day.[10]
However, when the estrogen output is reported as an E/C
ratio, the variation is reduced to 12% to 15%.
Interpretation
The rise of estriol levels in the blood and urine of a
pregnant woman is the result of an interplay of several
physiological and biochemical systems. The fetal adrenal
glands produce the biochemical precursor of estriol,
dehydroepiandrosterone sulfate (DHEAS) in increasing
amounts during development. The DHEAS is
hydroxylated to 16-hydroxydehydroepiandrosterone
sulfate (16-OH-DHEAS), which is desulfated to 16-OH-
DHEA by sulfatases in the placenta. Other placental
enzymes metabolize 16-OH-DHEA to estriol, which
crosses into maternal blood. The unconjugated maternal
estriol is rapidly conjugated to glucuronide and sulfate
by the maternal liver. Most of the estriol conjugates are
excreted by the maternal kidneys in urine. Some of the
conjugated estriol is excreted by the liver into the
gastrointestinal tract, where bacterial enzymes hydrolyze
the conjugates. Most of this estriol is returned to the
maternal plasma pool of unconjugated estriol by the
enterohepatic circulation.
The levels of unconjugated estriol in maternal serum
therefore reflect both a growing fetus and an intact
fetoplacental unit. However, these levels are also
dependent on maternal hepatic and renal function,
intestinal flora, and the enterohepatic circulation.
Decreased hepatic conjugation because of liver disease
might increase the plasma free-estriol pool and decrease
urine levels of estriol. Renal disease will decrease
excretion of estriol conjugates in urine but increase total
estrogen levels in maternal serum. If the mother is being
treated with ampicillin or other antibiotics that can
inhibit enteric flora, the enterohepatic return of estriol
will be diminished, decreasing the levels of conjugated
estriol in plasma. It is important that the status of
maternal organ function be considered when one is
interpreting the results of urine or serum estriol (total or
free) measurements, since they can cause changes in the
results that do not reflect the status of the fetoplacental
unit.
Given a healthy mother, serum or urine estriol levels can
be very helpful in the monitoring of a high-risk
pregnancy (as in diabetics, women over 35 years of age,
and drug addicts). The very broad range of serum
(Estriol: Figure 4), urine, and salivary estriol levels
indicates that the population variation is much greater
than the intraindividual variation. Thus, even with estriol
concentrations below the 95th percentile, good outcomes
can be obtained, though very low values in the last
month of pregnancy, less than 4 g/L (13.9 nmol/L) of
free estriol, are a cause for concern. Most often, rather
than be concerned with the absolute concentrations
within the reference interval, physicians follow
consecutive measurements. A decrease in serum free
estriol of 40% to 45% from the mean of the three
previous measurements has been considered to be an
early indicator of fetoplacental distress.[8] Similar
changes in urine are used to monitor pregnancies.
Salivary (unconjugated) estriol levels tend to parallel
plasma levels of this compound[63] and can also be used
to monitor pregnancies.[63-65] Although a saliva
specimen has the advantages of being readily available
and not requiring venipuncture, relatively little work has
been performed with it.
In addition to fetal death, there are several other
situations that can decrease maternal estriol levels. These
are suppression of fetal ACTH because of corticosteroids
administered to the pregnant woman, suppression of
DEAS synthesis if the fetus has pituitary aplasia or the
fetus is anencephalic, and deficiency in placental
sulfatase or 16--hydrolase activity.
Although there are reports that the measurement of the
individual conjugates of estriol in urine or serum can
provide clinically useful information, there is no
evidence that such measurements would provide
information not already obtained by measurement of free
or total estriol. With the recent advances in techniques
for measuring the conjugates[56,57] perhaps such
evidence will be forthcoming.
Estriol Performance Goals
The interlaboratory precision for urinary RIA, as
reported as 11% to 20% at concentrations of 4 to 6
mg/L, respectively, which is very similar to the %CV of
the spectrophotometric assay.
The chromatographic procedures used for specific
urinary estriol analysis appear to be both accurate and
precise. Between-day coefficients of variation of 3.7%
(mean, X, 15.9 mg/L)21 and 8.5% (X, 16.9 mg/L)[31]
have been reported for HPLC assays. The interlaboratory
525
Estriol
precision for serum estriol analysis has been found to be
approximately 23% to 29% CV (X, 1 to 2 ng/mL) and
6% to 9% CV (X, 25 to 28 ng/mL) for free serum estriol,
and 12% to 20% (X, 18 to 20 ng/mL) and 9% to 11% (X,
148 to 152 ng/mL) for total serum estriol.[66]
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Enzyme immunoassay of estrogen-like
substances in plasma with polyethylene glycol
as precipitant. Clin Chem 1979; 25: 716-718.
49 Carter JH, DeBernardi M. Double antibody
enzyme immunoassays for unconjugated and
total estriol in unextracted serum. Clin Chem
1984; 30: 1043(A).
50 Readio J, Freeman J, Lee C et al. An automated
enzyme immunoassay for the direct quantitation
of unconjugated estriol on the technicon
Immuno I system. Clin Chem 1997; 43: S190.
51 Kaplan LA, Hohnadel DC. Measurement of
unconjugated estriol in serum by liquid
chromatography with electrochemical detection
compared with radioimmunoassay. Clin Chem
1982; 29: 1463-1466.
52 Andreolini F, Borra C, DiCorcia A et al.
Improved assay of unconjugated estriol in
maternal serum or plasma by adsorption and
liquid chromatography with fluorometric
detection. Clin Chem 1984; 30: 742-744.
53 Kabra PM, Tsai FH, Marton LJ. Liquid
chromatography with fluorometric detection of
unconjugated estriol in serum of pregnant
women. Clin Chim Acta 1983; 128: 7-9.
54 Vanderbilt A, Spring T, Fino J, Shipchandler
M. A fluorescence polarization immunoassay
quantitating total estriol in serum and urine.
Clin Chem 1984; 30: 1043(A).
55 Nguyen HN, Huang NH, Besch NF, Besch PK.
Development and evaluation of four
radioimmunoassays for estriol in saliva. Clin
Chem 1983; 29: 1254(A).
56 Nambara T, Niwa T, Shimada K. Direct
radioimmunoassay for estriol 3-sulfate 16-
glucuronide using specific antiserum without
deconjugation. Clin Chim Acta 1985; 149: 275-
280.
57 Andreolini F, Borra C, Caccamo F, et al.
Estriol and its conjugates in late pregnancy
determined by extraction with Carbopack B and
liquid chromatography with fluorometric
detection. Clin Chem 1985; 31: 1698-1702.
58 Strickler HS, Stanchak PJ. Automated
fluorometry as applied to the assay of individual
estrogens in purified urinary extracts. Clin
Chem 1969; 15: 137-153.
59 Simkins A, Crawley M. Some chemical and
bacterial contributions to analytical variations in
527

Estriol

urinary oestrogen quantitations during 64 Truran PL, Read GF, Pearson JF. Salivary
pregnancy. Med Lab Sci 1978; 35: 325-334. oestriol in normal and abnormal pregnancies. Br
60 Schindler AE, Ratanasopa V, Herrmann WL. J Obstet Gynaecol 1984; 91: 1210-1215.
Acid hydrolysis of urinary estriol. Clin Chem 65 Kirkish LS, Compton AA, McCann DS.:
1967; 13: 186. Salivary estriol as an index to fetal well-being.
61 Worth HGJ. The effect of sugars on automated Clin Chem 1986; 32: 71-75.
urinary estrogen estimations in pregnancy. Clin 66 All conclusions and interpretations in the
Chim Acta 1973; 49: 53-57. Infobase with respect to the College of
62 Westergaard JG, Teisner B, Hau J et al. American Pathologists data base are those of
Placental protein and hormone measurements in the author of this section or the editors of the
twin pregnancy. Br J Obstet Gynaecol 1982; 92: Infobase, and not those of the College
72-76. 67 Shearman RP, Jools ND, Smith ID. Maternal
63 Evan JJ, Wilkinson AR, Aickin DR. Salivary and fetal venous plasma steroids in relation to
estriol concentrations during normal parturition. J Obstet Gynaecol Br Commonw
pregnancies and a comparison with plasma 1972; 79: 212-215.
estriol. Clin Chem 1984; 30: 120-121.

Tables

Estriol metabolites present in plasma and urine

Estrogen Urine (%) Plasma (%)
Unconjugated estriol 0 9
Estriol 3-sulfate 4 15
Estriol 3-glucuronide 13 15
Estriol 16-glucuronide 73 21
Estriol 3-sulfate 16-glucuronide 10 40
528

Estriol

Methods of estriol analysis

Method 1 (Urine): Kober reaction-direct or antibody, (NH4)2SO4 or dextrancharcoal
indirect (prior extraction) Usage: Frequent
Type of analysis: Spectrophotometric Comment: Requires hydrolysis to free
Principle of analysis: Estriol heated in the estriol; Specific
presence of sulfuric acid and hydroquinone Method 6 (Urine): Enzyme immunoassay
forms a pinkish red chromogen (absorbance (EIA)
maximum, 514 nm). Reaction can be Type of analysis: Spectrophotometric, end
performed with (indirect) or without (direct) point
prior extraction of estrogen. Principle of analysis: Competitive binding
Usage: Historically most popular method; assay; estriolenzyme conjugate separated from
frequently used today free enzyme and enzyme reaction (peroxidase)
Comment: Measures total estrogen; direct monitored at 463 nm
assay can have negative interference by glucose Usage: Rare
Method 2 (Urine): Ittrich Comment: Measures total estriol after
Type of analysis: Fluorometric hydrolysis
Principle of analysis: Chromophore Method 7 (Plasma): RIA free or total estriol
extracted and measured fluorometrically Type of analysis: see method 5
(excitation, 530 nm; emission, 550 nm) or Principle of analysis: See method 5;
colorimetrically extraction required for free estriol, hydrolysis
Usage: Rare for total
Comment: Measures total estrogen; Usage: Most frequent
automated on continuous-flow analyzers Comment: Very specific; automated
Method 3 (Urine): Gas-liquid chromatography Method 8 (Plasma): EIA
Type of analysis: Flame ionization Type of analysis: See method 6
detector Principle of analysis: See method 6
Principle of analysis: Volatile derivatives Usage: Rare
chromatographed on OV-1 with N2 mobile Comment: See method 6
phase and flame ionization detector Method 9 (Plasma): HPLC
Usage: Rare Type of analysis: Electrochemical or
Comment: Requires hydrolysis to free fluorescence
estriol; very specific Principle of analysis: Extracted
Method 4 (Urine): High-performance liquid unconjugated estriol is chromatographed by
chromatography (HPLC) reversed-phase chromatography and detected by
Type of analysis: Ultraviolet or electrochemical or fluorescence detection
fluorescence detection Usage: Rare
Principle of analysis: Estriol Comment: Very specific
chromatographed by reversed-phase (C18) Method 10 (Plasma): Fluorescence
chromatography, detected at 280 nm polarization immunoassay
(ultraviolet); fluorescence (excitation, 220 nm; Type of analysis: Fluorescence
emission, 608 nm); dansyl derivatives detected Principle of analysis: Fluorescent-labeled
by fluorescence estriol competes with sample estriol for binding
Usage: Rare sites; free label results in decreased polarized
Comment: Requires hydrolysis to fluorescence
unconjugated estriol; very specific Usage: Rare
Method 5 (Urine): Radioimmunoassay (RIA) Comment: Measures total estriol in plasma
Type of analysis: Liquid scintillation or and urine
gamma-ray counting
* 3H, tritium; 125I, radioactive iodine
Principle of analysis: Competitive binding
assay, employing 3H- or 125I-labeled estriol*;
free from bound label separated by second
529

Estriol

Comparison of representative assays for urine and serum estriol

Method 1 (Urine): Kober Linearity: 80 g/L
Reaction temperature: 100 C Assay time
Sample volume: 2 mL Incubation/extraction: 75 min
Label or detection system: Spectrophotometric; 514 nm Operational: 30 min
Linearity: 100 mg/L Sensitivity: 1000 ng/L
Assay time Final concentration of reagents: 5070 103 dpm/tube
Incubation/extraction: 20 min Reported precision (between run): X, %CV: 150 g/L,
Operational: 40 min 8.4%
Sensitivity: Interferences: Ampicillin
Final concentration of reagents: Hydroquinone: 180 Method 5 (Serum): RIA (free)
mmol/L; H2SO4; 24.5 mol/L Reaction temperature: Ambient
Reported precision (between run): X, %CV: Sample volume: 250 L
Interferences: Ampicillin Label or detection system: 125I
Method 2 (Urine): HPLC Linearity: 800 g/L
Reaction temperature: 60 C (hydrolysis) Assay time
Sample volume: 5 mL Incubation/extraction: 2.5 h/batch
Label or detection system: Spectrophotometric; 280 nm Operational:
Linearity: 100 mg/L Sensitivity: 100 ng/L
Assay time Final concentration of reagents: 76 103 dpm/tube
Incubation/extraction: 60 min Reported precision (between run): X, %CV: 3900 ng/L,
Operational: 15 min 8.8%
Sensitivity: 23 mg/L Interferences: Estriol 3-sulfate, ampicillin
Final concentration of reagents: Method 6 (Serum): HPLC
Reported precision (between run): X, %CV: 15.9 mg/L, Reaction temperature: Ambient
3.7% Sample volume: 2 mL
Interferences: Ampicillin Label or detection system: Chromatographic,
Method 3 (Urine): RIA* electrochemical detection
Reaction temperature: 27 C (hydrolysis) Linearity: 0.4-8 ng
Sample volume: 20 L Assay time
Label or detection system: 125I Incubation/extraction: 25 min
Linearity: 400 mg/L Operational: 25 min
Assay time Sensitivity: 1000 ng/L
Incubation/extraction: 15 min Final concentration of reagents:
Operational: 90 min Reported precision (between run): X, %CV: 14.9 g/L,
Sensitivity: 3 mg/L 9.9%
Final concentration of reagents: 90 103 dpm/tube Interferences: Ampicillin
Reported precision (between run): X, %CV: 100 mg/L, * Amersham Corp. Arlington Heights, IL.
4.3% Nuclear Medical Systems, Inc., Newport Beach, CA.
Interferences: Ampicillin Becton-Dickinson, Automated RIA, Rutherford, NJ.
Method 4 (Serum): RIA (total) dpm: Disintegrations per minute.
Reaction temperature: 37 C (hydrolysis)
Sample volume: 25 L
Label or detection system: 125I
530

Estriol

Reported reference intervals for total estriol[41,67]

Gestational week: 2830
Plasma, serum total estriol, ng/mL (nmol/L): 38140 (132485)
Urinary total estriol, mg/24 hours (mol/24 h): 518 (1762)
Gestational week: 30
Plasma, serum total estriol, ng/mL (nmol/L): 31140 (107485)
Urinary total estriol, mg/24 hours (mol/24 h): 518 (1762)
Gestational week: 32
Plasma, serum total estriol, ng/mL (nmol/L): 35330 (1211144)
Urinary total estriol, mg/24 hours (mol/24 h): 721 (2473)
Gestational week: 34
Plasma, serum total estriol, ng/mL (nmol/L): 45260 (156902)
Urinary total estriol, mg/24 hours (mol/24 h): 826 (2890)
Gestational week: 36
Plasma, serum total estriol, ng/mL (nmol/L): 48350 (1661214)
Urinary total estriol, mg/24 hours (mol/24 h): 1030 (35104)
Gestational week: 38
Plasma, serum total estriol, ng/mL (nmol/L): 59570 (2051977)
Urinary total estriol, mg/24 hours (mol/24 h): 1236 (42125)
Gestational week: 40
Plasma, serum total estriol, ng/mL (nmol/L): 95460 (3291595)
Urinary total estriol, mg/24 hours (mol/24 h): 1342 (45146)

Figures

Estriol: Figure 1

Ultraviolet absorbance
spectrum of estriol in
methanol.
531

Estriol

Estriol: Figure 2

Fluorescence spectrum of estriol in

methanol with excitation wavelength set
at 280 nm.

Estriol: Figure 3

Chromatograms of extracted sera.

nA, Nanoamperes. A, Serum
from nonpregnant woman spiked
with estriol (E3, 10 ng/mL) and
internal standard (I.S., 500
ng/mL). B, Serum from pregnant
woman (50 L injection). C,
Serum from pregnant woman
with spiked estriol (30 L
injection). (Modified from
Kaplan, L.A., and Hohnadel,
D.C.: Clin. Chem. 29:1463-1466,
1982.)
532

Estriol

Estriol: Figure 4

Mean (solid line) and

estimated 5th and 95th
percentiles (shaded area) for
plasma unconjugated estriol
during normal pregnancy.
Estriol patterns form three
actual pregnancy conditions
are shown.

8. Stock standard solution, 500 mg/L (1.73

Procedure: Total Urine Estrogen Determination [17] mmol/L). Dissolve 50 mg of estriol in 100 mL of
absolute ethanol. Stable for 6 months when capped
Principle tightly at 6 to 8 C.
Urine is acidified and saturated with sodium chloride to
9. Working standard, 20 mg/L (6.94 mol/L).
reduce the water solubility of estriol. Estrogens are then
Dilute 2 mL of stock solution to 50 mL with absolute
extracted into ethyl acetate. An aliquot of this extract is
ethanol. This is stable for 2 to 3 months when capped
evaporated, treated with hydroquinone in 64% sulfuric
tightly at 6 to 8 C.
acid, and heated in a boiling water bath to develop the
color. Absorbance readings are taken at 472, 514, and Assay
556 nm, and the Allen correction is used in the Equipment: A spectrophotometer with a band pass < 10
calculations. This method has no interference by glucose nm; ground-glass stoppered, 50 mL extraction tubes;
because of the initial extraction. centrifuge capable of 200 g.
1. Prepare the urine sample by
Reagents thoroughly mixing it and then centrifuging it to
1. 5 M HCl (5 mol/L). Dilute 486 mL of remove solid matter. The specific gravity of
concentrated HCl to 1 L with distilled water. This is each single-voided specimen should be
stable for 1 year at room temperature. measured with a refractometer and adjusted to
2. Crystalline NaCl, reagent grade. 1.015 or less. Prepare a 1:20 dilution of urine
3. Ethyl acetate, reagent grade. for creatinine determination.
4. Nitrogen gas. 2. To each extraction tube, add, in the
5. Hydroquinone in ethanol, 20 g/L (180 following order:
mmol/L). Dissolve 0.20 g of hydroquinone (Fisher, a. 2 mL of urine
purified) in 10 mL of absolute ethanol. Prepare fresh b. 0.4 mL of 5 M HCl
daily. c. 2 g of NaCl
6. Hydroquinone, 20 g/L (180 mmol/L) in 64% d. 2 mL distilled water
H2SO4 (24.5 mol/L). Add 664 mL of concentrated
e. 3 mL ethyl acetate
H2SO4 (98%) very slowly to 336 mL of distilled water
Cap the tube securely, and shake vigorously for
in an ice bath. Mix after each addition, and allow to cool 30 sec. Centrifuge for 5 min at 200 g at room
slightly before adding more acid. Add 20 g of temperature. The top layer is used for
hydroquinone to final solution, and heat to 50 C to subsequent analysis.
dissolve. Store in refrigerator. This solution is stable for 3. To each color development tube, add as shown
2 months. below:
7. Estriol standard (1,3,5,10-estratriene- Ethyl a
3,16,17-triol) (Mann Research Laboratories, New York,
NY).
533

Estriol

column, an ultraviolet detector, and an electrochemical

Ethyl acetate
extract (top
detector. The amount of serum estriol is calculated on
layer) the basis of peak height ratio.
2% Hydroquinone
Tube in ethanol Working standard Reagents
Blank 0.2 mL 1. Diethyl ether. Peroxide-free, reagent grade.
5 g of standard 0.2 mL 0.25 mL 2. Stock phosphateEDTA buffer (1.8 and
10 g of standard 0.2 mL 0.50 mL 0.085 mol/L, respectively), pH 6.5. Place 126.48 g of
Unknown 0.2 mL 0.5 mL
KH2PO4, 56.8 g of Na2HPO4, and 335 mg of
4. Place all tubes in a heating block set for no tetrasodium EDTA in a 1 L volumetric flask. Dissolve
more than 50 C, and evaporate all solvent from salts in approximately 800 mL of distilled water. Adjust
the tubes under a stream of nitrogen. the pH to 6.5, and bring to volume with additional
distilled water. Stable for 6 months at room temperature.
5. Add 2 mL of hydroquinone in 64% H2SO4, and
3. Working phosphateEDTA buffer (180 and
place in a boiling water bath. Swirl the tubes 8.5 mol/L, respectively), pH 6.5. Dilute 100 mL of
immediately to dissolve the dried extract. stock phosphateEDTA buffer to 1 L volumetrically
Stopper the tubes, and heat for 40 min. Cool the with distilled water. Prepare fresh daily.
tubes in tap water. 4. Mobile phase. Mix 370 mL of HPLC-grade
6. Add 1.7 mL of distilled water to each tube, mix acetonitrile with 1000 mL of working phosphate
thoroughly, and leave in the cold tap water until EDTA buffer, and filter solution through a 0.5 m FH
cool. Millipore filter. Degas under vacuum for 15 min with
7. Read the absorbance of each standard and occasional swirling.
unknown tube against the reagent blank at 472, 5. Estriol standards. Prepare a stock standard by
514, and 556 nm on the spectrophotometer. dissolving 100 mg of estriol (Sigma Chemical Co., St.
Louis, MO) in 1000 mL of methanol to give a
concentration of 100 mg/L (377 mol/L). This solution
Calculations is stable for at least a year at 4 to 8 C.
1. A corrected absorbance at 514 nm, A514 (corr),
should be calculated for each sample using the Prepare an intermediate standard by diluting the stock
Allen equation: standard 100-fold with methanol. The concentration of
this standard is 1000 ng/mL (3.47 nmol/mL). The stock
A514 (corr) = A514 _
A556 + A472 standard is diluted 1000-fold with stock internal standard
solution (see propiophenone below) to yield the working
2
chromatographic standard of 100 ng of estriol/mL (0.35
where A472, A514, and A556 are the absorbances
nmol/mL). This solution is stable for 1 month at 4 to 8
observed at designated wavelengths. C if kept in a tightly closed glass-stoppered container.
2. The concentration of estrogen (as estriol) in 6. Propiophenone, 500 g/L (3.73 mol/L).
urine is calculated by the equation: Dissolve 50 mg in 100 mL of methanol. This solution is
stable for up to a year at 4 to 8 C. This is the stock
g of estrogen/mL of urine = A514(corr) 3.6F internal standard solution.

where F = g of standard/A514 (corr) of standard. Assay

Equipment: This assay was established using a Waters
3. Milligrams of estrogen excreted per 24 h: (Waters, Inc., Division of Millipore, Milford, MA)
Radial-PAK cartridge (10 m particle size, 8 mm inner
mg of estrogen/24 h = diameter by 10 cm) containing C8 stationary phase and
g of estrogen/mL of urine Urine volume mL
an LC-4A electrochemical detector (ECD) from
4. Estrogen/creatinine ratio (E/C): Bioanalytical Systems, Inc. (West Lafayette, IN). A
Waters Bondapak C18 column worked equally well
E/C = g of estrogen/mL of urine
mg of creatinine/mL of urine with minor modifications of the experimental
system.[51] In addition, an injector, pump, dual pen
Procedure: High-Performance Liquid recorder, and ultraviolet spectrophotometer (254 nm) are
Chromatography Assay for Serum Unconjugated required to complete the HPLC system. The ECD was
Estriol [51] placed first in line to monitor the column eluate, which
then passed into the spectrophotometer. The signal
Principle output from each detector is passed to one of the pens of
Unconjugated estriol is extracted from serum with the dual pen recorder. A centrifuge capable of generating
diethyl ether. After evaporation of the solvent, the 800 g is also needed.
residue is redissolved with mobile phase containing an 1. For each sample or control to be analyzed, label
internal standard and analyzed by an HPLC system, two 50 mL glass-stoppered centrifuge tubes: T,
which employs a reversed-phase chromatographic test, and S, spike.
534

Estriol

2. Pipet 2 mL of specimen to be analyzed into IVunk, volume of sample injected into HPLC system
each centrifuge tube. Pipet a known amount of (mL)
the intermediate standard, for example, 10 to 50 0.8, factor to correct for only 80% of ether extract being
L (equal to 10 to 50 ng of spike) of the used in the analysis
intermediate standard to the tube labeled S. 2 mL, volume of serum extracted
3. Add 10 mL of diethyl ether to each tube. 4. Determine the percent recovery of the estriol
Thoroughly mix the contents of the tube by added (spike) to the second aliquot by using the
repeated inversion or vortex mixing for 10 min following calculation:
at room temperature. Centrifuge tubes for 5 min
at 800 g. Percent recovery =
4. Remove exactly 8 mL of the supernatant, and
(Cspike Cunspiked) 2 mL 100%
evaporate ether at 50 C under stream of
nitrogen gas. Cis Vs
5. Reconstitute residue in 200 L of internal where Cspike and Cunspiked are the estriol
standard solution (propiophenone). concentrations (ng/mL) calculated in step 3 for spiked
6. Analyze solution by injecting 10 to 50 L of the and unspiked samples; Cis is concentration of estriol in
reconstituted sample into the HPLC system. the intermediate estriol standard; and Vs is volume of
Also inject 10 to 50 L of the working estriol intermediate estriol standard added to spike tube (step 2
standard containing propiophenone. of assay directions).
7. Chromatographic system: 5. Correct the estriol concentration for unspiked
a. Chart speed, 0.5 cm/min. sample (step 3) for the percent recovery (step 4)
b. Electrochemical detector, 2 nA/V full- to yield the final estriol concentration.
scale sensitivity with the glassy carbon
electrode set at +0.75 V. Estriol (ng/mL) = Uncorrected estriol (ng/mL)
c. Ultraviolet detector, 0.005 AUFS Percent recovery
(absorbance units at full scale) at 254 Notes
nm. 1. The propiophenone is added to correct for
volume loss of the reconstituted sample because
d. Flow rate, 3 mL/min. The mobile
of solvent evaporation.
phase should be heated to
approximately 50 C and should be 2. The average recovery of added estriol was
constantly stirred. The column is kept 99.6% over the analytical range of 2 to 24
at room temperature. ng/mL. One could assume an average recovery
of 99.6% (or 100%) during the extraction
e. Analysis time, 10 min. See Estriol:
procedure and eliminate the need to determine
Figure 3 for example of
the recovery from each sample.
chromatograms.
3. The between-day precision of this assay (as
percent coefficient of variation) was 18.8% at
Calculations 5.1 ng/mL, and 9.9% at both 14.9 and 26.0
1. Measure the peak heights (in millimeters) of the ng/mL of estriol.
estriol peak (retention time approximately 5 4. Chromatograms essentially identical to those
minutes) and the internal standard peak for the shown in Estriol: Figure 3 were obtained using
working standard and each patient sample. the Waters Bondapak C18 columns.
2. Calculate the peak height ratio (PHR) for the 5. The HPLC system showed a linear response for
standard (std) and each unknown (unk): peak height (mm) versus nanograms of estriol
injected up to 8 ng at a 50 L injection volume,
PHR = Peak height of estriol (mm)____
and the sensitivity of the system was
Peak height of internal standard (mm)
approximately 0.4 ng of estriol (equivalent to
3. The amounts of estriol in the patients spiked
an estriol concentration of 1 ng/mL).
and unspiked samples are calculated. The example for
the unspiked is as follows:
std std
Estriol (ng/mL) = PHRunk (C ) IV (0.20 mL)
PHR std (IV unk ) (0.8) 2 mL

where Cstd, concentration of estriol standard (ng/mL)

IVstd, volume of working standard injected into HPLC
system (mL)
0.20 mL, volume of methanol used to reconstitute
extract
535
Ethylene Glycol

Ethylene Glycol
Ping Wang

Name: Ethylene glycol

Clinical significance: Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.

Molecular formula: C2H6O2

Molecular mass: 62.07 D
Merck Index: 3744
Chemical class: Alcohol
Structure:

Principles of Analysis and Current Usage colorimetry, spectrophotometry, or fluorimetry because

Ethylene glycol analysis is requested when a history of
ingestion of this compound is likely, or when a patient is
seen in a comatose or intoxicated state with an osmolar
gap, crystalluria, and a high anion gap indicative of
metabolic acidosis. The clinical toxicologist will
normally be called upon to either screen for the presence
of or determine the quantity of ethylene glycol in either
blood or urine. Depending on the circumstances, rapid
identification of this compound can prevent death many
hours after ingestion. Since ethylene glycol intoxication
is usually seen as an emergency situation, laboratories
should have the capability of identifying this compound
in a short time.
The subject of crystalluria associated with ethylene
glycol ingestion deserves special consideration as part of
the laboratory investigation for this poison. In 1946,
Milles referred to a 1930 report of oxalate crystals found
in the kidneys of a German soldier during an autopsy
performed after death from ethylene glycol poisoning
[1,2]. Later that year, Pons et al. reported on the deaths
of 10 soldiers, all of whom had oxalate crystals in their
urine [3]. Early reports such as these helped to establish
a link between crystalluria and the diagnosis of ethylene
glycol toxicity. This observation, coupled with the need
for rapid diagnosis of ethylene glycol ingestion, has
made the detection of crystalluria an important factor in
the diagnosis of ethylene glycol poisoning.
Techniques for the analysis of ethylene glycol fall into
two major groups: (1) oxidation with subsequent
measurement by colorimetry, spectrophotometry, or
fluorimetry and (2) various chromatographic methods.
Ethylene glycol cannot be measured directly by
i
Ethylene glycol
Previous and current authors of this method:
First edition: Not done
Methods edition: Dan Field
Second edition: Not updated
Third edition: Not updated
Fourth edition: Dan Field
Fifth edition: Ping Wang
the molecule does not absorb a sufficient amount of
ultraviolet light to allow detection by these methods. An
early but still often used technique employs the
oxidation of ethylene glycol by periodic acid to
formaldehyde [4-6]. The formaldehyde (HCHO) is
subsequently derivatized to a colorimetrically or
fluorimetrically measurable substance (Table 1, Methods
1 to 3).
HIO42 HCHO
OHCH2CH2OH
1. Harger and Forney colorimetric technique:
HCHO + Fuchsine reagent violet color
(max , 555 nm)
2. Rajagopal colorimetric technique:
3HCHO + 2K2Cr2O7 + 10H2SO4 + 2H+
(yellow orange)
2Cr2(SO4)4 + 2K2SO4 + 11H2O + 3HCOOH
(blue green)
(max , 550 nm)
3. Hantzsch reaction [6]:
Acetylacetone + HCHO NH4+Diacetyllutidine
derivative
(ex , 400 nm; em , 500 nm)
An enzymatic method that may be adapted to an
automatic platform uses glycerol dehydrogenase to
oxidize ethylene glycol to glycoaldehyde [7,8]. The
absorbance change at 340 nm associated with NADH
generation is proportional to the ethylene glycol
concentration (Table 1, Method 4).
glyceroldehydrogenase
Ethylene glycol +NAD+
glycoaldehyde + NADH + H+
536
Ethylene Glycol
Glycerol dehydrogenase enzymes isolated from different
organisms vary in their activity in this assay. The
enzyme from Cellulomonas species demonstrated high
activity. This method may detect ethylene glycol as low
as 10 mg/dL, and no interference has been observed
from ethanol, acetone, methanol, and isopropanol.
However, this method is subject to both false-positive
and false-negative interferences by a variety of
compounds listed in the section on Interferences below.
An alternative method using oxidation of ethylene glycol
by alcohol dehydrogenase enzyme [9] could be useful
(Table 1, Method 5).
alcohol dehydrogenase
HOCH2CH2OH + NAD+
HOCH2CHO + NADH + H + though there is ample research in animal studies. The
Ethylene glycol can also be estimated from the osmolal
gap obtained by subtraction of a calculated osmolality
from the osmolality measured by freezing-point
depression (Table 1, Method 6).
(Measured osmolality Osmocalc) 62.1 g/mol 0.92 =
milligrams of ethylene glycol per liter of serum, where
62.1 g/mol is the molecular weight of ethylene glycol,
Osmocalc is the calculated serum osmolality, and 0.92 is
the correction for volume of water in serum. This
technique is only of value if one can be certain that no
other compounds have been ingested that might be
contributing to the osmolal gap.
Various types of chromatography compose the second
major analytical approach to ethylene glycol analysis.
The thin-layer technique [10] (Table 1, Method 7) and
gas chromatography with a flame ionization detector are
most frequently used. Because of difficulties associated
with extraction and concentration of the compound, most
gas chromatographic techniques use the direct injection
method (Table 1, Method 8). Alternative approaches to
direct injection utilize dibenzoate [11] or boronate
derivatives [12] of ethylene glycol (Table 1, Method 9).
A high-performance liquid chromatographic method
utilizing ethylene glycol as a benzoyl ester derivative has
been reported (Table 1, Method 10) [13]. Another
method combines high-performance liquid
chromatography with mass spectroscopy for detection of
glycolic acid, a major metabolite of ethylene glycol
(Table 1, Method 11) [14]. Since rapid metabolism
clears the body of the parent ethylene glycol, this is a
potentially useful technique for providing a suitably
sensitive assay.
Reference and Preferred Methods
There is no reference method for ethylene glycol. The
choice of method depends on clinical needs and
availability of expertise and instrumentation.
Because some laboratories use the presence of
crystalluria as a screening test for ethylene glycol
toxicity, it is important to consider the role of
crystalluria in the diagnosis. A review of the literature
since the turn of the century reveals that crystalluria was
reported in less than 50% of the cases. On the other
hand, the test has a high specificity because oxalate
crystalluria is relatively rare. Taken together with the
usual clinical presentation of ethylene glycol toxicity,
the presence of oxalate crystals in the urine strongly
supports a diagnosis of ethylene glycol toxicity.
However, oxalate crystals may fail to take the classic
octahedral shape of the more commonly seen dihydrate
molecule and instead, in monohydrate form, may mimic
hippuric acid crystals (Figure 1). Such a
misinterpretation has led to misdiagnosis and death
[13,15]. No evidence exists in the literature to support
the often stated contention [16] that ethylene glycol
poisoning leads to hippuric acid crystalluria in humans,
formation of hippuric crystals has been documented in
studies of dogs and cats [10,17]. Thus analysis of
crystalluria remains a useful but limited technique.
With direct-injection gas chromatographic methods, the
sample needs little preparation; with 1 to 2 L of serum
or urine, the detection limits of these methods range
between 16 and 50 mg/L, but gas chromatographic
analyses employing these methods are said to suffer
from low sensitivity and poor reproducibility [14]. One
factor lowering the sensitivity of the direct injection
method is a characteristic of the flame ionization
detector itself. The sensitivity of the method is
proportional to the number of carbon atoms in the
compound being measured. In the boronate derivative
method, by derivatizing the ethylene glycol directly in
the specimen, extraction is avoided, and the method
gains some of the simplicity of direct injection, as well
as better precision and sensitivity. The HPLC benzoate
method has a lower limit of detection of 10 mg/L, and no
interference from other alcohols is reported [18].
The HPLC-mass spectrometric technique method uses
20 mL of urine; however this procedure is limited to
specialized laboratories with the necessary equipment.
With the enzymatic oxidation methods available, the
chemical oxidation assays are largely outdated. The
advantages of enzymatic assays are the simplicity of
reaction and the availability of required chemicals and
equipment.
Currently, no single method of ethylene glycol analysis
is preferred, but gas chromatography is, in one form or
another, most favored. The limits of detection of the
methods outlined above range between 10 and 100
mg/L, but since most toxic ingestions involve
concentrations at least a magnitude greater, the chosen
technique depends more on the available equipment and
the training level of the laboratory personnel. The direct-
injection gas-chromatography method presented here is
chosen for the simplicity of the procedure and the ready
availability of equipment. The method is rapid but not
suited to the analysis of large numbers of samples. In the
absence of gas chromatographic instrumentation, the
enzymatic procedures may be adapted to automatic
537
Ethylene Glycol
platforms for stat analysis in emergency cases. The
fluorimetric method also seems to have good specificity
[6].
Specimen
Serum, plasma, or urine are acceptable for analysis; it is
important to note that the appearance of ethylene glycol
in the urine may be delayed for many hours, depending
on the circumstances of ingestion. Samples should be
processed promptly to avoid time-dependent loss of
glycol. One report states that the concentration of
ethylene glycol in a sample stored in a refrigerator for 12
days decreased from 2850 to 800 g/L [4].
Interferences
A common interference in colorimetry and fluorometry
is interference from antibiotics and antitussives
containing 1,2-hydroxyl groups in sufficient quantity to
yield falsely positive results [19]. However, in one
method based on the Hantzsch condensation reaction,
when 36 commonly used drugs were examined, only
nystatin (Mycostatin) caused interference [6]. The
alcohol dehydrogenase reaction is not specific for
ethylene glycol. Other alcohols such as ethanol,
methanol, or isopropanol can also interfere. The glycerol
dehydrogenase method has the advantage of being free
of interference from some alcohols such as ethanol,
acetone, methanol, and isopropanol. However, high
concentrations of 2,3 butanediol, glycerol, and propylene
glycol may interfere. Although glycerol and propylene
glycol are widely used as solvents in IV medications,
they are usually not present in the patient specimen at
levels high enough to cause significant interference.
Samples from patients with high levels of lactate and
lactate dehydrogenase [20] and those with hepatotoxicity
caused by acetaminophen may also give elevated results.
In addition, lipemia may cause false-positive results.
Glycerol, propylene glycol, and hemolysis may generate
negative interference.
Ethylene Glycol Reference Interval
Any amount of ethylene glycol detected is abnormal.
Interpretation
The study of ethylene glycol toxicity has been
characterized by confusion and misinformation since the
compound was first developed as a nontoxic replacement
for glycerin at the turn of the century. Early investigators
went as far as drinking various quantities as proof of
nontoxicity, but by 1930, reports began to surface
questioning the benign nature of ethylene glycol [21].
Finally, the deadliness of the glycols became clear in the
Massengil Disaster, where greater than 100 people
eventually died after ingesting an elixir of sulfanilamide
in which a glycol served as the medicinal vehicle [22].
Ethylene glycol causes symptoms and organ damage
similar to that seen with ethanol toxicity. However, most
of the toxicities associated with ethylene glycol ingestion
are due to organ damage and acidosis caused by the
metabolites, especially oxalic acid and glycolic acid.
Initially, the measured serum osmolal gap becomes
significantly elevated, but rapid metabolism by alcohol
dehydrogenase causes the osmolality to fall and initiates
a high anion gap. Bicarbonate-resistant metabolic
acidosis is initially due to the production of glycolic acid
from ethylene glycol but is subsequently caused by
lactate and other organic acids formed as by-products of
an increased NADH/NAD ratio caused by the action of
alcohol dehydrogenase (see Figure 4). Oxalate, the end
product of ethylene glycol metabolism, precipitates
calcium to form crystals in the renal tubules, lungs, and
brain, which may cause significant damage to these
tissues.
The metabolic acidosis together with the precipitated
calcium crystals are part of a well-characterized
progression of signs and symptoms [23]. Beginning with
the central nervous system, the patient will appear
intoxicated or comatose, frequently without the smell of
alcohol but often with nausea, vomiting, ocular
abnormalities, and convulsions. After approximately 12
hours, the cardiopulmonary stage predominates and is
characterized by failure of these two systems. Finally,
approximately 36 hours after the initial ingestion, the
renal phase is chiefly characterized by oliguria. Death
can occur during any phase, but with appropriate
therapy, patients can survive massive ingestions [24].
The commonly quoted minimum lethal dose is stated to
be 100 mL (1.46 moles), but in fact as little as 30 mL
have been held responsible for death in adults [25]. The
actual number of deaths attributable to ethylene glycol is
unknown. However, in 1983, the National Data
Collection System [26] issued a report on deaths from
ethylene glycol taken from data collected on 11% of the
U.S. population. Taking into account extensive
underreporting by the participating centers (a fact
acknowledged by the authors) and the distribution of the
population, the extrapolated death figure from ethylene
glycol is less than 40 per year. However, there are no
reliable estimates for the nationwide deaths from
ethylene glycol.
Initial diagnosis and treatment should rest solely on the
criteria of history of ingestion, apparent intoxication or
coma without the stigma of alcohol (or inconsistent with
ethanol levels obtained and clinical course), an elevated
osmolal gap, and a metabolic acidosis with an anion gap.
As previously noted, crystalluria of any type is evidence
but if absent does not rule out ethylene glycol poisoning.
Induced emesis or gastric lavage is an important step
after recent ingestion of ethylene glycol. 4-MP
(fomepizole), a specific inhibitor of alcohol
dehydrogenase, can prevent further metabolism of
ethylene glycol into toxic metabolites and therefore
should be given as an antidote. In hospitals and clinics
where 4-MP is not available, intravenous ethanol should
be administered upon suspicion of ethylene glycol
ingestion. Ethanol has a tenfold greater affinity for
alcohol dehydrogenase than ethylene glycol has and
therefore causes a virtual shutdown of ethylene glycol
metabolism and can be considered a true antidote. This
administration of antidote can be coincident with both
emesis and subsequent charcoal treatment. Once
ethylene glycol is metabolized, hemodialysis becomes
538
Ethylene Glycol
crucial and should be instituted as early on in the
treatment as possible; hemodialysis removes all the toxic
agents while allowing for simultaneous correction of
acid-base and electrolyte abnormalities.
Ethylene Glycol Performance Goals
Ethylene glycol is an infrequent analyte included in the
Toxicology T survey of the College of American
Pathologists (CAP). Labs that carry out routine ethylene
glycol analysis may want to exchange samples with
other labs for proficiency testing. Because most assays
are noncommercial, home-developed tests, there is
generally poor standardization between laboratories. In a
CAP survey containing ethylene glycol, only 26.1% labs
correctly identified ethylene glycol in the specimen. Gas
chromatography was the method most commonly used
by the labs that correctly reported the presence of
ethylene glycol in the specimen. False-positive results of
ethanol, ibuprofen, and salicylates were reported by
individual labs. As many as 70.6% labs reported that no
drugs were detected. The interlaboratory CV by gas
chromatography was 13.4%. A Swedish study looking at
data from an external proficiency test found an
intralaboratory CV of 4.5% and an interlaboratory CV of
11.4% [27]. Results obtained using gas chromatography
and enzymatic methods were generally in good
agreement [27]. A U.K. study reported a between-
laboratory CV of 24% in 11 participants who measured
ethylene glycol in whole blood [28]. No Clinical
Laboratory Improvement Amendments required
performance goals have been set for ethylene glycol
proficiency testing.
References
1 Milles G. Ethylene glycol poisoning. Arch
Pathol 1946; 41: 631-637.
2 Boemke F. Beitrag zur Toxikologie und
Pathologie des thylenglykols (Glysantin).
Virchows Arch Pathol Anat 1943; 310: 106.
3 Pons C. Acute ethylene glycol poisoning: a
clinicopathologic report of 18 fatal cases. Am J
Med Sci 1946; 211: 544-552.
4 Harger RN, Forney RB. A simple method for
detecting and estimating ethylene glycol in
body materials: analytical results in six fatal
cases. J Forensic Sci 1959; 4: 136-143.
5 Rajagopal G, Ramakrishnan S. A new method
for estimation of ethylene glycol in biological
material. Anal Biochem 1975; 65: 132-136.
6 Meola J. Fluorometry of ethylene glycol in
serum. Clin Chem 1980; 26: 1709.
7 Standefer J, Blackwell W. Enzymatic method
for measuring ethylene glycol with a centrifugal
analyzer. Clin Chem 1991; 37: 1734-1736.
8 Hansson P, Masson P. Simple enzymatic
screening assay for ethylene glycol ( ethane-
1,2-diol) in serum. Clin Chim Acta 1989; 182:
95-102.
9 Eckfeldt J. Kinetic ethylene glycol assay with
use of yeast alcohol dehydrogenase. Clin Chem
1980; 26: 1278-1280.
10 Kramer J. Identification of hippuric acid
crystals in the urine of ethylene glycol
intoxicated dogs and cats. J Am Vet Med 1984;
184: 584.
11 Peterson RL. Gas chromatographic
determination of ethylene glycol in serum, Clin
Chem 1982; 28: 75-78.
12 Robinson S. A gas chromatographic procedure
for quantitation of ethylene glycol in
postmortem blood. J Anal Toxicol 1981; 5: 69-
72.
13 Gondolphin W. Unusual calcium oxalate
crystals in ethylene glycol poisoning. Clin
Toxicol 1980; 16: 479-486.
14 Hewlett T, Ray AC, Reagor JC. Diagnosis of
ethylene glycol (antifreeze) intoxication in dogs
by determination of glycolic acid in serum and
urine with high pressure liquid chromatography
and gas chromatography-mass spectrometry. J
Assoc Off Anal Chem 1983; 66: 276-283.
15 Berger J. Neurological complications of
ethylene glycol intoxication. Arch Neurol 1981;
38: 724-726.
16 Field DL. Acute ethylene glycol poisoning. Crit
Care Med 1985; 13: 872-873.
17 Riley J. Urine and tissue oxalate and hippurate
levels in ethylene glycol intoxication in the dog.
Vet Hum Toxicol 1982; 24: 331-334.
18 Gupta R. Liquid-chromatographic
determination of ethylene glycol in plasma. Clin
Chem 1982; 28: 32-33.
19 Doedens D. Methods for determination of
ethylene glycol. Vet Hum Toxicol 1983; 25: 96-
101.
20 Eder AF, Dowdy YG, Gardiner JAM, Wolf
BA, Shaw LM. Serum lactate and lactate
dehydrogenase in high concentrations interfere
in enzymatic assay of ethylene glycol. Clin
Chem 1996; 42: 1489-1491.
21 Anon. Possible death from drinking ethylene
glycol (Prestone). JAMA 1930; 94: 1940.
22 Geiling EMK, Cannon PR. Pathologic effects
of elixir of sulfanilamide (diethylene glycol)
poisoning. JAMA 1938; 111: 919-926.
23 Moriarity R. The spectrum of ethylene glycol
poisoning. Clin. Toxicol. 1974; 7:583-596.
24 Stokes J. Prevention of organ damage in
massive ethylene glycol ingestion. JAMA 1985;
243: 2065-2066.
25 Widmen C. A few cases of ethylene glycol
intoxication. Acta Med Scand 1946; 126: 295-
306.
26 Veltri J. 1983 Annual report of the American
Association of Poison Control Centers National
Data Collection System. Am J Emerg Med
1984; 2: 420-433.
27 Jones AW, Hrd L. How good are clinical
chemistry laboratories at analyzing ethylene
glycol? Scand. J. Clin. Lab. Invest t. 2004; 64:
629634.
28 Wilson JF, Toseland PA, Capps NE, Sandle
LN, Smith BL, Sweeney G et al. External
quality assessment of laboratory performance in
analysis of toxicological cases. Forens. Sci.
Intern. 2001; 121: 27-32.
539
Ethylene Glycol

Tables
Table 1: Methods of Ethylene Glycol Analysis
Method 1: Oxidation-periodic acid
Type of analysis: Colorimetric
Principle: Ethylene glycol is oxidized by acid to formaldehyde and measured as a colored derivative
Usage: Historical
Comments: Limited sensitivity
Method 2: Oxidation-periodic acid
Type of analysis: Colorimetric
Principle: As above; then chromotropic acid is used to form colored derivative
Usage: Limited
Comments: Required precipitation of many interfering substances with copper sulfatecalcium hydroxide
preparation
Method 3: Oxidationperiodic acid
Type of analysis: Fluorometric
Principle: Ethylene glycol is oxidized by acid to formaldehyde and then converted to a dihydrolutidine derivative
Usage: Alternative
Comments: As in method 2. Excessive interference by elevated triglycerides, limited range of linearity
Method 4: Oxidation-enzymatic (glycerol dehydrogenase)
Type of analysis: Spectrophotometric
Principle: Ethylene glycol is oxidized by glycerol dehydrogenase to glycoaldehyde, with concurrent reduction of
NAD+ to NADH; reaction monitored at 340 nm
Usage: Limited, good for stat analysis
Comments: Interference from 2,3 butanediol, glycerol, propylene glycol, lipemia, and hemolysis. Patients with
high levels of lactate and lactate dehydrogenase and patients with acetaminophen-induced hepatotoxicity may
have falsely elevated results.
Method 5: Oxidation-enzymatic (alcohol dehydrogenase)
Type of analysis: Spectrophotometric
Principle: Alcohol dehydrogenase oxidizes ethylene glycol to its aldehyde, with concurrent reduction of NAD+
to NADH; reaction monitored at 340 nm
Usage: Limited
Comments: Interference from ethanol, methanol, and isopropanol
Method 6: Osmolal measurement
Type of analysis: Freezing-point depression
Principle: Calculated osmolality is subtracted from measured osmolality; ethylene glycol estimated: (Osmolal
gap) 62.1 0.92
Usage: Possible screen
Comments: A quick method for estimating the quantity of ethylene glycol in a known or suspected ingestion;
mol. wt. of ethylene glycol, 62.1
Method 7: Thin-layer chromatography
Type of analysis: Chromatographic
Principle: Ether extraction; developed in one of three solutions, visualized with vanillin in H2SO4
Usage: Possible screen
Comments: Sensitivity, limit of detection not reported, use limited to few labs
Method 8: Gas chromatography (GC) (various researchers)
Type of analysis: Chromatographic
Principle: Flame ionization, direct injection
Usage: Common
Comments: Moderate sensitivity, widespread equipment, minimal preparation
Method 9: Gas-chromatography derivative
Type of analysis: Chromatographic
Principle: Extraction and derivatizationbenzoyl ester, N-butylboronate, phenylboronate
Usage: Alternative
Comments: Require extraction but offer increased sensitivity and precision
Method 10: High-performance liquid chromatography (HPLC) derivative (Gupta)
Type of analysis: Chromatographic
Principle: Benzoyl derivative prepared directly in plasma by Schotten-Baumann reaction to allow detection of
ultraviolet absorption spectra
Usage: Experimental
Comments: No extraction, good sensitivity, low interference potential
540
Ethylene Glycol

Method 11: HPLC; gas chromatography-mass spectrometry (GC/MS) (Hewlett)

Type of analysis: Chromatographic
Principle: Measures glycolic acid, the major metabolite; extraction with ketone and derivatization
Usage: Experimental
Comments: Advantage of diagnosis after ethylene glycol has been metabolized; GC/MS used to confirm glycolic
derivative

Figures
Figure 1: Ethylene glycol

Drawings of oxalate crystals. A, Calcium dihydrate

oxalate crystals. B, Calcium monohydrate oxalate
crystals. C, Hippurate crystals.

Figure 2: Ethylene glycol

Tracing of gas chromatographic analysis of ethylene glycol in

patient serum. A, Serum of healthy person. Notice endogenous
peak at 5.60 minutes. B, Serum spiked with 3000 g/mL of
ethylene glycol.
541
Ethylene Glycol

Figure 3: Ethylene glycol

Standard curve of ethylene glycol concentration in serum versus peak height of standard.

Figure 4: Ethylene glycol Metabolism

Schema of pathway of ethylene glycol

metabolism. Boxes, Enzymatic reactions;
LDH, lactate dehydrogenase.
542
Ethylene Glycol

Procedure: Gas Chromatographic Analysis months.

Principle Use a fresh control with each run. Bring an aliquot to

A 1:1 ratio of volatile ethylene glycol (EG) and internal room temperature prior to use, taking care to make sure
standard is injected into a carbowax column within a gas that the sample is homogeneous prior to dilution with
chromatograph, where it is separated from other internal standard and injection.
volatiles. Using a flame ionization detector, the ethylene
glycol may then be quantitated by measurement of the D. 12 75 glass culture tubes
peak height. The amount of EG is quantitated by a E. 100 L Eppendorf pipette
comparison of peak height ratio to that obtained with a F. Hamilton 10 L fixed syringe for
known 1:1 ratio of an EG standard to Internal Standard. injections

Specimens of Choice Assay

Serum (1 mL) collected in a red-top tube with or without Equipment: Gas chromatographic system with
serum separator. Samples should be collected during the flame ionization detector; 6-foot column containing 5%
acute phase of toxicity. Urine (1 ml) may be used for Carbowax 20 M.
qualitative testing only. Samples should be collected in 1. Chromatograph parameters:
the acute phase of toxicity. Specimens may be Chart speed, 0.5 cm/min
transported at room temperature. Serum should be Settings for the Shimadzu gas chromatograph:
separated from the clot upon arrival. If testing will not be A. Column: 6 ft by 2 mm glass column
performed within 3 hours (non-stat), store specimens packed with 5% carbowax 20 M
frozen (<20C); do not heat upon thawing. B. Column temp 150C
C. Injector temp 240C
Indications D. Detector temp 240C
Ethylene glycol is a major constituent of E. Nitrogen flow tank 40 psi (approx 30
antifreeze. The test is often requested based on a positive mL/min)
ingestion history, acidosis of unknown etiology, or the F. Air flow tank 20 psi (approx 300
presence of oxalate crystals seen during urinalysis. The mL/min)
test is available and usually performed on a stat basis. G. Hydrogen flow tank 20 psi (approx 30
mL/min)
Reagents and Materials H Attenuation 32
A. Internal Standard: 2-Ethoxyethanol. I. Range 10
Using a 50 L Hamilton spiking syringe, deliver 20 L J. Mode single
of liquid internal standard into a 100 mL volumetric
flask, and dilute to the mark with deionized water. Ethylene glycol peak, approximately 3.0 cm (or
Prepare fresh daily as needed. Store at room 6 minutes)
temperature. Discard after 24 hours. 2. Set instrument conditions. Allow column to
B. Working Ethylene Glycol Standard bake out for at least 30 minutes, and establish a flat
(111 mg/dL). Using a glass 0.2 mL serological pipette, baseline.
deliver 0.1 mL of EG into a 100 mL volumetric flask, 3. Inject 1 L of ethylene glycol stock standard
and dilute with deionized water to bring to volume. into the instrument. Record retention time of glycol.
Prepare fresh every 24 hours. Store at room temperature. Peak height of standard should be at least 50%
Linearity Check Standards. Procedure linearity should be of full scale.
established monthly, or whenever a new column is put 4. Inject (in duplicate) 1 L of sample, standard,
into service, by analyzing the following curve prepared or control. Record retention time of all peaks.
by adding stock EG in the amounts indicated to 100 mL Figure 2 shows a representative chromatogram
volumetric flasks and diluting to the mark with for a standard and a serum sample.
deionized water.
Procedure
Concentration Stock EG Analytical runs will consist of the following:
(mg/dL) (L) 111 mg/dL working EG Standard
223 200 In-house Control
111 100 Patient sample
56 50 If patient sample is positive; a deionized water
28 25 blank
Reinjection of positive patient
C. In-House Frozen Ethylene Glycol
Control. (56 mg/dL) To a 100 mL volumetric flask, add Note: Rinse syringe with methanol between
50 l of EG stock solution. Dilute to volume with pooled each injection.
negative serum. Mix well, then aliquot 0.2 mL portions A. With an Eppendorf syringe, add 100 l of
into 1275 glass tubes. Store at 20C. Stable for 6 internal standard solution to a glass culture tube.
543
Ethylene Glycol

B. To the same tube, add 100 l of standard, Interpretation

control, or patient sample, and mix the solution.
C. Inject 1 l of the mixture into the gas
chromatograph.
D. Immediately press [start] on the HP integrator
to initiate the run. Repeat this process for mixtures of the
standard, control, and all patient or unknowns. The
internal standard should have a peak height of
approximately one half the width of the recorder paper.
Adjust injection amount or attenuation of the recorder as
needed.
E. Note that occasionally it may be necessary to
inject the internal standard and ethylene glycol
individually to determine the retention time of each
substance.
F. Proceed to the calculation section.
Calculations and Quality Control
A. Compare the retention time of EG
standard with patients retention times. The unknown is
positive for EG if it possesses a peak with a relative
retention time 2% of the standard, with a peak height at
least 2 times greater than peaks in the H2O blank.
See Figure 3 as an example of a typical standard curve.
NOTE: A positive test result is indicated by a
concentration of 5 mg/dL or greater. For urine
specimens, further quantitative calculations are not
undertaken. Report as positive.
B. If the integrator was programmed for
calculating the amounts of EG in the sample, enter the
control value into the QC computer to determine if the
assay is within QC specification. If within acceptable
limits, proceed with reporting of the patient serum
quantitative results unless greater than upper limit of
calibration.
A. Any detectable amount of EG should be
considered abnormal. Therefore, critical values are noted
to be >5 mg/dL. Levels are generally between 50 and
300 mg/dL in serum or urine during acute toxicity,
although the compound dissipates rapidly. Generally,
EG will not be detectable in serum beyond 12 hours post
ingestion, but this is concentration dependent.
B. Specificity: There are no known interfering
substances, although unknown metabolic endogenous
substances have been noted to present false-positive
results in other laboratories (see D below). Ethanol,
methanol, isopropanol, and acetone do not interfere (they
appear before EG). Occasionally, small peaks with the
RT of EG will be noted (see Sensitivity below).
C. Sensitivity: 5 mg/dL. DO NOT REPORT
CONCENTRATIONS BELOW 5. Report as <5 mg/dL.
D. Results may be reported up to 446 mg/dL.
Report results greater than this as >446 mg/dL.
Notes
1. Ethanol, methanol, isopropanol, and acetone
will not interfere.
2. The method is capable of detecting as little as
50 mg/L.
3. In view of the rapid conversion of ethylene
glycol to unmeasured metabolites, any detectable
amount should be considered clinically significant.

C. If the integrator was not programmed to

calculate the results, proceed with manual calculations of
control and patient as follows:
1. Using a millimeter ruler,
determine the peak heights of the ethylene glycol and
internal standard peaks. Form a ratio of these values by
dividing the EG peak height by the I.S. peak height.
2. Use the same procedure for
the rest of the samples. Then divide the ratio of the
control or patient samples by the ratio of the EG
standard, and multiply this number by the concentration
of EG in the standard (111 mg/dL). The result will be in
mg/dL units.

Concentration mg/dL =
peak height ratio of patient or control 111 mg/dL
peak height ratio of EG working standard

D. For integrated or manual calculated results

>223 mg/dL, perform a 1:1 dilution of patient serum
with deionized water, then repeat analysis, multiply
resulting concentration 2. There is no need to perform
additional dilutions
544
Fecal Electrolytes and Osmolality
Fecal Electrolytes and Osmolality
Felix O. Omoruyi, Anthony O. Okorodudu
Names: Electrolytes and osmolality
Clinical significance: Loss of fluids and electrolytes from the gastrointestinal system lead to
diarrhea. Excessive lost electrolytes interrupt acid-base balance.
Refer to Chapter 34, The Pancreas: Function and Chemical Pathology, and Chapter 35
Gastrointestinal Function and Digestive Disease in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Principles of Analysis and Current Usage
The measurement of fecal electrolytes is most often
performed for the assessment of intestinal salt losses in
cases of diarrhea. Thus most fecal samples for which
electrolyte measurements are requested are usually fairly
liquid, with undigested fat and solid-food material
suspended in the water portion. For these most
frequently encountered stool samples, a measured
aliquot of the liquid portion is removed and centrifuged
at 500 to 1000 g to remove particulate matter.
Sodium and potassium can be easily measured by
dilutional or non-dilutional ion-selective electrode
analysis. Chloride can be measured by the ferric
thiocyanate method, by an ion-selective electrode
method, or by coulometry (Chloridometer). Osmolality
can be measured with freezing-point or vapor-pressure
osmometers.
More rarely, electrolyte analysis may be requested on a
stool specimen that is well formed, without a separate
liquid phase. It is not recommended that analysis be
performed on such a sample, because the result will have
little meaning.
To distinguish between osmotic and secretory diarrhea
(see Interpretation below), it is necessary to calculate
and measure fecal water osmolality, looking for an
osmolar gap.
Fecal Osmolar Gap
If poorly absorbed material is retained within the gut,
water moves into the gut and an osmotic diarrhea results.
This provides the rationale for the calculation of fecal
osmolar gap [1,2]. Osmolar gap is estimated by the
following calculation: Osmolar gap = measured
osmolality 2 (Na + K). Note that sodium and
i the measured and calculated osmolality which is
Fecal electrolytes and osmolality
Previous and current authors of this method:
First edition: Not done
Methods edition: Lawrence A. Kaplan
Second edition: Not updated
Third edition: Not updated
Fourth edition: Lawrence A. Kaplan
Fifth edition: Felix O. Omoruyi, Anthony O.
Okorodudu
potassium are the predominant cations in stool water.
When electrolytes constitute most of luminal osmolality,
the calculated fecal osmolar gap will be low (<50
mOsm/kg). When poorly absorbable substances are
present, the fecal osmolar gap will be large (>100
mOsm/kg).
Reference and Preferred Methods
There is no reference method for the analysis of stool
electrolytes. The preferred methods are ISE & freezing-
point osmometry.
Specimen
The preferred sample is a diarrheic stool specimen with a
distinct water phase. A minimum of 1 mL can be
assayed. The physician should be advised if the
laboratory receives a well-formed, solid stool specimen;
such a sample is inappropriate for analysis.
The stool specimen should be collected for a 72-h period
in a pre-weighed can (i.e., paint can). On completion of
the collection, weigh the can with the specimen, and
record the weight. The weight of the feces is obtained by
subtracting the weight of the can (recorded on the can)
from the total weight. The sample should be assayed
without delay, because bacterial contamination will be
present, and metabolism will occur.
Interferences
Substances that might interfere with the analysis of
electrolytes can be found in the method description for
serum electrolytes and osmolality. The laboratory should
ensure that the final specimen is free of particulate
matter and is not too viscous so as not to adversely affect
the instruments used to make the analyses.
Fecal Electrolytes and Osmolality Reference Interval
Fecal electrolyte composition can be very variable. It is
important.
Interpretation
The value in measuring electrolytes in a stool sample
from a person with diarrhea is to distinguish secretory
from osmotic diarrhea. Secretory diarrhea occurs when
there is excess secretion of solute within the gut.
Osmotic diarrhea occurs when there is an accumulation
within the gut of poorly absorbed non-ionic particles, as
545

Fecal Electrolytes and Osmolality

may be seen in conditions such as lactase deficiency.

The two conditions can be differentiated by measuring
osmolality and calculating the osmolar gap. It will be
large in osmotic diarrhea, since the non-ionic substances
will not contribute to the calculated osmolality [3].

References
1 Castro-Rodriquez JA, Salazar-Lindo E, Leon-
Bama R. Differentiation of osmotic and
secretory diarrhea by stool carbohydrate and
osmolar gap measurements. Arch Dis Child
1997; 77: 201-205.
2 Ladefoged K, Schaffalitzky de, Muckadell OB,
Jarnum S. Fecal osmolality and electrolyte
concentrations in chronic diarrhea: do they
provide diagnostic clues. Scand J Gastroenterol
1987; 22: 813-820.
3 Walmesley RN, White GH. A guide to
diagnostic clinical chemistry. Blackwell
Scientific, 1994, p387.
546

Fecal Electrolytes and Osmolality

Methods of Stool Electrolyte and Osmolality

Analyses

Principle
The liquid portion is filtered or centrifuged to remove
particulate matter. The clarified specimen is then
analyzed for electrolytes by standard procedures.
Reagents
1. Distilled water. Class I, electrolyte free.
2. Filter paper. Whatman No. 1, or equivalent.

Assay
Equipment: Centrifuge capable of generating 1000 to
1500 g, filtering funnels, commercial paint can shaker.
1. Weigh the preweighed paint can containing the
collected stool sample.
2. If the sample is already quite liquid, mix it
thoroughly to achieve a homogeneous sample. A
few minutes of mixing in a paint shaker will be
sufficient.
3. Centrifuge at 1000 g for 15 min at room
temperature.
4. If the supernatant is not particle-free and clear,
filter through Whatman No. 1 filter paper, allowing the
filtrate to drip into a clean test tube.
5. Analyze the sample using the usual laboratory
method available for measuring bicarbonate,
osmolality, sodium, potassium, and chloride.
6. Calculate and measure the osmolality of the
stool water. An osmolar gap < 50 mOsm/kg suggests
that the patient has a secretory diarrhea, and an osmolar
gap > 100 mOsm/kg suggests the patient has an
osmotic diarrhea.
547

Fecal Fat and Fat Absorption

Fecal Fat and Fat Absorption

Lawrence A. Kaplan

Name: Fat absorption

Clinical significance: Refer to Chapter 34, The Pancreas: Function and Chemical Pathology, in
the 5th Edition of Clinical Chemistry: Theory, Analysis, Correlation.
Chemical class: Triglycerides

Principles of Analysis and Current Usage Isotope Tests

An assessment of fat absorption is important in the
assessment of malabsorption [1-5]. The most specific
assessment of this function is the accurate chemical
quantitation of fat in a 72-hour stool collection.
Fecal Fat Screening Test
A successful fat screening method is the microscopic
fat evaluation of a single sample. This has been done
by application of fat stains to a smear of a
representative stool sample on a microscope slide,
followed by visual semiquantitation. To perform the
method, an aliquot of stool on a slide is mixed with
two drops of 95% ethanol and two drops of a
saturated ethanolic solution of Sudan III. Under a
microscope, neutral fats appear as large orange or red
drops. In some methods, fatty acids appear as lightly
staining flakes or needle-like crystals. An abnormal
number of fat droplets per high-power field (the
number varies, depending on the method) indicates
malabsorption.
While this procedure is usually used as a qualitative
screening test, its use as a quantitative procedure in
place of the 72-hour fecal fat quantitation has been
proposed [6]. In this procedure, fecal fat in a stool
sample is stained with Sudan III and assessed
microscopically by counting the number of drops and
measuring the size of the fat globules to produce a
quantitative result.
i suggested that the electrical capacitance of a solvent
i extract of stool be measured (Table 1, Method 2)
Fecal Fat and Fat Absorption
Previous and current authors of this method:
First edition: Not done
Methods edition: Michael D.D. McNeely
Second edition: Not updated
Third edition: Not updated
Fourth edition: Michael D.D. McNeely
Fifth edition: Lawrence A. Kaplan
The first isotope-based test for the assessment of fat
absorption was the 131I-triolein test [7]. In this test,
the patient ingested a triglyceride labeled with 131I.
At prescribed time intervals after ingestion, blood
was collected for quantitation of radioactivity. In
general, the more efficient the fat absorption, the
greater the radioisotope count.
A 14C isotope breath test has proved superior to
iodine-labeled methods. It was first described by Abt
in 1966, who measured the recovery of 14C-labeled
lipids [8]. One drawback to the use of this test was
the increased number of false-positive results
observed in obese patients. A popular method based
upon this approach was developed by Kaihara, who
used 14C-tripalmitin [9,10].
A 13C stable-isotope breath test has been developed
as well [11-14]. The advantage of this test over the
14C isotope breath test is the use of non-radioactive
test material. However, this method requires
measurement of 13C by a mass spectrometer or by
NMR (see below).
Fecal Fat Quantitation
The oldest available quantitative method is a
gravimetric technique (Table 1, Method 1). In this
method, the fat from stool is extracted into an organic
solvent, which is then evaporated, and the residue is
weighed to determine the fat content. It has been
[15]. The principle of this test is that the presence of
fatty acids in the extract will decrease electrical
capacitance; the greater the change in capacitance as
measured by change in frequency of a radio-oscillator
circuit, the more fat was extracted from the stool.
Bangs acid dichromate reagent [16] has been used in
a spectrophotometric analysis (Table 1, Method 3).
The basis of this reaction is that fatty acids, released
548
Fecal Fat and Fat Absorption
from extracted triglycerides by saponification, reduce
dichromate to form blue-green chromous ions by the
following reaction:
K2Cr2O7 + 5H2SO4 + 3H+ + R-COOH
(yellow-orange)
Cr2(SO4)4 + K2SO4 + CO2 + 7H2O
(blue-green)
The absorbance of the chromous ions measured at
600 nm is proportional to the amount of fat in the
stool.
Thin-layer chromatography and gas-liquid
chromatography have been applied to the quantitation
of fatty acids in organic extracts of stool (Table 1,
Methods 4a and 4b) [17,18]. Although often used for
research, these methods are very rarely used for
routine analysis, because they are technically
demanding.
A convenient and rapid spectrophotometric method
using the sulfovanillin reaction [19,20] has been
shown to be useful with unsaturated fatty acids but
not C8 or C10 saturated fatty acids. The reaction
(Table 1, Method 5), as suggested by Knight et al.
[21], involves the formation of a carbonium ion at the
double bond by the action of H2SO4 and the reaction
of an aromatic phosphovanillin with the carbonium
ion to form a stable carbonium complex absorbing at
approximately 525 nm.
An interesting approach is the steatocrit, in which
homogenized stool is drawn into a capillary tube
(Table 1, Method 6). The tube is sealed at one end by
a flame and centrifuged for 15 minutes. The top
layer, representing lipid, is measured with calipers
and expressed as a percentage of the total volume
(steatocrit) [22,23].
The most commonly used method to measure fecal
fat is based on the procedure of van de Kamer et al.
[24]. This method hydrolyzes fats by reflux of a
weighed fecal sample in alcoholic KOH. The sample
is then acidified and the fatty acids extracted into
petroleum ether. The solvent is then evaporated, the
residue dissolved in ethanol, and the fatty acids
determined by titration against standardized NaOH
using thymol blue as an indicator (Table 1, Method
7). Many modifications of the van de Kamer
technique have been employed. These include the
conditions of saponification (i.e., alcohol
concentration), choice of extraction solvent, indicator
(i.e., the use of methyl red), and choice of standard.
Most methods employ pure tripalmitin as the
standard.
Near-infrared reflectance spectrophotometry
(NIRRS; Table 1, Method 8) has been used to
quantitate fecal fat concentration [25]. In this
technique, radiation in the near-infrared spectrum that
is scattered by the surface of a homogenized fecal
specimen is measured. Measurement of the scattered
radiation at different wavelengths enables the
determination of total nitrogen content, total fat, and
hydrolyzed fat. With the availability of the NIRRS
equipment, this technique is both rapid and relatively
simple to perform.
Another method for the determination of total fecal
fatty acids is the use of nuclear magnetic resonance
(NMR; Table 1, Method 9). The common unsaturated
fatty acids (oleic, linoleic, linolenic) can be
quantified using 1H-NMR by utilizing the area-per-
proton (determined by integration) technique
(http://www.lipidlibrary.co.uk/nmr/nmr.html) [26].
Equations can then be used to estimate the amounts
of the unsaturated fatty acids. NMR can also be used
to detect 13C-labeled fat [27].
The gold standard, and thus still the most commonly
used procedure for the determination of fecal fat for
the diagnosis of malabsorption syndromes, remains
the 72-hour quantitative fat estimation, using
methods based on van de Kamer et al. [24].
Reference and Preferred Methods
There are no reference methods for fecal fat analysis
(http://www.bipm.org/jctlm/), although literature on
this topic almost always refers to 72-hour fecal
quantitative fatty acid titratimetric analysis (Table 1,
Method 7) as the gold standard. This method is also
the easiest to establish and requires little specialized
equipment or specialized laboratory skills. For this
reason, it remains the most frequently performed and
recommended method.
For those laboratories that want to screen feces
before performing the quantitative analysis, the
microscopic technique for fat malabsorption is
recommended. It is simple to perform and reasonably
sensitive. Drummey [27] reported that the technique
gave no false-negative results for anyone ingesting
more than 15% of their diet as fat. Simko [28]
evaluated this approach and reported that problems
cited in the past may have been attributable to poor
technique. The method was shown to be less reliable
when there were increased levels of soaps and when
free fatty acids were present, because these do not
stain.
549
Fecal Fat and Fat Absorption
The quantitative approach to the microscopic
screening technique reported by Fine and Ogunji [6]
was reported to compare well with the 72-hour fat
quantitation method. However, there has been no
other study reported using this method, so its clinical
efficacy remains uncertain and can not be
recommended.
The NMR method with 1H proton analysis is used by
large reference laboratories in the United States. With
no extraction required and rapid analysis time, the
capital expense for the NMR equipment seems
worthwhile if large numbers of samples are presented
to these laboratories. These costs will most likely be
prohibitive for small laboratories with few samples.
The remaining methods seem not to be in routine use
and may be better suited for research purposes.
The isotopic methods for estimation of fat absorption
have the disadvantage of the need to deal with
radioisotopes. Also, the iodine-labeling process
renders the triglyceride chemically unstable, and the
radioactive iodine can be severed from the lipid by
the gut mucosa and absorbed independently of lipid.
The labeled triglyceride is often contaminated with
radioactive free fatty acids, fatty acid esters, and
other fragments that have variable rates of
absorption. To avoid these problems, 14C label has
been increasingly used instead of the 125I label. West
[29] compared 14C-tripalmitin test results to changes
in serum turbidity and serum triglyceride after a fat
meal. Only four false-positive results out of 28
healthy persons were observed, and 32 out of 32
abnormals were identified.
Meeker [30] devised a variation of the test in which 5
Ci of tripalmitin-carboxyl-14C was mixed with 35
to 40 mL of an intravenous fat emulsion and
administered orally to the 124 patients first with, and
then several days later without, Viokase (650 U of
lipase). Compared to results obtained by fecal fat
measurement, this method showed 15% false
positives and 7% false negatives. The Viokase
modification was judged useful to separate pancreatic
from nonpancreatic disease and was helpful in
determining the best course of therapy. Other authors
have stated that the test does not discriminate
adequately [31]. However, Newcomer [10] found that
distinguishing patients with and without pancreatic
disease was improved when triolein was used rather
than tripalmitin. Ghoos [32] employed a mixed
triglyceride-1,2-dioleyl-2-14C-decanoyl glycerol and
considered it better than either trioleic or tripalmitic
acids.
Butler and Gehling [33] demonstrated success with a
4-hour, 14C breath test. Tomkin examined the
possibility of measuring blood 14C but found this
more variable than breath analysis [34]. A number of
workers have recognized the problem of low levels of
radioactivity (false-positive results) in obese patients.
Although this has been said to be caused by delayed
gastric emptying, it is probably caused by increased
insulin. To correct for some of the inconsistencies,
Strange et al. [35] added factors for age, body weight,
metabolic rate, and respiratory quotient but did not
believe these improved the diagnostic power of the
test.
A number of reports have demonstrated that the 13C
breath test compared well with the standard 72-hour
fecal fat determination. The advantage of using a
non-radioactive tracer is certainly attractive.
However, most of the recent reports that suggest high
clinical utility of this non-isotopic breath test have
been performed outside the United States [11-14].
Thus far, the only non-isotopic breath test to be
approved by the U.S. Food and Drug Administration
is the 13C-urea breath test [36].
The steatocrit test has the advantage of rapidity and
simplicity when estimating the amount of fat in stool.
However, this tests clinical efficacy has been
questioned [37], and the steatocrit remains a rarely
performed procedure.
The near-infrared reflectance spectroscopy (NIRRS)
procedure seems to be an accurate and reproducible
method [38,39]. Although NIRRS has the advantage
of not requiring stool extraction, this technique
requires specialized equipment and knowledge and
thus remains an interesting but not widely used
procedure.
Major drawbacks of the gravimetric method are the
need to work with dangerous solvents (e.g.,
petroleum ether, hexane, xylene, and carbon
tetrachloride) and the overestimation of lipid content
by extraction of nonpolar, nonlipid matter. There may
be underestimation of lipid when medium-chain
triglycerides are lost during the evaporation step [40].
The electrical capacitance approach has been
criticized [41] because petroleum ether extracts of
medium-chain triglyceride (or medium-chain fatty
acid) and long-chain triglyceride (or long-chain fatty
acid) have distinctly different electrical capacitances.
In fact, if triolein is used as a standard, any medium-
550
Fecal Fat and Fat Absorption
chain triglyceride present is underestimated by 27%,
whereas medium-chain fatty acid is overestimated by
35%. These methods are rarely used in routine
laboratories.
The sulfovanillin method produces decreasing
amounts of color as the number of double bonds in
the fatty acids increases, thus yielding varying results
as the fatty acid composition changes [20]. In
addition, many alcohols can be dehydrated by the
reaction conditions and subsequently enter into the
reaction. Nevertheless, the method demonstrated
good recovery of added lipid (approximately 99.6%)
and good precision.
Several workers, including van de Kamer himself
[42], have noted that the fatty acid titration method
recovers only about 60% of the medium-chain
triglyceride. Recently this has become particularly
important when the success of medium-chain
triglyceride administration is being monitored for the
treatment of fat malabsorption and
abetalipoproteinemia; it is of questionable importance
in routine work.
Specimen
Diet
For all direct methods of fat measurement in stool, it
is important that the patient ingest a normal daily
diet containing a minimum of 100 g of triglyceride
[42]. Rather than prescribing a complex diet, it is
advisable to insist that the patient ingest 1 ounce of
corn oil and a glass of whole milk with each meal for
3 days preceding the stool collection and for the 3
days of the collection.
A 72-hour collection is considered essential.
Defecation is highly variable from day to day (up to
several hundred percent), and only this collection
time will average out the usual variations. Workers
have reported using markers such as charcoal, Congo
red, chromic oxide, barium sulfate, and cuprous
thiocyanate to keep accurate track of the transit time.
These markers are not generally necessary.
It is difficult to establish criteria by which a stool can
be rejected for analysis. Some laboratories do not
wish to analyze specimens that are formed. However,
although fatty stools are usually fluid, semifluid, or
soft, bulky, foul, or foamy borderline conditions may
produce relatively normal samples.
Stool Collection
Whatever technique is selected for analysis, the most
important steps in any fecal analysis are the
collection and homogenization procedures.
An elaborate but effective method has been described
in which the feces are kept deep frozen using a
portable toilet [43]. A simpler approach is to use a
portable campers toilet, to which can be fixed a
plastic bag. Each collection is then contained in a
separate bag and frozen.
Preweighed 5-gallon paint cans are the most
commonly used collection containers, since they are
relatively inexpensive and easy to use and can also be
used as the extracting vessels (see below).
For many years, dry sampling techniques were used
in fecal fat analysis. These involved thorough mixing
of the entire stool collection into a homogeneous
paste, removal of an aliquot, and drying of the aliquot
in an oven or under a heat lamp. In addition to being
a rather repugnant procedure, there is very little
clinical significance to a result expressed as units of
fat per unit of dry feces. Furthermore, the application
of intense heat may result in some fatty acids being
volatilized [44].
Wet techniques, which involve the addition of a
known amount of liquid to the specimen, mixing, and
removal of an aliquot, are most commonly used.
Manual mixing or the use of a blender have been
suggested and are used, but these techniques are not
only very messy but are probably also less efficient
than those of Newell [45] and Jover and Gordon [46],
in which the entire collection is placed in a
preweighed 5-gallon paint can. This can be done
directly (as above), or the frozen aliquots may be
added separately and allowed to thaw. The weight of
the container and complete collection can then be
determined, and the weight of the collection can be
determined by the difference. The contents of the can
may then be diluted with a homogenizing solution
(such as isopropanol) and the entire mixture
homogenized by vigorous agitation on a paint shaker.
Interferences
Oil-containing cathartics can cause false-positive
results. In addition, ingestion of Olestra, the artificial
dietary fat, may also cause a false-positive result
[47]. The most important cause of false-negative
results is insufficient fat ingestion during the test
period. Contamination of the fecal collection with
urine should be avoided.
551
Fecal Fat and Fat Absorption
Fecal Fat Reference Interval
For adults, less than 5 g of fat are normally excreted
in 24 hours after ingestion of 50 g of fat in the diet.
Steatorrhea is generally defined as the excretion of
greater than 7 g of fat per day. Using the near-
infrared reflectance spectroscopy technique, one
group has published reference values for stool fat for
ages < 0.5, > 0.5 to 1.5, > 1.5 to 4, and > 4 to 14
years [48].
For more detailed information, see the detailed
procedures at the end of this chapter.
Interpretation
Both the total weight and fatty acid excretion per 24
hours should be reported. If the daily fat excretion is
greater than 17 mmol, fat malabsorption is occurring.
If the daily fat excretion is between 10 and 17 mmol,
the patient is not normal and may be suffering from
mild fat malabsorption. The fecal weight is
important, since it indicates to the clinician the
seriousness of the diarrhea, and very heavy
specimens with a low fat content may indicate watery
diarrhea.
Fecal fat analysis is a primary test to diagnose fat
malabsorption in a wide variety of disorders in which
pancreatic insufficiency is a major part of the disease
state [49-53].
Fecal Fat Performance Goals
There are no established performance goals for fat
absorption analysis, either from proficiency programs
or from clinical professional groups. A laboratory
performing quantitative fecal fat analysis needs to
base acceptable recovery of extracted fat and
acceptable precision on within-lab data, as well as
guidance form their user-physicians.
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51 Kalivianakis M, Minich DM, Bijleveld
CMA, van Aalderen WMC, Stellaard F,
Laseur M et al. Fat malabsorption in cystic
fibrosis patients receiving enzyme
replacement therapy is due to impaired
intestinal uptake of long-chain fatty acids.
Am J Clin Nutr 1999;69:127-134.
52 Steer ML, Waxman I, Freedman S. Chronic
Pancreatitis. N Eng J Med 1995;332:1482-
1490.
53 Holt PR. Diarrhea and malabsorption in the
elderly. Gastroenterol Clin North Am
2001;30:427-44.
554

Fecal Fat and Fat Absorption

Tables
Table 1: Methods of Quantitative Fecal Fat Measurement
Method 1: Gravimetric
Principle of analysis: Fat-soluble material is extracted, and after solvent removal, is weighed.
Comments: Historical; nonspecific; requires handling of dangerous solvents
Method 2: Capacitance
Principle of analysis: Fatty acids present in a solvent extract of stool decrease electrical capacitance of the
solvent.
Comments: Historical; requires special equipment; difficult to standardize properly
Method 3: Dichromate reduction
Principle of analysis: Potassium dichromate is reduced by saponified, extracted fatty acids to form blue-
green chromous ions and measured spectrophotometrically at 600 nm.
Comments: Rare; nonspecific; different color yield with different fatty acids
Method 4: Chromatographic
a. Thin-layer chromatography
b. Gas-liquid chromatography
Principle of analysis: Saponified, extracted fatty acids are separated and quantitated.
Comments: Research only; requires special equipment; results have limited clinical significance
Method 5: Sulfovanillin reaction
Principle of analysis: Saponified, extracted, unsaturated fatty acids react with vanillin to form a colored
carbonium ion complex whose absorbance can be measured at 525 nm.
Comments: Rare; color yield dependent on number of double bonds in fatty acids; good precision
Method 6: Steatocrit
Principle of analysis: Homogenized stool specimen is drawn into a capillary tube and centrifuged.
Volume of fatty layer is described as percentage of total.
Comments: Rare; nonspecific
Method 7: Titration
Principle of analysis: Extracted, saponified fatty acids are titrated using thymol blue as an indicator.
Comments: Most common; can underestimate amount of medium-chain fatty acids
Method 8: Near-infrared reflectance assay
Principle of analysis: Radiation in the near-infrared spectrum that is scatted by the surface of a fecal
sample is measured.
Comments: Simple and reliable method; allows determination of dry weight, total fat, and hydrolyzed fat
Method 9: Nuclear magnetic resonance (NMR)
Principle of analysis: The nuclear magnetic resonance spectra of 1H protons of individual fatty acids
measured and the total amounts of fatty acids calculated.
Comments: Does not require hydrolysis or extraction of the sample.
555

Fecal Fat and Fat Absorption

Figures
Figure 1: Fecal Fat Analysis

Standard curve for fecal fat analysis: absorbance at 502 nm versus amount of tripalmitin standard.

Procedure: Microscopic Screen (Simko 1981 5. Mix thoroughly with a glass rod.
Modified) [27] 6. Fill a hemocytometer chamber with the
mixture.
Principle 7. Warm for 2 min on a 37C heating block to
Stool sample is mixed with a fat-staining dye. The hydrolyze the fecal soaps into fatty acids.
colored droplets are counted by microscopic 8. While it is still warm, count the pink fat
examination. If the number of droplets exceeds droplets in 10 high-power (400) fields, and express
normal limits, malabsorption is strongly indicated. the results using the table below:

Reagents
1. Sudan III dye reagent, 5 g/L. Dissolve 1 g Calculation and Interpretation
of Sudan III dye reagent in 100 mL of 95% ethanol Average number Approximate
and 100 mL of glacial acetic acid to form a saturated of fat droplets fat content
solution. Stable for 6 months at room temperature. 10 5%
20 10%
30 15%
Assay 35 20%
Equipment: hemocytometer, heating block, >40 >30%
and a microscope.
1. Collect stool as a single sample or as a
prolonged collection. Fat Absorption Reference Interval
2. Mix thoroughly. Normal: Less than 5% fat content.
3. Place a 500-mg aliquot of the sample in a
100-mL beaker.
4. Add 0.5 mL of a Sudan dye reagent.
556
Fecal Fat and Fat Absorption
Procedure: Chemical Quantitation of Fecal Fat
Output [52]
Principle
A well-mixed stool sample is saponified by treatment
with alcoholic KOH at 80C. The free fatty acids are
extracted and quantitated by spectrophotometric
observation of their reaction with methyl red. The
amount of fat present in the sample is calculated by
comparison of the unknown with a tripalmitin
standard.
Reagents
1. Alcoholic potassium hydroxide, 0.357
mol/L of ethanol. Place 2.0 g of KOH in a 100-mL
volumetric flask, dissolve, and dilute to volume with
absolute ethanol. Prepare fresh each day.
2. Sodium hydroxide, 1.0 mol/L. Place 4.0 g
of NaOH pellets in a 100-mL volumetric flask. Add
approximately 80 mL of distilled water to dissolve
pellets. Allow solution to cool, add water to mark,
and mix well. Store in plastic container at room
temperature for up to 6 months.
3. Hydrochloric acid, 0.6 mol/L. Place 50
mL of concentrated HCl in a liter volumetric flask
containing 800 mL of distilled water, and add
distilled water to mark. Mix well, and store at room
temperature for up to 6 months.
4. Petroleum ether (boiling point 39C to
53C), redistilled.
5. Methyl red (Fisher Scientific Co.,
Pittsburgh), 2 g/L. Dissolve 200 mg of powder in
100 mL of 95% ethanol. Solution proceeds slowly.
Store at 4C to 8C for up to 1 year.
6. Sodium acetate, 1 mol/L. Place 13.6 g of
sodium acetate trihydrate into a 100-mL volumetric
flask. Add approximately 80 mL of distilled water,
and dissolve salt. Add water to mark, and mix well.
Store at room temperature for up to 2 months.
7. Buffered methyl red reagent. Prepare this
reagent by photometric measurement to avoid
variations in dye lots. Add 10 mL of 1 mol/L NaOH
to 1000 mL of 95% ethanol. Add enough indicator
(10 to 13 mL) to bring absorbance to a range of 0.095
to 0.100 at 502 nm. Add 2.0 mL of 1 M sodium
acetate solution. Using a magnetic stirring device,
add 1 mol/L HCl by drops until a faint orange-red
tinge persists. Carefully add more acid until the
solution has an absorbance of 0.195 0.005. If this
point is passed, titrate back with dilute NaOH, being
very careful not to dilute the reagent excessively.
8. Tripalmitin standards. Dissolve 2.5 mg of
tripalmitin in 200 mL of heptane. A standard curve
may be prepared by addition of 0.25, 0.50, 1.00, 1.50,
and 2.00 mL of this solution to 15 150-mm screw-
topped culture tubes and drying with a stream of
warm air. These standards are equivalent to 5, 10, 20,
30, and 40 mmol of fatty acid per liter of homogenate
or 10, 20, 40, 60, and 80 mmol of fatty acid per total
specimen. It is convenient to prepare several sets of
standards at one time and store them, tightly capped,
at room temperature. They are stable for several
months.
9. Carboxymethyl cellulose. Add 4 g of
carboxymethyl cellulose (sodium salt) to 1 L of
distilled water. Mix to dissolve, and store at 4C to
8C for up to 6 months.
Assay
Equipment: Preweighed paint cans, paint shaker,
water bath (50C to 60C), and spectrophotometer
(band pass 10 nm) capable of reading at 502 nm.
Sample preparation:
1. The patient ingests a high-fat diet as
previously described.
2. Feces collected into plastic bags are placed
in preweighed paint cans. This is easily done when
the specimens are frozen.
3. When the analysis is to be performed, the
can and its contents are weighed and the weight of
the fecal collection is determined by subtraction.
4. The cans contents are diluted to 970 g with
water, and 1 L of isopropanol is added.
5. Next, 2 g of NaOH pellets and 100 mL of 4
g/L carboxymethyl cellulose solution are added. A
handful of washed pebbles or metal washers is also
included.
6. The can is placed on a commercial paint
shaker and agitated vigorously for 15 min.
7. Without delay, the can is opened, and an
aliquot of the newly formed emulsion is removed by
use of a pipet with the tip removed or a plastic
drinking straw. The aliquot may be stored
indefinitely in the frozen state.
8. If the sample weighs more than 970 g, or a
very high fat content is suspected, an additional
dilution should be made by addition of water to a
weight of 1825 g, 4 g of NaOH, and 200 mL of
carboxymethyl cellulose solution, followed by 1890
mL of isopropyl alcohol. Shake for 30 min, and
multiply the final result by 1.9.
Analysis:
1. Remix aliquot of fecal emulsion vigorously.
2. Pipet 0.20 mL into a 15 150-mm screw-
topped culture tube.
3. Add 2.0 mL of alcoholic KOH, and cap
tightly with a Teflon-lined cap.
4. Incubate for 60 min at 80C.
5. Add 3.6 mL of 0.6 M HCl, and cool to room
temperature.
557
Fecal Fat and Fat Absorption
6. Add 10 mL of petroleum ether, and shake
vigorously for 2 full min.
7. Centrifuge at 790 g for 5 min.
8. Transfer 5.0 mL of petroleum ether (upper
phase) to a 15 125 mm screw-capped culture tube,
carefully avoiding lower phase.
9. Place tubes in water bath (50C to 60C),
and evaporate ether with a gentle stream of air.
10. Add 3.0 mL of buffered methyl red reagent
to dry residue, stopper tightly, warm for 2 min in
water bath (50 to 60C), and shake vigorously to
ensure solution.
11. Read absorbance of colored solution against
isopropanol at 502 nm in a spectrophotometer.
Calculation
Plot absorbance of tripalmitin standards against
concentration on rectilinear graph paper to obtain
standard curve, and read concentration of unknowns
directly from the curve. An example of a standard
curve is shown in Fat Absorption Figure 1. Divide
result by 3 to obtain total fatty acid excretion per 24
h.
Fat Absorption Reference Interval
Less than 5 g of fat are normally excreted in 24 h
when a person has ingested 50 g of fat in a diet.
Procedure: Breath Test (Butler) [32]
Principle
14C-Triolein is ingested with a fat load, and one
measures the amount of 14CO2 produced by trapping
expired air and counting dissolved CO2 in a liquid
scintillation counter.
Reagents
14C-Triolein. Obtained from Amersham
1.
(Arlington Heights, IL) in a capsule form.
2. Trapping reagent. click here
3. Scintillation cocktail. click here
4. Collection device. click here
Assay
1. Administer 5 Ci of 14C-triolein
(Amersham) in a gelatin capsule followed by 40 mL
of fat (corn oil emulsion; 30% oleic acid).
2. Exhale through a 2 mL solution of hyamine
hydroxide (2 mmol/L). (See the 14C-CO2 breath test
by McNeely.)
3. Take duplicate samples for the baseline
value and each hour after the isotope has been
administered.
4. Add 10 mL (6 g/L PPO in toluene) to each
vial.
5. Count for 10 min using a 1:10 dilution of the
administered dose as a standard.
6. Express the result as a percentage of the
original dose per mmol of CO2 per kilogram of body
weight.
Calculation
2- to 4-h rate = 4-h cumulative 2-h cumulative
2
Fat Absorption Reference Interval
Normal: (2- to 4-h rate) > 2%/h.
Fat Stain (for Examining Fat Droplets in Stool)
I. Principle of Test and Clinical Significance
The microscopic examination of feces for fat
is performed as a screen to assess digestive function.
Increased fat in the stool is an indication of
steatorrhea or malabsorption of fat. Neutral fat
droplets are stained with alcoholic Sudan III,
appearing as yellow-orange or red droplets.
II. Sample (Specimen)
A stool specimen is collected in a clean, fat-
free container (glass, Pyrex, or polyethylene). Waxed
containers are not suitable.
III. Reagents
A. Sudan III dye. Fisher certified
biological stain, catalog #5669. Store at room
temperature. Stable to expiration date on vial.
B. 95% ethanol (190 proof). Obtain
from UH Pharmacy. Store stock container in
flammable cabinet at room temperature. Stable to
expiration date on bottle.
C. Sudan III stain, a saturated solution
in 95% ethanol. Add Sudan III dye to 100 mL 95%
ethanol. Swirl to mix. Solution is saturated when dye
will no longer dissolve. Stable 1 year at room
temperature.
IV. Equipment
A. Microscope
B. Microscope slides and coverslip
C. Wooden applicator sticks
V. Calibration: N/A
VI. Quality Control
VII. Procedure and Methodology
A. Place a small aliquot of stool
558

Fecal Fat and Fat Absorption

suspension on a slide with a wooden applicator stick.

Add 2 drops of 95% ethanol and 2 drops of saturated
Sudan III, and mix with the applicator stick. IX. Expected Result: Negative
A. Reference range 311 mIU/mL
B. Place a coverslip over the B. No critical values.
specimen, and examine under high power (400).
Neutral fats appear as large, yellow-orange or red
X. Procedure Notes: None
droplets.
VIII. Calculations (Derivation of Results)
XI Procedure Performance: N/A
The presence of 60 or more stained droplets
of neutral fat per high-power field is considered
positive.
 559
Fecal Occult Blood
Fecal Occult Blood
R. Swaminathan
Name: Fecal occult blood
Clinical significance: Refer to Chapter 35, Gastrointestinal function, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
Fecal occult blood testing is performed to detect bleeding
in the gastrointestinal tract. As the name implies, it refers
to bleeding that is not apparent to the patient.
When blood enters the upper gastrointestinal tract, the
globin part of the hemoglobin molecule is completely
digested by the proteolytic enzymes, and the heme is
converted by bacterial action to porphyrins. Hemoglobin
entering the lower part of the large intestine is largely
undigested. In normal subjects, the amount of blood lost
from the gastrointestinal tract is 0.5 to 1.5 mL per day
[1,2,3]. This amount of blood is not usually detected by the
fecal occult blood test.
Common methods for detection of occult blood are based
on the detection of hemoglobin or its breakdown products.
Blood in feces can also be detected by macroscopic
examination of feces for blood cells or hematin crystals or
spectroscopic identification of hemoglobin and its
derivatives [4,5].
Tests commonly used in clinical laboratories are based on
the detection of hemoglobin, heme, or heme-derived
porphyrins. Hemoglobin is detected by immunological
methods, heme by guaiac-based methods utilizing the
pseudoperoxidase activity of heme, and porphyrins by
fluorimetry.
Guaiac-Based Methods
The most common method for detection of fecal occult
blood is that based on the detection of heme. In these
methods, pseudoperoxidase activity of heme liberates
nascent oxygen from hydrogen peroxide. The liberated
oxygen oxidizes a chromogen. Historically benzedrine and
o-tolidine were used as the chromogen [6]. These methods
are sensitive, but benzedrine and o-tolidine are
carcinogenic and are no longer used in clinical laboratories
[7]. Other chromogens used in this type of test are
imipramine hydrochloride and desipramine hydrochloride
[8]. The most common chromogen used now is guaiac, a
natural resin extracted from Guaiacum officinale. The
sensitivity of the guaiac method is less than that based on
i stool [14]. These methods are also more specific for blood
Fecal Occult Blood
New method
Fifth edition: R. Swaminathan
o-tolidine [5]. By employing a stabilizer, the sensitivity of
the method has been improved.
These tests consist of a card containing a high-quality filter
paper impregnated with guaiac. This is stable for long
periods because the guaiac is not in solution. The
developing solution is stabilized hydrogen peroxide in an
aqueous alcoholic solution. Hemoglobin and its iron-
containing degradation products, due to the
pseudoperoxidase activity, release oxygen from hydrogen
peroxide. The oxygen then oxidizes alpha-guaiaconic acid,
a phenolic compound present in guaiac. A quinine
structure is formed, and this rearranges to a blue dye by
internal electron transfer [9].
Immunological Methods
These methods are more specific and use antibodies
against one of the components in blood, most commonly
against the globin chain of the hemoglobin. Hemoglobin
forms a complex with a conjugate of an antibody to
hemoglobin. The conjugate consists of a monoclonal or
polyclonal antibody attached to a dye or enzyme which
will produce a colored product from the substrate present
in the system.
A variety of immunochemical detection systems have been
described. These include enzyme immunoassays (EIA)
[10], hemagglutination [11], latex agglutination [12], and
colloidal gold agglutination assay [13].
Many commercial kits have been developed for the
detection of blood by immunochemical method, and some
of these are automated. Immunochemical methods can be
performed as a point-of-care method at the bedside by a
health care professional, by the patient (methods such as
Hemoccult ICT), or performed in the laboratory.
Immunochemical methods do no require dietary restriction
and therefore are better accepted by the public. Some
immunochemical methods can be automated and therefore
are more reproducible. Immunochemical methods are also
more sensitive, that is, the detection limits are lower than
the guaiac-based methods. With automated systems, the
reproducibility is increased further as the subjective nature
of visual reading of a result is removed. These methods are
capable of detecting as little as 0.3 mL of blood added to
from the lower gastrointestinal tract, especially the colon,
 560
Fecal Occult Blood
because the globin released from hemoglobin in the upper
gastrointestinal tract is hydrolyzed by proteolytic enzymes.
In one device, the MonoHaem, a combination of
immunological and guaiac-based tests is used. Hemoglobin
is immobilized by a monoclonal antibody, and the
hemoglobin is then visualized by the guaiac-based
reaction. Non-hemoglobin peroxidases will give a
background blue color which is discounted.
Heme-Porphyrin Test
This test is based on the fact that the heme in the
hemoglobin entering the gastrointestinal tract is converted
to porphyrins, probably by gut bacteria. In this method,
porphyrins in the feces are extracted and then quantitated
by spectrofluorimetry. This allows exact quantitation of
the hemoglobin entering the gastrointestinal tract. This
method is better at detecting bleeding in the upper
gastrointestinal tract. Guaiac and immunological methods
are unreliable in detecting upper gastrointestinal tract
bleeding, because the hemoglobin may be digested by
proteolytic enzymes in the gut [15].
In addition to hemoglobin, other heme-containing proteins
will also contribute to the amount of porphyrin detected by
this method. Furthermore, the conversion of heme and
other heme proteins to porphyrins is an incomplete process
and depends on colonic transit time, site of bleeding, and
amount of luminal heme [16]. Quantification of fecal
hemoglobin is therefore likely to underestimate the amount
of hemoglobin from the bleeding. Comparison of fecal
heme-porphyrin (HemoQuant) with estimation of blood
loss by the 51Cr-labelled red cell method in patients with
osteoarthritis on aspirin showed that the HemoQuant
underestimated the blood loss [17]. Recovery of ingested
blood by healthy volunteers was only 63% by HemoQuant,
showing that absorption of heme may be a problem [18].
HemoQuant measures heme and dicarboxylic porphyrins
by fluorimetry. However, iron in the heme molecule can
cause quenching, and a reduction step with oxalic acid is
usually introduced to remove the iron [19]. In patients with
the rare inherited disorders of porphyrin metabolism, this
test will give false-positive results.
Other Methods
Methods based on the detection of haptoglobin [20] and
calprotectin [21] have been described. These methods have
not been fully evaluated. Hoff et al. found that the
calprotectin method was not as good as the FlexSure occult
blood test [21]. Recently a method using matrix-assisted
laser desorption ionization/time of flight mass
spectrometry (MALDI-TOF-MS) has been described [22].
In this method, fecal sample was mixed with water,
ultrasonicated, and after centrifugation, the supernatant
was mixed with a matrix solution and used for MALDI-
TOF-MS. Blood was detected by identifying the water-
soluble and chains of hemoglobin. The sensitivity of
the method was 10 to 100 times higher than conventional
methods, and there was no interference from plant
peroxidases or dietary red meat. The application of this
method for routine use or screening has not been shown.
This instrument is expensive; despite its many advantages,
it may not be practical to use it in clinical laboratories at
present. The sensitivity (lower detection limit) of this
method is 0.01 mg/g feces compared to 3 to 10 mg
hemoglobin/g feces for the Hemoccult method [15].
An ELISA method to detect transferrin and globin in feces
has also been described [23].
Reference and Preferred Methods
There is no reference method for fecal occult blood
determination.
Many commercial methods based on guaiac are available.
These include Hemoccult (Beckman-Coulter), Hema
Screen (Immunostics, Inc), Hemoccult SENSA (Beckman-
Coulter), ColoScreen (Helena lab), and HEMDETECT
(DIPROmed, Handels GmbH, Weigelsdorf, Austria). All
guaiac methods are qualitative, and the sensitivity of these
methods is not as good as the immunochemical methods.
In a recent evaluation of some of these kits, the lower
detection limit was found to be 0.65, 0.9, 0.6, and 0.3 mg
Hb/g feces for ColoScreen, Hema Screen, HEMDETECT,
and Hemoccult, respectively [24]. Hemoccult SENSA has
a slightly lower detection limit than Hemoccult and is also
reported to be more reliable [25]. To improve the
sensitivity of the method, it has been shown that fecal
rehydration improves the sensitivity but with loss of
specificity. Rehydration is done by adding about 25 L of
water to each fecal smear 1 minute before adding the
developer. It has been shown that fecal hemoglobin
concentrations must exceed 10 mg/g feces, equivalent to
10 mL of daily blood loss, before the Hemoccult II test
will be positive 50% of the time [26]. However, stools
containing as little as 1 mg of Hb/g of feces can give
positive results [27].
Many commercial kits based on immunological methods
are also available. These include Hemoccult (Beckman
Coulter Inc, USA), HemeSelect (Beckman-Coulter Inc,
USA), MonoHaem ( Nihon pharmaceutical, Japan),
FlexSure OBT (Beckman-Coulter Inc, USA). Of these kits,
some such as HemeSelect and FlexSure OBT are no longer
available. Hemoccult ICT is a colloid agglutination
method and HemeSelect is a hemagglutination assay.
Many of these methods, like guaiac-based methods, are
qualitative or semi-qualitative. Quantitation is possible
with immunological methods, and automated systems are
also available. The Magstream 1000/Hem SP and the OC-
Auto Micro 80 are automated systems utilizing
immunochemical methods to detect occult blood [28,29].
Heme-porphyrin testing is a quantitative method requiring
expensive instrumentation. A comparison of methods is
given Table 1.
 561
Fecal Occult Blood
The selection of a method depends on the purpose of the
assay, as well as cost and convenience. Occult blood tests
can be used diagnostically to detect bleeding in the
gastrointestinal tract or as a screening test to detect
colorectal neoplasms. As shown in Table 2, heme-
porphyrin testing is more useful for detecting gastric
bleeding, whereas guaiac or immunological methods are
better for the detection of bleeding from the distal bowel
(colon and rectum).
The selection of a method for screening for colorectal
neoplasms depends on the sensitivity and specificity of the
method, as well as cost and acceptability of the method to
the population to be screened. A systematic review of the
literature by the Centre for Reviews and Dissemination
(CRD) on the accuracy of the immunological and guaiac
methods came to the following conclusions:
There is not enough evidence to suggest that one
method has better diagnostic accuracy.
There is limited evidence suggesting that
immunochemical methods may have better
overall performance, but more evidence is
required.
There is very little choice between different
guaiac-based methods in detecting colorectal
neoplasia [30].
However, some guaiac-based methods perform poorly in
external quality-assurance programs (see below).
Specimen
Feces should be sampled before they come into contact
with the toilet water, since hemoglobin will leach out of
the sample, and toilet sanitizers may affect the results.
Because blood may not be uniformly distributed within the
stools, and bleeding may be intermittent, it is important to
collect samples from more than one area, and the
collection should be repeated on 3 different days [31]. It is
best not to expose the sample to extreme heat or humidity;
dried samples can be stored at room temperature for 14
days [32]. If there is a delay between sample collection and
analysis, false-negative results may be seen because of the
degradation of pseudoperoxidase activity of heme in moist
feces. If samples are collected directly onto the filter paper
in the test kit and allowed to dry, this problem can be
prevented [33]. In some immunological methods,
collection devices with liquid preservatives are used, and
samples from patients can be collected by smearing the
stools onto the card provided. For most methods, some
patient preparation is required to give optimum results.
Patient Preparation and Guidelines
Samples should not be collected if blood is visible in the
stools or urine (e.g., menstruation, active hemorrhoids, or
urinary tract infection). Each stool sample should be
collected before the stools come into contact with toilet
water. For 3 days before and during the stool collection,
avoid red meat (beef, lamb, and liver) (see below). A high-
residue diet is recommended for 2 days before and during
the collection. For guaiac-based tests, raw fruits and
vegetables which contain high levels of peroxidase (e.g.,
turnip, broccoli, horseradish, cauliflower, parsnip, and
radish) should be avoided.
For 7 days before and during the collection period, it is
best to avoid nonsteroidal antiinflammatory drugs
(NSAIDs) such as aspirin or ibuprofen. Vitamin C in
excess of 250 mg per day should be avoided for 3 days
before and during collection.
Interferences
Guaiac-based methods are prone to interferences from
many sources. One of these is plant peroxidases. Raw
fruits and vegetables such as turnips, broccoli, horseradish,
cauliflower, cantaloupe, parsnip, and red radish contain
high concentrations of peroxidases [34]. These peroxidases
are heme proteins and have the prosthetic group ferri-
protoporphyrin IX (hemin) and cause false-positive results
in guaiac-based tests. However, it has been shown that
cooking vegetables at 100C for 20 min inactivates the
plant peroxidase activity [34]. Unlike hemoglobin, because
these peroxidases are within the cellulose cell wall, they
escape digestion by proteolytic enzymes during the
passage down the gastrointestinal tract. Inclusion of
moderate amounts of raw vegetables had no effect on the
Hemoccult test [35]. Furthermore, gastric acid denatures
peroxidase activity, so in patients with normal gastric-acid
secretion, ingestion of raw vegetables should not be a
problem [36]. Ingestion of 750 g of raw, peroxidase-rich
fruit and vegetables daily can cause false-positive results,
but this amount of vegetables is unusually large to be eaten
daily.
Delaying the development of the slide by 48 hours will
reduce interferences from plant peroxidases [37].
Nevertheless, many manufacturers of guaiac-based tests
recommend exclusion of high-peroxidase-containing fruits
and vegetables prior to and during collection of samples.
Ingestion of red meat can cause false-positive results due
to peroxidase activity of heme in the meat [38]. The false-
positive rate was higher when the rehydration method was
used before analysis [35]. Even after cooking the meat,
some peroxidase activity can be detected [34]. Studies in
healthy volunteers have shown that it takes about 3 days
for the false positive due to ingested meat to disappear
[39]. Hence the recommendation is made that red meat
should be avoided for at least 3 days prior to testing.
However, the length of time it takes for the false-positive
results to disappear after stopping red meat may vary in
different clinical situations and with people with altered
bowel movements. However, others have not found any
significant effect of red meat on occult blood tests [40]. A
meta-analysis of available data until 1999 concluded that
dietary restriction may not be necessary for guaiac-based
fecal occult blood tests [41].
 562
Fecal Occult Blood
Ascorbic acid (Vitamin C) can also cause a negative effect
on the oxidation of alpha-guaiaconic acid, because vitamin
C is a reducing agent. Ingestion of 1 to 2 g of vitamin C
daily can cause a false-negative result [42]. In-vitro studies
suggest that a normal intake of vitamin C is unlikely to
cause false-positive results [43]. In a recent study, it was
found that consumption of 60 mg of vitamin and 500 mL
of orange juice (350 mg of vitamin C) produced variable
results. In subjects taking high-dose supplements, false-
negative results were seen [24]. It is therefore
recommended by the manufacturers of all occult blood
testing kits that vitamin C intake should not be more than
250 mg/day (for Hema Screen) or 500 mg/day (for
Hemoccult SENSA).
Ingestion of iron supplements may cause false-positive
results [44]. However, others have not confirmed these
findings [45]. Some test manufacturers caution against the
use of iron supplements (Hema Screen), but others do not
(Hemoccult SENSA).
The use of povidone-iodine antiseptic solution was
associated with false-positive results with guaiac-based
assays [46]. In-vitro studies showed that that as little as
0.005 mL of 1:1000 dilution of this solution will give a
false-positive result. This interference is due to the iodine
in the antiseptic causing oxidation of the alpha-guaiaconic
acid [47]. The use of such antiseptic solutions on the
perianal area or during urinary catheterization should be
avoided prior to fecal occult blood testing using guaiac
methods.
Drugs such as aspirin, other antiinflammatory drugs (such
as ibuprofen, naproxen, corticosteroids, phenylbutazone),
cancer chemotherapeutic agents, and alcohol in excess can
all cause a positive reaction due to loss of blood from
gastric irritation. Recent studies, however, suggest that
low-dose aspirin has no effect on the test [48]. In a
comparison of subjects taking aspirin or other NSAIDs
with those not on these drugs, no difference in the rate of
positive fecal occult blood test results was found, and it
was therefore suggested that avoidance of these drugs
before fecal occult blood testing may not be necessary
[49]. Toilet sanitizers may cause false-positive results.
Chlorine-generating sanitizers will give false-positive
results with guaiac methods [50]. Nonchlorine-generating
sanitizers reduce the immunological detection of
hemoglobin [51].
The heme-porphyrin method cannot distinguish porphyrins
of hemoglobin origin from other heme-containing proteins
of non-human origin. Ingestion of red meat was shown to
cause a 375% increase in fecal porphyrin measured by
HemoQuant [19]. However, plant-derived peroxidases do
not interfere with this assay.
Interpretation
Normal subjects lose about 1.5 mL of blood per day, based
on radio chromium (51Cr-labeled red cells) methods [52].
In patients with carcinoma of the large bowel or adenomas,
bleeding is often microscopic but even so can be in excess
of 1.5 mL per day [35,53,54]. However, bleeding can be
intermittent and not always present. Thus it is not always
possible to distinguish normal from pathological bleeding
by measuring fecal blood loss [35]. Furthermore, the
amount of blood loss from tumors can vary from day to
day [55,56], and there is non-uniform distribution of
hemoglobin and its products in the feces [57]. Peptic ulcer,
hemorrhoids, and diverticula are also causes of fecal occult
blood loss; bleeding from these conditions is highly
variable. Of all the methods available, heme-porphyrin
testing is more likely to detect small amounts of blood loss
from the upper gastrointestinal tract, such as that seen due
to aspirin [17].
Fecal occult blood testing is performed to detect
gastrointestinal bleeding and is most often used as a
screening test to detect colorectal cancer, which is one of
the most common cancers in many parts of the world. This
is also an important investigation in patients with iron-
deficiency anemia to rule out gastrointestinal bleeding as
the cause of anemia. Since bleeding from any part of the
gastrointestinal tract can cause iron deficiency, it is
important to select the appropriate test. Testing based on
guaiac or immunological methods is less likely to detect
upper gastrointestinal tract bleeding; heme-porphyrin
testing is better at detecting upper gastrointestinal tract
bleeding [58] and was able to detect 90% of upper
gastrointestinal tract bleeding. As little as 5 mL of blood
loss per day can be detected.
There have been many studies on the use of fecal occult
blood testing for early detection of colorectal cancer.
Colorectal cancer is the second leading cause of cancer
death in United States. Studies have shown that screening
for colorectal cancer by fecal occult blood testing can
reduce cancer incidence and mortality [59]. Although
immunological methods have been suggested to have
better sensitivity, review of the available evidence does not
favor one type of test over the other [60]. Combination of
the two tests did not do any better than the immunological
method [61]. A working party from the World Health
Organization and the World Organization for Digestive
Endoscopy suggested that the immunochemical method
may be more amenable to standardization and quality
control and more acceptable to patients, because dietary
restriction is not required [62].
Reference Interval
The amount of blood lost from the normal gastrointestinal
tract is approximately 0.5 to 1.5 mL per day [1,2]. When
using a quantitative test, a positive result may be seen in
2% to 16% of the population, depending on the method
used and the population studied [63,64].
 563
Fecal Occult Blood
Performance Goals
Evidence from the UK External Quality Assurance
Programme suggests that some of the guaiac-based
methods show poor analytical performance [65]. Guaiac-
based tests are qualitative, and errors can arise in
interpreting the color changes [66]. Because of the poor
performance of these test kits and the possible effects of
dietary factors, it has been recently suggested that guaiac-
based fecal occult blood tests should not be used in the
clinical setting and only used in screening for colorectal
cancer in asymptomatic individuals [67]. Furthermore,
several national guidelines state that for the investigation
of symptomatic patients, fecal occult blood testing has no
value [68].
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2 Dybdahl JH, Daae LN, Larsen S. Occult faecal
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3 St John DJ, Young GP. Evaluation of
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11 Turunen MJ, Liewendhal K, Paratanen P,
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12 Vaananen P, Tenhunen R. Rapid
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13 Nagata M, Tanaka T. Detection of fecal blood by
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14 Saito H. Screening for colorectal cancer by
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18 Young GP, St John DJ, Lynch NM, McHutchison
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19 Rose IS, Young GP, St John DJ, Deacon MC,
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20 Xing PX, Young GP, Ho D, Sinatra MA, Hoj PB,
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blood testing based on the detection of
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21 Hoff G, Grotmol T, Thiis-Evensen E, Bretthauer
M, Gondal G, Vatn MH. Testing for faecal
calprotectin (PhiCal) in the Norwegian Colorectal
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SY, Shiea J. Diagnosis of occult blood in human
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23 Uchida K, Matsuse R, Miyachi N, Okuda S,
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24http://www.surrey.ac.uk/GMEC/Pages/GuildfordEvaluat
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%20survey.pdf Accessed 10-3-2008.
25 St John DJ, Young GP, Alexeyeff MA, Deacon
MC, Cuthbertson AM, Macrae FA, Penfold JC.
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1993;104:1661-8.
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26 Stroehlein JR, Fairbanks VF, McGill DB, Go VL.
Hemoccult detection of fecal occult blood
quantitated by radioassay. Am J Dig Dis
1976;21:841-4.
27 Rockey DC. Occult Gastrointestinal Bleeding. N
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28 Greenwald B. From guaiac to immune fecal
occult blood tests: the emergence of technology in
colorectal cancer screening. Gastroenterol Nurs
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29 Khoury RH, Gandhi AH, Salmon BP, Gudaitis P
Patel SN, Gudaitis D. Performance of the OC-
Auto micro80 for the determination of fecal
Occult blood. Clin Chem 2007;53 Suppl A107.
30 Burch JA, Soares-Weiser K, St John DJ, Duffy S,
Smith S, Kleijnen J, Westwood M. Diagnostic
accuracy of faecal occult blood tests used in
screening for colorectal cancer: a systematic
review. J Med Screen 2007;14:132-7.
31 Thomas WM, Pye G, Hardcastle JD, Mangham
CM. Faecal occult blood screening for colorectal
neoplasia: a randomized trial of three days or six
days of tests. Br J Surg 1990;77:277-9.
32 Young SP, St John DJB. Faecal occult blood
tests: choice, usage and clinical applications. Clin
Biochem Revs 1992;13:161-167.
33 Young GP, Sinatra MA, St. John DJ. Influence of
delay in stool sampling on fecal occult blood test
sensitivity Clin Chem 1996;42:1107 - 1108.
34 Caligiore P, Macrae FA, St John DJ, Rayner LJ,
Legge JW. Peroxidase levels in food: relevance to
colorectal cancer screening. Am J Clin Nutr
1982;35:1487-9.
35 Macrae FA, St John DJ, Caligiore P, Taylor LS,
Legge JW. Optimal dietary conditions for
hemoccult testing. Gastroenterology
1982;82:899-903.
36 Meyer GW, Komadina K, Perucca P. Vegetable
peroxidase is denatured by gastric acid: fresh
vegetables do not cause false-positive stool
Hemoccults in normal subjects. Gastroenterology
1991;101:871.
37 Sinatra MA, St John DJ, Young GP. Interference
of plant peroxidases with guaiac-based fecal
occult blood tests is avoidable. Clin Chem
1999;45:123-6.
38 Rose S, Young GP, St John DJ, Deacon MC,
Blake D, Henderson RW. Effect of ingestion of
hemoproteins on fecal excretion of hemes and
porphyrins. Clin Chem 1989;35:2290-6.
39 Feinberg EJ, Steinberg WM, Banks BL, Henry
JP. How long to abstain from eating red meat
before fecal occult blood tests. Ann Intern Med
1990;113:403-4.
40 Norfleet RG. Effect of diet on fecal occult blood
testing in patients with colorectal polyps. Dig Dis
Sci 1986;31:498-501.
41 Pignone M et al. Meta-analysis of dietary
restriction during fecal occult blood testing.
Effect Clin Pract 2001;4:150-6.
42 Jaffe RM, Kasten B, Young DS, MacLowry JD.
False-negative stool occult blood tests caused by
ingestion of ascorbic acid (vitamin C). Ann Intern
Med 1975;83:824-6.
43 Garrick DP, Close JR, McMurray W. Detection
of occult blood in faeces. Lancet 1977;2:820-1.
44 Lifton LJ, Kreiser J. False-positive stool occult
blood tests caused by iron preparations. A
controlled study and review of literature.
Gastroenterology 1982;83:860-3.
45 Anderson GD, Yuellig TR, Krone RE Jr. An
investigation into the effects of oral iron
supplementation on in vivo Hemoccult stool
testing. Am J Gastroenterol 1990;85:558-61.
46 Orchard JL, Lawson R. False-positive Hemoccult
reaction caused by Betadine. N Engl J Med
1984;311:199.
47 Blebea J, McPherson RA. False-positive guaiac
testing with iodine. Arch Pathol Lab Med
1985;109:437-440.
48 Greenberg PD, Cello JP, Rockey DC.
Relationship of low-dose aspirin to GI injury and
occult bleeding: a pilot study. Gastrointest Endosc
1999;50:618-622.
49 Kahi CJ, Imperiale TF. Do aspirin and
nonsteroidal anti-inflammatory drugs cause false-
positive fecal occult blood test results? A
prospective study in a cohort of veterans. Am J
Med 2004;117:837-841.
50 Ahlquist DA, Schwartz S, Isaacson J, Ellefson M.
A stool collection device: the first step in occult
blood testing. Ann Intern Med 1988;108:609-12.
51 Imafuku Y, Nagai T, Yoshida H. The effect of
toilet sanitizers and detergents on immunological
occult blood tests. Clin Chim Acta 1996;253:51-
9.
52 Pierson RN Jr, Holt PR, Watson RM, Keating RP.
Aspirin and gastrointestinal bleeding. Chromate
blood loss studies. Am J Med 1961;31:259-65.
53 Herzog P, Holtermller KH, Preiss J, Fischer J,
Ewe K, Schreiber HJ, Berres M. Fecal blood loss
in patients with colonic polyps: a comparison of
51
measurements with chromium-labeled
erythrocytes and with the Hemoccult test.
Gastroenterology 1982;83:957-62.
54 Dybdahl JH, Daae LN, Larsen S, Myren J. Occult
faecal blood loss determined by a 51Cr method
and chemical tests in patients referred for
colonoscopy. Scand J Gastroenterol 1984;19:245-
54.
55 St John DJ, Young GP, McHutchison JG, Deacon
MC, Alexeyeff MA. Comparison of the
specificity and sensitivity of Hemoccult and
HemoQuant in screening for colorectal neoplasia.
Ann Intern Med 1992;117:376-82.
 565

Fecal Occult Blood

56 Farrands PA, Hardcastle JD. Accuracy of occult
blood tests over a six-day period. Clin Oncol
1983;9:217-25.
57 Rosenfield RE, Kochwa S, Kaczera Z, Maimon J.
Nonuniform distribution of occult blood in feces.
Am J Clin Pathol 1979;71:204-9.
58 Harewood GC, McConnell JP, Harrington JJ,
Mahoney DW, Ahlquist DA. Detection of occult
upper gastrointestinal tract bleeding: performance
differences in fecal occult blood tests. Mayo Clin
Proc 2002;77:23-8.
59 Mandel JS, Church TR, Bond JH, Ederer F,
Geisser MS, Mongin SJ et al. The effect of fecal
occult-blood screening on the incidence of
colorectal cancer. N Engl J Med 2000;343:1603-
7.
60 Burch JA, Soares-Weiser K, St John DJ, Duffy S,
Smith S, Kleijnen J, Westwood M. Diagnostic
accuracy of faecal occult blood tests used in
screening for colorectal cancer: a systematic
review. J Med Screen 2007;14:132-7.
61 Allison JE, Sakoda LC, Levin TR, Tucker JP,
Tekawa IS, Cuff T et al. Screening for colorectal
neoplasms with new fecal occult blood tests:
update on performance characteristics. J Natl
Cancer Inst 2007;99:1462-70.
62 Young GP, St John DJ, Winawer SJ, Rozen P.
Choice of fecal occult blood tests for colorectal
cancer screening: recommendations based on
performance characteristics in population studies:
a WHO (World Health Organization) and OMED
(World Organization for Digestive Endoscopy)
report. Am J Gastroenterol 2002;97:2499-2507.
63 Levin B, Hess K, Johnson C. Screening for
colorectal cancer. A comparison of 3 fecal occult
blood tests. Arch Intern Med 1997;157:970-6.
64 Mandel JS, Bond JH, Church TR, Snover DC,
Bradley GM, Schuman LM, Ederer F. Reducing
mortality from colorectal cancer by screening for
fecal occult blood. Minnesota Colon Cancer
Control Study. N Engl J Med 1993;328:1365-71.
65 Duncan A, Hill PG. A review of the quality of
gastrointestinal investigations performed in UK
laboratories. Ann Clin Biochem 2007;44:145-58.
66 Selinger RR, Norman S, Dominitz JA. Failure of
health care professionals to interpret fecal occult
blood tests accurately. Am J Med 2003;114:64-7.
67 Fraser CG. Faecal occult blood testseliminate,
enhance or update? Ann Clin Biochem
2008;45:117-21.
68 NICE. Clinical Guidance 27. Referral for
Suspected Cancer 2005. Available at
www.nice.org.uk/nicemedia/pdf/cg027niceguideli
ne.pdf.

Table 1: Comparison of Features of Fecal Occult Blood Tests

Guaiac-Based Immunochemical Heme-porphyrins
Biochemical basis Peroxidase activity Immunoreactive Hb Fluorescent porphyrins
Compounds detected Hb Globin (human) Porphyrins
Myoglobin Myoglobin
All heme All heme
Non-heme porphyrins porphyrins form haem

Method Chromogen indicator Heme agglutination, ELISA, Solvent extraction,

latex fluorimetry
Quantitation No Possibly Yes
Drugs affecting Vitamin C No No
Interference/interpretatio Ingestion of meat No Ingestion of meat
n Plant peroxidases
Sample required Fecal smear on filter paper Smear of a scoop of feces Small scoop of feces
Equipment None Depends on method Fluorimeter
Time for sample Few minutes 15 min-2 hrs, depending on 4-8 hrs
processing method
Modified from Young and St. John (Clin Biochem Rev 1992;13:161-167)
 566

Fecal Occult Blood

Table 2: Hb and Hb Products in Feces According to the Site of Bleeding

Gastric Proximal large bowel Distal bowel
Intact hemoglobin - + ++
Intact heme + + ++
Heme-derived porphyrins +++ ++ +
 567
Ferritin
Ferritin
Hassan M.E. Azzazy
Name: Ferritin
Clinical significance: Measure of body iron stores Refer to Chapter 39, Iron, Porphyrin
and Bilirubin Metabolism, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass (apoferritin): 445,000 D
Merck Index: 3957
Chemical class: Protein, ferroprotein
Principles of Analysis and Current Usage
Until 1972, no method with sufficient sensitivity was
available to measure ferritin in normal serum. It was
therefore assumed that ferritin, known to be a storage form
of iron found in many tissues, did not circulate. The advent
of assays using radiolabeled antibodies demonstrated not
only the presence of ferritin in normal serum but also its
usefulness in the diagnosis of disorders resulting in either
low or high amounts of storage iron.
The first assay for measurement of ferritin in serum [1]
was an immunoradiometric assay (IRMA). This
methodology is similar to radioimmunoassay (RIA) but
uses a labeled antibody rather than a labeled antigen. In the
IRMA method (Table 1, Method 1), serum is incubated
with an excess of 125I-labeled antibody. Serum ferritin and
antibody form a soluble complex that is separated from
unreacted antibody by means of an immunoabsorbent
consisting of ferritin coupled to cellulose particles. The
ferritin-cellulose absorbs unreacted labeled antibody and
removes it from solution after a centrifugation step. The
radioactivity remaining in solution represents antibody
bound to serum ferritin and is directly related to serum
ferritin concentration. A modification of the IRMA method
called the two-site IRMA was introduced for the
measurement of serum ferritin in 1974 by Miles et al.
(Table 1, Method 2) [2]. A third type of assay for serum
ferritin used a competitive radioimmunoassay technique
(Table 1, Method 3) [3]. Neither the two-site IRMA nor
the competitive radioimmunoassay procedures are in
common use today.
Another method more frequently employed to quantitate
serum ferritin is a sandwich, solid-phase enzyme
i described for a centrifugal analyzer [7]. This turbidimetric
Ferritin
Previous and current authors of this method:
First edition: Robert S. Franco
Methods edition: Robert S. Franco
Second edition: Not updated
Third edition: Not updated
Fourth edition: Harini Patel
Fifth edition: Hassan Azzazy
immunoassay (EIA) (Table 1, Method 4). The solid phase,
such as beads or plastic tubes, is coated with antiferritin
antibodies and then incubated simultaneously with serum
and enzyme conjugated to antiferritin antibodies. Enzymes
employed as markers include horseradish peroxidase
(HRP) and alkaline phosphatase (AP). The ferritin in the
serum binds to the antibody on the bead, and the
conjugated antibody then binds to the immobilized ferritin.
After washing to remove unbound materials, a substrate
for horseradish peroxidase (such as a complex
azosulfonate dye [4] or o-phenylenediamine) or alkaline
phosphatase (p-nitrophenyl phosphate, 4-
methylumbelliferyl phosphate, or adamantyl dioxetane
phosphate) is added and allowed to react with the bound
enzyme. The amount of reaction product present is directly
proportional to the amount of ferritin in the serum
specimen.
Another immunoassay procedure employs a radial
partition sandwich enzyme immunoassay [5]. The assay is
similar to the EIA previously described. Antibodies to
ferritin are immobilized, in this case, on glass-fiber filter
paper. The radial-partition technique employs diffusion to
wash away any material (in serum or in the reagents) that
does not bind to the filter paper during the formation of the
sandwich. The amount of enzyme (alkaline
phosphatase)-antiferritin antibody in the sandwich can be
determined by adding 4-methylumbelliferyl phosphate and
measuring the rate of release of the product, 4-
methylumbelliferone, fluorometrically.
A latex particle-agglutination immunoassay employing
continuous flow automation and a particle counter has
been described [6]. A similar automated latex-particle
agglutination immunoassay for serum ferritin has been
immunoassay, also known as a microparticle-enhanced
turbidimetric immunoassay, has enhanced sensitivity by
attaching the antibody to microscopic latex particles.
Measurement of ferritin by a two-site, solid-phase
(sandwich) chemiluminometric immunoassay (Table 1,
Method 5) has also been described [8]. This assay uses a
constant amount of two antiferritin antibodies. The labeled
 568
Ferritin
antibody is coupled to a chemiluminescent molecule,
acridinium ester. The capture antiferritin antibody is
covalently bound to paramagnetic particles, which serve as
the solid phase. The complex is separated from unbound
materials by placing the tubes in a magnetic rack where the
bound fraction aggregates in the area of the magnets, and
the free fraction is removed. Oxidization of the acridinium
ester occurs rapidly, coupled with the emission of light,
which is measured by a luminometer. The ferritin
concentration is directly proportional to the relative light
unit (RLU).
A cloned-enzyme donor immunoassay (CEDIA) has been
introduced [9] for ferritin measurement (Table 1, Method
6) which can be run on many open, random-access
analyzers. This is a homogeneous assay for ferritin which
utilizes recombinant DNA technology to split the enzyme
-galactosidase into two inactive fragments, enzyme
acceptor (EA) and enzyme donor (ED). The ferritin
antibody is covalently attached to the ED fragment in such
a way that it allows the recombination of the two
fragments to form the active enzyme. The reassociation of
the EA and ED molecules is inhibited by the binding of the
ferritin antibodyED conjugate to ferritin in the sample.
Hence less ED-conjugate is available to combine with EA
to form active -galactosidase. The enzyme activity is
inversely proportional to the concentration of ferritin in the
sample.
Reference and Preferred Method
There is no reference method for serum ferritin. According
to a 2007 College of American Pathologists (CAP)
proficiency testing program, 100% of participating
laboratories reported using non-isotopic ferritin assays.
Fully automated ferritin immunoassays are now available
and may be preferred over manual ones because they have
several advantages, including better precision, less
operator dependence, and faster throughput analysis.
Proper standardization of the ferritin assay is a significant
problem. Ferritin is present as many isoforms [10]. The
most acidic form (pI 4.6) contains 24 H-subunits, whereas
the most basic isoferritin molecule (pI 5.8) consists of 24
L-subunits [11]. Diverse ferritin forms (isoferritins) react
differently in different immunoassays [12]. Variability in
commercial assays has been documented over the years,
and no reference method is generally accepted [13]. Much
of the lack of consensus about ferritin levels can probably
be attributed to the absence of an accepted, well-defined
standard [14]. The introduction of a third National Institute
for Biological Standards and Control/World Health
Organization (NIBSC/WHO) Recombinant Ferritin
International Standard 94/572 appears to have reduced but
not eliminated the variability in commercial assays
[15,16].
Specimen
Serum is the recommended sample type for this assay. No
patient preparation is required. Blood should be drawn in
plain red-top tubes or serum gel tubes. Specimens are
stable for at least 1 week when kept refrigerated at 4C
[17] and for 6 months at 20C. Frozen specimens should
not be thawed at 37C, and repeated freezing and thawing
should be avoided, as well as violent mixing, which may
denature ferritin.
Interferences
A disadvantage of the two-site IRMA for ferritin is the
high-dose hook effect [18]. This is a paradoxical
decrease in radioactive counts at high concentration of
antigen, resulting in a maximum in the curve of
radioactivity versus concentration. Since the hook effect
takes place at very high serum ferritin concentration, this
problem does not affect samples with levels of ferritin in
the normal range. However, some patients can have
extremely elevated ferritin levels; therefore, it is good
practice to run two dilutions of each sample and compare
the results. If the calculated ferritin concentration of the
more dilute sample is significantly higher than that of the
less dilute sample, it could be because of the hook effect.
EIAs, competitive RIA, and some commercially available
IRMAs can perform satisfactorily at high ferritin
concentrations [18]; however, each method should be
verified for the absence of the hook effect. Most
manufacturers give hook-effect limits in their package
inserts.
Few interferences have been described for the ferritin
assay. Immunonephelometric methods for ferritin have
been shown to be adversely affected by the presence of
increased turbidity or lipemia [19]. The presence of
heterophilic antibodies can interfere with immunoassay
procedures. Patient samples that give results not consistent
with the clinical status may require further investigation to
rule out interference due to heterophilic antibodies. In-vivo
interferences such as ethanol, iron salts, and oral
contraceptives may cause elevated values. Ferritin level is
decreased by erythropoietin [20].
Ferritin Reference Interval
In general, the reference interval is approximately 20 to
200 ng/mL (about 0.05 to 0.5 nmol/L) for adult males.
However, normal values are both age- and sex-related,
with premenopausal females and children under 16
exhibiting lower values as a result of physiologically lower
iron stores. Postmenopausal women have higher values
when compared with females who have not reached
menopause [21]. Release of ferritin from cultured alveolar
macrophages, recovered from cigarette-smoker lungs by
bronchoalveolar lavage, was increased compared with
nonsmokers [22]. Ferritin concentrations in capillary
(finger-prick) blood samples have been reported to be
significantly lower than ferritin concentrations in venous
 569
Ferritin
blood [23]. Serum ferritin concentration may be increased
(as much as two- to threefold) after several days of fasting
[24].
In a study of infants aged 9 months [25], the reference
interval for ferritin in capillary samples was reported to be
between 7.1 and 224 ng/mL. The ferritin levels were
positively related to birth weight, and females had higher
concentrations than males. In other studies [26,27],
pediatric reference intervals from newborns to 19-year-
olds have been reported by using different methods. There
are decreased serum ferritin levels in prepubertal and early
pubertal boys due to extra iron being mobilized through
redistribution to red blood cells [28]. Population norms for
serum ferritin are discussed in recent literature [29].
Normal ranges need to be determined per the used assay.
The conventional serum ferritin reference intervals are
presented in the table below.
Table 2. Serum Ferritin Reference Intervals [30,31]
Age Ferritin Value (ng/mL)
Newborn 25-200
1 month 200-600
2-5 months 50-200
6 months-15 years 7-140
Adult male 20-250
Adult female 10-120
Iron overload (adult male) >400
Iron overload (adult female) >200
Interpretation
Ferritin consists of a protein shell (apoferritin) containing a
variable amount of iron in the hollow core. The iron
content, in the form of colloidal hydrous ferric oxide
phosphate micelles, varies from almost none to 30%.
Serum ferritin is a sensitive indicator of body iron stores; it
has been shown to correlate with stainable bone-marrow
iron [23,32]. Its concentration reflects iron stores in normal
individuals and those with iron deficiency where serum
ferritin concentration of 1 ng/mL is equivalent to 8 mg of
stored iron [20]. It should be noted, however, that in the
presence of significant tissue destruction or rapid cellular
turnover, the serum ferritin concentration is
disproportionately high when compared to bone-marrow
iron. Therefore, the serum ferritin level cannot be used to
help in the diagnosis of iron deficiency in the presence of
inflammation, liver disease, or malignancies such as acute
leukemia or Hodgkins disease. Ferritin concentration is
significantly increased in cases of iron-overload conditions
such as hemochromatosis [33].
A serum ferritin of less than 10 ng/mL almost always
indicates iron deficiency. Although there is some overlap
between the normal and iron-deficient population, this is
not usually a diagnostic problem, because serum ferritin is
usually increased in anemia resulting from other causes.
Serum ferritin is especially useful in distinguishing iron
deficiency from the anemia of chronic disorders, because
in the latter, ferritin levels are increased [24,34]. Serum
ferritin is also increased in other anemias [35], including
aplastic anemia, sideroblastic anemia, and chronic
hemolytic anemias. In idiopathic hemochromatosis and in
multiply transfused patients, the serum ferritin may be
extremely high. Ferritin has also been suggested as a
nonspecific marker for many neoplastic malignancies.
Serum ferritin concentrations in adult Stills disease
(ASD), an acute, systemic inflammatory disorder, are
higher in active ASD than in inactive ASD and other
systemic diseases [36]. In addition, elevation in serum
ferritin (SF) and red-cell ferritin (RCF) have been reported
in HIV-infected patients [37]. SF levels are higher in
patients with more clinical symptoms, and RCF is
significantly higher in asymptomatic AIDS patients and
those treated with zidovudine (AZT).
It has been suggested [38] that in hematological
malignancies, tissue-specific isoferritins may cross-react,
resulting in poor correlations when different immunoassay
methods are used. Hence monitoring of ferritin should be
performed using the same method.
A study of pregnant women [39] reported that plasma
ferritin concentration is significantly greater in smokers
than in nonsmokers, suggesting that plasma/serum ferritin
values should be carefully interpreted when assessing iron
status in pregnant women who smoke. Falsely high values
in these women may not detect patients with iron
deficiency, who require prenatal iron supplementation. It
may be desirable to establish a revised cutoff point for
normal ferritin values for smokers at different stages of
gestation.
Ferritin Performance Goals
The current CLIA performance goal for measurement of
ferritin is for laboratories to be within 3 standard
deviations of the peer-group mean. According to the 2007
CAP Survey, % coefficient of variation (CV) for all
ferritin methods at a mean value of 192.4 g/L (SD 22.2)
was 11.6%.
The normal day-to-day variation in ferritin has been found
to be approximately 14 ng/mL in men and 26 ng/mL in
menstruating women [40]. Long-term intraindividual
variation in healthy adults measured over a 5-month period
has been found to be less than 20% [41].
Steele et al. [42] determined the long-term within-
laboratory and between-laboratory variation of ferritin
assays in a study that was based on a CAP fresh frozen
serum study. Five of seven of ferritin methods met within-
laboratory imprecision goals based on biological criteria.
The largest contribution to the total imprecision was the
total within-laboratory component and not the between-
laboratory component of variation. The desirable analytical
 570
Ferritin
quality specifications for imprecision, bias, and total error
as derived from biological variation are 7.1%, 5.2%, and
16.9%, respectively [43].
References
1 Addison GM, Beamish MR, Hales CN, Hodgkins
M, Jacobs A, Llewellin P. An immunoradiometric
assay for ferritin in the serum of normal subjects
and patients with iron deficiency and iron
overload. J Clin Pathol 1972;25:326-329.
2 Miles LE, Lipschitz DA, Bieber CP, Cook JD.
Measurement of serum ferritin by a 2-site
immunoradiometric assay. Anal Biochem
1974;61:209-224.
3 Marcus DM, Zinberg N. Measurement of serum
ferritin by radioimmunoassay: results in normal
individuals and patients with breast cancer. J Natl
Cancer Inst 1975;55:791-795.
4 Revenant MC. Sandwich enzyme immunoassay
for serum ferritin with polypropylene test tubes as
the solid phase. Clin Chem 1983;29:681-683.
5 Timmons R, Soto AA. Radial partition sandwich
enzyme immunoassay for ferritin [abstract]. Clin
Chem 1984;30:984.
6 Bernard AM, Lauwerys RR. Continuous-flow
system for automation of latex immunoassay by
particle counting. Clin Chem 1983;29:1007-1011.
7 Simo JM, Joven J, Cliville X, Sans T. Automated
latex agglutination immunoassay of serum ferritin
with a centrifugal analyzer. Clin Chem
1994;40:625-629.
8 Stacy DL, Han P. Serum ferritin measurement and
the degree of agreement using four techniques.
Am J Clin Pathol 1992;98:511-515.
9 Engel WD, Khanna PL. CEDIA in-vitro
diagnostics with a novel homogeneous
immunoassay technique. Current status and future
prospects. J Immunol Methods 1992;150:99-102.
10 Drysdale JW, Kohgo Y, Watanabe N. Ferritin
phenotypes. In: Albertini A, ed.
Radioimmunoassays of Hormones, Proteins, and
Enzymes. Amsterdam: Excerpta Medica;
1980:213.
11 Forman DT, Parke SL. The measurement and
interpretation of serum ferritin. Ann Clin Lab Sci
1980;10:345-349.
12 van Suijlen JD, van Noord PC, Leijnse B.
Accuracy of serum ferritin determinations in
tissue preparations and human serum. J Clin
Chem Clin Biochem 1990;28:43-48.
13 Bock JL, Endres DB, Elin RJ, Wang E,
Rosenzweig B, Klee GG. Comparison of fresh
frozen serum to traditional proficiency testing
material in a College of American Pathologists
survey for ferritin, folate, and vitamin B12. Arch
Pathol Lab Med 2005;129:323-327.
14 Iacobello C, Ghielmi S, Belloli S, Arosio P,
Albertini A. Use of a reference standard to
improve the accuracy and precision of seven kits
for determination of ferritin in serum. Clin Chem
1984;30:298-301.
15 Thorpe SJ, Walker D, Arosio P, Heath A, Cook
JD, Worwood M. International collaborative
study to evaluate a recombinant L ferritin
preparation as an International Standard. Clin
Chem 1997;43:1582-1587.
16 Lotz J, Hafner G, Prellwitz W. Reference values
for a homogeneous ferritin assay and traceability
to the 3rd International Recombinant Standard for
Ferritin (NIBSC code 94/572). Clin Chem Lab
Med 1999;37:821-825.
17 Dale JC, Pruett SK. Phlebotomy: a minimalist
approach. Mayo Clin Proc 1993;68:249-255.
18 Ng RH, Brown BA, Valdes R. Three commercial
methods for serum ferritin compared and the
high-dose hook effect eliminated. Clin Chem
1983;29:1109-1113.
19 Borque L, Rus A, del Cura J, Maside C, Escanero
J. Automated quantitative nephelometric latex
immunoassay for determining ferritin in human
serum. J Clin Lab Anal. 1992;6:239-44.
20 Wu AHB, ed. Tietz Clinical Guide to Laboratory
Tests. 4th ed. Philadelphia: Saunders; 2006.
21 Rubin C, Wood PJ, Archer T, Rowe DJ. Changes
in serum ferritin and other acute phase proteins
following major surgery. Ann Clin Biochem
1984;21:290-294.
22 Wesselius LJ, Nelson ME, Skikne BS. Increased
release of ferritin and iron by iron-loaded alveolar
macrophages in cigarette smokers. Am J Respir
Crit Care Med. 1994;150:690-5.
23 Harju E, Pakarmen A, Larmi TA. Comparison
between serum ferritin concentration and the
amount of bone marrow stainable iron. Scand J
Clin Lab Invest 1984;44:555-556.
24 Lipschitz DA, Cook JD, Finch CA. A clinical
evaluation of serum ferritin as an index of iron
stores. N Engl J Med 1974;290:1213-1216.
25 Edmond AM, Hawkins M, Pennock C, Golding J.
Hemoglobin and ferritin concentrations in infants
at 8 months of age. Arch Dis Child 1996;74:36-
39.
26 Murthy JN, Hicks JM, Soldin SJ. Evaluation of
the Technicon Immuno 1 random-access
immunoassay analyzer and calculation of
pediatric reference ranges for endocrine tests, T-
uptake and ferritin. Clin Biochem 1995;28:181-
185.
27 Soldin SJ, Morales A, Albalos F, Lenherr S, Rifai
N. Pediatric reference ranges on the IMx for FSH,
LH, prolactin, TSH, T4, T3, free T4, Free T3, T-
uptake, Ig E and ferritin. Clin Biochem
1995;28:603-606.
28 Anttila R, Siimes MA. Serum transferrin and
ferritin in pubertal boys: relations to body growth,
 571
Ferritin
pubertal stage, erythropoiesis, and iron
deficiency. Am J Clin Nutr 1996;63:179-183.
29 Custer EM, Finch CA, Sobel RE, Zettner A.
Population norms for serum ferritin. J Lab Clin
Med 1995;126:88-94.
30 Seamonds B, Anderson K, Whitaker B. Reference
intervals for ferritin: age dependence. Clin Chem
1980;26:1515-1516.
31 Williams WJ, Beutler E, Erslev AJ et al, eds.
Hematology 3rd ed. New York: McGraw-Hill;
1983.
32 Forman DT, Vye MV. Immunoradiometric serum
ferritin concentration compared with stainable
bone marrow iron as indices to iron stores. Clin
Chem 1980;26:145-147.
33 Halliday JW, Russo AM, Cowlishaw JL, Powell
LW. Serum-ferritin in diagnosis of
haemochromatosis. A study of 43 families. Lancet
1977;2:621-624.
34 Bentley DP, Williams P. Serum ferritin
concentration as an index of storage iron in
rheumatoid arthritis. J Clin Pathol 1974;27:786-
788.
35 Alfrey CP. Serum ferritin assay. CRC Crit Rev
Clin Lab Sci 1978(9)3:179-208.
36 Van Reeth C, Le Moel G, Lasne Y, Revenant
MC, Agneray J, Kahn MF, Bourgeois P. Serum
ferritins and isoferrtins are tools for diagnosis of
active adult Stills disease. J Rheumatol
1994;21:890-5.
37 Riera A, Gimferrer E, Cadafalch J, Remacha A,
Martin S. Prevalence of high serum and red cell
ferritin levels in HIV-infected patients.
Haematologica 1994;79:165-167.
38 Vernet M, Renversez JC, Lasne Y, Revenant MC,
Charlier de Bressing C, Guillemin C et al.
Comparison of six serum ferritin immunoassays
and isoferritin spectrotypes in malignancies. Ann
Biol Clin (Paris) 1995;53:419-427.
39 Tamura T, Goldenberg RL, Johnson KE, DuBard
MB. Effects of smoking on plasma ferritin
concentrations in pregnant women. Clin Chem
1995;41:1190-1191.
40 Borel MJ, Smith SM, Derr J, Beard JL. Day-to-
day variation in iron-status indices in healthy men
and women. Am J Clin Nutr 1991;54:729-735.
41 Gallagher SK, Johnson LK, Milne DB. Short-term
and long-term variation of indices related to
nutritional status. I. Ca, Cu, Fe, Mg, and Zn. Clin
Chem 1989;35:369-373.
42 Steele BW, Wang E, Palmer-Toy DE, Killeen
AA, Elin RJ, Klee GG. Total long-term within-
laboratory precision of cortisol, ferritin,
thyroxine, free thyroxine, and thyroid-stimulating
hormone assays based on a College of American
Pathologists fresh frozen serum study: do
available methods meet medical needs for
precision? Arch Pathol Lab Med 2005;129:318-
322.
43 Westgard QC. Desirable specifications for total
error, imprecision, and bias, derived from biologic
variation. Available at
<http://www.westgard.com/biodatabase1.htm>
 572

Ferritin

Table 1: Summary of Ferritin Methods

Method 1: Immunoradiometric assay (IRMA) (one-site)

Principle of analysis: Antibody is labeled, serum ferritinantibody complex is formed, and unreacted antibody is removed;
remaining radioactivity is directly related to serum ferritin concentration.
Comments: Not commercially available for clinical use; first method with enough sensitivity to measure normal levels,
historical usage
Method 2: IRMA (two-site)
Principle of analysis: Serum ferritin is bound to solid-phase antibody, and labeled antibody is then bound to immobilized
ferritin; after washing, the radioactivity of the bound labeled antibody is directly related to serum ferritin
concentration.
Comments: Commercially available for clinical use; hook effect at high concentration (see text)
Method 3: Radioimmunoassay (RIA)
Principle of analysis: Serum ferritin and labeled ferritin compete for antibody; antigen-antibody complexes are precipitated
and counted; radioactivity is inversely related to serum ferritin concentration.
Comments: Commercially available for clinical use; requires larger sample than the two-site IRMA does; not used for routine
measurement
Method 4: Enzyme immunoassay (EIA)
Principle of analysis: Antiferritin antibody coated on solid phase is incubated with serum and antiferritin antibody conjugated
to alkaline phosphatase or horseradish peroxidase. Serum ferritin binds to the immobilized antibody and is in turn
bound by the antibodyenzyme conjugate. Appropriate enzyme substrate is added, and products of the reaction could
be measured colorimetrically or fluorometrically.
Comments: Commercially available for clinical use; microprocessor-controlled reader available from manufacturer
Method 5: Chemiluminometric assay
Principle of analysis: In this two-site chemiluminometric immunoassay, the first ferritin antibody is labeled with acridinium
ester, and the second antibody is covalently bound to a paramagnetic particle (solid phase). Binding of the two
antibodies to ferritin forms a complex that is separated and measured with a luminometer. The light signal is directly
proportional to ferritin in the sample.
Comments: Very sensitive; very frequently used
Method 6: Cloned-enzyme donor immunoassay (CEDIA)
Principle of analysis: The enzyme -galactosidase is split into two inactive fragments, enzyme acceptor (EA) and enzyme
donor (ED), using recombinant DNA technology. Ferritin antibody is covalently attached to the ED fraction that is
free to combine with EA fraction, when not bound to ferritin, to form the active enzyme. When ED-antibody reacts
with ferritin, the association with EA fragment is inhibited, and the active enzyme is not formed. The enzyme activity
is inversely proportional to the concentration of ferritin.
Comments: Homogeneous assay; frequently used
573
Folic Acid and Folate

Folic Acid and Folate

Sheila Dawling

Name: Folic acid, pteroylglutamic acid, PGA

Clinical significance: Refer to Chapter 41, Human Nutrition, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C19H19N7O6
Molecular mass: 441.40 D convert molar to mass units ng/mL or g/L = nmol/L 0.4414
Merck Index: 4221
O H
O C
O
N C
H
OH
OH HN
N
N
Folic Acid
H2N N N

Name: Tetrahydrofolic acid (THF)

Clinical significance: Refer to Chapter 41, Human Nutrition, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C19H23N7O6
Molecular mass: 445.40 D convert molar to mass units ng/mL or g/L = nmol/L 0.4454
Merck Index:
O H
O C
O
N C
H
OH
OH HN
H
N
N
Tetrahydrofolic Acid
H2N N N
H

Name: N-5-Methyltetrahydrofolic acid (MTHF)

Clinical significance: Refer to Chapter 41, Human Nutrition, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular formula: C20H25N7O6
Molecular mass: 459.40 D convert molar to mass units ng/mL or g/L = nmol/L 0.4594
Merck Index:
O H
O C
O
N C
H
OH
OH CH3 HN
N
N
N-5-Methyltetrahydrofolic Acid
H2N N N
H

Name: N-5-Formyltetrahydrofolic acid (Folinic acid, FTHF)

574
Folic Acid and Folate
Clinical significance: Refer to Chapter 41, Human Nutrition, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C20H23N7O7
Molecular mass: 473.44 D convert molar to mass units ng/mL or mcg/L = nmol/L
0.4734
Merck Index: 4222
O H
O C
O
N C
H O H
OH
OH C HN
N
N
N-5-Formyltetrahydrofolic Acid
H2N N N
H
Principles of Analysis and Current Usage
Folate species present in physiological fluids are a group
of chemically and biologically related B vitamer
compounds [1]. They function as coenzymes in the
metabolism of one-carbon compounds, in purine and
pyrimidine synthesis, and in the degradation of histidine
to glutamic acid (Figures 1 and 2). The measurement of
folate in serum and whole blood is important in the
differential diagnosis of megaloblastic anemia. However,
much of the current understanding of folate metabolism
stems from research into the relationship of folate status
to the incidence of fetal neural tube defects [2]. There
are several different approaches to folate measurement,
including microbiological assays, competitive folate
binding protein (FBP) assays, and chromatographic
techniques.
Microbiological assays were developed first, but FBP
methods are now standard in most laboratories. All these
methods aim to measure total folates. As mass
spectrometry (MS) becomes more widely available,
high-performance liquid chromatography (HPLC)-
tandem MS will likely be adopted for reference methods,
which will enable differentiation of the discrete folate
species present in serum or RBCs.
Microbiological assays (Table 1, Method 1) utilize the
folate-dependent bacterium Lactobacillus casei [3,4].
Microtiter plates containing folate-depleted growth
medium inoculated with L. casei are incubated for 18 to
40 hours at 37C with patient serum. The amount of L.
casei growth permitted is determined by measurement of
turbidity at 600 to 700 nm and reflects the folate content
i
Folate
Previous and current authors of this method:
First edition: Michael D.D. McNeely
Methods edition: Michael D.D. McNeely
Second edition: Not updated
Third edition: Steven C. Kazmierczak
Fourth edition: Michael D.D. McNeely
Fifth edition: Sheila Dawling
of the serum. The absorbance is compared to the growth
permitted by standard solutions of
methyltetrahydrofolate (MTHF).
FBP assays utilize high-affinity folate-specific binding
proteins (Table 1, Method 2). Early assays used radio-
labeled folate (125I) and a limited amount of naturally
produced FBP to quantitate the concentration of
unlabeled folate in the patient sera in a standard
competitive binding assay format. Phase separation was
typically with dextran-coated activated charcoal, but if
the FBP can be immobilized in some way, then the
phases can be separated by a simple washing step.
Incubation times could be reduced to as little as 1 hour,
and assays were also arranged for simultaneous
measurement of vitamin B12 and folate [5,6]. Today,
FBP assays enjoy widespread use because they are rapid,
precise, and easily automated and are available in a
variety of formats suitable for modern immunoassay
analyzers. Their properties are compared in Table 2 and
Figures 3 and 4.
As a first step in all FBP assays, the natural binders
present in the sample must be destroyed and the folate
species released, which are subsequently all reduced to
pteroylmonoglutamic acid (PGA). Initially these steps
were performed off-line by boiling at alkaline pH, but
they are now performed chemically and incorporated
into the automated process. Popular reagents are NaOH
or KOH, then HCl, with either 2-mercaptoethanol,
monothioglycerol, dithiothreitol and KCN.
Once the de-binding and reduction are completed, the
folate-protein binding step must be carried out at pH 9.3
to ensure equivalent binding of PGA and MTHF, either
as a single- or two-stage process. The one-stage assay is
a conventional competitive binding assay in which
sample, limited quantity of FBP, and tracer are
combined simultaneously. Radio-labeled assays are
seldom used nowadays, as titrated tracers have low
specific activity and require liquid scintillation for beta-
particle emission and 125I tracers, while offering much
greater counting activity and the added ease of gamma-
ray counting are not readily available. The two-stage
575
Folic Acid and Folate
assay is noncompetitive and involves sequential
incubations. Excess FBP is first incubated with sample,
and subsequently tracer-bound folate is added to occupy
the remaining available binding sites. This approach is
more sensitive. As shown in Table 2 and Figures 3 and
4, the tracer may be an enzyme (alkaline phosphatase or
horseradish peroxidase), a luminescent, or a
chemiluminescent label. Later developments in
heterogeneous assay formats utilize streptavidin and
biotin, and one assay uses a specific folate-binding
protein antibody and allows for the tracer to be attached
to not just the folate but also to the folate-binding protein
or an FBP antibody.
The traditional binding agent was obtained from
unpurified milk powder or partially purified -
lactoglobulin [7]. Modern techniques in protein
purification have improved assay reproducibility.
However, it is important to recognize that milk binders
have different binding affinities for the different
chemical forms of folate. Furthermore, binding is pH-
dependent, affecting the various folate species
differentially [8]. The pH effect is so sensitive that the
amount of CO2 absorbed from the atmosphere by open
reagents may be sufficient to shift the binding curve
significantly [9,10]. To complicate matters further, FBP
exhibits marked aggregation tendencies, which also
affect its folate binding characteristics [11,12]. Assays
using FBP as the carrier for the tracer molecule (e.g.,
Table 2, Method 2f and Figure 4) should determine the
effect of the addition of the tracer on the binding
characteristics.
Intracellular folate (e.g., RBC), by contrast, is
predominantly polyglutamated species and is present in
much higher concentrations than in serum but can be
measured by the same analytical techniques. The folate
in red blood cells more closely reflects tissue folate
stores and is less variable as a consequence of recent
folate ingestion [13,14]. To liberate the intracellular
folate, freezing and thawing are considered more
complete than incubation with ascorbic acid [15],
although the latter is much more amenable to automation
and so is more frequently used. Incubation times are
typically 90 minutes (Table 2). The hematocrit should be
determined on the sample so the results can be expressed
as a function of red blood cell mass.
Standardization of FBP folate assays is problematic and
is often cited as a major source of variation among
analytical methods [16]. In theory, standardization of
folate assays where compounds are commercially
available in pure and stable forms should be easier than
for a peptide analyte where no traceability to SI units is
available. MTHF is the most rational compound to use,
since this is the predominant serum form, but it is prone
to oxidation. The molecule has two asymmetric carbons,
and the physiologically produced and active
diastereomer is L-MTHF [6S,S]. Until recently, only
racemic MTHF was commercially available, and the
different forms do not react equally with FBP [17]. It has
long been recognized that the chemical nature of the
calibration material can influence the results obtained
[18]. Given carefully controlled conditions at pH 9.3, the
reaction can be manipulated so that PGA and MTHF
react equally with the FBP. The purity of MTHF can be
assessed spectrophotometrically at 290 and 245 nm [13].
PGA has been advocated as a standard because it is
stable, well characterized spectrophotometrically,
inexpensive, and easy to purify [13]. For these reasons,
most commercial assays use PGA as the calibration
material.
Standardization of immunoassays in the absence of an
accepted primary reference method and standardized
reference materials has been discussed recently [19].
One approach is that of harmonization or desired
outcome standardization, in which assays are calibrated
to read the same, ideally against a best available
candidate reference method. While this is not ideal,
given that absolute standardization is so far away, it may
at least provide comparability between patient values
and allow collection of objective data for clinical studies
[20].
Development of a new standard reference material for
folate in serum (SRM 1955) [21] will enable reagent
manufacturers to address some of these problems. This
standard will be available at three concentrations, with
assigned values for PGA, MTHF, 5-
formyltetrahydrofolic acid (FTHF), and total folate
(together with vitamin B12 and total homocysteine).
Chromatographic techniques, by contrast, aim to
differentiate the folate species present in physiological
fluids, targeting either solely the active MTHF form or
PGA and FTHF in addition to MTHF. Gas
chromatography-mass spectrometry achieved sufficient
sensitivity for RBC folate but required complex sample
preparation and derivatization; high temperatures
introduce the possibility of folate degradation [22].
High-performance liquid chromatography (HPLC)
methods with electrochemical oxidation detection gave
improved specificity over microbiological and
immunoassays, but until recently they lacked sensitivity
and had no internal standard and so could not be
considered high-order reference methods [23-25].
Recent developments in food science resulting from
grain-supplementation initiatives introduced the first
LC/MS methods for both supplemented (PGA) and
naturally occurring folates [26]. With the availability of
suitable deuterated internal standards and price
competitiveness of triple quadrupole instruments
LC/MS/MS (tandem LC/MS), applications for
physiological fluids are appearing in the literature [27-
29]. With detection limits around 0.5 ng/mL from a 0.25
to 0.5 mL specimen, these are competitive with
immunoassays and are currently being considered as
reference methods. Sample preparation is, however,
labor intensive in that folates must first be removed from
their serum binding proteins as described earlier.
Subsequently, protonated and stabilized folates in acidic
solutions are then adsorbed onto C18 solid phase
extraction columns. After washing, folate species are
eluted from the columns and injected onto the HPLC
analytical column [25,27,28]. An alternative isolation
approach is affinity chromatography. Here, folate
species from the plasma are trapped in a column
576
Folic Acid and Folate
containing FBP on a solid support, impurities are washed
off, and then the purified folates are eluted onto the
analytical column using an automated column-switching
technique [23]. Sample throughput (30 to 100 samples
per day maximum) is still inferior to automated FBP
assays, and the methods are unlikely to translate into
mainstream analysis in the foreseeable future. Similar
methods for RBCs are also reported [30].
Reference and Preferred Methods
At present there is no accepted reference method for
folate. HPLC/tandem MS is the candidate reference
method [27], but it is unlikely to translate into
mainstream analysis in the foreseeable future due to lack
of speed and high cost. The microbiological assay is not
suitable for routine work but is still considered a gold-
standard procedure. However, the technique is difficult
to perform, requires specialized microbiological
equipment, and may take up to 3 days to complete.
Clinical laboratories almost exclusively use FBP assays
(Table 1, Method 2), which are available in a variety of
formats suitable for modern immunoassay analyzers.
These assays are rapid, precise, and all steps, including
the denaturing of endogenous binders and de-
glutamination, are easily automated. Assay formats are
summarized and compared in Table 2, with examples
shown in Figures 3 and 4. Standardization of FBP folate
assays is problematic, but the development of a new
standard reference material for homocysteine and folate
in serum (SRM 1955) will enable reagent manufacturers
to address some of these problems. Possible interference
with these assays is addressed in the section below.
RBC folate is present in much higher concentrations
than in serum but can be measured by the same
analytical techniques. Incubation with ascorbic acid for
90 minutes liberates intracellular folate. The hematocrit
should be determined on the sample so the results can be
expressed as a function of red blood cell mass.
Specimen
Samples for serum (or plasma) folate should be collected
in the fasting state, since folate concentrations reflect
recent food content. In the case of ingestion of folate-
fortified foods or vitamin supplements, some of the
plasma content may be inactive PGA if > 300 g folic
acid has been ingested rather than active MTHF [31].
Stability is maintained for 24 hours at 4C. If kept longer
before assay, the serum should be kept frozen at 20C.
Serum specimens should be protected from light,
because there is an approximate 10% and 20% loss of
folate after 8 and 24 hours of light exposure, respectively
[32].
Some studies have reported decreased folate in plasma
when compared with serum. Since 95% of whole-blood
folate is found in red blood cells, hemolyzed samples
should not be accepted for serum analysis, and these can
be detected and rejected by mainframe chemistry
analyzers that determine hemolysis indices.
RBC folate content is not dependent on recent food
intake and is advocated as a better measure of body
folate load; consequently, samples can be collected at
any time of day. Samples should be anticoagulated with
powdered EDTA, heparin, or oxalate. Liquid
anticoagulants are not suitable, because they dilute out
the RBCs. Some automated assays list only one or two
permitted anticoagulants, and reference should be made
to the package insert for the specific assay. Unspun
whole blood must be carefully maintained at 4C; it is
stable at this temperature for 3 days. It is recommended
that samples be protected from light, because there is no
evidence that the presence of RBCs in the sample affords
protection from the degradative effects seen in separated
serum described above.
Calculation of RBC Folate from Lysate Folate
Concentration
Since whole blood is processed for the RBC assay, the
hematocrit (packed cell volume) must be determined by
an approved method. To obtain red blood cell folate
expressed as ng folate /mL packed RBCs, a calculation
is required. For platforms that have individual tests for
RBC and serum folate, the RBC lysate value is usually
corrected automatically for the dilution made during the
hemolysis step. If this is not the case, then the instrument
value must be multiplied by the dilution factor used.
Subsequently, all values must be corrected for the
hematocrit, since whole blood (rather than packed red
cells) has been used for the analysis. Strictly speaking,
the correct formula to apply is:
RCF = WBF [SF (100 H)] 100
100 H
where RCF = red cell folate
WBF = whole blood folate
SF = serum folate
H = % hematocrit
In practice, serum folate is often not measured
simultaneously, and the part of the formula in brackets is
in most cases is so small as to be insignificant, so the
calculation is simplified to:
RCF = WBF 100
H
where RCF = red cell folate
WBF = whole blood folate
H = % hematocrit
Interferences
In general, hyperbilirubinemia (below 20 mg/dL) or
lipemia (below 1000 mg/dL triglycerides) do not
interfere in any of the reported FBP methods. The
presence of hemoglobin (below 0.5 g/dL) does not per se
interfere in the assay but will artifactually elevate the
serum folate because of the much higher concentration
of folate in RBCs. The hemolysis index as measured by
many mainframe chemistry analyzers could be used to
reject such samples. Although FBP assays share many
features of immunoassays, the small range of specimen
concentrations encountered (as opposed to a tumor
marker assay, for example) exclude the requirement to
monitor for a Hook effect caused by excess antigen in
577
Folic Acid and Folate
the patient sample saturating the binding sites on the
capture protein. Likewise, interference by heterophile
antibodies might be expected in those FBP assays that
also contain animal antisera (Table 2, Methods 2a, 2b,
2d, 2e,) and unlikely in those assays that do not (Table 2,
Methods 2c, 2f, and 2g ).
Heterophile antibodies should be suspected when serial
specimens from the same patient or repeat analysis of the
same sample produce markedly different results, or
when recovery on dilution is not linear. Analysis by an
alternative method is usually sufficient to align the
measured result with the clinical picture, but in some
cases, it may be necessary to prove that these antibodies
exist. In this case, a broad spectrum passive precipitant
such as PEG can be used, but it is preferable to use an
active blocking agent such as The Scantibodies
Heterophilic Blocking Reagent Tubes (HBRT).
Although FDA-approved for this purpose, the value
obtained from a sample treated with HBR should not be
used as a reportable result.
For the assays (Table 2, Methods 2f and 2g) that have
biotin-streptavidin components, specimens from patients
receiving high-dose biotin supplements (>5mg/day) may
be problematic. It is recommended that a minimum of 8
hours elapse between biotin ingestion and specimen
collection for prolactin analysis. Isolated cases of high-
titer antibodies against streptavidin are also mentioned in
the literature, but supporting data are difficult to locate.
The presence of antibodies to the cross-linker used for
attachment of the ruthenium ion in the Roche assay
(Table 2, Method 2f) has been documented in a few
patients with the thyroid assays but is equally applicable
to all assays with this structure [33]. FBP assays are also
prone to cross-reactivity with other species that can also
be bound by the folate-binding protein. Methotrexate
(amethopterin), aminopterin (4-aminopteroic acid), and
folinic acid (5-formylTHF, leucovorin, citrovorum) are
chemotherapeutic agents whose structures are similar to
that of folate. The mechanism of action of methotrexate
is to inhibit cellular folate uptake and its subsequent
reduction to active forms. Cross-reactivity data from the
package inserts are summarized in Table 3.
Superficially, cross-reactivity appears marginal (<1%),
but it should be remembered that while a serum folate
concentration is of the order of 10 ng/mL, a
methotrexate concentration after initial administration is
may exceed 50,000 ng/mL and about 2000 ng/mL at 48
hours [34]. This translates to an apparent serum folate of
500 and 20 respectively at 1% cross-reactivity. RBC
methotrexate concentrations are only 3% of these in
plasma with acute dosing, but on long-term, low-dose
oral therapy, methotrexate gradually replaces
intracellular folate [35]. Even so, RBC folate
measurement is unlikely to be adversely affected by
methotrexate therapy [36]. Leucovorin is metabolized to
MTHF intracellularly and therefore repletes intracellular
active folate.
Chromatographic assays are not susceptible to these
types of interferences.
Folic Acid Reference Interval
Reference intervals (normal ranges) vary somewhat by
method and are rather higher in folate-supplemented
populations. There is, however, consensus over what
constitutes folate deficiency.
Serum folate:
Reference interval 3 to 18 ng/mL (6.8 to 40.8 nmol/L)
Deficiency <3 ng/mL (<6.8 nmol/L)
Red blood cell folate:
Reference interval 200 to 1000 ng/mL packed cells
(453.1 to 2266 nmol/L)
Deficiency <150 ng/mL packed cells (<340 nmol/L)
Interpretation
Folic acid (pteroylglutamic acid, PGA) is stable and is
therefore the form used in dietary supplements. Once
absorbed, it must be reduced to the naturally occurring
bioactive form tetrahydrofolic acid (THF). Addition of a
methyl group and subsequent reduction produces N-5-
methyltetrahydrofolate glutamate (MTHF). This is taken
up by the bloodstream and constitutes 80% to 95% of
circulating folate. MTHF is transported into cells, where
it is polyglutamated, preventing its efflux, and it thus
accumulates within cells (including RBCs). Here it
functions as both a methyl donor and acceptor and
consequently exists in a variety of oxidized and reduced
states (Figure 1). Folate in the blood is 90% unbound.
Ten percent is bound to albumin, 2-macroglobulin, and
to a specific folic-acid-binding protein, FBP. FBP
increases as the body store of folate declines, probably
by regulating gene expression in a mechanism similar to
many other serum binding proteins.
The primary indication for ordering a serum or RBC
folate measurement is the differential diagnosis of a
megaloblastic anemia. Iron deficiency may also be
present, which may tend to normalize RBC size and thus
confound the picture. Folate is also measured during
pregnancy, because low folate concentrations increase
the risk of neural tube defects in the fetus [2]. However,
many pregnant women are folate-supplemented
automatically without the expense of a folate assay. A
diet deficient in folate can lead to folate deficiency after
some 2 to 4 months of zero dietary intake. Folate
deficiencies are often associated with chronic alcoholism
due to concomitant poor nutritive state. Other conditions
that place patients at risk for subnormal folate levels are
pregnancy and lactation, hypermetabolic states such as
sepsis or burns, and malignancy (and its treatment).
Many intestinal disorders will result in decreased folate
absorption, and in extreme cases, folate injections may
be necessary. Oral contraceptives, phenytoin, and other
anticonvulsants also decrease folate absorption from the
intestines.
Folic Acid Performance Goals
With the considerations outlined above, such as the
nature of the calibration materials, removal of binders,
and the chemical-reduction processes, it is not surprising
that there is poor agreement between different assays
and different laboratories. Recent attempts at
standardization with the provision of SRM 1955 at three
578
Folic Acid and Folate
different concentrations and harmonization of assays are
making some headway into improving performance [21].
Worldwide specimen exchange events have shown poor
performance, with coefficient of variations (CVs) of
18% to 41% and two- to ninefold differences, with red
cell assays significantly underperforming their serum
counterparts [37,38]. Increased awareness of the
importance of folate has galvanized action to improve
individual assay performance and also to standardize
between assays. Acceptable performance criteria (CLIA-
88) for measurement of serum and RBC folate requires
that laboratories be accurate to within 3 SD of the
peer-group mean. Although this is for the most part
achieved, wide bias is seen between the specific assay
formats, as shown in Table 4. Precision data from the
College of American Pathologists 2006-7 Participant
Summary Reports for serum folate are shown in Table 4
for 1725 laboratories returning results for FBP assays to
CAP in 2006-7 [39,40]. Only 40% of these laboratories
participated in the serum
calibration/linearity/verification surveys, and the spread
of these data shows the bias between methods. Abbott
AxSYM (Table 2, Method 2a) gave the highest values in
all surveys, with a bias of about +26%, while Bayer
Centaur (Table 2, method 2c) gave the lowest values,
with a bias of about 13%. Comparative data are shown
for the 255 laboratories participating in the 2006 United
Kingdom National External Quality Assurance Scheme
(UKNEQAS), showing imprecision and bias. In this
survey, Beckman-Coulter and Abbott Architect (Table 2,
Methods 2d and 2b and Figure 3) showed the most
negative bias, with Roche (Table 2, Method 2f and
Figure 4) and DPC (Table 2, Method 2e) showing
positive bias. Bias varied with folate concentration, with
largest difference at the clinical decision cut-off point.
Imprecision around the deficiency cut-off (3 ng/mL) is
still poor, with most methods returning a CV of 13% to
25%, while most methods have a CV below 10% to 12%
at 6 ng/mL. The difference between the two Abbott
methods ranging from + to bias is due to the Architect
being calibrated by harmonization, compared to the
traditional PGA calibration of the AxSYM method [20].
Relatively few U.S. laboratories are participating in RBC
folate schemes compared to serum folate (14%),
indicating either a smaller number of laboratories
offering this test or a lack of satisfaction with
performance. Calibration/linearity/verification surveys
are not offered for RBC folate. Results are summarized
in Table 4. In the United Kingdom National External
Quality Assessment Scheme (UKNEQAS), RBC folate
participation shows a continued decline over recent
years, with participation currently at 53% of that of
serum folate. Performance is poor in both schemes, with
an overall CV of almost 40% and individual method
CVs of 15% to 25%. In contrast with the serum data,
RBC folate bias does not trend in a predictable manner
with concentration but is variable from one sample to
another. In general, the Architect shows a negative bias,
with Bayer and Beckmann a less pronounced positive
bias [39].
Desirable specifications for analytical imprecision of
serum folate, derived from studies of biological
variation, indicate an assay imprecision of no greater
than 12% and a total error of no greater than 39%. For
RBC folate, an assay imprecision of no greater than 6%
and a total error of no greater than approximately 27% is
desirable. The average intra-individual variation of both
serum and RBC folate is approximately 24% [40].
References
1 Uddin E. Blood folic acid studies. VI.
Chromatographic resolution of folic acid-active
substances obtained from blood. J Biol Chem
1959; 234: 2373-6.
2 Blom HJ, Shaw GM, den Heijer M, Finnell RH.
Neural tube defects and folate: case far from
closed. Nature Reviews Neuroscience 2006; 7:
724-731.
3 Molloy AM, Scott JM. Microbiological assay
for serum, plasma, and red cell folate using
cryopreserved, microtiter plate method.
Methods Enzymol 1997; 281: 43-53.
4 OBroin S, Kelleher B. Microbiological assay
on microtiter plates of folate in serum and red
cells. J Clin Pathol 1992; 45: 344-7.
5 Gutcho S, Mansbach L. Simultaneous
radioassay of serum vitamin B12 and folic acid.
Clin Chem 1977; 23: 1606-14.
6 Bio-Rad Laboratories. Quantaphase assay
instruction manual. Hercules, CA. 1993.
7 Chitis J. The folate binding in milk. Am J Clin
Nutr 1967; 20: 1-4.
8 Givas JK, Gutcho S. pH dependence of the
binding of folates to milk binder in radioassay
of folates. Clin Chem 1975; 21: 427-8.
9 Billen J, Zaman Z, Claeys G, Blanckaert N.
Limited dynamic range of a new assay for
serum folate. Clin Chem 1999; 45: 581-2.
10 Wilson DH, Williams G, Herrmann R, Wiesner
D, Brookhart P. Issues in immunoassay
standardization: The ARCHITECT folate model
for intermethod harmonization. Clin Chem
2005; 51: 684-7.
11 Hansen SI, Holm J, Lyngbye J, Pedersen TG,
Svendsen IB. Dependence of aggregation and
ligand affinity on the concentration of the
folate-binding protein from cows milk. Arch
Biochem Biophys 1983; 226: 636-42.
12 Salter DN, Scott KJ, Slade H, Andrews P. The
preparation and properties of folate-binding
protein from cows milk. Biochem J 1981; 193:
469-76.
13 Mortensen E. Determination of erythrocyte
folate by competitive protein binding assay
preceded by extraction. Clin Chem 1976: 22:
982-92.
14 Mortensen E. Effect of storage on the apparent
concentration of folate in erythrocytes, as
measured by competitive protein binding
radioassay, Clin Chem 1978; 24: 663-668.
15 Netteland B, Bakke OM. Inadequate sample-
preparation technique as a source of error in
determination of erythrocyte folate by
579
Folic Acid and Folate
competitive binding radioassay. Clin Chem
1977; 23: 1505-6.
16 Owen WE, Roberts WL. Comparison of five
automated serum and whole blood folate assays.
Am J Clin Pathol 2003; 120:121-6.
17 Shane B, Tamura T, Stokstad R. Folate assay: a
comparison of radioassay and microbiological
methods. Clin Chim Acta 1980; 100: 13-9.
18 Mitchell GA, Pochron SP, Smutny PV, Guity R.
Decreased radioassay values for folate after
serum extraction when pteroylglutamic acid
standards are used, Clin Chem 1976; 22: 647-9.
19 Stenman UH. Immunoassay standardization: is
it possible, who is responsible, who is capable?
Clin Chem 2001; 47: 815-20.
20 Lequin RM. Standardization: comparability
and traceability of laboratory results. Clin
Chem 2002; 48: 391-3.
21 Satterfield MB, Sniegoski LT, Sharpless KE,
Welch MJ, Hornikova A, Zhang N-F et al.
Development of a new standard reference
material: SRM 1955 (homocysteine and folate
in human serum). Anal Bioanal Chem 2006;
385: 612-22.
22 Cheruppolil R, Santhosh-Kumar CR, Kolhouse
JF. Molar quantitation of folate by gas
chromatography-mass spectrometry. Methods
Enzymol 1997; 261: 26-34.
23 Bagley PJ, Selhub J. Analysis of folate form
distribution by affinity followed by reversed-
phase chromatography with electrochemical
detection. Clin Chem 2000; 46: 404-11.
24 Belz S, Frickel C, Wolfrom C, Nau H, Henze
G. High-performance liquid chromatographic
determination of methotrexate, 5-
methyltetrahydrofolic acid and folinic acid in
serum and cerebrospinal fluid. J Chromatogr B
1994; 661: 109-18.
25 Lucock MD, Hartley R, Smithells RW. A rapid
and specific HPLC-electrochemical method for
the determination of endogenous 5-
methyltetrahydrofolic acid in plasma using solid
phase sample preparation with internal
standardization. Biomedical Chromatography
1989; 3: 58-63.
26 Pawlosky RJ, Flanagan VP. A quantitative
stable-isotope LC-MS method for the
determination of folic acid in fortified foods. J
Agric Food Chem 2001; 49:1282-6.
27 Pfeiffer CM, Fazili Z, McCoy L, Zhang M,
Gunter EW. Determination of folate vitamers
in human serum by stable-isotope-dilution
tandem mass spectrometry and comparison with
radioassay and microbiologic assay. Clin Chem
2004; 50: 423-32.
28 Nelson BC, Satterfield MB, Sniegoski LT,
Welch MJ. Simultaneous quantification of
homocysteine and folate in human serum or
plasma using liquid chromatography/tandem
Table 1: Folate Methods Summary
Method 1: Microbiological
Type of analysis: Bioassay
mass spectrometry. Anal Chem 2005; 77:
3586-93.
29 Garbis SD, Melse-Boonstra A, West CE, van
Breemen RB. Determination of folates in
human plasma using hydrophilic interaction
chromatography-tandem mass spectrometry.
Anal Chem 2001; 73: 5358-64.
30 Fazili Z, Pfeiffer CM, Zhang M, Jain R.
Erythrocyte folate extraction and quantitative
determination by LC/MS/MS: comparison of
results with microbiologic assay. Clin Chem
2005; 51: 2318-25.
31 Kelly P, McPartin J, Goggins M, Weir D, Scott
JM. Unmetabolized folic acid in serum: acute
studies inn subjects consuming fortified food
and supplements. Am J Clin Nutr 1966; 65:
1790-5.
32 Mastropaolo W, Wilson MA. Effect of light on
serum B12 and folate stability. Clin Chem
1993; 39: 913.
33 Ando T, Yasui J, Inokuchi N, Usa T, Ashizawa
K, Kamihara S, Eguchi K. Non-specific
activities against ruthenium crosslinker as a
new cause of assay interference in an
electrochemiluminescent immunoassay. Jap J
Intern Med 2007; 46: 1225-9.
34 Treon SP, Chabner BA. Concepts in use of
high-dose methotrexate therapy. Clin Chem
1996; 42:1322-1329
35 Kremer JM, Galivan J, Streckfuss A, Kamen B.
Methotrexate metabolism analysis in blood and
liver of rheumatoid arthritis patients:
association with hepatic folate deficiency and
formation of polyglutamates. Arthritis Rheum
1986; 29: 832-5.
36 Grim J, Chladek J, Martinkova J.
Pharmacokinetics and pharmacodynamics of
methotrexate in non-neoplastic diseases. Clin
Pharmacokinet 2003; 42:139-51.
37 Van den Berg H, Finglas PM, Bates C. FLAIR
intercomparisons on serum and red cell folate.
Int J Vitam Nutr Res 1994; 64: 288-93.
38 Gunter EW, Bowman BA, Caudill SP, Twite
DB, Adams MJ, Sampson EJ. Results of an
international round robin for serum and whole
blood folate. Clin Chem 1996; 42: 1689-94.
39 Blackmore S, Hamilton MS. UKNEQAS
Annual Report 2006 for haematinic assays and
intrinsic factor antibodies. February 2007
(Personal Communication).
40 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV, et al. Current
databases on biologic variation: pros, cons and
progress. Scand J Clin Lab Invest 1999 ; 59:
491-500.
41 Mincey EW, Wilcox E, Morrison RT.
Estimation of serum and red cell folate by a
simple radiometric technique. Clin Biochem
1973; 6: 274-84.
580
Folic Acid and Folate

Principle: Growth of folate-dependent bacteria related to specimen folate concentration; growth monitored by
turbidimetry
Usage: Rare, historical reference method
Comments: False-negative because of presence of antibiotics or antibodies to target organism; false-positive
results because of turbidity
Method 2: Folate Binding Protein (FBP) Assays
a. Type of analysis: Competitive protein binding assay
Principle: Folates in sample compete with labeled ligand for binding to milk proteins (such as lactoglobulin);
separation of bound and free labeled-ligand by dextran-coated charcoal, pre-binding to magnetic particles or
vessel walls
b. Type of analysis: Immunometric protein binding assay (see Table 2)
Principle: Pre-incubation of specimen and FBP, with sequential addition of tracer. Tracer-labeled ligand occupies
remainder of FBP sites not occupied by serum folate. Separation of bound and free labeled-ligand by pre-binding
to magnetic particles or vessel walls. Signal generated may be radioactivity, chemiluminescence, fluorescence, or
an enzyme-generated product.
Usage: Frequently used
Comments: Easily automated, must destroy endogenous folate binders
Method 3: Liquid Chromatography (HPLC)
Type of analysis: Chromatographic separation after isolation, using solid-
phase or affinity extraction
Principle: Signal generated may be electrochemical or Mass Spectrometric
Usage: Specialist laboratories only
Comments: Proposed reference method, low throughput

Table 2: Folate Binding Protein Methods Summary

ULOQ ng/mL
Pre-treatment Detection Label / Separation LLOQ ULOQ
Method Solid phase / conjugate Assay Mechanism Detection System Extended by
System Bound to: method ng/mL ng/mL
Automatic Dilution

2 step
anionic microparticles /
DTT then 0.8 M ALP-folate binds to ALP / ionic attraction by 4-Methylumbelliferyl-phosphate
2a mc mouse anti-FBP 0.9 20 no
KOH excess conjugated folate fiber filter Fluorescent product
coupled to FBP
FBP
2 step Acridinium (N-sulfonyl)
paramagnetic particles
Acridinium-folate binds Acridinium ester / carboxamide yes for serum
2b DTT then KOH / mc mouse anti-FBP magnet 0.8 20
to excess conjugated folate Chemiluminescence with x2
coupled to FBP
FBP Peroxide and NaOH trigger
DTT 95 mg/L competitive binding for Acridinium ester
paramagnetic particles Acridinium ester / yes for serum
2c then 1.1 M limiting biotin-labeled magnet Chemiluminescence with 0.35 24
/ avidin folate x2, x5
NaOH FBP Acid and NaOH trigger

0.1 M HCl paramagnetic particles

competitive binding for ALP / Chemiluminescent product with
2d neutralized with / goat anti-mouse / mc magnet 0.5 20 no
limiting FBP folate Lumi-Phos 530 substrate
0.5 M NaOH mouse anti FBP

competitive binding for Luminescent product with

DTT then polystyrene bead / ALP / yes for serum
2e limiting FBP between centrifugal wash Adamantyl-1,2-dioxetane 0.8 24
NaOH/KCN mc mouse anti-FBP anti-ligand x2, x5
folate and folate-ligand phosphate substrate

monothioglycerol 2 step immunometric Electrochemiluminescence with Ru

paramagnetic particles Ruthenium complex
2f 53.3 g/L / NaOH folate-biotin binds to magnet Electron amplification with 0.6 20 no
/ streptavidin / FBP
37 g/L excess FBP Tripropylamine
Luminol derivative substrate
competitive binding for
well wall / HRP / Peracid salt peroxide generator
2g 1.2 M NaOH limiting biotin-labeled wash 0.2 20 no
streptavidin folate Electron amplification with 3-
FBP
Chloro,4-hydroxy acetanilide

a Abbott Axsym ALP Alkaline phosphatase

b Abbott Architect HRP Horseradish Peroxidase
c Bayer Centaur * mc = monoclonal; pc = polyclonal
d Beckman-Coulter Access / DxI FBP Folate Binding protein
e Diagnostic Products Immulite Series DTT Dithiothreitol
f Roche Elecsys Systems
g Vitros ECi

Table 3: Cross Reactivity Data for Folate Binding Protein Assays

581
Folic Acid and Folate

Manufacturer and Instrument ng/mL Added % Cross Reactivity

Methotrexate Leucovorin Aminopterin
Abbott Axsym 100 <2 <3 <1
Abbott Architect 100 1.1 -0.3 1.2
Bayer Centaur not stated --- --- ---
Beckman-Coulter Access / DxI 57 <1 <1 0.2
Diagnostic Products Immulite Series 100 0.9 --- ---
Roche Elecsys Systems not stated 2.3 2.3 2.7
Vitros ECi 500 <0.04 <0.04 <0.04

Data extracted from manufacturer package inserts, experiments performed according to NCCLS Protocol EP7

Table 4: Performance of Folate Binding Assays

Survey CAP 2006 - 7 UKNEQAS 2006

Average of 6 Spot Summary n = 135 Labs
n=242 Labs
Challenges
Method % of Labs mean ng/mL cv % % of labs % cv

2a 2.5 --- --- --- ---

2b --- --- --- 20.7 ---
2c 47.9 316.3 14.1 20.0 ---
2d 22.3 254.6 12.0 39.3 ---
2e 8.3 210.1 28.8 8.1 ---
2f 11.6 405.9 14.0 6.0 ---
2g 5.0 148.4 22.4 --- ---
Others 2.4 6 ---
All Results 100.0 299.9 39.3 100.0 35 - 40

a = Abbott AxSYM; b = Abbott Architect; c = Bayer Centaur; d = Beckman-Coulter Access/DxI; e = Diagnostic Products
Immulite Series; f = Roche Elecsys Systems; g = Vitros ECi.
582
Folic Acid and Folate

Figure 1. Structures and metabolic pathways for folates.

Folic acid

Pteroic acid
L-Glutamic acid
p-Aminobenzoic
acid O H
O C
2-Amino,
O
4-hydroxy-
pteridine N C
H
10 R
OH HN R Name

4a N 7 OH Monoglutamyl folate
N3 4 5 6
2 1 -glutamyl}n Polyglutamyl folate
9
H2N N 8a N

Reduction
R'
5 10
One Carbon Entity
OH HN N N

N Oxidized H CHO N-10-Formyl-

N
CHO H N-5-Formyl-
H2N N N H CHNH H N-5-Formimino-
H
CH N-5,10-Methenyl-
7,8-Dihydrofolate
CH2 N-5,10-Methylene-
Reduction
Reduced CH3 H N-5-Methyl-

OH HN 10
H OH R' HN
N One-Carbon N
N
H Addition N 5

H2N N N H
H2N N N
H H

5,6,7,8-Tetrahydrofolate
583
Folic Acid and Folate

Figure 2. Diagram shows inter-relationship of transmethylation (red) and remethylation (black)

pathways and the dependence on Vitamins B12 and B6.

CS, Cystathionine--synthase; DHF, dihydrofolate; MS, methionine synthase; MTHF,

methyltetrahydrofolate; SAM, S-adenosylmethionine; SHM, serine hydroxymethyl transferase; THF,
tetrahydrofolate; TS, thymidylate synthase
584
Folic Acid and Folate

Figure 3. Schematic for an immunometric folate binding protein assay (Table 2, Method 2d).
Reproduced with permission from http://www.beckman.com/products/testmenu/access.asp

In this assay, folic acid conjugated to alkaline phosphatase competes with folic acid released from the patient sample for
limited amount of milk FBP. After incubation with paramagnetic particles coated with antibodies to FBP, unbound entities
are removed by washing. The amount of enzyme bound to the particles is inversely proportional to the patient folate
concentration. Enzyme substrate, dioxetane-P (4-methoxy-4-(3-phosphatephenyl)spiro [1,2-dioxetane-3,2-adamantane] is
converted to a meta-stable anion, which fragments further to form an excited-state anion that emits a steady glow of light at
477 nm as it reverts to the ground state. Lumi-Phos* 530 is a micellar formulation containing dioxetane phosphate,
magnesium chloride, cetyltrimethylammonium bromide (CTAB) in 2-amino-2-methyl-1-propanol buffer (pH 9.6), together
with a fluorescent surfactant as an additional signal enhancer. This co-surfactant system promotes energy transfer to the
fluorescent head groups, causing the generation of luminescence with a broad maximum at 530 nm. It provides
ultrasensitive detection and is 10,000 times more sensitive than colorimetric substrates in solution.

H2O +
+ + H2 O
+
+
O O
+ +
C C
+ H2O +
+ +
+
H2 O

Fluorescein head group

+ CTAB
585
Folic Acid and Folate

Figure 4. Two-step electrochemiluminescent immunometric assay. (Roche Diagnostics Table 2,

Method 2f.)

1st Incubation

Folate-Biotin
Folate in
Excess Ru-FBP conjugate
patient sample

Wash
off

Streptavidin Magnetic
coated magnetic Capture
micro-particles

Ru(tpy)2 2+
Ruthenium bis(2,2:6,2-
terpyridine)

Photons

e- TPA
H+
Ru2+ 3+
RuTPA TPA
base state
TPA+
Electron donor
e-
(Tripropylamine, TPA)
+ Platinum Electrode + ensures that the reaction
Magnet continues to cycle,
multiplying the signal

In the first step, patient sample is incubated with biotinylated folate and an excess of ruthenium-labeled folate binding
protein (FBP). Next, streptavidin-coated paramagnetic microparticles are added and incubated. A sandwich complex is
formed, which becomes bound to the magnetic particles by the interaction of streptavidin and biotin. After washing to
remove unbound components, the magnetic particles are attracted out of solution by a magnet onto the electrode.
Application of a voltage to the electrode then induces chemiluminescent emission, which is measured by the
photomultiplier. The addition of an electron donor (tripropylamine) ensures that the reaction continues to cycle, thus
improving the sensitivity.
 586
Follicle-Stimulating Hormone
Follicle-Stimulating Hormone
Angela Ferguson
Name: Follicle-stimulating hormone, FSH, or follitropin
Clinical significance: Refer to Chapter 50, The Gonads, in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Molecular weight: 30 kDa
Chemical class: Glycoprotein
Reference method: None
Principles of Analysis and Current Usage
Follicle-stimulating hormone (FSH) is a 30-kDa
glycoprotein produced by the pituitary. FSH, also called
follitrop1n, contains two subunits to which are covalently
linked carbohydrate groups, including fructose, mannose,
galactose, glucosamine, galactosamine, and sialic acid.
The -subunit is chemically similar to the -subunits of
luteinizing hormone (LH), thyroid-stimulating hormone
(TSH), and human chorionic gonadotropin (hCG). The -
subunits of these hormones are biochemically distinct and
impart their hormonal and immunological specificity.
Whereas isolated -subunits are biologically inactive, free
-subunits retain some activity, but both subunits must be
present and recombined for full activity.
In women, FSH is released from the pituitary gland in a
pulsatile fashion throughout the menstrual cycle. There is a
small peak of FSH at the end of the cycle (starting on
about day 26) that begins the follicular maturation of the
next cycle. Serum FSH concentrations fall and remain low
during the follicular phase but peak again at midcycle. The
importance of the mid-cycle peak is not entirely clear. In
males, FSH is also secreted in a pulsatile fashion and acts
on Sertoli cells to stimulate spermatogenesis.
Reference and Preferred Methods
Immunoassays have become the method of choice for
measuring serum FSH, with all 1650 labs participating in
the 2007 College of American Pathologists (CAP) survey,
using two-site sandwich immunoassays with direct
chemiluminometric technology [1]. Typically these two-
site sandwich assays consist of one antibody against the
alpha chain and one antibody against the beta chain. These
assays are very specific, and cross-reactivity with other
pituitary glycoproteins such as LH, TSH, and hCG is
effectively eliminated. Claimed analytical sensitivities are
1
Follicle-Stimulating Hormone
Previous and current authors of this method:
First edition: Not done
Methods edition: Not done
Second edition: Not done
Third edition: Not done
Fourth edition: Amadeo J. Pesce
Fifth edition: Angela Ferguson
as low as 0.05 mIU/mL for some instruments (e.g. the
Abbott Architect method). Radioimmunoassays (RIA) are
available (Siemans offers the Coat-A-Count IRMA), but
they are no longer widely used.
Historically, serum concentrations and therapeutic
preparations of FSH were measured by in-vivo bioassays
such as the Steelman-Pohley method, which relies upon
the linear relationship between the amount of FSH
administered and ovarian weight in female rats [2,3].
Bioassays have poor precision, sensitivity, and specificity,
and they are time consuming and expensive. High-
performance liquid chromatography is being used more
frequently now to quantify different recombinant
preparations of FSH and decrease the variability between
batches [3,4].
Because of the great heterogeneity of FSH created by the
different combinations of carbohydrate modifications, it is
difficult to classify it as a single molecule; it is more
correct to consider FSH as a mixture of closely related
compounds. This presents a problem in comparing
methods, because assay-specific antibodies recognize the
various FSH isoforms differently [5]. The earliest
reference material used to standardize FSH assays was
prepared from the urine of postmenopausal women. This
calibrator was not appropriate for serum measurements and
was later replaced with purified pituitary extracts.
Recombinant gonadotropin calibrators are now available.
Most assays currently use the World Health Organization
(WHO) 2nd IRP 78/549 (interim replacement code
94/632).
Specimen
Serum is the preferred specimen for measuring
gonadotropins. FSH in serum is stable at room temperature
for 8 days and for 14 days at 4C [6]. Serum should be
kept frozen at or below 20C for long-term storage.
Plasma FSH is stable for at least 8 days at 4C and at room
temperature and is not affected by up to 5 freeze/thaw
cycles [7]. Urine FSH is stable for up to 32 weeks at both
4C and 20C, but the stability declines after that [8].
Because of the episodic and cyclic secretion of FSH,
pooled or serial blood specimens or timed urine collection
can sometimes be useful [9]. Urinary gonadotropin
measurements are used to diagnose pubertal and pituitary
 587
Follicle-Stimulating Hormone

disorders in children, owing to the low levels of Monitoring FSH is important both in the diagnosis and
gonadotropins found in serum and the less invasive nature treatment of infertility in women. Increased
of urine collection [10]. concentrations of serum FSH are an indication of
diminished ovarian reserve, and depending on the age of
Interferences the woman, it can also suggest how she will respond to
As with all sandwich immunoassays, heterophile ovarian stimulation protocols [16]. Day 3 FSH
antibodies are a possible interferent in FSH assays [11]. concentrations > 15 mIU/mL are considered elevated and
Plasma samples containing EDTA can give a falsely associated with poor reproductive outcome, as are basal
elevated result when using the Biomerieux Vidas, and estradiol concentrations > 75 pg/mL [17,18]. The diagnosis
sodium citrate in plasma can cause results to be falsely of polycystic ovarian syndrome (PCOS) can be aided by
decreased by 20% when using the Roche Elecsys 1010 and measurement of serum testosterone, DHEA-S, LH, and
2010 and MODULAR ANALYTICS analyzers [12]. FSH. Concentrations of LH are often elevated, with low to
low-normal concentrations of FSH. Some utilize an LH:
Reference Intervals FSH ratio of > 2 to indicate PCOS [19].
Reference intervals vary with assay manufacturer. Given
here are serum FSH reference intervals for three of the five During menopause, the decrease in estradiol concentration
most widely used methods according to CAP. results in increased serum LH and FSH concentrations (see
Table in Reference Intervals) [20]. Urine-based home FSH
Table 1. kits are now available which are intended to help
ADVIA Abbott DPC determine menopausal status. These tests are not
Centaur AxSym Immulite definitive, owing to the fluctuations in FSH concentrations,
(mIU/mL) (mIU/mL) 2000 and can only suggest a trend; there have been no published
(mIU/mL) peer-reviewed studies examining their utility.
Women
Follicular Phase 2.5-10.2 4-13 2.8-11.3
Children
Mid-Cycle Peak 3.4-33.4 5-22 5.8-21
In children, FSH measurement can aid in the diagnosis of
Luteal Phase 1.5-9.1 2-13 1.2-9.0
Postmenopausal 23.0- 20-138 21.7-153
delayed or precocious puberty. Most patients present with
116.3 failed or arrested puberty due to low plasma concentrations
Men 1.4-18.1 1-8 0.7-11.1 of LH, FSH, and testosterone. The gold standard for the
diagnosis of central precocious puberty is the GnRH
Interpretation and Performance Goals stimulation test. A peak serum concentration of LH > 8
Measurements of serum FSH are used in men, women, and IU/L after administration of GnRH is considered
children to evaluate pituitary function and differentiate diagnostic for precocious puberty. It is also useful to
between hypergonadotropic and hypogonadotropic compare the ratio of FSH concentration to LH
hypogonadism. concentration after stimulation; a predominant LH
response is indicative of a pubertal response [21]. In girls
Males with peripheral precocious puberty, the secretion of sex
In men, concentrations of FSH greater than 120 mIU/mL steroids is independent of pituitary gonadotropic release,
along with decreased testosterone are indicative of primary thus the response to the GnRH stimulation test is
testicular failure. Klinefelters syndrome is associated with repressed, and concentrations of LH and FSH are low. If
dysgenesis of the seminiferous tubules, a lack of sperm there are high concentrations of FSH and LH in
production, and marked elevation of serum FSH and LH conjunction with low Tanner stages, this can be a sign of
concentrations [13]. Decreased concentrations of FSH and delayed puberty and hypergonadotropic hypogonadism.
testosterone suggest hypogonadotropic hypogonadism.
References
Women 1 College of American Pathologists. Participant
Hypergonadotropic hypogonadism arises from primary summary. Y-A Ligands (Special). 2007:6-7.).
gonadal failure. The most common cause for 2 Rose MP, Gaines Das RE, Balen AH. Definition
hypergonadotropic hypogonadism is Turners Syndrome, and measurement of follicle stimulating hormone.
which is defined as a 45 X0 karyotype and female Endocrine Reviews 2000; 21: 5-22.
phenotype [14,15]. Moderately high concentrations of FSH 3 Driebergen R, Baer G. Quantification of follicle
and LH are also found in cases of androgen insensitivity, stimulating hormone (follitropin alfa): is in vivo
where the patient has a 46 XY karyotype but is bioassay still relevant in the recombinant age?
phenotypically female. In patients without signs of Curr Med Res Opin 2003; 19: 41-46.
hyperandrogenism, elevated concentrations of FSH and 4 Loureiro RF, de Oliveira JE, Torjesen PA,
LH could indicate ovarian failure. Bartolini P, Ribela, MTCP. Analysis of intact
human follicle-stimulating hormone preparations
by reversed-phase high-performance liquid
 588
Follicle-Stimulating Hormone
chromatography. J Chromatogr 2006; 1136: 10-
18.
5 Sturgeon CM, Ellis AR. Standardization of FSH,
LH and hCG-Current position and future
prospects. Mol Cell Endocrinol 2007; 260-262:
301-309.
6 Kubaski NP, Ricotta M, Hunter T, Sine, HE.
Effect of duration and temperature of storage on
serum analyte stability: examination of 14
selected radioimmunoassay procedures. Clin
Chem 1982; 28: 164-165.
7 Livesey JH, Hodgkinson SC, Roud HR, Donald
RA. Effect of time, temperature and freezing on
the stability of immunoreactive LH, FSH, TSH,
growth hormone, prolactin, and insulin in plasma.
Clin Biochem 1980; 13: 151-155.
8 Brindle E, Miller RC, Shofer JB, Klein NA,
Soules MR, OConnor KA. Urinary beta-
luteinizing hormone and beta-follicle stimulating
hormone immunoenzymometric assays for
population research. Clin Biochem 2006; 39:
1071-1079.
9 Demers LM. General Endocrinology. In:Kaplan
LA, Pesce AJ, Kazmierczak SC, editors. Clinical
Chemistry: Theory, Analysis, Correlation. St.
Louis: Mosby; 2003.p826.
10 Demir A, Dunkel L, Stenman U-H, Voutilainen.
Age-related course of urinary gonadotropins in
children. J Clin Endocrinol Metab 1995; 80:
1457-1460.
11 Boscato LM, Stuart MC. Heterophilic antibodies:
a problem for all immunoassays. Clin Chem
1988; 34: 27-33.
12 Youngs Effects Online [database on the internet].
Washington DC: AACC. c2005 [updated 2007
Jun 13; cited 2007 Jun 27]. Available from:
http://www.fxol.org/
13 Bojesen A, Gravholt CH. Klinefelter syndrome in
clinical practice. Nat Clin Pract Urol. 2007; 4:
192-204.
14 Slap GB. Menstrual disorders in adolescence.
Best Pract Res Clin Obstet Gynaecol 2003; 17:
75-92.
15 The Practice Committee of the American Society
for Reproductive Medicine. Current evaluation of
amenorrhea. Fertil Steril 2006; 86(Suppl 4):
S148 155.
16 Barad DH, Weghofer A, Gleicher N. Age-specific
levels for basal follicle-stimulating hormone
assessment of ovarian function. Obstet Gynecol
2007; 109: 1404-1410.
17 Macklon NS, Fauser BC. Ovarian reserve. Semin
Reprod Med. 2005; 23: 248-256.
18 Licciardi FL, Liu HC, Rosenwaks Z. Day 3
estradiol serum concentrations as prognosticators
of ovarian stimulation response and pregnancy
outcome in patients undergoing in vitro
fertilization. Fertil Steril. 1995; 64: 991-994.
19 Carr BR. Disorders of the ovary and female
reproductive tract. In: Williams RH, Foster DW,
Kronenberg HM, Larsen, PR, Wilson JD, eds.
Williams textbook of endocrinology. 9th ed.
Philadelphia: WB Saunders, 1998:751-817.
20 Burger HG, Dudley EC, Hopper JL, Groome N,
Guthrie JR, Green A et al. Prospectively
measured levels of serum follicle-stimulating
hormone, estradiol, and the dimeric inhibins
during the menopausal transition in a population-
based cohort of women. J Clin Endocrinol Metab
1999; 84: 4025-4030.
21 Lee PA. Central precocious puberty. An overview
of diagnosis, treatment, and outcome. Endocrinol
Metab Clin North Am. 1999; 28: 901-918.
589
Free Thyroxine and Free Triiodothyronine

Free Thyroxine and Free Triiodothyronine

Greg Ward

Name: Free T4 (fT4) and free T3 (fT3)

Clinical significance: Refer to Chapter 49, Thyroid, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Merck Index: 9262 (thyroxine), 5337 (3,5,3-triiodothyronine)
Chemical class: Amino acid

Structure:
Thyroxine

3,5,3-Triiodothyronine
i amount of protein-bound hormone would only
Principles of Analysis and Current Usage
The measurement of free thyroid hormones
presents two major problems. One is analytical, the
other is clinical. The analytical problem is that
serum concentration of the free (non-protein-
bound) fractions of both physiologically active
thyroid hormones, thyroxine (T4) and
triiodothyronine (T3), is extremely low (both in the
pmol/L range). The clinical problem is related to
the continuing controversy regarding the
physiological role of nonprotein/nonpeptidetype
free hormones in general. The clinical use of free
hormone measurements assumes the validity of the
free hormone hypothesis, according to which only
the free (i.e., non-protein-bound) fraction of the
hormone is able to enter the target cell and to exert
physiological activity [1-4]. Thus the much larger
i of free thyroid hormone began in the 1960s.
Free Thyroxine and Free Triiodothyronine
Previous and current authors of this method:
First edition: Not done
Methods edition: Andre van der Walt
Second edition: Not updated
Third edition: Not updated
Fourth edition: Gyorgy Csako
Fifth edition: Greg Ward
serve as a passive reservoir to maintain the free
hormone concentration within set limits. However,
there is evidence that protein-bound hormones may
also be available for transport to target tissues.
Physiological factors that strongly affect the
analysis of free thyroid hormones are their plasma
concentration and the mode of transport of the
entire T4 and T3 pool. In man, approximately
0.03% of 7.4 g/dL (or 96 nmol/L) of plasma T4 is
found free (about 2.2 ng/dL or 29 pmol/L), whereas
0.4% of the total 100 ng/dL (or 1.54 nmol/L)
plasma T3 is in the free form (about 400 pg/dL or
6.2 pmol/L) under standard thermodynamic
equilibrium conditions [5].
Laboratory assessment of the serum concentration
Similar to other free (non-protein-bound)
hormones, the free thyroid hormones initially were
measured by indirect equilibrium dialysis (tracer
dialysis) or indirect ultrafiltration [6-16].
Although several radionuclides (e.g., 3H) could be
considered theoretically for radiolabeling of thyroid
hormones, for obvious reasons the isotopic
590
Free Thyroxine and Free Triiodothyronine
methods for free thyroid hormones involved and
continue to involve the use of radio-iodinated
(131I- or 125I-labeled) thyroid hormones. Since the
1970s, a wide variety of relatively inexpensive,
technically simple, often automated non-isotopic
free T4 (fT4) and free T3 (fT3) assays have become
available. The basic concept, performance, and
evolution of assays for the measurement of free
thyroid hormones have been reviewed repeatedly
[1,2,16-33]. Virtually all of these assays involve
the use of an antibody or antibodies at some stage
of the analysis; that is, they are all at least in part
immunoassays.
Before single-test, free-thyroid-hormone ligand
assays became widely available in routine
laboratories, the free-thyroid-hormone status had
been routinely evaluated by the T3 uptake (T3U)
test. The T3U test is now named the thyroid
hormonebinding ratio (THBR). Originally
described in the late 1950s [29,34], this test
measures the number of unoccupied thyroid
hormonebinding sites on serum proteins,
primarily on TBG. If the proportion of thyroid
hormone carried by TBG is at least 60%, the T3U
correlates with the fT4 and fT3 fractions in the
serum, and determination of the T3U, in
combination with total thyroid hormone
determinations, allows indirect estimation of the
fT4 and fT3 concentrations in terms of indices (free
T4 index, fT4I [or less correctly, fTI], and free T3
index, fT3I) [35,36]. The total thyroid hormone
concentrations correct for variations in binding
protein such as TBG concentrations that are
relatively often encountered in everyday clinical
situations.
Free-Thyroid-Hormone Techniques
Free-thyroid-hormone assays are now classified as
follows:
Index Methods which require two
unrelated measurements from which the
concentration of the free hormone is
derived. One is the unsaturated binding
protein capacity, the total TBG
concentration, or the free hormone
fraction; the other is the total hormone
concentration, usually measured by an
immunoassay. Examples include classic
fT4I, T4/TBG ratio, and indirect (tracer)
equilibrium dialysis.
Single-test ligand assays. A single
measurement for free hormone
concentration is performed and requires
no subdivision of the test sample (e.g.,
one-step [analog] and two-step
immunoassays).
Physical separation methods which
separate free from bound thyroid hormone
prior to direct assay of the free fraction.
Examples include direct equilibrium
dialysis (ED) and ultrafiltration (UF). In
ED, the free-thyroid-hormone
concentration is determined at the time
when thermodynamic equilibrium is
attained across a membrane or partition
phase in the assay system. The final
measurement depends on the distribution
of hormone between the free and bound
state or the absolute amount of hormone in
the free moiety.
According to the type of calibration involved, the
assay can be:
1. Absolute. Calibrated by gravimetrically
established quantities of (pure) hormone
dissolved in buffer.
2. Comparative. Calibrated by serum standards
that in turn are traceable in calibration to absolute
methods.
Techniques for Free-Thyroid-Hormone Estimates
Index Methods Using Free Hormone Fraction
Determination
As mentioned earlier, indirect (tracer) equilibrium
dialysis (Table 1, Method 1) was the first method
capable of measuring the absolute fT4
concentration [6]. For assay, first a minute amount
of radiolabeled (usually 131I or 125I) thyroid
hormone (T3 or T4) was preincubated with the
serum sample to provide uniform labeling of the
total thyroid hormone. The sample typically was
used diluted. The free moiety of hormone was
separated from the protein-bound fraction by
dialysis through a semipermeable membrane, and
the amount of radiolabeled T4or T3 in the dialysate
was measured. At equilibrium, the product of the
dialyzable (free) fraction of total radiolabeled T4 or
T3 and the total T4 or T3 concentration of the
sample was the mass of fT4 or fT3 in the test
system. Correction for sample dilution yielded the
free hormone concentration in the patients serum.
In euthyroid individuals, the %fT4 or %fT3
determined by equilibrium dialysis is inversely
proportional to the respective thyroid hormone
binding capacity, but the mass of fT4 or fT3 in the
system remains essentially the same. The
separation of radioiodinated thyroid hormones from
contaminating iodines in the dialysate was
achieved by a variety of methods. However,
because of the many elution or wash steps
involved, these methods never became suitable for
use in routine clinical laboratories. Even the use of
the most practicable and popular separation
591
Free Thyroxine and Free Triiodothyronine
technique based on MgCl2 precipitation of thyroid
hormones [12,37] remained restricted to reference
and research laboratories.
Index Methods Using TBG Measurement
(Calculated fT4 and fT3 ) (Table 1, Method 2)
Since the free-thyroid-hormone concentration is
governed by the law of mass action, knowledge of
the total hormone concentrations, binding
constants, and binding-site concentrations (i.e.,
binding-protein concentrations and the number of
binding sites per binding-protein molecule) allows
the calculation of both the free hormone
concentration and the distribution of hormone
among various binding proteins. A number of
different models based on the mass action law have
been developed for computer-assisted calculations
of fT4 and/or fT3 concentrations [38-42].
Techniques for Measuring Thyroid Hormone
Binding Ratio(THBR) (Formerly Known as T3 or
T4 Uptake [T3U or T4U])
Classic Isotopic THBR or T3U (and T4U) Tests
The T3U test was developed originally by
Hamolsky et al. [34,43] in the late 1950s as an
isotopic in-vitro diagnostic test of thyroid function.
In the original T3U tests, a fixed amount of
radioactive T3 was incubated with whole blood,
and the binding of radioactive T3 by red blood cells
(RBC) was measured by counting the radioactivity
in the separated erythrocytes obtained after
centrifugation and washing:
counts in RBC(i.e.,matrixboundcounts
T3U(%)
totalcounts
Under these circumstances, the radioactivity taken
up by red blood cells (the T3 uptake) was
inversely proportional to the radioactivity bound to
T4-binding serum proteins, especially TBGthat
is, a high T3 uptake meant high TBG saturation
and reduced available T4-binding sites.
Nonisotopic THBR or T3U and T4U (T
Uptake) Tests
Since the late 1970s, non-isotopically labeled T3
and T4 have been increasingly used as tracers in a
variety of fully automated uptake tests [44-49].
Whereas in heterogeneous immunoassay
formulations, the antibodies continue to require
binding to a solid phase and thus form a classic
secondary binder, in the homogeneous T-uptake
immunoassays, all reactions take place in solution,
that is, the secondary binder is a liquid-phase
antibody. The solid phase may be a glass fiber filter
with an immobilized second antibody,
magnetizable (paramagnetic) particles with a
second antibody, or a multilayer thin film. For
signal detection, the tracers may be labeled with an
enzyme (e.g., alkaline phosphatase, glucose-6-
phosphate dehydrogenase) or with a more directly
measurable compound (e.g., fluorescein, an
undisclosed type of fluorescent compound, or
acridinium ester). The signal detection may be
based on colorimetry (e.g., p-nitrophenol or
chromogen from a patented substrate), ultraviolet
spectrophotometry (e.g., reduced nicotine adenine
dinucleotide, NADH), fluorescence (e.g., 4-
methylumbelliferone, or fluorescent label),
fluorescent polarization (e.g., fluorescein), and
chemiluminescence (e.g., acridinium, unstable
intermediates from the phosphate ester of
adamantyl dioxetane, or Lumi-Phos 530).
Index Methods Using Free-Thyroid-Hormone
Indices
Classic Free T4 and Free T3 Indices (fT4I and
fT3I) (Table 1, Method 3)
In 1966, Clark and Horn [35] proposed first that
because of its good correlation with the serum fT4
concentration, the product of T3U (obtained by
their anion-exchange resin uptake method) and
serum protein-bound iodine (PBI, as a measure of
the total thyroid hormone concentration) should be
called the free thyroxine index (fTI). It is now
generally agreed that the product of THBR and the
total T4 or T3that is, the fT4I or fT3Iis a
reliable indirect estimate of the fT4 or fT3
concentration in many patients [25,36,50-56]. Thus
if the THBR is to be determined, then the total
thyroid hormone (T4 or T3) concentration should
also be measured and the fT4I or fT3I calculated
[57]. Since these indices are products of the total
T3 or T4 concentration and a ratio, they
theoretically have concentration units. However,
the Committee on Nomenclature of the ATA [57]
recommended that in order to avoid confusion with
serum T4 or T3 results, the free-thyroid-hormone
index units be either omitted or simply termed
index units.
Single Test Ligand Assays for Estimation of fT4
or fT3
Ligand assays for the fT4 (or FT3) fraction of serum
are designed not to disrupt the equilibrium between
free and protein-bound thyroxine. In practice,
thyroxine is dissociated from the bound fraction
[31]. However the more important factors for free
thyroxine estimates are (1) whether the dissociation
of hormone occurs identically in samples and
standards and (2) whether samples and standards
592
Free Thyroxine and Free Triiodothyronine
show identical protein binding of the assay tracer
[30].
Two-Step Labeled Hormone (Sequential/Back-
Titration) Immunoassay (Table 1, Method 4)
Early coated-tube RIAs for free thyroid hormones
employed a sequential, two-stage method
[21,22,58-60]. A commercial version of this
methodology was introduced which entailed the
binding of free hormone to polyclonal antibodies
coated to the walls of assay tubes
(immunoextraction). Since then, other solid-
phase materials such as micro- and macrobeads
have also been used for immobilizing the
antithyroid-hormone antibodies. In a basic two-step
assay, after decanting and washing of serum, the
unoccupied antibody-binding sites are
(sequentially) back-titrated with the respective
labeled thyroid hormone. As a consequence, (1) the
sample and the labeled hormone are never in
contact with each other in a classic two-step assay,
and (2) the more binding of labeled hormone
during the second step, the less free hormone was
present, that is, the signal and the free-thyroid-
hormone concentration are inversely related. In this
assay, the reaction between serum proteins and
labeled thyroid hormone is avoided. The Abbott
AxSYM, Abbott ARCHITECT, Beckman DxI and
Wallac Delfia free-thyroid-hormone assays are all
two-step nonisotopic immunoassays.
Labeled Hormone-Analog ( One-Step )
Immunoassays (Table 1, Method 5)
Since their conception in the late 1970s [1,61-64],
the isotopic and recently the automated non-
isotopic, one-step (analog) immunoassays became
the most common methods for free-thyroid-
hormone measurements. Despite their controversial
performance [1,2,31,65-78], they allowed the
widespread measurement of free thyroid hormones
in routine diagnostic laboratories. In their initial
isotopic form, these competitive assays for free
thyroid hormone invariably required only a single
incubation step, hence the name one-step. The
initial labeled analogs were small molecules, but
these compounds bound strongly to albumin. To
avoid protein binding, the Siemens ADVIA
Centaur free thyroxine method uses a high
molecular weight IgG-T4 compound labeled with
acridinium ester analog [78]. The Roche Elecsys or
E170 assays are one-step assays which have a two-
step incubation [30].
Labeled Antibody Method (Table 1, Method 6)
This method is also known as the solid-phase
antigen-linked technique (SPALT). In its original
isotopic version, the method is one step, with the
sample to be tested simultaneously incubated with
solid phasebound thyroid hormone analog and a
limited amount of labeled antithyroid-hormone (T4
or T3) antibody. The free thyroid hormone and the
immobilized hormone (macro) analog molecules
[2] compete for binding sites on the labeled
antibody. The Ortho Vitros ECi thyroid hormone
assays and the Siemens ADVIA Centaur FT3
assays are examples of this immunoassay format.
Equilibrium Dialysis (Table 1, Method 7)
In direct equilibrium dialysis, the free thyroid
hormones are first separated from the protein-
bound moieties. After dialysis, the (free) T4 and/or
T3 in the dialysate is directly measured by a
sensitive T4 or T3 immunoassay [20,79], by gas
chromatography (GC) with electron capture
detection [80,81], or by mass spectrometry.
Although the latter technique has the advantage of
providing concomitant, absolute analysis for free
(dialyzable) T4 and T3 in the sample, it is both
cumbersome and expensive, with little potential for
routine diagnostic use.
In indirect symmetric dialysis, the unknown serum
containing a minute quantity of radiolabeled
thyroid hormone is dialyzed against the same
unspiked serum. The initial rate of entry of the
radiolabeled thyroid hormone into the second
compartment is proportional to the free hormone
concentration in serum. With proper calibration,
the results can be expressed in concentration of the
free hormones. In the original applications, the
serum was used undiluted [82,83], but now serum
samples are usually diluted for analysis [83-86].
A method has recently been described which uses a
96-sample well dialysis plate followed by liquid
chromatographytandem mass spectrometry (LC-
MS/MS) [85]. This assay is used for routine
analysis of serum free thyroid hormones and has
been described as a major step forward in the
analysis of free thyroid hormones [86].
Ultrafiltration (Table 1, Method 8)
FT4 and fT3 concentrations also can be measured
by ultrafiltration of diluted or undiluted serum
samples, either in dialysis bags or special
ultrafiltration devices. As with equilibrium dialysis,
ultrafiltration may be direct or indirect. In direct
ultrafiltration, the free-thyroid-hormone
concentration is measured directly, usually by a
highly sensitive immunoassay, in the ultrafiltrate
[87-89]. In indirect ultrafiltration [8,12,13,90], the
test sample is enriched with trace amounts of
radiolabeled thyroid hormone prior to
centrifugation, then the radioactivity is determined
in the resultant ultrafiltrate. Using 125I and 131I
for the labeling of T4 and T3, respectively,
simultaneous determination of fT4 and fT3 is also
possible. Compared to equilibrium dialysis
593
Free Thyroxine and Free Triiodothyronine
methods, ultrafiltration methods are faster to
perform. They are, however, also cumbersome and
difficult to run in large batches. Further, because of
the very high ratio between protein-bound and free
thyroid hormones, even small protein leakage
through the ultrafiltration membrane [87,88,91,92]
readily invalidates the free hormone measurement.
Centrifugal ultrafiltration followed by isotope
dilution MS/MS was first described by Soldin et al.
[91]. This procedure was used for fT4 but lacked
the sensitivity for fT3 determination. The technique
was modified [92] to improve sensitivity, allowing
simultaneous measurement of fT4 and fT3.
Gel Filtration/Gel Bead Dialysis (Table 1,
Method 9)
Polyacrylamide [93] and Sephadex [94-96] gel
filtration and gel-bead dialysis [97] have been also
employed for the measurements of free thyroid
hormones. Although these methods are faster than
equilibrium dialysis, they are also tedious and
cumbersome for large numbers of samples, and in
their usual indirect version, they are subject to error
arising from radioactive impurities of the tracer.
Besides, these methods may not always yield a true
free-thyroid-hormone fraction but rather a fraction
that is only proportional to the free-thyroid-
hormone concentration [95].
Column Adsorption Chromatography (Table 1,
Method 10)
Although this method was once considered by
some investigators as a version of a resin uptake
test, it is better described as a direct equilibrium
method for the real measurement of free thyroid
hormones [1,20]. In this method, Sephadex LH-20
chromatography is used to separate free from
protein-bound hormone after equilibrium has been
reached on the column [84,98,99]. The protein-
bound thyroid hormones (T4 or T3) emerge from
the column first and are discarded before elution of
the free-thyroid-hormone (T4 or T3) fraction,
which is then measured.
Critical Analytical Issues
Issues that are particularly critical for valid free-
thyroid-hormone measurements are discussed
below. For further details, see reports by Ekins
[1,2,66,100], Nelson and Wilcox [101], Midgley
[31], Stockigt [30] and Baloch [32].
Incubation Temperature
Because the temperature coefficients governing
thyroid-hormone binding to various transport
proteins differ (TBG being the most sensitive), the
relative change in free hormone concentrations at
37C and at room temperature will vary between
standards and samples that contain different
relative amounts of transport proteins. The
consequences of testing at room temperature
instead of the more appropriate 37C have been
well documented recently for a two-step
immunoassay for fT4 [102,103].
Incubation Time
Obviously, all equilibrium assays require that the
incubation time be sufficiently long to reach
equilibrium in the assay system. Nevertheless,
historically there were several instances when the
incubation times were found to be inadequate for
fT4 immunoassays, and the manufacturers were
eventually forced to correct this design deficiency.
Witherspoon et al. [104] showed that similar
problems exist for isotopic T3U assays as well.
Incubation Milieu
Different ions have been shown to have different
effects on the binding of thyroid hormones to their
specific proteins. For instance, both chloride and
phosphate ions inhibit the binding of T4 to serum
proteins [105]. Barbital ions preferentially inhibit
the binding of T3 to TTR (transthyretin,
prealbumin) [106]. In addition, both endogenous
substances (e.g.,, various phenol compounds in
uremia, FFAs in diabetes) and drugs (e.g.,
salicylates, furosemide) are known to specifically
inhibit thyroid-hormone binding to serum proteins
and to raise the apparent fT4 and fT3
concentrations [106].
Hormone Sequestration
The loss of thyroid hormone from the serum
compartment (hormone sequestration) into
another compartment is a general feature of all
assays for free thyroid hormone [1,2], including
direct equilibrium dialysis with undiluted sera
[1,50,107]. This hormone loss may be part of the
assay design (e.g., sequestration in a dialysis cell or
by an antithyroid-hormone antibody) or may be
incidental to the assay (sequestration to extraneous
sites such as adsorption of hormone to glass)
[1,101].
Serum Dilution
Except for ultrafiltration of undiluted sera, all
methods of free hormone measurements involve
sample dilution with buffer [1,2]. In accordance
with the mass action law, dilution of serum
containing protein-bound and free thyroid hormone
causes the free hormone concentration to fall, but
the large protein-bound pool of thyroid hormones
has a large reservoir capacity to sustain the free
hormone level in the diluent. For instance, a 100-
fold dilution causes only an approximately 2%
depletion of the protein-bound T4 hormone pool
and a similar depression of the ambient fT4
concentration in the diluted sample [1]. Because
594
Free Thyroxine and Free Triiodothyronine
the fT3 concentration is higher than the fT4
concentration relative to the protein-bound, the
dilution causes a somewhat more conspicuous drop
in the fT3 concentration (5 to 20% decrease at a
1:100 serum dilution) [1,2,108-113].
Reference and Preferred Method
There are no reference methods for either fT4 or
fT3. However, both equilibrium dialysis (ED) and
ultrafiltration (UF) have been proposed as
separation techniques for a reference measurement
procedure. Problems with UF include non-
equilibrium, absorption, protein leakage, and
temperature control. Problems with ED are dilution
and dialysis-buffer composition [108]. Free fatty
acids and drugs which can displace thyroid
hormones from the binding proteins are potential
confounders for the separation of free from bound
thyroid hormone prior to definitive analysis [108].
A reference measurement system for free thyroxine
has been proposed which uses ED followed by a
trueness-based measurement procedure [109,110].
An ED LC-MS/MS procedure has recently been
proposed which could be considered a candidate
reference method for FT4 [111]. There is currently
no proposed reference method for fT3, however the
new LC-MS/MS methods should facilitate
development.
According to the 2007 General Ligand Survey by
the College of American Pathologists (CAP), all
participating laboratories used non-isotopic
methods for fT4 and fT3 assays. Greater than 99%
of these assays have imprecision of less than 10%
and less than 8%, respectively, for fT4 and fT3,
which is satisfactory imprecision for routine
clinical use.
There has been considerable discussion over many
years on the validity of immunoassay estimates of
free-thyroid-hormone concentration. A recent study
[85] demonstrated that two non-isotopic
immunoassays for FT4 correlated well with ED LC-
MS/MS but had quite different results which could
be attributed to standardization issues. Other
studies using LC-MS/MS have shown poor
correlation with modern automated non-isotopic
immunoassay [91,92]. The development of
reference methods for free thyroid hormones will
allow for better standardization of these analytes
with improved analytical accuracy.
Specimen
The preferred specimen is a serum sample that has
been removed from the clot as rapidly as possible.
Fresh serum samples may be stored at room
temperature for up to 1 week without appreciable
changes in the fT4, fT3 or T-uptake values, but
storage at 4C is preferred if the test is not
performed within 24 hours. FT4 and FT3 in serum
appear to be stable for 24 hours in whole blood
stored at temperatures between 15C and 35C
[112]. No change in estimated FT4 concentration
has been observed in samples stored at 4C to 8C
for either 5 days as whole blood or for 13 days as
stored serum [113]. If the time between sample
collection and analysis is longer than 6 days, the
sample should be stored frozen at 20C or below
in non-cycling freezers. Frozen samples should be
mixed well by inversion after thawing and before
assay. Repeated freezing and thawing and mixing
by vortexing or vigorous agitation should be
avoided, since this may result in denaturation of
proteins. Ideally, blood should be collected in the
total absence of heparin [114], and clinicians
should be made aware that thyroid function tests
are contraindicated in heparin-treated patients. The
popularity of non-isotopic assays for free-thyroid-
hormone measurement presents a special problem
with respect to specimen acceptability.
Consequently, particular attention should be paid to
the manufacturers specific recommendations for
specimen collection when non-isotopic methods are
involved in testing. Finally, because the law of
mass action governs the free-thyroid-hormone
concentration, it is of great importance that
specimens with results above the assay range
should not be diluted for re-assay for any of the
free-thyroid-hormone or hormone-binding tests but
should be reported as such.
Interferences
The use of hemolyzed or lipemic samples is not
recommended. Siliconized collection tubes have
been claimed to produce artifactual results in some
solid-phase fT4 assays, but these observations could
not be confirmed in later studies [24,115-127].
Apart from heparin, most blood-collection-tube
additives have little if any effect on the
performance of isotopic assays. However, some of
the non-isotopic assays are profoundly affected by
certain tube additives. For instance, some fT4
methods are incompatible with the presence of
EDTA, citrate, or fluoride.
Patients on heparin therapy may have increased
free fatty acids in stored serum samples as a result
of heparin-induced lipoprotein lipase activity.
These free fatty acids displace T4 from TBG and
may cause an artifactual increase in the FT4
concentration [30,32].
Drugs such as furosemide, disalicylic acid,
phenytoin, and carbamazepine, which displace T4
from TBG, cause dilution artifacts in FT4 assays
which dilute samples as part of the analytical
procedure. Underestimation of FT4 is observed, and
595
Free Thyroxine and Free Triiodothyronine
this is greatest in methods which have the least
sample dilution [30,115].
Abnormal binding proteins can cause artifactual
results. Examples include autoantibodies,
rheumatoid factor, and familial dysalbuminemic
hyperthyroxinemia [32]. Autoantibodies can cause
either false-positive or false-negative results,
depending on the assay design [116]. Autoantibody
interference can be assessed by incubating patient
serum with radiolabeled thyroid hormone, followed
by polyethylene glycol (PEG) precipitation of
autoantibody-bound radiolabel [116-118]. It has
recently been demonstrated [118] that samples
could be re-assayed for FT4 after PEG treatment in
a patient with autoantibody interference. In general,
analog ligand assays are the most susceptible to
thyroid hormone autoantibodies. It is generally
assumed that two-step assays are not as susceptible
because protein is removed prior to incubation with
tracer. Recently the results of seven different
automated immunoassay systems were compared
with ED/RIA in a patient with thyroid hormone
autoantibodies [117]. The one-step assays ( DPC
IMMULITE 2500, Siemens ADVIA Centaur,
Tosoh AIA 1800, Roche Elecsys E170) all had
artifactually elevated results. The Wallac Delfia
two-step assay results were consistent with
ED/RIA, while the Abbott ARCHITECT and
Beckman DXi 800 had comparative results that
were considered borderline low. One suggestion for
the latter observation was incomplete washing
away of patient sample prior to the addition of
tracer in the two automated immunoassay systems.
Nonspecific interference by ruthenium cross-
linking causing artifactual elevation of results has
been observed in the Roche E170
electrochemiluminescent one-step assays for FT4
and FT3 [119,120].
Free Thyroxine and Free Triiodothyronine
Reference Intervals
As with most clinical diagnostic tests, commonly
cited reference intervals for free thyroid hormones
and thyroid-hormone binding are based on the
study of healthy adult males and nonpregnant
females. For correct interpretation, it is important
to note, however, that a number of factors (e.g.,
age, gender, pregnancy) can alter the concentration
of free thyroid hormones under physiological
conditions. Therefore, a brief review of these
factors is also necessary [113]. It is important to
recognize that FT4 and total T4 population-based
reference intervals are wide when compared to the
narrow reference interval for individuals. Thus a
measurement of FT4 might be outside an
individuals own set point, indicative of an
abnormality, but the result could still be within the
limits of the population-based reference interval
[113,121].
FT4 Concentration
Reference Interval for (Nonpregnant) Adults:
0.72.0 ng/dL (926 pmol/L)
(two-step and one-step immunoassays)
0.82.3 ng/dL (1030 pmol/L)
(indirect [tracer] equilibrium dialysis)
0.82.7 ng/dL (1035 pmol/L)
(direct equilibrium dialysis)
A trend for defining the normal range between 0.8
and 1.9 ng/dL (10 to 25 pmol/L) is evident, but it is
also apparent that the reference intervals vary not
only with the type of methodology (see earlier
discussion), but also among kits based on the same
analytical concept. This reinforces the need to
establish, or at least verify, the reference interval
for the local population. Reference intervals for
thyroid hormones have been determined for a
number of non-isotopic immunoassay platforms
and MS/MS methods, including DPC IMMULITE
[122]; ADVIA Centaur [123]; Abbott
ARCHITECT [124,125]; Beckman Access [126];
Roche E170 [127]; UF/LC-MS/MS [92]; and
ED/LC-MS/MS [85].
FT3 Concentration
Reference Range for (Nonpregnant) Adults:
143-468 pg/dL ( 2.2-7.2 pmol/L)
(one-step immunoassay)
250-550 pg/dL ( 3.8-8.5 pmol/L)
(one-step immunoassay)
260-480 pg/dL ( 4.0-7.4 pmol/L)
(indirect [tracer] equilibrium dialysis)
Like fT4, the reference range for fT3 varies with
both the methodology and the actual assay kit.
Therefore, verification or establishment of a
reference interval based on the local population is
necessary.
In a large study with analog fT3 RIA, results from
41,655 successive patients were analyzed to obtain
the distribution curve for the population [18].
Using fT3 alone as a discriminator, 14% of the
patients were classified as hyperthyroid when
applying a reference interval of 3 to 8 pmol/L.
When the borderline hyperthyroid range was
defined as 7 to 9 pmol/L, 16.8% of the cases were
included in this category, whereas when the
borderline cases were defined as those results
falling between 8 and 9 pmol/L, 7.4% of the
patients were included in this category.
T3 and T4 Uptake, Thyroid HormoneBinding
Ratio (THBR), and fT4 and fT3 Indices
596
Free Thyroxine and Free Triiodothyronine
Reference Range for (Nonpregnant) Adults:
THBR: 0.831.17 (unitless)
fT4I: 4.213.0 (unitless)
The Committee on Nomenclature of the American
Thyroid Association (ATA) [51] strongly
recommends expression of the T3U as a ratio
(THBR) and considers it mandatory when one is
calculating the fT4I or (possibly) fT3I. The
Committee also recommended that if the THBR is
reported directly, it should always be reported with
a serum total T4 concentration so that the fT4I can
be calculated. In other words, the T3U or T4U test
or, more appropriately, the THBR is useful only as
a means of calculating the fT4I (or, by using total
T3, the fT3I):
fT4I = THBR total T4 concentration
Therefore, if the reference range of the THBR is
0.83 to 1.17 and that of total T4 concentration is 45
to 115 g/L (or 58 to 148 nmol/L), the reference
range of the fT4I would be 37.4 to 134 g/L (or 48
to 173 nmol/L). The ATA Committee [57]
recommended omission of the units from the fT4I
(or fT3I) to avoid confusion with measurements of
T4 (or T3) itself.
Physiological Variations in Free Thyroid
Hormones
Age
There are considerable differences in fT4 and fT3
concentrations among various age groups
[17,38,128-133]. The most dramatic changes occur
at birth. Within 24 to 28 hours after birth, the fT4
concentration more than doubles, and the fT3
concentration increases threefold. Full-term infants
have higher concentrations of both fT4 and fT3
than preterm neonates [130]. Under physiological
conditions, the serum fT4 concentration is highest
during the immediate postnatal period then falls
rapidly during the first few weeks of life in full-
term infants. The adult concentration of fT4 is
established only at the beginning of the third
decade of life. The age-related changes are
comparatively small in the THBR. The THBR is
lower in newborns (mean = 0.90) than adults (mean
= 1.00). The THBR is the highest (mean value
about 1.15) during the first few days of life, then
apart from a small decrease between the first and
fourth months, it is maintained at adult levels from
the end of the first week of life. Since the serum
total T concentration varies markedly with age
(peaks at a level about twice as high as the adult
during the first few days of life, then gradually
decreases to adult levels by the end of puberty), the
fT4I will also change with age. In general, higher
values occur in children of all ages than in adults.
Gender
Whereas no significant gender-related differences
were found in children [133], the differences
between males and females were statistically
significant between 16 and 49 years [129].
Although these differences were relatively small,
the fT4 and fT3 concentrations and the T4/TBG
ratios were lower in women during that period than
in men. The differences are apparently related to
hormonal differences between males and women,
with an estrogen effect in women during the years
of fertility.
Race, Posture, Immobilization, Dietary
Influences, Nutrition
At the present time, there is no evidence of an
appreciable effect of these variables on free thyroid
hormones [113,134].
Pregnancy
Although the magnitude of changes varied from
0% to 65% with widely different techniques, a
consistent decrease was observed with most
methods in fT4 and fT3 values toward the final
trimester of pregnancy [17,38,128,134-145]. The
decreased fT4 concentrations probably represent
physiological adaptation of the mother to increased
requirements of the fetus for thyroid hormones
[143,145]. Recently, UF/LC-MS/MS has been used
to determine reference intervals for pregnant
subjects [145]. In this study, the free thyroxine
levels similarly decreased through the pregnancy.
Lactation
Up to 16% decreases have been reported in the
serum fT4 concentrations of healthy lactating
women [146].
Menstrual Cycle
Although both serum TBG and total thyroid
hormone concentrations may fluctuate slightly with
the estrogen effect during the normal menstrual
cycle, no definitive data exist for a possible effect
on concentrations of free thyroid hormone [147].
Diurnal Variations
It is well established that serum thyrotropin levels
are markedly affected by the time of the day when
the sample is collected for analysis (levels are
highest around midnight [during the so-called
acrophase] and lowest in the morning). This
pattern is opposite to the diurnal secretion of
cortisol and is likely caused by inhibition of
thyrotropin secretion. Cortisol also produces a
decrease in TBG and an increase in transthyretin
597
Free Thyroxine and Free Triiodothyronine
(TTR), with a likely fall in both total T4 and T3.
Interestingly, compared to young adults, the elderly
had a significantly diminished 24-hour circadian
rhythm assessed by either the amplitude or mesor
(24-hour mean value) for thyrotropin, but the fT4
mesor was not significantly different between the
older and younger groups (19.5 2.1 pmol/L versus
19.4 3.6 pmol/L). Although these observations are
intriguing, further studies are needed to verify the
findings and establish the possible underlying
mechanism(s). A practical conclusion is that the
use of defined sampling times can improve the
diagnostic utility of free-thyroid-hormone
measurements.
Seasonal Changes
The seasonal and annual fluctuations in the
concentrations of free thyroid hormones are minor
[113].
Physical Exercise
The fT4 concentrations are elevated in trained
athletes and after prolonged physical exercise
[148]. This latter effect has been shown to be
related to the enhanced release of free fatty acids
(FFA) and their competition with thyroid hormones
for binding sites on transport proteins [148,149].
Interpretation
Based on the assessment of available diagnostic
tests [51], current guidelines of the ATA [150,151]
recommend that the principal laboratory tests for
thyroid disease should be estimation of fT4 and
measurement of TSH with a sensitive assay. For
correct interpretation of test results, it is therefore
important to understand the relationship between
these two key laboratory tests of thyroid function.
In most patients with an intact hypothalamic-
pituitary axis, there is a strong inverse log-linear
correlation between TSH and fT4 [27,115,152-155].
The relationship is such that an approximately 100-
fold change in serum TSH concentration is
associated with only a twofold change in
circulating fT4 concentration. This indicates that if
the hypothalamic-pituitary axis is normal, TSH is
generally a more sensitive indicator of thyroid
status than fT4 [115,121,153]. Subclinical thyroid
disease has been defined as when a patient has FT4
within the population reference interval but an
abnormal TSH concentration [154]. One
explanation for subclinical thyroid disease is the
narrow intraindividual biological variation [155].
When the thyroid function spontaneously changes
or the level of thyroid hormone therapy is altered,
the pituitary TSH response may require several
weeks for full expression. The time lag needed to
achieve full TSH response to changes in circulating
fT4 (and fT3) requires a minimum of 4 and as long
as 8 weeks to occur [27,152,156]. Obviously,
knowledge of this time-lag response is particularly
critical when adjusting thyroid hormone dosage.
Discordant thyroid function test results may also be
related to the large difference between the half-
lives of T4 (1 week) and TSH (1 hour) and
sampling under non-standard conditions.
In terms of both diagnostic performance and cost,
there is still an ongoing debate on the best approach
to screen for, diagnose, and monitor thyroid
disease. Initially, TSH alone, fT4 alone, and TSH
and fT4 in combination, either simultaneously or
sequentially, were all proposed as a simplified
screening strategy [157-167]. There is now
argument that TSH alone could be potentially
misleading in some patients [167]. Because of the
high prevalence of both thyroid disease and non-
thyroidal illness (NTI) in hospitalized patients
[168,169], newer schemes usually follow different
approaches for healthy populations (e.g., newborns,
elderly subjects) [170-173] and ambulatory and
hospitalized patients [27,174,175]. Hence caution
is advised in the interpretation of thyroid-function
test results in hospitalized or acutely ill patients,
since the results can be misleading. In addition to
TSH and fT4, in selected cases, the measurement
of fT3 and other thyroid-related analyte may be
also needed. Since the development of simple and
economic immunoassays for the measurement of
total T4, the THBR (formerly T3U or T4U) test by
itself is seldom used as a general screening test for
thyroid function, because the serum total T4
concentrations show much less overlapping of
values among euthyroid, hypothyroid, and
hyperthyroid states than the THBR values do.
However, the THBR test can provide information
regarding the status of serum binding proteins for
thyroid hormones, particularly if used in
combination with total T4 and the fT4I (or fT3I) is
calculated [50,150,176-179]. In hyperthyroid
patients, serum total T4 concentrations are
elevated. TBG is more saturated with T4, and thus
the THBR (T3U or T4U) is increased. On the other
hand, in hypothyroid patients, serum total T4
concentrations are abnormally low, TBG is less
saturated with T4, and the THBR is reduced.
Although the estimation of unoccupied binding
sites on T4-binding serum proteins (primarily
TBG) still may be of some value in assessing the
thyrometabolic status of the patient, the widespread
availability of relatively inexpensive, fast,
automated, non-isotopic assays for free thyroid
hormones made this approach largely obsolete.
TSH is considered the best screening test for
hypothyroidism in ambulatory patients, and a
useful follow-up test for those with a confirmed
598
Free Thyroxine and Free Triiodothyronine
elevation of TSH is measurement of thyroid
peroxidase antibodies [32].
Besides considerable physiological variations due
to age, pregnancy, and other conditions (see
earlier), free thyroid hormone results can be
difficult to interpret because of patient overall
disease state, medications, and other less common
factors. Undoubtedly, the use of free hormone
measurements during pregnancy and in the elderly
has gained prominence, but NTI, various drugs
such as heparin, binding-protein abnormalities,
human thyroid-hormone autoantibodies, and
heterophile, immunization-induced, or other human
antibodies usually are the most challenging for the
interpretation of free-thyroid-hormone results. A
number of strategies have been described for the
identification or interpretation of discordant thyroid
function tests [33,115,177-179].
Non-Thyroidal Illness
Non-thyroidal illness (NTI) remains a somewhat
ill-defined concept but introduces problems
associated with the evaluation of true thyroid
status. NTI refers to the anomaly of patients who
display abnormal results in their thyroid function
tests but do not actually appear to suffer directly
from thyroid disease [180-187]. These include
those patients who are mildly to severely unwell or
stressed and those to whom certain
pharmacological agents have been administered.
Among the thyroid abnormalities, most prominent
are low serum T3 (and often elevated reverse T3)
concentrations, leading to the description low T3
syndrome [181]. In patients suffering from more
severe NTI, both serum T3 and T4 are depressed,
and this condition was originally described as
euthyroid sick syndrome and referred to by some as
low T4 syndrome. The fall in serum T4 may be
severe and possibly indicates a poor outcome
[185,188]. In mild cases of NTI, serum free and
total T4 both tend to be higher than normal (high
T4 syndrome). Immunoassay FT4 results from
different methods are quite variable in hospitalized
patients, and it has been suggested that correct
interpretation of results should also take into
account the assay method [188].
NTI patients present two types of diagnostic
challenges. One is differentiation of abnormal
thyroid results caused by NTI from those caused by
a treatable thyroid disorder. The other is the
treatment and monitoring of thyroid patients,
particularly those with hypothyroidism whose
disease is obscured by the presence of NTI.
Performance Goals
Survey data from the 2007 CAP Participant
Summary Report show imprecision values (%
coefficient of variation) for FT4 ranging from
approximately 3.8% to 10.8% at mean
concentrations ranging from 1.9 ng/dL to 2.7
ng/dL. In the case of FT3, the summary report
shows imprecision values ranging from
approximately 3% to 9% at mean concentrations of
0.6 ng/dL to 1.0 ng/dL. The imprecision targets for
FT4 and FT3 are 9.5% and 7.9%, respectively [32].
Thus most laboratories are attaining the desired
imprecision target.
Of greater concern are the accuracy and validity of
immunoassay methods. The development of LC-
MS/MS and reference methods will aid in the
resolution of these issues. Thienpont [86] has
expressed concern at the potential proliferation of
free-thyroid-hormone methods using either UF or
ED followed by LC-MS/MS procedures which
have not been validated against a reference
measurement procedure. This is a critical point
because it should not be accepted that any
procedure using MS techniques automatically
qualifies as an accepted method. It is likely that the
future routine measurement of free thyroid
hormones lies with MS technology.
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146 Iwatani Y, Amino N, Tanizawa O, Mori
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147 Burrow GN, Fisher DA, Larsen PR.
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148 Liewendahl K, Helenius T, Naveri H,
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149 Mendel C, Frost PH, Cavalieri RR. Effect
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150 Hay ID, Bayer MF, Kaplan MM, Klee
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151 Surks MI, Chopra IJ, Mariash CN,
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153 Stockigt JR. Case finding and screening
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158 Penney MD, OSullivan DJ. Total or free radioimmunoassay. Clin Chem
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163 Helfand M, Crapo LM. Screening for 176 Csako G, Zweig MH, Ruddel M,
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164 Kaptein EM. Clinical application of free patients with nonthyroidal illness. III.
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illness: the euthyroid sick syndrome. altered thyroid hormone indices to

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607
Free Thyroxine and Free Triiodothyronine

Tables
Table 1. Free Thyroxine and Free Triiodothyronine Estimate Methods
Method 1: Index methods using indirect (tracer) equilibrium dialysis
Principle of analysis: Semipermeable membrane used to separate free thyroid hormone, including
tracer amount of iodinated, from protein-bound thyroid hormone. Percent free determined by
proportion of tracer in dialysate. Levels of free thyroid hormone determined using proportion of
measured total T4 of dialysate.
Comments: Historical research tool. Unwieldy. Purification of iodinated thyroid hormone critical.
Method 2: Index methods using TBG measurement
Principle of analysis: Calculation of free thyroid hormone based on law of mass action using
measurements of total T4 and TBG
Comments: Number of derived models, requires accurate T4 and TBG. Historical, not in routine use.
Method 3: Index methods using free thyroid hormone indices
Principle of analysis: Calculation of free thyroid hormone index (fTI) based on product of THBR and
total T4
Comments: No unit, indirect estimate of free hormone level
Method 4: Two-step, labeled hormone immunoassay
Principle of analysis: First step is reaction of patient sample with solid-phase antibody. Solid phase is
washed to remove patient serum prior to addition of tracer dose of labeled hormone.
Comments: Binding of tracer to serum proteins is avoided, which should eliminate protein-based
interferences.
Method 5: Labeled hormone analog (one-step) immunoassay
Principle of analysis: Immunoassay using analog tracer which does not bind to thyroid binding
proteins or disturb equilibrium between free and protein-bound thyroid hormone
Comments: Early, low-molecular analog binds to albumin; these methods are not recommended.
Large-molecular-weight analogs do not bind albumin but have autoantibody interference.
Method 6: Labeled antibody immunoassay
Principle of analysis: Tracer amount of labeled antibody competes with free thyroid hormone and
solid phasebound thyroid hormone analog.
Comments: Autoantibodies interference
Method 7: Equilibrium dialysis
Principle of analysis: Semipermeable membrane used to separate free from protein-bound thyroid
hormone. Free hormone level in dialysate determined by either immunoassay or LC-MS/MS.
Comments: Initially cumbersome, but recent developments with dialysis plate technology make
method amenable for routine use.
Method 8: Ultrafiltration
Principle of analysis: Centrifugation using semipermeable membrane used to separate free from
protein-bound thyroid hormone. Free hormone level in filtrate determined by either immunoassay or
LC-MS/MS.
Comments: Ultrafiltration cumbersome, not amenable to large batches
Method 9: Gel filtration/gel-bead dialysis
Principle of analysis: Separation based on molecular sieving prior to assay
Comments: Historical
Method 10: Column adsorption chromatography
Principle of analysis: Separation based on molecular weight
Comments: Historical
608
Free Thyroxine and Free Triiodothyronine
609
Gammaglutamyl Transferase (GGT)

Gammaglutamyl Transferase (GGT)

Danyal B. Syed

Name: Gammaglutamyl transferase, GGT, GGT1, GGT2, GGT3, -glutamyl

transpeptidase, GGTP, -GTP, a-glutamyl transpeptidase,
L--glutamyltransferase
Clinical significance: Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Systematic name: (5L-glutamyl)-peptide:amino-acid 5-glutamyltransferase
Clinical significance: click here <page 5, line 21>
Enzyme number: EC 2.3.2.2
Molecular mass: 90,000 D to 110,000 D (gel filtration)
Number of subunits: Dimer
Chemical class: Enzyme, protein, glycoprotein
Known isoenzymes: Multiple forms are known, but their identity is still in question.
Chromosomal location: 22q11.1-11.2; 22[1]; 22q11.23 [1,2]
Biochemical reaction:
-Glutamyl-L-peptide + amino acid EC 2.3.2.2 -Glutamyl amino acid + peptide
NRSCL Reference Method: NCCLS RS17

Principles of Analysis and Current Usage
-Glutamyltransferase (GGT) catalyzes the transfer of a
-glutamyl group to an amino acid or peptide (external
transpeptidation), to another substrate molecule (internal
transpeptidation) or to water (hydrolysis). The enzyme is
involved in the following metabolic pathways: (1)
arachidonic acid metabolism, (2) cyanoamino acid
metabolism, (3) glutathione metabolism, (4)
selenoamino acid metabolism, and (5) taurine and
hypotaurine metabolism. Because of its involvement in
the glutathione cycle, glutathione was used as the donor
substrate in the earlier methods of GGT activity
measurement. Substrate disappearance or product
formation was detected by manometric,
chromatographic, or ultraviolet techniques.
(ammediol) buffer, pH 8.6, because enzyme activity is
not affected over a broad range of buffer concentrations,
and the donor substrate has a higher solubility in this
buffer [4]. The glutamyl-group acceptor in this selected
method is glycylglycine, 50 mmol/L. Although other
substances, such as methionine, glutamine, and cystine,
can act as acceptor substrates, glycylglycine is the
acceptor of choice [6].
The temperature used in the selected method is 25C or
30C, with the reaction followed at 405 nm where the
hydrolysis product, 4-nitroaniline, absorbs, but the
substrate does not [4]. The day-to-day imprecision of
this method in the manual mode measured as the
coefficient of variation (CV) is 5.5% at a level of 23.6
U/L for 22 days.

Modern methods use either (-L-glutamyl)-4-nitroanilide
or (-L-glutamyl)-3-carboxy-4-nitroanilide as substrates
because these compounds allow the direct measurement
of reaction rate without deproteinization or any chemical
treatment of the cleavage products (Table 1, Methods 1
and 3). Orlowski and Meister introduced the use of (-L-
glutamyl)-4-nitroanilide in 1963 [3]. Szasz used this
substrate (4.0 mmol/L) in a kinetic method [4] that was
published as a selected method by the American
Association for Clinical Chemistry [5]. Szasz preferred
100 mmol/L of 2-amino-2-methyl-propane-1,3-diol
i
Gammaglutamyl Transferase
Previous and current authors of this method:
First edition: Steven Gendler
Methods edition: Steven Gendler, Amadeo J. Pesce
Second edition: Not updated
Third edition: Not updated
Fourth edition: Steven Gendler, Amadeo J. Pesce
Fifth edition: Danyal B. Syed
A modification of the Szasz procedure was also
published by the Committee on Enzymes of the
Scandinavian Society for Clinical Chemistry and
Clinical Physiology (Table 1, Method 2) [7]. The (-L-
glutamyl)-4-nitroanilide concentration for this method is
4 mmol/L. The buffer used is Tris-HCl, 100 mmol/L, pH
7.6. MgCl2 is added to a concentration of 10 mmol/L to
increase the solubility of the substrate. Glycylglycine is
used at 75 mmol/L, with the reaction carried out at 37C.
Fluorometric methods for GGT analysis have also been
reported [8,9] (Table 1, Method 4). Imprecision is
similar to that of spectrophotometric GGT analyses, but
sensitivity is 20 times greater. This allows the use of
smaller sample volumes and shorter incubation times.
These substrates have not come into common usage,
probably because of the equipment requirements.
Table 2 compares the various methods for GGT
determination.
610
Gammaglutamyl Transferase (GGT)
Reference and Preferred Methods
The reference method for GGT utilizes -L-glutamyl-3-
carboxy-4-nitroanilide (LGCN) as substrate [10]. Most
of the problems encountered in GGT catalytic activity
measurement include:
1. The purity of the reagents; for example, some
investigators report the presence of an impurity when
the LGCN is synthesized by certain manufacturers
[11]. This requires a pyridine extraction to remove the
impurity, which acts as a competitive inhibitor [12].
2. Because of the low solubility of LGCN, it is
necessary to use a supersaturated solution to achieve
a maximum reaction rate. This supersaturated
solution is stable for only a few hours [7].
3. Optimization of the assay is difficult because of the
inhibitory effect of excess donor or acceptor
substrates. This double inhibition is caused by the
kinetic mechanism of GGT [13]. Donor substrate
inhibition with both 4-nitroanilide compounds is pH-
dependent: maximal at acid pH, slight at pH 8.5, and
absent at more alkaline pH [14]. Thus assay
conditions must strike a balance that takes into
account maximal substrate inhibition, which is both
pH and substrate-concentration dependent, and
maximum reaction rate, which is also both pH and
substrate-concentration dependent.
The selected methods (Table 1, Methods 1 and 2) do not
use the currently IFCC-preferred substrate, LGCN. This
chemical is the preferred substrate because it is much
more soluble than (-L-glutamyl)-4-nitroanilide.
Spontaneous hydrolysis of the carboxy derivative is not
detectable. Further, kinetic constants and serum GGT
activities with the new substrate are comparable to those
of the old substrate. However, controls supplemented
with beef kidney GGT are on average 19% lower with
the carboxylated substrate. Thus the (-L-glutamyl)-4-
nitroanilide has been more frequently used, since higher
GGT activation and greater sensitivities are achieved
with this substrate. The use of the carboxy derivative is
increasing, however.
Several studies were done to optimize the GGT reaction
by use of the carboxylated substrate [14-16]. Given the
complicated kinetics of the enzyme, it is not surprising
that several optimized methods have been proposed.
Some inhibition of GGT by LGCN was reported. The
extent of the reported inhibition varied with the
investigator and the instrument used. Significant
inhibition by the carboxylated donor substrate was
reported with a concentration as low as 3.5 mmol/L [17],
but most data showed no inhibition until a concentration
of 10 to 12 mmol/L is achieved [14].
In the past, equations that modeled the GGT assay were
developed. Equations derived using kinetic theory [15]
and equations derived using a computerized optimization
scheme (called response surface methodology) have
been shown to accurately model the enzyme assay over a
broad range of GGT concentrations. Response surface
methodology has shown that maximal GGT activity is
achieved within a range of assay conditions: pH 7.8 to
8.5, glycylglycine 129 to 250 mmol/L, and LGCN 6.6
to 10.2 mmol/L, given a serum-to-reagent ratio of 1:11,
100 mmol/L Tris-HCl buffer, at a temperature of 30C.
However, the latest recommendation of IFCC is to
measure the enzymatic activity at 37C. Response
surface methodology has shown that maximal activity is
achieved using the given buffer, temperature, and
fraction of sample volume with a pH of 8.16, a
glycylglycine concentration of 190 mmol/L, and a
substrate concentration of 8.4 mmol/L. These conditions
are proposed in the recommended method that follows
[16]. The IFCC Primary Reference Procedure utilizes pH
of 7.70, the glycylglycine concentration of 150 mmol/L,
and a substrate (LGCN) concentration of 6 mmol/L
[10].
The absorbance of the hydrolysis product, 2-nitro-5-
aminobenzoic acid, is followed at 410 nm. This is a
slightly longer wavelength than is used for the non-
carboxylated substrate, since the carboxy derivative has
a higher absorbance than the non-carboxylated substrate
[17]. The longer wavelength reduces the blank
absorbance.
In order to overcome the concerns mentioned above, the
IFCC primary method has well-defined measurement
conditions. Besides, there is an international effort
requiring the manufacturers of in-vitro diagnostic
medical devices to meet the metrological traceability of
values assigned to the calibrators by using primary
reference procedures and reference materials [18]. The
standard ISO 15189 requires that when possible, the
results should also be traceable to SI units. To meet this,
an International Standard on traceability was put
forward, describing the order of measurement
procedures and calibration materials [19]. Another
related standard, ISO 18153, deals with the traceability
of the enzyme catalytic concentrations [20]. The IFCC
Committee on Reference Systems for Enzymes (C-RSE)
has published the recommendations for establishing a
global reference system for enzymes. The system
comprises primary reference procedures to measure the
catalytic enzyme concentrations, a network of well-
established laboratories and primary calibrators (certified
enzyme reference materials [ERMs]) [21]. For GGT, the
certified ERM, AD-452 /IFCC 452, is available for use
as primary standard/calibrator [22]. Commercial
calibrators which can be traced to this primary reference
material are also available [23,24]. Some instrument and
kit manufacturers use a calculation factor to provide
traceability to the IFCC GGT reference measurement
procedure.
Specimen
Serum or plasma (lithium heparin or Na heparin) is the
recommended specimen for the determination of the
catalytic concentration of GGT [5,7]. Either specimen
can be collected by employing normal phlebotomy
procedures. The plasma or serum should be separated
from the contact with cells within 2 to 4 hours of
specimen collection.
These specimens give the same results. EDTA and
citrate do not interfere with analysis, and serum GGT
611
Gammaglutamyl Transferase (GGT)
activity is stable at room temperature or 2C to 8C for
at least 7 days and for at least 2 months when frozen at
or below 18C [25]. When refrigerated or frozen, the
specimen must first be brought to room temperature and
then mixed well prior to analysis.
Interferences
The presence of hemolysis and prolonged contact with
cells does not influence GGT values, according to one
review [25]. However, some methods require that
hemolyzed specimens with hemoglobin concentration of
150 mg/dL should be avoided, whereas others have set
this limit to 500 mg/dL or higher. Some investigators
have shown fluoride, oxalate, and citrate at a
concentration of 1 g/L to inhibit GGT by approximately
15%. The presence of lipemia or icterus has been noted
to cause decreased activity with some methods but only
above 2000 mg/dL of triglycerides and 40 mg/dL of total
bilirubin, respectively. Divalent ions like calcium and
magnesium and monovalent ions like sodium and
potassium have no effect on the catalytic activity of
GGT.
GGT Reference Interval
It is recommended that each laboratory establish its own
reference interval based on methodology, temperature of
GGT activity measurement and population. The
reference interval is sex and age dependent [25].
Activities in women are typically less than those in men
of the same age. Individuals who have increased body
mass index (BMI) by 30% or more typically show higher
enzyme activities [26,27]. The within-day variation has
been reported to be between 10% and 15%, which is
effectively independent of age and gender [28-30].
Americans of African origin have been shown to exhibit
GGT activity almost twice that of the corresponding
European-origin population [31]. Smoking a pack of
cigarettes a day will increase the GGT activity by 10%
[27,32], and alcohol consumption has a direct
relationship with increased GGT activity [27,32,33]. The
values presented in Table 3 are from a selected
population that excluded persons who consumed more
than 1 L/day of wine or beer and women on oral
contraceptives. GGT activities are much higher during
the first months of life as compared with older children,
as seen in Table 4. Oral contraceptives resulted in a 20%
increase in GGT activity, and a large increase in GGT
activity was noted for children (400%) and adults
(200%) on anticonvulsant drug (phenytoin and
phenobarbital) therapy. Other commonly used drugs
associated with increased GGT activity include
carbamazepine, cimetidine, furosemide, methotrexate,
heparin, isotretinoin, and valproic acid [34]. Full-term
neonates exhibit GGT activity levels six to seven times
the upper limit of the adult reference range; values
decline and approach adult levels by the age of 5 to 7
months [35].
Interpretation
Gamma-glutamyltransferase is an enzyme present
primarily in the kidney, pancreas, liver, and prostate
[36]. GGT in serum has a molecular mass of 90,000 D,
as measured by electrophoresis [37]. This molecular
mass and the hydrophilic properties are similar to those
of papain-solubilized liver GGT. Serum GGT shows an
electrophoretic mobility and lectin-affinity reaction
identical to those of the liver enzyme but different from
those of GGT from the kidney, urine, and pancreas.
However, an antibody raised against liver GGT has the
same reactivity against liver GGT as it does against
kidney and pancreatic GGT. The data are consistent with
(1) the isoenzymes having similar structures but
differing primarily in their carbohydrate content, similar
to the alkaline phosphatase isoenzymes, and (2) serum
GGT originating from the hepatobiliary system [38].
Larger forms of GGT are reported with molecular
masses greater than 200,000 D. This varies according to
the detergent used for solubilization and molecular mass
determination, since the enzyme is known to aggregate
with lipoproteins and membrane fragments. Given the
confusion in GGT characterization, it is prudent to wait
for more evidence before any conclusions are drawn
[39].
It has been proposed that GGT participates in the
transport of amino acids into cells. This accounts for its
membrane-bound structure, and because transport into
the renal proximal tubules is important to conserve
amino acids, this concept fits its high activity in this
organ. The enzyme is universally distributed in tissue but
with considerable variance in content per gram of tissue,
as shown in Table 5 [40,41]. The problem of whether
this reaction and enzyme is the major enzyme pathway
of amino acid transport into the cell is not completely
settled [42].
The enzyme appears in serum after chronic, excessive
ethanol ingestion and drug intake, as well as in the
presence of hepatobiliary disease. The reference interval
population was selected to eliminate these factors, since
it is well known that ethanol ingestion increases serum
GGT. The enzyme by itself has poor specificity for the
differential diagnosis of liver disease. For example,
when 1040 adult hospital patients were studied, 139
were found to have increased serum GGT levels [43]. Of
the 139 cases, only 45 had primary hepatobiliary disease.
Twenty-one percent of the elevated GGT values
occurred in patients with malignancy and 16% in
patients with cardiac disease. Thus elevations of the
enzyme are not sufficiently specific to render it useful as
the sole criterion for establishing hepatobiliary disease.
GGT levels were reported to be increased an average of
12-fold in obstructive liver disease when compared to
ALP, which increased only 3-fold, so GGT is slightly
more sensitive than ALP in this regard. GGT activity
increases in cholestasis via the same mechanism as does
ALP [44-46]. It has been reported that increased GGT
activity level in children may be a reliable index of bile
duct damage and that the serum GGT may provide a
useful indicator in separating the two forms of idiopathic
cholestasis, with or without a bile duct involvement [47].
It has been noted that in infants diagnosed with biliary
atresia and managed surgically, the GGT levels stay high
in the blood if the infant is breastfed. This is due to the
612
Gammaglutamyl Transferase (GGT)
high level of GGT in human breast milk for at least 4
weeks postpartum [48].
In three comprehensive European cohort studies
involved in EUROSTROKE, the association of GGT as
a marker for alcohol consumption related to fatal and
nonfatal hemorrhagic and ischemic stroke was evaluated.
The results showed that an increased GGT, as a marker
of alcohol consumption, is associated with increased risk
of stroke, especially hemorrhagic stroke [49].
GGT activity measurement has been found to be useful
in differentiating the source of ALP activity increase,
whether it is of bone or liver origin, because GGT
activity is increased only in cases of hepatic injury,
whereas ALP is increased when either liver, bone, or
both are involved. It can also be used for similar
differentiation among Duchenne muscular dystrophy
patients who are treated with potentially hepatotoxic
drugs [50].
In a perspective study, the association between GGT and
the risk for chronic kidney disease (CKD) in nondiabetic
and nonhypertensive Korean workers was evaluated.
From the findings of this study, it was concluded that
GGT may be an early predictor of CKD independent of
other well-known baseline factors [51].
Gammaglutamyl Transferase Performance Goals
The short-term variation of GGT in healthy subjects has
been shown to be approximately 15% (within-subject)
[52]. Performance goals for GGT are based on biological
variation and analytical precision. The total error, based
on biological variation and analytical imprecision and
bias, are all approximately 20% to 22% [53,54]. The
National Academy of Clinical Biochemistry (NACB)
recommends that (1) assays for GGT should have a total
error of 20% at the upper reference limit, (2) fasting
blood sample is recommended, (3) single upper
reference limit is appropriate for adult men, but for
children and adult women, separate reference limits
based on age are needed, and (4) GGT activity
measurement should be reserved for specific indications
to determine the source of high ALP activity level [54].
GGT is not a regulated analyte according to the Clinical
Laboratory Improvement Amendments (CLIA)
(493.931, 42 CFR Ch. IV PP1022-23[10-1-2004
edition]) rules and regulations. The College of American
Pathologists (CAP) acceptability criterion for
proficiency testing is a peer-group mean of 3 SD. With
increasing use of the IFCC primary reference procedure
and the international reference material, as well as the
efforts by instrument/kit manufacturers to trace their
calibration to the primary reference method, the
analytical intralaboratory variation has been reduced to
less than 3%, while interlaboratory variation is now less
than 5% (CAP Participants Summary C-B,
Chemistry/Therapeutic Drug monitoring, Surveys 2007).
References
1 Entrez Gene: GGT1 gamma-
glutamyltransferase 1 [Homo sapiens] Gene Id
2678. Available at
<http://www.ncbi.nlm.nih.gov/sites/entrez?db=
gene&cmd=retrieve&dopt=default&rn=1&li>
2 Collins JE, Mungall AJ, Badcock KL, Fay JM,
Dunham I. The organization of the -Glutamyl
transferase genes and other low copy repeats in
human chromosome 22q11. Genome Research
1997;7:522-31.
3 Orlowski M, Meister A. -Glutamyl-p-
nitroanilide: a new convenient substrate for
determination and study of L- and D--glutamyl
transpeptidase activities. Biochim Biophys Acta
1963;73:679-81.
4 Szasz, G. A kinetic photometric method for
serum -glutamyltranspeptidase. Clin Chem
1969;15:124-36.
5 Szasz, G. Reaction-rate method for -
glutamyltransferase activity in serum. Clin.
Chem. 1976;22:2051-55.
6 Shaw LM, London JW, Petersen LE. Isolation
of -glutamyltransferase from human liver and
comparison with the enzyme from human
kidney. Clin Chem1978;24:905-15.
7 Stromme JH. Chairman of the Committee on
Enzymes of the Scandinavian Society for
Clinical Chemistry and Clinical Physiology:
Recommended method for the determination of
-glutamyltransferase in blood. Scand J Clin
Lab Invest 1976;36:119-25.
8 MacQueen JD, Driscoll RC, Gargiulo RJ. New
substrate for fluorometric determination of -
glutamyltransferase activity in blood serum.
Clin Chem 1977;23:879-881.
9 Prusak E, Siewinski M, Szewczuk A. A new
fluorometric method for the determination of -
glutamyltransferase activity in blood serum,
Clin Chim Acta 1980;107:21-26.
10 Schumann G, Bonora R, Ceriotti F, Ferard G,
Ferrero CA, Franck P FH et al. IFCC primary
reference procedures for the measurements of
catalytic activity concentrations of enzymes at
37C: part 6 reference procedure for the
measurement of catalytic concentration of -
glutamyltransferase. Clin Chem Lab Med
2002;40:734-38.
11 Rowe JA, Tarlow D, Rosalki SB. Impurity in -
glutamyl-p-nitroanilide used as a substrate for
D-glutamyl transferase [letter]. Clin
Chem1973;19:435-36.
12 Huseby NE, Stromme JH. Practical points
regarding routine determination of -glutamyl
transferase (-GT) to serum with a kinetic
method at 37C. Scand J Clin Lab
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13 Stromme JH, Theodorsen L. -Glutamyl-
transferase: substrate inhibition, kinetic
mechanism, and assay conditions. Clin Chem
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14 Schiele F, Artur Y, Bagrel D, Bagrel C, Siest G.
Measurement of plasma gamma
glutamyltransferase in clinical chemistry:
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Clin Chim Acta 1981;112:187-95.
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Gammaglutamyl Transferase (GGT)
15 Shaw LM, London JW, Fetterolf D, Garfinkel
D. -Glutamyltransferase: kinetic properties and
assay conditions when -glutamyl-4-nitroanilide
and its 3-carboxy derivative are used as donor
substrates. Clin Chem 1977;23:79-85.
16 London JW, Shaw LM, Theodorsen L,
Stromme JH. Application of response surface
methodology to the assay of gamma-
glutamyltransferase. Clin Chem 1982;28:1140-
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17 Theodorsen L, Stromme JH. -Glutamyl-3-
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transferase activity in serum? Clin Chim Acta
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18 International Organization for Standardization.
Medical Laboratories: Particular Requirements
for Quality and Competence. ISO 15189.
Geneva: ISO; 2003.
19 International Organization for Standardization.
In-Vitro Diagnostic Medical Devices.
Measurement of Quantities in Biological
Samples: Metrological Traceability of Values
Assigned to Calibrators and Control Materials.
ISO17511. Geneva: ISO; 2003.
20 International Organization for Standardization.
In-Vitro Diagnostic Medical Devices.
Measurement of Quantities in Biological
Samples: Metrological Traceability of Values
for Catalytic Concentration of Enzymes
Assigned to Calibrators and Control Materials.
ISO 18153, Geneva: ISO; 2003.
21 Siekmann L, Bonora R, Burtis CA, Ceriotti F,
Clerc-Renaud P, Frard G et al. IFCC primary
reference procedures for the measurement of
catalytic activity concentrations of enzymes at
37C. Part 1. The concept of reference
procedures for the measurement of catalytic
activity concentrations of enzymes. Clin Chem
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22 Linsinger T, Kristiansen N, Schimmel H,
Pauwels J, Siekmann L, Ceriotti F et al.
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-Glutamyltransferase (GGT) Determined by
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Material ERM-AD452/IFCC. Report EUR
19577 EN. Luxembourg: Office for Official
Publications of the European Committees;
2005.
23 Canalias F, Camprubi S, Snchez M, Gella FJ.
Metrological traceability of values for catalytic
concentration of enzymes assigned to a
calibration material. Clin Chem Lab Med
2006;44:333-9.
24 Infusino I, Bonora R, Panteghini M.
Traceability in clinical enzymology. Clin
Biochem Rev 2007;28:155-161.
25 Ladenson JH. Nonanalytical sources of
variation in clinical chemistry results. In:
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Vol. 1. 8th ed. St Louis: Mosby; 1980.
26 Salaspuro M. Characteristics of laboratory
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gamma-glutamyltransferase in people aged 40-
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29 Holzel WGE. Intra-individual variation in
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30 Fraser CG, Cummings ST, Wilkinson SP,
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31 Manolio TA, Burke GL, Savage PJ, Jacobs DR
Jr, Sidney S, Wagenknecht LE et al. Sex- and
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CARDIA study. Clin Chem 1992;38:1853-59.
32 Nilssen O, Helge-Forde O, Brenn T. The
Tromso Study: distribution and population
determinants of -glutamyltransferase. Am J
Epidemiol 1990;132:318-26.
33 Moussavian SN, Becker RC, Piepmeyer JL,
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of alcohol ingestion and liver disease. Dig Dis
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34 Young DS. Effect of Drugs on Clinical
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35 Cabrera-Abreu JC, Green A. Gamma-
glutamyltransferase: value of its measurement
in paediatrics. Ann Clin Biochem 2002;39:22-5.
36 Rosalki SB. Gamma-glutamyl transferase. Adv
Clin Chem 1975;17:53-10.
37 Tsuji A, Matsuda Y, Katunuma N.
Characterization of human serum -
glutamyltransferase. Clin Chim Acta
1980;104:361-6.
38 Huseby NE. Separation and characterization of
human -glutamyltransferases. Clin Chim Acta
1981;111:39-45.
39 Henderson AR. Clinical enzymology. Annu
Rev Clin Biochem 1980;1:233-70.
40 Goldberg JA, Friedman EM, Pineda EP, Smith
EE, Chatterji R, Stein EM, Ruttenburg AM. The
colorimetric determination of -glutamyl
transpeptidase with a synthetic substrate. Arch
Biochem Biophys 1960;91:61-70.
41 Orlowski M, Szewczuk A. Colorimetric
determination of -glutamyl transpeptidase
activity in human serum and tissues with
synthetic substrate. Acta Biochemica Polonica
1961;8:189-200.
42 Goldberg DM. Structural, functional and
clinical aspects of -glutamyl transferase. Crit
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43 Burrows S, Feldman W, McBride F. Serum
gamma-glutamyl transpeptidase: evaluation in
screening of hospitalized patients. Am J Clin
Pathol 1975;64:311-314.
44 Anciaux ML, Pelletier AG, Attali P, Meduri B,
Liguory C, Etienne JP. Prospective study of
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45 Sheeman M, Maythorn P. Predictive value of -
glutamyl transpeptidase in various diseases. In:
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1979:184-7.
46 Ratanasavanh D, Tazi A, Gaspart E. Hepatic -
glutamyl transpeptidase release: effect of the
bile salt and membrane structure modification.
Adv Biochem Pharmacol 1982;3:93-103.
47 Maggiore G, Bernard O, Hadchouel M,
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serum gamma-glutamyl transpeptidase activity
in children. J Pediatr Gastroenterol Nutr
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48 Colagiovanni DB, Meyer DJ, Wolf J, Hart K,
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(GGT) activity in human breast milk confounds
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nursing infant with liver disease. Clin Chem
2005;51:1750-51
49 Bots ML, Salonen JT, Elwood PC, Nikitin Y,
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glutamyltransferase and risk of stroke: the
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50 Rosales XQ, Chu ML, Shilling C, Wall C,
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glutamyltransferase (GGT) in differentiating
skeletal muscle from liver damage. J Child
Neurol 2008;23(7):748-51.
51 Ryu S, Chang Y, Kim D-I, Kim WS, Suh B-S.
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nondiabetic Korean men. Clin Chem
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52 Ricos C, Arumi EG, Rubio RR, Schwartz S.
Eficia de un programa interno de control de
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53 Rics C, Alvarez V, Cava F, Garcia-Lario JV,
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54 Dufour RD, Lott JA, Nolte FS, Gretch DR,
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55 Moss D, Henderson R. Clinical enzymology. In:
Burtis CA, Ashwood ER, eds. Tietz Textbook
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56 Schiele F, Guilmin AM, Detienne H, Siest G.
Gamma-glutamyltransferase activity in plasma:
statistical distributions, individual variations,
and reference intervals. Clin Chem
1977;23:1023-8.

Tables
Table 1: Methods for -Glutamyltransferase (GGT) Determination
Method 1: (Szasz [5]) Reaction-rate method for -glutamyltransferase activity in serum; quantitative, kinetic
Principle of analysis: (-L-Glutamyl)-4-nitroanilide 2-nitroaniline (405 nm)
Comments: Most frequently used; selected method of American Association for Clinical Chemistry; donor-
substrate at supersaturating concentration

Method 2: (Scandinavian Society for Clinical Chemistry [SSCC] [7]) Modified Szasz; quantitative, kinetic
Principle of analysis: As for Method 1
Comments: Similar to Method 1; different pH, buffer, and substrate concentrations

Method 3: Substrate, modified SSCC [14]; quantitative, kinetic

Principle of analysis: (-L-Glutamyl)-3-carboxy-4-nitroanilide 2-nitro-5-aminobenzoic acid (410 nm)
Comments: Also the principle of analysis for the IFCC Primary Reference Method*; recommended method
because of more soluble substrate donor and well-defined assay conditions

Method 4: Fluorometric; quantitative [8,9]

Principle of analysis:
Comments: Not in current use; most rapid, sensitive technique; requires specialized instrumentation
615
Gammaglutamyl Transferase (GGT)

Table 2: Comparison of Methods for GGT Determination

Reaction condition: Temperature SSCC-Szasz: 0.0909

AACC-Szasz*: 25C or 30C Recommended method: 0.0909
SSCC-Szasz: 37C IFCC Primary Reference Method: 0.0909
Recommended method: 30C (1:11)
IFCC Primary Reference Method: 37.0C
0.1C Reaction condition: Wavelength
Reaction condition: Final concentration of AACC-Szasz*: 405 nm
reagents SSCC-Szasz: 405 nm
a. Substrate donor Recommended method: 410 nm
b. Substrate acceptor IFCC Primary Reference Method: 410 nm 1
c. Buffer nm
d. pH
e. Other Reaction condition: Interferences
AACC-Szasz*: AACC-Szasz*: Gross hemolysis
a. (-L-Glutamyl)-4-nitroanilide: 4 SSCC-Szasz: Gross hemolysis
mmol/L Recommended method: Gross hemolysis
b. Glycylglycine: 50 mmol/L IFCC Primary Reference Method: Gross
c. 2-Amino-2-methyl-propane-1,3-diol: hemolysis
100 mmol/L
d. 8.6 Reaction condition: Precision (at upper limit of
SSCC-Szasz: normal) (%CV)
a. (-L-Glutamyl)-4-nitroanilide: 4.0 AACC-Szasz*: 5.5%
mmol/L SSCC-Szasz:
b. Glycylglycine: 75 mmol/L Recommended method:

c. Tris-HCl: 100 mmol/L IFCC Primary Reference Method :< 2.5
d. 7.6
e. MgCl2: 10 mmol/L Reference interval
Recommended method: a. Men
a. (-Glutamyl)-5-carboxy-4-nitroanilide: b. Women
8.4 mmol/L AACC-Szasz*:
b. Glycylglycine: 190 mmol/L a. 5-30 U/L
c. Tris-HCl: 100 mmol/L b. 3-20 U/L
d. 8.16 SSCC-Szasz:
IFCC Primary Reference Method: a.
a. -L-Glutamyl-3-carboxy-4-nitroanilide: 6 b.
mmol/L Recommended method:
b. Glycylglycine: 150 mmol/L a.
c. Sodium hydroxide; 2 mol/L (for adjusting b.
IFCC Primary Reference Method
pH)
a. 55 U/L or 0.92 kat/L (upper 97.5 percentiles)
d. pH 7.70 0.05
b. 38 U/L or 0.63kat/L (upper 97.5 percentiles)
Reaction condition: Fraction of sample
volume
AACC-Szasz*: 0.0909 (1:11)
*Selected method, American Association for Clinical Chemistry. From Szasz G. Reaction-rate method for -
glutamyltransferase activity in serum. Clin Chem 1976;22:2051-2055.
Committee on Enzymes of the Scandinavian Society for Clinical Chemistry.[7]
Kaplan and Pesces Clinical Chemistry
International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) , Scientific Division, Committee on
Reference Systems on Enzymes(C-RSE)[10]
Certification Report: Certified Reference Material ERM-AD452/IFCC European Commission, Directorate general
Joint Research Center Institute for Research Materials and Measurements (IRMM) Geel (BE).[22]
616
Gammaglutamyl Transferase (GGT)

Table 3: -Glutamyltransferase Activity as a Function of Sex and Age for a Selected Population
Age Males (Percentiles) Females (Percentiles)
in Years
2.5 50 90 95 97.5 2.5 50 90 95 97.5
Activity, U/L
8-12 8.4 14.0 19.8 23.8 27.8 8.0 13.9 19.4 21.7 24.7
12-16 10.1 15.4 20.0 23.6 26.4 6.4 13.7 19.8 23.9 31.0
16-20 9.2 17.5 29.1 35.0 45 7.0 13.4 19.5 21.6 24.4
20-30 11.5 23.6 48.4 63.8 76.1 7.0 15.7 25.8 32.2 41.5
30-40 12.1 28.6 64.4 81.2 94.0 9.0 17.4 33.9 43.9 55.0
40-50 13.1 32.4 68.1 85.6 105 8.9 18.5 42.2 53.2 71.3
50-60 14.6 32.6 66.7 86.3 103 10.3 20.2 47.4 59.4 83.2
>60 13.8 28.6 61.7 72.5 83.6 10.7 22.5 40.5 48.5 60.2

From [56] With permission of the American Association for Clinical Chemistry.

Table 4: -Glutamyltransferase Activity During Early Childhood Development

Age Mean (U/L) Range (U/L)

0-1 month 71 0-151
1-2 months 48 0-114
2-4 months 33 0-81
4-7 months 17 0-34
7 months-2 years 11 0-24

Ref: [35,55]

Table 5: Tissue Distribution of -Glutamyltransferase

Tissue Units/mg of Wet Units/g of Wet Ratio to Serum
Tissue Tissue
Kidney 44 2225 7420
Pancreas 13 184.5 614
Liver 4 86.5 288
Spleen 2 34 113
Heart * 1 3
Skeletal muscle * 1.5 5
Lung 6 7 23
Brain * 11 37
Intestine 1 21 70
Sera 0.3 1
*<1 unit/mg tissue.

Ref:[40,42]
617
Gammaglutamyl Transferase (GGT)

Procedure: Kinetic Measurement of GGT Activity Calculations

Using the molar absorptivity, , of 2-nitro-5-
Principle
aminobenzoic acid as 9500 Lcm-1mol-1, one can
(-L-Glutamyl)-3-carboxy-4-nitroanilide + glycylglycine
calculate the enzyme activity for a 1-cm pathlength with
EC 2.3.2.2 the following formula:
(-L-glutamyl)glycylglycine + 2-nitro-5-aminobenzoic acid
or A for t min total volume 1 106 mol
(-L-Glutamyl)-3-carboxy-4-nitroanilide + H2O U/L
t min sample volume mole
EC 2.3.2.2
glutamic acid + 2-nitro-5-aminobenzoic acid where A = difference between the initial and final
absorbance over analysis time,
This reaction is monitored at 410 nm. t, in minutes
= molar absorptivity of 2-nitro-5-aminobenzoic acid
Reagents 106 = factor to convert concentration to micromoles per
1. Buffer/substrate solution. Tris (100 liter
mmol/L), 12.11 g; glycylglycine (190 mmol/L), 25.10 g;
and (-L-glutamyl)-3-carboxy-4-nitroanilide (8.4 For the assay, the values are as follows:
mmol/L), 2.91 g are added to about 800 mL of distilled
water. Adjust pH to 8.16 with 1 mol/L of HCl, and dilute A for t min 2.20 mL 1 106
U/L
to 1000 mL. This solution is stable 2 days at room 5 min 0.2 mL 9500(L/mol cm)
temperature or 5 days at 2C to 8C. U / L A 231.6

Assay Note: The use of enzyme reference materials (ERM) as

Equipment: spectrophotometer (10 nm bandpass) calibrators with well-defined assay values is being
equipped with cuvette heated at 37C and a 410 nm recommended by IFCC and ISO 18153 for uniformity of
filter. results across methods and instruments and for
1. Place 2 mL of buffer/substrate solution and traceability of the results to the primary reference system
0.20 mL of specimen (control, enzyme comprising primary reference method and ERM with
reference material/standard/calibrator or assigned value. See Tables 1 and 2 for IFCC primary
unknown patient serum or plasma) in a 3-mL reference procedure principle and reaction conditions.
cuvette. For details of IFCC primary reference procedure for the
2. After 3 minutes of preincubation at 37C, measurement of catalytic activity of GGT, reader is
follow absorbance at 410 nm for 5 minutes. referred to Clin Chem Lab Med 2002;40:734-738.
3. The serum sample volume fraction is 0.091
(1:11).
618
Gastric Fluid Analysis
Lawrence A. Kaplan
Name: Gastric fluid
Clinical significance: Refer to Chapter 34, The Pancreas: Function and Chemical Pathology,
and Chapter 35 Gastrointestinal Function and Digestive Disease in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
Gastric fluid analysis is a vague term that can be used to
include analysis of a number of electrolyte and protein
(enzymes) components of gastric juices. The most
commonly performed analysis of gastric fluid is the
measurement of the total acid output.
Historically,[1] total gastric acid was defined as the sum
of the combined acid (that acid combined with
physiological buffers, such as proteins and salts) and the
free acid (the acid that exceeded the buffering capacity
of gastric fluid). Subsequent investigations have
demonstrated that most pure gastric fluid closely
resembles hydrochloric acid[2] but has some additional
minor buffering activity, perhaps reflecting weak organic
acids such as lactic, carbonic, and others.[3]
Because of the division of total gastric acid into
combined (the weak organic acids) and free (HCl),
the earlier analyses used a two-stage titration (method 1,
Gastric Fluid Analysis Methods Analysis Table). A
mixed indicator solution, consisting of Topfers reagent
(5 g/L alcoholic solution of p-
dimethylaminoazobenzene) and phenolphthalein (10 g/L
in ethanol), was added to the gastric fluid sample.[4]
Sodium hydroxide (0.1 mol/L) was added from a buret
to titrate both of these indicators; Topfers reagent was
titrated to a pH range of 2.8 to 3.5 (red to colorless) and
the phenolphthalein was titrated to a pH range of 8.2 to
10 (colorless to red). Results were often reported as the
number of milliliters of 0.1 M NaOH needed to titrate
100 mL of gastric fluid. Alternatively, the more
meaningful total milliequivalents (mEq) of acid secreted
can be calculated as follows:
where Vg = volume of gastric fluid titrated (mL)
VNaOH = volume of NaOH needed to titrate
the gastric fluid (mL)
M = molarity of NaOH (usually 0.1 mol/L or 0.1
Eq/L)
Other alternative titration methods include the titration
of total acidity using a phenolphthalein end point and
titration to a neutral pH (or pH 7.4) using phenol red
indicator (1 g/L)[5] or a pH meter.[3,5,6]
As an alternative to titration methods, Moore and
Scarlata[3] determined total gastric acid content by
direct measurement with a pH glass electrode (method 2,
Gastric Fluid Analysis Methods Summary Table). They
determined the actual hydrogen-ion concentration by
dividing the pH by the hydrogen-ion activity
coefficient.[7] The H+ activity coefficient, which is the
thermodynamic activity of the hydrogen ions,[7] is
dependent on the molar concentration of other ions
present in the solution, such as sodium or potassium
ions. Tables providing the relationship between the H+
activity coefficient and the concentration of H+ and
other ions are available.[8] As an approximation, one
can assume an average total concentration of Na and K
of 50 mEq/L and perform a simplified calculation.[4]
The measurement of total gastric acidity by methods 1
and 2 (Gastric Fluid Analysis Methods Summary Table)
requires intubation of the patient with a gastric tube and
aspiration of gastric fluids. To avoid the necessity for
intubations, indirect measurements of gastric acidity
have been suggestedthe tubeless gastric analysis.
These tests employ a carboxylic-cationic resin in which
some of the hydrogen ions have been replaced by a
water-soluble dye (method 3, Gastric Fluid Analysis
Methods Summary Table). The resin is ingested, and
some of the dye is displaced by gastric acid, absorbed by
the small intestines, and excreted in urine. The amount
of dye excreted in the urine, measured
spectrophotometrically, is proportional to the total
gastric acidity.[1] The most commonly used dye is
azure A (3-amino-7-dimethylaminophenazathionium
chloride). The presence of this dye can be measured by
spectrophotometry at 630 nm.[9,10] Heating the urine in
the presence of CuSO4 has been recommended so that
colorless, reduced forms of azure A can be converted
back to the blue form.[10]
It is important to measure gastric acid secretion both
under a basal, resting, condition (basal acid output,
BAO) and under a stimulation condition (maximum acid
output, MAO). Various foodstuffs have historically been
employed to provide such stimulation. These test meals
have varied considerably.[1] refined chemical
stimulants include histamine, insulin-induced
hypoglycemia, betazole (Histalog, a histamine analog),
porcine gastrin, and recently pentagastrin, the artificially
synthesized active portion of the gastrin molecule.
The most commonly used procedures for measurement
of gastric acid content are the titration methods.
619
Gastric Fluid Analysis
Reference and Preferred Methods
The concept of combined and free gastric acid is
presently considered inaccurate. The procedure
employing both Topfers reagent and phenolphthalein is
not considered to provide any additional useful
information. For measurement of total acidity, one can
use either a titration method or the pH electrode method
of Moore and Scarlata.[3] Titrating to neutrality (or pH
7.4) with a pH meter or phenol red indicator, or to pH 8
to 10 with phenolphthalein, provides sufficient clinical
information and employs readily available laboratory
equipment. Although the method of Moore and Scarlata
may provide a more exact measurement of gastric
acidity, the greater accuracy may not be clinically
necessary. In addition, a pH meter able to measure pH to
the nearest thousandth (0.001) will probably not be
available in many laboratories. Also, to be most
accurate, one needs to measure sodium and potassium
as well as pH, or else one must make assumptions
concerning the concentration of Na+ and K+ that may
not always be valid.
Tubeless gastric analysis using azure A has not been
widely accepted because of the large number of false-
positive (excess acid production, 1% to 3%) and false-
negative results (10% to 15%) obtained with this
method.[1,11] A large number of physiological causes
of erroneous results have been reviewed,[1] though
false-negative results may be reduced if one employs a
proper gastric stimulator before the patient ingests the
resin. Nevertheless, direct comparisons of the azure A
procedure to a quantitative assessment of total acid
output have shown the azure A test to be
unreliable.[12,13]
The recommended procedure for routine use is a
titrimetric quantitation of total acidity or the direct
measurement of gastric acidity with a pH meter.
The recommended gastric stimulant is pentagastrin.[14]
This substance provides a more reproducible stimulus
than histamine or betazole (Histalog), is more potent
than these two, and is virtually free of undesired side
effects.[15] Histamine can cause drowsiness, headache,
tachycardia, erythema, and hypertension, even in the
presence of antihistamines.[16] Although the use of
betazole (Histalog) can avoid most of these side effects,
cases of life-threatening syncope and shock have been
reported.[17] Since the use of pentagastrin affords an
inexpensive, reliable alternative, it is used almost
exclusively as the stimulator for gastric secretion. The
use of intragastric electrodes is the latest technique to
measurement of gastric pH. These systems can record
the pH at defined intervals and allow changes in gastric
acid pH due to the effects of various substances to be
monitored.
Specimen
A gastric sample is usually obtained from a patient who
has fasted overnight (12 h). It is important to prevent
dilution of gastric fluid with saliva or water during the
procedure. The intubation tube is introduced (usually
through the nose) into the lowest portion of the stomach.
After removal of residual gastric fluid, sampling of
gastric fluid from the stomach continues for the next 60
min, with samples collected in four 15-min sampling
periods. After the collection of the basal gastric
secretions, the pentagastrin (usually 5 to 6 g/kg of body
weight) is administered, and gastric secretions are again
collected for 60 minutes in four 15-min sampling
periods. Each gastric fluid sample should be placed in a
suitable, clean container, closed, and sent to the
laboratory for analysis. The specimen should be clear
and without sediment. Centrifugation of specimens may
be necessary to remove particulate matter. The samples
are stable for several days at room or refrigerated
temperatures.
Gastric Fluid Reference Interval
Gastric residue (stomach contents after a 12-h fast) has a
volume of 20 to 100 mL. A volume greater than 100 mL
may be considered abnormal. This can be caused by
delayed emptying, increased secretion, duodenal ulcer,
ZollingerEllison syndrome, or a mixture of regurgitated
material containing bile from the duodenum. Gastric
juice may be fluid or viscous (mucus increases the
viscosity) and usually has a sour odor. It is usually
colorless. If it is yellow green, bile is probably present. If
brown or red, the presence of blood should be suspected.
Acid content should increase after stimulation.
The pH of the basal gastric fluid should be less than 3,
though very low basal pH values are also associated with
excessive parietal cell activity. A basal pH greater than 6
is almost certainly caused by anacidity, a condition of
subnormal parietal cell secretion. The pH of gastric
secretions after gastric stimulation should fall to less
than 2 with normally functioning parietal cells.
Values for maximal acid output are listed in Gastric
Fluid Analysis Table: Computed range of peak acid
output as determined by age, weight, and sex.[18]
Interpretation
Anacidity (achlorhydria) is associated with pernicious
anemia, and the presence of sufficient gastric acidity
rules out this disease. Insufficient HCl production by the
parietal cell is denoted by an elevated pH of the basal
gastric secretions and by the failure of the gastric fluid
pH to fall below 2 after pentagastrin stimulation. Other
diseases associated with achlorhydria include gastric
carcinoma, hypochronic anemia, rheumatoid arthritis,
and myxedema.
Ulcers, on the other hand, are associated with excessive
HCl production in the basal period (pH < 3) and an
MAO greater than 35 (Gastric Fluid Analysis Table:
Pentagastrin stimulation test), especially in a patient
older than 45 years of age.
The ZollingerEllison syndrome is an extreme form of
excessive acid production resulting from a gastrin-
secreting tumor, usually of the pancreas (gastrinoma).
Diagnosis of a gastrinoma can be made by
620

Gastric Fluid Analysis

demonstration of very high gastric acidity (10 to 100
BAO or a MAO that is only 40% to 60% higher than the
BAO). If the BAO/MAO ratio is greater than 60%, the
person is almost certain to have the ZollingerEllison
syndrome.
References
1 Cannon DC, Winkleman JW, editors. Henrys
Clinical chemistry: principles and techniques.
2nd ed. New York: Harper & Row; 1974. p
1557-63.
2 Bock OA. The concepts of free acid and
total acid of the gastric juice. Lancet
1962;2:1101-2.
3 Moore EW, Scarlata RW. The determination of
gastric acidity by the glass electrode.
Gastroenterology 1965;49:178-88.
4 Bauer JD. Clinical laboratory methods. 9th ed. 9,
St. Louis: C.V. Mosby Co; 1982. p. 780-2.
5 Lubran M. Measurement of gastric activity.
Lancet 1966;2:1070-1.
6 Baron JH. Measurement and nomenclature of
gastric acid. Gastroenterology 1963;45:118-21
7 Weber SG. Electrode-based measurements. In:
Kaplan LA, Pesce AJ. editors: Clinical
chemistry: theory, analysis, and correlation, 3rd
ed. St. Louis: C.V. Mosby Co; 1984 p. 253.
8 Moore EW. Determination of pH by the glass
electrode: pH meter calibration for gastric
analysis. Gastroenterology 1968;54:501-7.
9 Klein B, Weisman M. Quantitative
determination of azure A in urine. Clin Chem
1959;5:115-118.
10 Rosenthal HL, Bascaglia S. Tubeless gastric
analysis. JAMA. 1958;168:409.
11 Correia JP, DeMoura MC, Da Costa CG.
Tubeless gastric analysis. Br J Clin Pract
1967;211:227-31.
12 Galambos JT, Kirsner JB. Tubeless gastric
analysis. Arch Intern Med 1955;96:752-6.
13 Segal HL, Miller LL, Plumb EJ. Tubeless
gastric analysis with an azure A ion-exchange
compound. Gastroenterology 1955;28:402-8.
14 Abernathy RJ, Gillespie IE, Lowrie JH.
Pentagastrin as a stimulant of maximal gastric
acid response in men: a multicentre pilot study.
Lancet 1967;1:291-5.
15 Konturek SJ, Lenkosz J. Pentapeptide infusion
test. Scand J Gastroenterol 1967;2:112-7.
16 Callender ST, Retief FP, Witts LJ. The
augmented histamine test with special reference
to achlorhydria. Gut 1960;1:326-36.
17 Blum NI, Mayoral LG, Kalser MH.
Augmented gastric analysisa word of
caution. JAMA. 1965;19:339-41.
18 Blackman AH, Lambert DL, Thayer WR,
Martin HF. Computed normal values for peak
acid output based on age, sex, and body weight.
Am J Dig Dis 1970;15:783-9.

Tables

Gastric Fluid Analysis: Methods of Gastric Acid Output Analysis

Method 1: Titration
Principle of analysis: Acid is titrated using a pH indicator to indicate end point; Tpfers reagent for pH ~3,
phenol red for pH 77.4, or phenolphthalein for pH 812. Amount of total acid present calculated from volume of
base consumed
Comments: Most common; use of phenolphthalein indicator is sufficient; recommended method
Method 2: pH electrode
Principle of analysis: Acid concentration determined by pH electrode and corrected for presence of other ions by
calculation or estimation of the H+ activity coefficient
Comments: Rare; high degree of accuracy; probably greater than needed clinically
Method 3: Tubeless dye test
Principle of analysis: Dye, bound to ingested cationic resin, is displaced by gastric acid, absorbed by intestine,
and excreted in urine. Amount of dye, measured spectrophotometrically, is related to total gastric activity
Comments: Common; poor accuracy; not recommended
621

Gastric Fluid Analysis

Gastric Fluid Analysis: Computed Range of Peak Acid Output (PAO, mEq/hour) as Determined by Age,
Weight, and Sex

Age Weight Weight Male Female

(yr) kg lb
1540 45.4 100 9.9 18.7 5.9 18.7
1540 50.0 110 11.1 19.9 6.6 15.4
15-40 54.5 120 12.3 21.1 7.4 16.2
1540 59.0 130 13.5 22.3 8.2 16.9
1540 63.6 140 14.7 23.4 9.0 17.8
1540 68.2 150 15.9 24.7 9.8 18.6
1540 72.7 160 17.1 25.9 10.6 19.4
1540 77.3 170 18.2 27.0 11.4 20.1
1540 81.8 180 19.4 28.2 12.1 20.9
1540 86.4 190 20.6 29.4 12.9 21.7
1540 90.9 200 21.8 30.6 13.7 22.5
1540 95.5 210 23.0 31.8 14.5 23.3
1540 100.0 220 24.4 32.9 15.3 24.0
1540 104.5 230 16.1 34.2 16.1 24.9
1540 109.1 240 26.6 35.4 16.9 25.6
1540 113.6 250 27.8 36.6 17.7 26.4
1540 118.8 260 28.9 37.7 18.4 27.2
1540 122.7 270 30.2 38.9 19.2 28.0

Age Weight Weight Male Female

(yr) kg lb

45 45.4 100 6.6 16.6 4.5 13.3

45 50.0 110 7.6 17.6 5.2 13.9
45 54.5 120 8.6 18.6 5.8 14.6
45 59.0 130 9.5 19.6 6.5 15.2
45 63.6 140 10.5 20.6 7.1 15.9
45 68.2 150 11.5 21.6 7.8 16.5
45 72.7 160 12.5 22.5 8.4 17.2
45 77.3 170 13.5 23.6 9.1 17.9
45 81.8 180 14.5 24.5 9.7 18.5
45 86.4 190 15.5 25.5 10.4 19.2
45 90.9 200 16.5 26.5 11.0 19.8
45 95.5 210 17.4 27.5 11.7 20.5
45 100.0 220 18.4 28.5 12.3 21.1
45 104.5 230 19.4 29.5 12.9 21.8
45 109.1 240 20.4 30.5 13.6 22.4
45 113.6 250 21.4 31.5 14.3 23.1
45 118.8 260 22.4 32.4 14.9 23.7
45 122.7 270 23.4 33.4 15.6 24.4
622

Gastric Fluid Analysis

Age Weight Weight Male Female

(yr) kg lb

50 45.4 100 5.6 15.7 3.9 12.7

50 50.0 110 6.5 16.6 4.5 13.3
50 54.5 120 7.4 17.5 5.1 13.8
50 59.0 130 8.3 18.4 5.7 14.4
50 63.6 140 9.2 19.3 6.3 15.0
50 68.2 150 10.1 20.2 6.8 15.6
50 72.7 160 11.0 21.1 7.4 16.2
50 77.3 170 11.9 21.9 8.0 16.8
50 81.8 180 12.8 22.9 8.6 17.4
50 86.4 190 13.7 23.7 9.2 17.9
50 90.9 200 14.6 24.6 9.8 18.6
50 95.5 210 15.5 25.5 10.4 19.2
50 100.0 220 16.4 26.4 10.9 19.7
50 104.5 230 17.3 27.3 11.6 20.3
50 109.1 240 18.1 28.2 12.1 20.9
50 113.6 250 19.0 29.1 12.7 21.5
50 118.8 260 19.9 30.0 13.3 22.1
50 122.7 270 20.8 30.9 13.9 22.7

Age Weight Weight Male Female

(yr) kg lb

55 45.4 100 4.4 14.5 3.1 11.9

55 50.0 110 5.2 15.3 3.6 12.4
55 54.5 120 5.9 16.0 4.1 12.9
55 59.0 130 6.8 16.8 4.6 13.4
55 63.6 140 7.5 17.6 5.1 13.9
55 68.2 150 8.3 18.4 5.6 14.4
55 72.7 160 9.7 19.1 6.2 14.9
55 77.3 170 9.8 19.9 6.7 15.4
55 81.8 180 10.6 20.7 7.2 15.9
55 86.4 190 11.4 21.5 7.7 16.5
55 90.9 200 12.2 22.2 8.2 16.9
55 95.5 210 12.9 23.0 8.7 17.5
55 100.0 220 13.7 23.8 9.2 17.9
55 104.5 230 14.5 24.6 9.7 18.5
55 109.1 240 15.3 25.3 10.2 19.0
55 113.6 250 16.0 26.1 10.8 19.5
55 118.8 260 16.8 - 26.9 11.3 20.0
55 122.7 270 17.6 27.6 11.8 20.5
623

Gastric Fluid Analysis

Age Weight Weight Male Female

(yr) kg lb

60 45.4 100 2.9 13.0 2.1 10.9

60 50.0 110 3.6 13.7 2.5 11.3
60 54.5 120 4.2 14.3 2.9 11.7
60 59.0 130 4.9 14.9 3.4 12.1
60 63.6 140 5.5 15.5 3.8 12.6
60 68.2 150 6.1 16.2 4.2 12.9
60 72.7 160 6.7 16.8 4.6 13.4
60 77.3 170 7.4 17.4 5.0 13.8
60 81.8 180 7.9 18.1 5.4 14.2
60 86.4 190 8.6 18.7 5.9 14.6
60 90.9 200 9.2 19.3 6.3 15.0
60 95.5 210 9.9 19.9 6.7 15.5
60 100.0 220 10.5 20.6 7.1 15.9
60 104.5 230 11.1 21.22 7.5 16.3
60 109.1 240 11.7 21.9 7.9 16.7
60 113.6 250 12.4 22.4 8.3 17.1
60 118.8 260 12.9 23.0 8.7 17.5
60 122.7 270 13.6 23.7 9.2 17.9

Age Weight Weight Male Female

(yr) kg lb

65 45.4 100 1.2 11.3 0.9 9.8

65 50.0 110 1.7 11.8 1.3 10.1
65 54.5 120 2.2 12.2 1.6 10.4
65 59.0 130 2.6 12.7 1.9 10.7
65 63.6 140 3.1 13.1 2.2 10.9
65 68.2 150 3.5 13.6 2.5 11.3
65 72.7 160 3.9 14.0 2.8 11.6
65 77.3 170 4.4 14.5 3.1 11.9
65 81.8 180 4.9 14.9 3.4 12.2
65 86.4 190 5.3 15.4 3.7 12.5
65 90.9 200 5.8 15.8 3.9 12.8
65 95.5 210 6.2 16.3 4.3 13.1
65 100.0 220 6.7 16.8 4.6 13.4
65 104.5 230 7.1 17.2 4.9 13.7
65 109.1 240 7.6 17.7 5.2 13.9
65 113.6 250 8.1 18.1 5.5 14.3
65 118.8 260 8.5 18.6 5.8 14.6
65 122.7 270 8.9 19.0 6.1 14.9
From Blackman, A.H., et al.: Am. J. Dig. Dis. 15:783-789, 1970.
624

Gastric Fluid Analysis

Gastric Fluid Analysis: Pentagastrin Stimulation Test

Adult men reference interval:
Basal acid output (BAO) (mmol/h):
Under age 30 2.22.7
Over age 30 2.22.7
Maximal acid output (MAO) (mmol/h):
Under age 30 1442
Over age 30 333
Adult women reference interval:
Basal acid output (BAO) (mmol/h): 11.5
Maximal acid output (MAO) (mmol/h): 720
ZollingerEllison syndrome
Basal acid output (BAO) (mmol/h): 10100 (or more)
Maximal acid output (MAO) (mmol/h): 40%-60% above BAO
Ulcer predisposition
Maximal acid output (MAO) (mmol/h):
Likely >35
Highly likely >45
Low risk<11
5. If the specimen volume is less than 10 mL,
Procedure: Measurement of Total Gastric Acidity titrate a 1 mL aliquot with 0.01 M NaOH
Principle (dilute 0.1 M NaOH 1:10 with distilled water).
Pentagastrin is a synthetic product that has the Calculations
same physiological action as gastrin. It is a powerful
stimulus for HCl secretion in the stomach. Acid present
in the stomach contents is titrated with 0.1 M NaOH
with phenolphthalein as a pH indicator. Total gastric
acidity is calculated under basal (basal acid output,
BAO) and maximal (maximum acid output, MAO)
stimulation conditions.
For a pentagastrin stimulation, record each acid value,
Reagents and then report the following:
1. 0.1 M NaOH. Place 4 g of NaOH in a 1 L
volumetric flask, and add approximately 900 mL of Basal I + Basal II = mEq of acid per h in basal juice
distilled water to dissolve the pellets. When the solution 0.5
cools to room temperature, bring the volume to 1 L with
additional distilled water, and mix. Stable in plastic where basal I and II are the two highest values obtained.
bottle for 2 years at room temperature. This is the BAO.
2. 1% phenolphthalein, 10 g/L (31.2 mmol/L).
Dissolve 100 mg of phenolphthalein in 10 mL of Stim I + Stim II + Stim III + Stim IV =
absolute ethanol. Stable for 1 year at room temperature. mEq of acid per h in stimulated juice

Assay Alternatively, one can use the two highest consecutive

Equipment: pH meter, buret.
1. Measure and record the volume of each
specimen. Use a graduated cylinder.
2. Measure the pH of each specimen using a pH
meter that has been calibrated at pH 4 and 7.
3. If the specimen contains food particles,
centrifuge at 500 g for 15 min. Discard pellet.
4. Put a 10 mL aliquot of the specimen in a 50 mL
Erlenmeyer flask. Add 3 drops of
phenolphthalein, and mix. Titrate, with 0.1 M
NaOH delivered from a buret, to the first pink
color that remains. Record the volume (in
milliliters) of NaOH used.
15-min stimulated samples and multiply the results by 2.
This is the MAO.
Notes
1. The color of the gastric fluid should be noted on
the report form. Normal gastric fluid is almost colorless.
Contamination of fluid by bile will result in a yellow-
green color, whereas blood contamination will color the
gastric fluid a red to coffee-ground brown color.
2. Mucus can interfere in the analysis. Excess
mucus, found in cases of gastritis and pyloric
obstruction, can be removed by centrifugation of the
sample at 1500 g for 10 min.
Gastric Fluid Reference Interval
click here
 625
Gentamicin and other Aminoglycosides

Gentamicin and other Aminoglycosides

Danni L. Meany and William Clarke

Name: Gentamicin and other aminoglycosides

Clinical significance: Refer to Chapter 14, Therapeutic Drug Monitoring, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation
Molecular formula: See Table 1
Molecular weight: See Table 1
Merck Index: See Table 1
Chemical class: Aminoglycosides
Structure:

NH
O
NH2
H 2N NH2
O

O
HO

HN
HO OH

Principles of Analysis and Current Usage

Aminoglycosides are polycationic agents that are used
primarily to treat serious infections caused by aerobic
gram-negative bacilli [1-4]. Nine aminoglycosides
(gentamicin, tobramycin, amikacin, streptomycin,
neomycin, kanamycin, paromomycin, netilmicin, and
spectinomycin) have been approved for clinical use in the
United States. Gentamicin is the aminoglycoside used
most often because of its low cost and reliable activity
against gram-negative aerobes [4]. Tobramycin and
amikacin are also frequently prescribed. In general,
gentamicin, tobramycin, and amikacin are often used
interchangeably in similar circumstances [3]. Other
aminoglycosides, however, are used only for selected
indications (e.g., paromomycin for protozoa and
spectinomycin for Neisseria gonorrhoeae).
i onto sterile paper disks and placed onto an agar plate that
Gentamicin and Other Aminoglycosides
Previous and current authors of this method:
First edition: Joseph R. DiPersio
Methods edition: Joseph R. DiPersio
Second edition: Joseph R. DiPersio
Third edition: Joseph R. DiPersio
Fourth edition: Joseph R. DiPersio
Fifth edition: Danni L. Meany, William Clarke
Monitoring of serum aminoglycoside concentration is
essential for both efficacy and avoidance of toxicity, since
aminoglycosides have clearly defined therapeutic ranges,
and nephrotoxicity and ototoxicity are frequently
experienced after exposure to high concentrations. Many
assays have been developed for quantitative
aminoglycoside measurement. In this chapter, current
methodologies for quantitative gentamicin measurement
will be described, which serves as a prototype for
measurement of other aminoglycosides.
Microbiological Assay
Microbiological assays were commonly used for analysis
of gentamicin prior to the 1970s. Among them, agar disk
diffusion assay was the most frequently used [4-8]. In this
assay, gentamicin standards prepared in pooled human sera
or serum specimens containing gentamicin were pipetted
was prepared with antibiotic medium and seeded with
indicator bacteria (e.g., Klebsiella pneumoniae) [8]. The
agar plate was then incubated at 37C for 4 to 18 hours,
depending on the antibiotic medium and indicator
organism used. During this period, the indicator organism
grew throughout the agar medium, producing a visual haze
of growth. However, zones of growth inhibition formed
around the antibiotic-containing disks because of diffusion
 626
Gentamicin and other Aminoglycosides
of drug into the agar. After incubation, the diameters of
each zone of inhibition were measured with a vernier
caliper. A calibration curve was established using the
diameters of zone of inhibition versus the gentamicin
concentration in standards, which was then used to
calculate the gentamicin concentration in a clinical
specimen.
Microbiological assays were inexpensive, easy to set up,
and required no instrumentation. However, their
turnaround time was long (e.g., 17 hrs), and their accuracy
varied substantially [6,9]. In addition, they are known to be
affected by the presence of other antimicrobial agents such
as colistin, nalidixic acid, polymyxin B, amikacin, and
tobramycin [8]. To minimize these interferences, an
indicator organism resistant to the antibiotics or techniques
that inactivate these interfering drugs could be used.
Microbiological assays only measure biological activity of
gentamicin against the indicator organism, not necessarily
against bacteria infecting patients. Because of these
shortcomings, microbiological assays have been largely
replaced by immunoassays for monitoring serum
aminoglycoside concentrations. Nevertheless, they are still
used in microbiology laboratories for aminoglycoside
susceptibility testing.
Bioluminescent Assay
Bioluminescent assays utilize the rapid effect of
gentamicin on bacterial ATP levels. This antibiotic causes
dose-dependent decreases in total and intracellular ATP
and dose-dependent increases in extracellular ATP [10-
13]. In the bioluminescent assay that measures
extracellular ATP [10], serum specimens containing
gentamicin are incubated with a day-old bacterial culture
at 37C for 45 minutes. After incubation, luciferase is
added directly into the culture. It reacts with extracellular
ATP and emits luminescence. In this case, the recorded
light intensity is proportional to the gentamicin
concentration. In the bioluminescent assay that measures
total [13] or intracellular ATP [12], extraction of
intracellular ATP is required before addition of luciferase,
and the recorded light intensity is inversely proportional to
the gentamicin concentration. Bioluminescent assays have
the same drawbacks as microbiological assays because of
their similarities. Likewise, they are still used in
microbiology laboratories for aminoglycoside
susceptibility testing. Because of the chemiluminescence
detection scheme employed, they offer better sensitivity
than microbiological assays. In addition, bioluminescent
assays require shorter incubation times [10].
Radioimmunoassay
Radioimmunoassays (RIAs) were developed to measure
gentamicin in 1972 [14]. In an RIA, unlabeled gentamicin
competes with radio-labeled gentamicin for antibody. Then
antibody-bound radioactivity is precipitated by addition of
a second antibody and is measured in the precipitate.
Unlabeled gentamicin is determined by the extent to which
it competes with radio-labeled gentamicin and thus reduces
precipitation of radioactivity [14-16]. The technical
simplicity, sensitivity, and specificity of RIA made it
superior to microbiological assays. For the first time,
gentamicin could be measured in patients receiving
multiple antibiotics. However, because of continuous
changes in the counting rate of radioisotope standards due
to the short half-life of the isotope, a calibration curve
must be generated with each run. This inconvenience has
caused replacement of RIA by non-isotopic homogenous
immunoassays.
Radioenzymatic Assay
Radioenzymatic assays (REAs) were introduced in an
attempt to overcome the drawbacks of widely used
microbiological assays and radioimmunoassays in the
1970s [17]. An REA utilized enzymes that are known to
inactivate gentamicin by transfer of radio-labeled
substrates to gentamicin through adenylation [18,19] or
acetylation [17]. One example of an REA is the gentamicin
3-transferase assay [17]. In this assay, serum specimens
containing gentamicin were incubated in a buffer solution
with [acetyl-14C] acetyl-coenzyme A and gentamicin 3-
transferase. This enzyme transferred the radio-labeled
acetyl group to gentamicin. After incubation, the reaction
mixture was pipetted onto phosphocellulose paper that the
radio-labeled gentamicin bound very tightly. The excess
[acetyl-14C] acetyl-coenzyme A was washed out, and the
radioactivity left on the paper was measured by a liquid
scintillation counter. A control for non-specific binding of
[acetyl-14C] acetyl-coenzyme A to phosphocellulose paper
was run in the absence of gentamicin 3-transferase. This
assay was known to be affected by sisomicin, a structural
analog of gentamycin; netilmicin and tobramycin are also
detected to a small extent [17]. REA bears the same
drawback as RIA because of the short half-life of the
radioisotopes, and REA has also been replaced by non-
isotopic homogeneous immunoassays.
Non-Isotopic Homogeneous Immunoassays
Non-isotopic homogenous immunoassays were first
developed in the late 1970s [20]. Now they have become
the most popular methods for measuring gentamicin and
other aminoglycosides in rapid-response and core
laboratories, because they are easily coupled with
automated multiple-channel analyzers. All non-isotopic
homogenous immunoassays are competitive binding
immunoassays.
Particle-Enhanced Turbidimetric Inhibition
Immunoassay
The particle-enhanced turbidimetric inhibition
immunoassay (PETINIA) is based on the coupling of
gentamicin to a latex particle which reacts with anti-
gentamicin antibody to form aggregates that increase
turbidity of the solution [21]. Unlabeled gentamicin from a
sample competes for antibody and thus decreases the rate
of aggregation. Since turbidity of the solution is measured
 627
Gentamicin and other Aminoglycosides
by a turbidimeter as the intensity of light transmitted
through the medium, the lower the turbidity is, the higher
the recorded light intensity and the higher the unlabeled
gentamicin concentration in the sample. In other words,
the recorded light intensity is proportional to the unlabeled
gentamicin concentration.
A nephelometer can also be used to measure turbidity of a
solution [22]. However, because it measures the scattered
light at right angles to the incident light rather than the
transmitted light in the direct path, nephelometric
measurements cannot be as easily integrated with
automated analyzers as turbidimetric measurements. For
this reason, turbidimetric inhibition immunoassays are
more popular than nephelometric inhibition
immunoassays.
Fluorescence Polarization Immunoassay
A fluorescence polarization immunoassay (FPIA)
measures polarized fluorescence intensity from
competitive binding of unlabeled and fluorescein-labeled
gentamicin to anti-gentamicin antibodies [23-30]. When
fluorescein-labeled gentamicin binds to an antibody, the
complex has high polarized emission upon excitation by
polarized laser because of its slow molecular rotation.
However, when fluorescein-labeled drug is free in
solution, it has low polarized emission because of its fast
rotation. Therefore, the greater the amount of unlabeled
gentamicin in the sample, the less polarization of emitted
lightthat is, polarized fluorescence intensity is inversely
proportional to the unlabeled gentamicin concentration in
the sample.
Enzyme-Multiplied Immunoassay Technology
The enzyme-multiplied immunoassay (EMIT) was the first
homogeneous immunoassay to be developed for clinical
use [20]. In this assay, unlabeled gentamicin competes for
anti-gentamicin antibody with gentamicin linked to
glucose-6-phosphate dehydrogenase [24,26-30]. When the
enzyme-linked gentamicin binds to an antibody, activity of
the enzyme is reduced. Consequently, only when the
enzyme-linked gentamicin is free in solution can it convert
NAD+ to NADH, which is measured photometrically at
340 nm. The absorbance at 340 nm is proportional to the
unlabeled gentamicin in the sample.
Cloned-Enzyme Donor Immunoassay
The cloned-enzyme donor immunoassay (CEDIA) is based
on bacterial enzyme -galactosidase which has been
genetically engineered into two inactive fragments:
enzyme donor (ED) and enzyme acceptor (EA) [31]. These
fragments spontaneously reassemble to form fully active
enzyme that cleaves a galactopyranoside substrate,
generating a color change. In CEDIA, gentamicin is tagged
to ED, which does not interfere with enzyme activity.
However, when ED-linked gentamicin binds to an
antibody, it interferes with reassembly of ED and EA and
thus eliminates the activity of the enzyme. Unlabeled
gentamicin in the sample regulates the reassembly of ED
and EA by competing for a limited amount of antibody.
Therefore, the amount of signal produced is proportional to
the concentration of unlabeled gentamicin.
Direct Chemiluminescence Immunoassay
A direct chemiluminescence immunoassay is analogous to
RIA, except the label is a chemiluminescent acridinium
ester [4]. In this assay, solid-phase antibody is incubated
with unlabeled and acridinium-labeled gentamicin. After
incubation, antibody-coated beads are separated using a
magnetic field, and the amount of antibody-bound
acridinium ester is measured by photomultiplier detection
of flash emission upon addition of an oxidizing reagent.
Fluorescence Immunoassay
A fluorescence immunoassay (FIA) is based on
competitive binding between unlabeled gentamicin in a
sample and a known amount of gentamicin labeled with a
fluorogenic substrate (e.g., a derivative of umbelliferyl--
D-galactoside) for an antibody [9,10]. Binding of the
labeled gentamicin to the antibody prevents hydrolysis of
the fluorogenic substrate by -galactosidase. Therefore, -
galactosidase only hydrolyses the unbound substrate to
produce a fluorescent product, which is measured
fluorometrically. FIA is similar to EMIT, except it is the
enzyme substrate that is linked to gentamicin.
High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) has
been used for determination of gentamicin mostly in
pharmacokinetic and drug stability studies [4]. It is not
frequently used in clinical laboratories, because the
protocol is labor intensive: (1) gentamicin needs to be
extracted from serum, and (2) ultraviolet absorption of
gentamicin is not sensitive enough for detection, so it
needs to be fluorescently derivatized with o-
phthalaldehyde [7,32]. Recently, HPLC coupled with mass
spectrometry has been used for gentamicin analysis, which
has become the most powerful method for confirmation of
antibiotic residues in food [4,33].
A summary of gentamicin methods is presented in Table 2.
Reference and Preferred Methods
Currently there is no reference method for determination
of gentamicin in serum or plasma. Non-isotopic
homogeneous immunoassays are the most widely used
methods because they are rapid, sensitive, accurate,
precise, and easily automated. Radioimmunoassay and
radioenzymatic assay are not practical in rapid-response
and core laboratories for two reasons: (1) a calibration
curve should be generated with each run due to decay of
radioisotopes, and (2) gentamicin-specific radioactivity
should be separated (e.g., precipitation or phosphocellulose
paper) from nonspecific radioactivity. In comparison,
calibration curves for homogeneous non-isotopic
immunoassays are stable for up to 2 weeks, and they do
 628
Gentamicin and other Aminoglycosides
not require separation steps. Among all the non-isotopic
immunoassays, PETINIA is the most popular, followed by
FPIA. According to the 2007 College of American
Pathologists (CAP) Participant Summary Report, 40% of
participants use the former, and 19% use the latter.
Specimen
Serum and plasma are the most commonly used specimens
for therapeutic drug monitoring of gentamicin, which are
obtained after patients have received 2 to 3 doses after
initiation of therapy or after adjustment of the dose.
Trough concentrations are measured within 30 min of the
next dose, and peak concentrations 30 to 45 min after the
end of an intravenous infusion or approximately 60 min
after an intramuscular injection. Gentamicin administration
times and the time that specimens are obtained are
essential in interpreting the results. Serum gentamicin
concentrations should be monitored periodically
throughout therapy. Routine measurement is not necessary
with prophylactic therapy given for less than 48 to 72 hrs.
Microbiological assays and bioluminescent assays can be
used for antimicrobial susceptibility testing in any body
fluids. HPLC may be used to assay gentamicin in other
body fluids. However, there are no concrete data on the
general use of immunoassays in fluids other than serum or
plasma.
Interferences
Microbiological and bioluminescent assays are prone to
interferences by antimicrobial agents present in samples
[4,8,9,17]. Therefore, they cannot be used for patients
receiving multiple antibiotics. Compounds that are
structurally related to gentamicin, such as netilmicin,
sisomicin, and sagamicin, may cross-react with antibodies
to gentamicin in immunoassays. However, with
improvement of antibody specificity, minimum cross-
reactivity with other aminoglycoside (e.g., less than 10%)
has been achieved by most available immunoassays
[21,25,29]. In addition, they are slightly affected by
hemolysis, lipemia, icterus, rheumatoid factor, and
heterophilic antibodies (e.g., less than 10%) [21].
Gentamicin and Other Aminoglycosides Therapeutic
Range
Since gentamicin and other aminoglycosides display peak
concentrationdependent killing of microorganisms, the
therapeutic goal is to achieve a concentration in serum
such that the indicated organisms are killed, but the host
remains undamaged. Because the indicated organisms are
variable and become resistant to certain drugs, treatment
with specific aminoglycosides agent should always be
directed by susceptibility testing [2]. Therefore, desired
peak concentrations vary with indicated organisms.
Two different approaches have been used for
administration of aminoglycosides: traditional multiple-
daily dosing and consolidated once-daily dosing. In the
traditional dosing, aminoglycosides are administered every
8 hrs in patients with normal renal function; in patients
with impaired renal function, loading and maintenance
dose are reduced and/or the dosing interval extended
because aminoglycosides have prolonged half-life in them.
In the case of gentamicin, the loading dose for adults is
typically 2 to 3 mg per kilogram of lean body weight, and
the maintenance dosing is 1.7 mg per kilogram. The
recommended dosing interval is 8 hrs with creatinine
clearance > 90 mL/min, 12 hrs with creatinine clearance
between 50 and 90 mL/min, and 24 to 48 hrs with
creatinine clearance between 10 and 50 mL/min [3]. In the
traditional dosing, maintenance dose is selected based
upon a percentage of the loading dose to maintain effective
peak concentration. Trough gentamicin concentrations
should also be monitored to avoid concentration-related
toxicity (see Table 3).
Consolidated dosing, however, administers
aminoglycosides in higher dose (e.g., 7mg/kg) every 24 hrs
in patients with normal renal function or longer in patients
with impaired renal function. It targets a peak
concentration of ~ 20 g/mL, and trough concentration is
often undetectable because of the short half-life of the
drugs compared to the long dosing interval [34]. In this
dosing regime, a specimen for drug monitoring is obtained
6 to 14 hours after the first dose, and the result from this
measurement is then used to predict the necessary dosing
interval [35]. See Table 4 for consolidated once-daily
dosing and desired serum concentrations for
aminoglycosides.
Consolidated dosing utilizes two pharmacodynamic
properties of aminoglycosides: concentration-dependent
killing and post-antibiotic effect, which is defined as the
time required for an organism to demonstrate viable
regrowth following the removal of an antibiotic. Higher
dosage of aminoglycosides results in higher peak
concentration, which increases both the rate and extent of
bacterial cell death. In addition, the higher the dosage, the
greater the post-antibiotic effect, up to a certain maximal
response [36]. The potential benefits of the consolidated
dosing over the traditional one are (1) possibility of
decreased nephrotoxicity, (2) ease of administration and
therefore less expensive, and (3) a lesser intensity of
monitoring of serum drug concentrations [37]. Currently,
consolidated dosing may be considered an option in
treatment of moderate and severe infections due to gram-
negative aerobic bacteria.
Interpretation
Aminoglycosides are used as the treatment for a wide
range of aerobic gram-negative bacilli, as well as
staphylococci and enterococci when used in combination
with beta-lactams [2,3]. The antibacterial properties of
aminoglycosides result from (1) irreversible binding to the
30S bacterial ribosome, thus inhibiting bacterial protein
synthesis, and (2) creating fissures in the outer bacterial
 629
Gentamicin and other Aminoglycosides
membrane, resulting in the leaking of intracellular contents
and increasing antibiotic uptake [38,40]. Since the uptake
of aminoglycosides is energy dependent, and anaerobes
have less energy available, they are intrinsically resistant
to aminoglycosides. As a result, beta-lactams are used
synergistically to facilitate uptake of aminoglycosides into
anaerobes such as enterococci. In contrast to the intrinsic
resistance is acquired resistance of aminoglycosides. Two
major mechanisms account for this: (1) bacterial
production of enzymes that inactivate the drug [41-43] and
(2) decreased accumulation of the drug due to an efflux
system [44]. Because of structural differences, amikacin is
not inactivated by the enzymes that inactivate gentamicin
and tobramycin. Therefore, amikacin is usually reserved
for serious gram-negative infections that are resistant to
gentamycin and tobramycin. Although acquired resistance
to aminoglycosides is becoming increasingly prevalent
owing to repeated and long periods of use of one type, it is
still less frequently encountered than other antibiotics,
which confirms their clinical importance despite the
introduction of newer and less toxic antimicrobials.
Because aminoglycosides are very polar and poorly
absorbed through the gastrointestinal tract, they are
administered intravenously or intramuscularly. Peak serum
concentrations are measured 30 to 60 min after
intravenously infusions or 30 to 90 min after intramuscular
injections. In addition to blood, adequate drug
concentrations are also found in bone, synovial fluid,
peritoneal fluid, and other body fluids [3]. Since
aminoglycosides are primarily distributed within the
extracellular fluid, disease states and drugs that alter fluid
balance (e.g., renal diseases and diuretics) require
modifications to dosage to avoid adverse effects of
aminoglycosides [3].
The main adverse effects of aminoglycosides are
nephrotoxicity and ototoxicity (Table 5). Aminoglycosides
are not metabolized in human bodies, and they are quickly
excreted by glomerular filtration, with an average half-life
of 2.5 hrs with normal renal function [2]. However, their
half-life in the renal cortex is approximately 100 hrs [3].
Therefore, repetitive dosing may result in renal
accumulation of aminoglycosides and hence
nephrotoxicity. Another important aspect of nephrotoxicity
of aminoglycosides comes from their high intracellular
concentrations in proximal renal tubule cells.
Aminoglycosides are absorbed from the glomerular filtrate
by proximal renal tubular cells. The very high intracellular
levels lead to subsequent tubular necrosis.
Aminoglycosides may produce clinically evident
nephrotoxicity in 2% to 8% of patients, which is usually
reversible with discontinuation of therapy. Moreover,
periodic monitoring of serum creatinine concentrations
may alert to developing nephrotoxicity. By comparison,
ototoxicity is usually irreversible. Aminoglycoside-
induced ototoxicity may result in either vestibular or
cochlear damage. Manifestations of the former include
vertigo, disequilibrium, light-headedness, nausea,
vomiting, and ataxia; the symptoms of the latter are
tinnitus and hearing loss. Subclinical ototoxicity may be
detected in 10% to 20% of patients treated with
aminoglycosides for long periods, but clinically overt
ototoxic reactions may occur in only 1% to 3% of patients.
The pathogenesis of nephrotoxicity is mainly attributed to
the cationic amino groups on aminoglycosides. When
taken up into proximal renal tubule cells, aminoglycosides
are accumulated within lysosomes. This may be a charge-
mediated process. Inhibition of lysosomal function may be
responsible for subsequent aminoglycoside-induced
cellular injury [45]. Another hypothesis of nephrotoxicity
postulates that aminoglycosides induce cell death by
stimulating the extracellular calcium-sensing receptor on
the surface of proximal tubular cells [46,47]. On the other
hand, the pathogenesis of induced ototoxicity with hearing
loss is less well understood. Sustained or excessive peak
serum concentrations are thought to be a risk factor. In
order to minimize toxicity, potential risk factors that
predispose to aminoglycoside toxicity should be identified
and when possible corrected (Table 6). Aminoglycosides
should only be used when their unique antibiotic potency
is needed, and once the infecting organism has been
determined, it should be replaced by a potentially less
toxic antibiotic.
Gentamicin Performance Goals
Acceptable performance (accuracy) for gentamicin assays
according to the Clinical Laboratory Improvement
Amendments of 1988 (CLIA-88) is the target value 25%
of the peer group mean. The same criteria are applied to
amikacin and tobramycin. According to the 2007 CAP
survey, the imprecision for all the gentamicin
measurements ranged from 4% to 11% for specimens, with
an average gentamicin concentration of 5.05 g/mL. The
imprecision for tobramycin and amikacin ranged from 3%
to 9% for an average tobramycin concentration of 3.07
g/mL and 4% to 15% for an average amikacin
concentration of 5.83 g/mL, respectively. Based on the
data, current aminoglycoside assays demonstrate
acceptable performance with regard to laboratory
regulations.
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24 Cheng AF, Lam AW, French GL. Comparative
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27 Ngui-Yen JH, Doyle PW, Smith JA. Comparative
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28 Pohlod DJ, Saravolatz LD, Somerville MM.
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29 Andrews JM, Wise R. A comparison of the
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30 Araj GF, Khattar MA, Thulesius O, Pazhoor A.
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31 Henderson DR, Friedman SB, Harris JD,
Manning WB, Zoccoli MA. CEDIA, a new
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32 Anhalt JP. Assay of gentamicin in serum by high-
pressure liquid chromatography. Antimicrob
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34 Nicolau DP, Wu AH, Finocchiaro S, Udeh E,
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35 Nicolau DP, Freeman CD, Belliveau PP,
Nightingale CH, Ross JW, Quintiliani R.
Experience with a once-daily aminoglycoside
program administered to 2184 adult patients.
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36 Ferriols-Lisart R, Alos-Alminana M.
Effectiveness and safety of once-daily
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37 Barza M, Ioannidis JP, Cappelleri JC, Lau J.
Single or multiple daily doses of
aminoglycosides: a meta-analysis. BMJ (Clin
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38 Montie T, Patamasucon P. Aminoglycosides: the
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clinical applications. Eur J Clin Microbiol Infect
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39 Mingeot-Leclercq MP, Glupczynski Y, Tulkens
PM. Aminoglycosides: activity and resistance.
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40 Fourmy D, Recht MI, Blanchard SC, Puglisi JD.
Structure of the A site of Escherichia coli 16S
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aminoglycoside antibiotic. Science.
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41 Davies J, Wright GD. Bacterial resistance to
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42 Hon WC, McKay GA, Thompson PR, Sweet RM,
Yang DS, Wright GD et al. Structure of an
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43 Daigle DM, McKay GA, Wright GD. Inhibition
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44 Westbrock-Wadman S, Sherman DR, Hickey MJ,
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Characterization of a Pseudomonas aeruginosa
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45 Olbricht CJ, Fink M, Gutjahr E. Alterations in
lysosomal enzymes of the proximal tubule in
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46 Ward DT, McLarnon SJ, Riccardi D.
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receptor. J Am Soc Nephrol. 2002;13:1481-1419.
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Riccardi D. Aminoglycosides induce acute cell
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Nephrol. 2005;16:1236-1244.
 632
Gentamicin and other Aminoglycosides

Table 1: Commonly Used Aminoglycoside Antibiotics

Merck Route of
Discovery Molecular Molecular Index Administration*
Name Date Formula Mass Number
Streptomycin 1944 C21H39N7O12 581.58 8685 IM
Neomycin 1949 6300 PO
Neomycin A C12H26N4O6322.36 6283
Neomycin B C23H46N6O13 614.65 6300
Neomycin C C23H46N6O13 614.65 6300
Kanamycin 1957 5118 IM,IV
Kanamycin A C18H36N4O11 484.50 5118
Kanamycin B C18H37N5O10 483.52 5118
Kanamycin C C18H36N4O11 484.50 5118
Gentamicin 1963 4251 IM,IV
Gentamicin A C18H36N4O11 484.50 4251
Gentamicin C1 C21H43N5O7477.60 4251
Gentamicin C2 C20H41N5O7463.57 4251
Gentamicin C1a C19H39N5O7449.55 4251
Tobramycin 1967 C18H37N5O9467.54 9318 IM,IV
Amikacin 1972 C22H43N5O13 585.62 405 IM,IV
Netilmicin 1976 C21H41N5O7475.60 6322 IM,IV

* IM, Intramuscular; PO, by mouth; IV, intravenous.

Neomycin is no longer used parenterally because of toxicity.
 633
Gentamicin and other Aminoglycosides

Table 2: Gentamicin Methods Summary Table

Method 1: Microbiological assay (agar plate diffusion)
Principle of analysis: Diameter of bacterial growthinhibition zone is proportional to gentamicin concentrations.
Advantages: Inexpensive, easy to set up, and requires no instrumentation
Disadvantages: Long turnaround time (4 to 18 hrs), variable accuracy, interfered by other antibiotics
Method 2: Bioluminescent assay; photometry
Principle of analysis: Dose-dependent effects of gentamicin on bacterial ATP levels that are measured upon addition
of luciferase
Advantages: Inexpensive, easy to set up
Disadvantages: Interfered by other antibiotics
Method 3: Radioimmunoassay (RIA); radiometry
Principle of analysis: Competitive binding of radio-labeled gentamicin and unknown for antibody
Advantages: Sensitive, specific, accurate
Disadvantages: Labor-intensive, establishment of calibration curves every run, centrifugation needed, specific
instrumentation, not appropriate in rapid response and core laboratories
Method 4: Radioenzymatic assay (REA); radiometry
Principle of analysis: Enzymatic transfer of radio-labeled substrate to gentamicin
Advantages: Sensitive, specific, accurate
Disadvantages: Labor-intensive, establishment of calibration curves every run, centrifugation needed, special
instrument, not appropriate in rapid-response and core laboratories
Method 5: Particle-enhanced turbidimetric inhibition immunoassay (PETINIA); turbidimetry
Principle of analysis: Competitive binding of particle-labeled gentamycin and unknown for antibody
Advantages: Sensitive, specific, accurate, automated instrumentation
Disadvantages: Expensive instrumentation, not suitable for low workload
Method 6: Fluorescence-polarization immunoassay (FPIA); polarization analyzer
Principle of analysis: Competitive binding of fluorescein-labeled gentamicin and unknown for antibody
Advantages: Sensitive, specific, accurate, automated instrumentation
Disadvantages: Expensive instrumentation, not suitable for low workload
Method 7: Enzyme-multiplied immunoassay technique (EMIT); spectrophotometry
Principle of analysis: Competitive binding of enzyme-labeled gentamicin and unknown for antibody
Advantages: Sensitive, specific, accurate, automated instrumentation
Disadvantages: Expensive instrumentation, not suitable for low workload
Method 8: Cloned-enzyme donor immunoassay (CEDIA); spectrophotometry
Principle of analysis: Competitive binding of enzyme-donor-labeled gentamicin and unknown for antibody;
reassembly of enzyme donor and receptor gains the full enzyme activity
Advantages: Sensitive, specific, accurate, automated instrumentation
Disadvantages: Expensive instrumentation, not suitable for low workload
Method 9: Direct chemiluminescence immunoassay; photometry
Principle of analysis: Competitive binding of acridinium ester-labeled gentamicin and unknown for antibody
Advantages: Sensitive, specific, accurate
Disadvantages: Moderate to expensive equipment
Method 10: Fluorescence immunoassay; spectrophotometry
Principle of analysis: Competitive binding of fluorogenic substrate-labeled gentamicin and unknown for antibody
Advantages: Sensitive, specific, accurate
Disadvantages: Moderate to expensive equipment
Method 11: High-performance liquid chromatography (HPLC); chromatography, fluorometry
Principle of analysis: Chromatographic separation followed by derivatization of gentamicin to fluorescent product
for detection
Advantages: Sensitive, highly specific, accurate, versatile; can handle small workloads
Disadvantages: Not appropriate for large workloads, expensive equipment, labor-insensitive sample preparation
 634
Gentamicin and other Aminoglycosides

Table 3: Traditional Multiple-Daily Doses and Desired Serum Concentrations for Aminoglycosides
Gentamicin, Tobramycin
Loading dose*: 2-3 mg/kg
Maintenance dose*: 1.7 mg/kg
Dosing interval*: every 8 hrs
Peak: 5-8 g/mL
Trough: <2 g/mL
Toxic peak range: >10 g/mL
Toxic trough range: >2 g/mL
Amikacin
Loading dose*: 7.5 mg/kg
Maintenance dose*: 7.5 mg/kg
Dosing interval*: every 12 hrs
Peak: 15-25 g/mL
Trough: 5-10 g/mL
Toxic range peak: >30-55 g/mL
Toxic range trough: >10 g/mL
Netilmicin
Loading dose*: 2-3 mg/kg
Maintenance dose*: 1.7 mg/kg
Dosing interval*: every 8 hrs
Peak: 4-10 g/mL
Trough: <2 g/mL
Toxic range peak: >10-20 g/mL
Toxic range trough: >2 g/mL
Streptomycin
Loading dose*: 7.5 mg/kg
Maintenance dose*: 7.5
Dosing interval*: every 12 hrs
Peak: 15-30 g/mL
Trough: 5-10 g/mL
Toxic range peak: >10-20 g/mL
Toxic range trough: >2 g/mL
* For adults with normal renal function.
Peak concentration determined 1 hr after intramuscular dose or 30-45 min after intravenous dose.
Trough determined 30 min before the next dose.

Table 4: Consolidated Once-Daily Doses and Desired Serum Concentrations for Aminoglycosides
Gentamicin, Netilmicin, and Tobramycin
Loading dose*: 5-7 mg/kg
Dosing interval*: every 24 hrs
Serum concentration for dosing every 24 hrs: <3 g/mL
Trough: 0.5-1.0 g/mL
Amikacin
Loading dose*: 15 mg/kg
Dosing interval*: every 24 hrs
Serum concentration for dosing every 24 hrs: <8 g/mL
Trough: <5 g/mL

* For adults with normal renal function.

Serum concentration determined 12 hrs after the start of the 60-min transfusion.
Trough determined 30 min before the next dose.
From Gonzalez LS 3rd, Spencer JP [3]. Am Fam Physician. 1998;58:1811
 635
Gentamicin and other Aminoglycosides

Table 5: Relative Aminoglycoside Toxicity

Relative Ototoxicity Relative
Aminoglycoside Vestibular Cochlear Nephrotoxicity
Streptomycin 3+ 1+ 2+
Neomycin 1+ 3+ 3+
Kanamycin 1+ 2+ 2+
Gentamicin 2+ 1+ 2+
Tobramycin 1-2+
Amikacin 2+ 1+ 2+
Netilmicin 1-2+

From Korzeniowski OM, Hook EW. In: Mandell GL, Douglas RG, Bennet JE, eds. Principles and Practice of Infectious
Diseases. New York: Wiley; 1979. .

Table 6: Risk Factors Predisposing Aminoglycoside Toxicity

Potentially Alterable Factors
Use of diuretics
Radiographic contrast exposure
Effective circulating volume depletion
Use of ACE inhibitors
Use of NSAIDs
Use of other nephrotoxic medications
Concomitant use of amphotericin (Fungizone IV)
Use of cisplatin (Platinol)
Unalterable Factors
Age
Preexisting renal diseases

From Gonzalez LS 3rd, Spencer JP [3]. Am Fam Physician 1998;58:1811.

 636

METHODS in CLINICAL
CHEMISTRY
An accessory work to the 5th edition of

Kaplan and Pesces : Clinical Chemistry:

Theory, Analysis, Correlation*
Published by Pesce Kaplan Publishers 2009

Editors
Peter E. Hickman, MB BS, PhD, FRCPA
Methods Editor
Associate Professor
Australian National University Medical School;
Director of Chemical Pathology
The Canberra Hospital
Australian Capital Territory, Australia

Gus Koerbin, BAppSci, AFAIM

Methods Associate Editor
Principal Scientist ACT Pathology
Adjunct Professional Associate
University of Canberra
ACT Pathology
The Canberra Hospital,
Garran, Australian Capital Territory, Australia

A work of 144 Methods of Analysis describing current methodology.

*Published by Mosby, an affiliate of Elsevier; 2009

637

METHODS in CLINICAL
CHEMISTRY
An accessory work to the 5th edition of

Kaplan and Pesces : Clinical Chemistry:

Theory, Analysis, Correlation*
Published by Pesce Kaplan Publishers 2009

Volume II
 638

Pesce Kaplan Publishers

Methods Clinical Chemistry

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Notice

Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our knowledge, changes in (i) on state-of-the-art methodology and measurement technique, (ii) effect of
interferences, or (iii) interpretation of results may become necessary or appropriate. Readers are advised
to check the most current information in the relevant scientific literature. It is the responsibility of
the clinical laboratory practitioner, relying on their own experience and knowledge of the patient, to
determine the best procedure to take all appropriate safety precautions. To the fullest extent of the law,
neither the Publisher nor the Editors assumes any liability for any injury and/or damage to persons or
property arising out of or related to any use of the material contained in this book.
The Publisher

Previous copyrighted printed editions: 1987, electronic editions 1996, 2003

Library of Congress Cataloging-in-Publication Data

Methods Clinical chemistry [edited by] Lawrence A. Kaplan, Amadeo J. Pesce.

Bound version and electronic version.

Printed in the United States of America

 639
Contributors to Methods of Analysis
Zakaria Ahmed, PhD
Clinical Chemist
Department of Pathology and Laboratory
Medicine
Rochester General Hospital
Rochester, New York
Hassan M. E. Azzazy, PhD, DABCC, FACB
Chairman and Associate Professor
Department of Chemistry
The American University in Cairo
Cairo, Egypt
Tony Badrick, BAppSc, BSc, BA, MLitSt
(Math), MBA, PhD, FAIMS, FAACB, FQSA,
FAIM, FACB, FRCPA (Hon)
Executive ManagerLaboratories
Sullivan Nicolades Pathology
Taringa, Queensland, Australia
John Beilby, BSc(Hons), PhD, FAACB,
MHGSA, ARCPA
Principal Scientist
PathWest
Nedlands, Western Australia, Australia
Marion Black, BSc(Hons), Dip. Ed, MAACB
Senior Scientist
Clinical Biochemistry, Alfred Pathology
Service
Victoria, Australia
John R. Burnett, MB ChB, MD, PhD,
FRCPA, FAHA
Clinical Professor
PathWest Laboratory Medicine
Royal Perth Hospital
Perth, Western Australia, Australia
Kevin Carpenter, PhD, FHGSA
Principal Scientist and Head of
Department
NSW Biochemical Genetics Service
The Childrens Hospital at Westmead
Westmead, New South Wales, Australia
Kee Cheung, BSc(Hons), PhD, GradCert.
Mgt.
Manager
Pathology QueenslandPrincess Alexandra
Hospital
Woolloongabba, Queensland, Australia
William Clarke, PhD, MBA, DABCC
Director, TDM and Toxicology, Director,
CPOCT
Department of Pathology
Johns Hopkins School of Medicine
Baltimore, Maryland
Paul F. Coleman, PhD
Research Fellow
Infectious Disease Core R&D
Abbott Diagnostics
Abbott Park, Illinois
Joe DAgostino, BSc, Grad Dip FMI,
MAACB
Senior Scientist
Clinical Biochemistry Unit
Alfred Pathology Service
Alfred Hospital, Melbourne, Australia
Sheila Dawling, PhD, CChem, FRSC
Associate Professor of Pathology
Director, Toxicology
TDM Laboratory,
Associate Director
Clinical Chemistry
The Vanderbilt Clinic
Nashville, Tennessee
Joris R. Delanghe
Professor of Clinical Chemistry
Department of Clinical Chemistry
Ghent University Hospital
Gent, Belgium
Goce Dimeski, BSc
Supervising Scientist
Pathology QLD
Princess Alexandra Hospital
Woolloongabba, Brisbane, Australia
Angela Ferguson, PhD
Clinical Chemistry Fellow
Washington University School of
Medicine
Department of Pathology and Immunology
St. Louis, Missouri
Michael J. Figursk, PhD
Research Associate
University of Pennsylvania
Hospital of University of Pennsylvania
Philadelphia, Pennsylvania
John Galligan
Supervising Scientist
Pathology Queensland Central Laboratory
Royal Brisbane Hospital
Herston, Queensland, Australia
 640
Karen Golemboski, PhD, MT(ASCP)
Program Director and Chair
Clinical Laboratory Science
Bellarmine University
Louisville, Kentucky
Ronda F. Greaves, BSc, Grad Dip Ed,
MAppSc, PhD, MAACB
Senior Scientist
Complex Biochemistry Department
The Royal Childrens Hospital
RCH Laboratory Services
Parkville, Victoria, Australia
Kathryn Green, BSc(Hons), MSc(Med)
Senior Scientist
NSW Biochemical Genetics Service
The Childrens Hospital at Westmead
Westmead, New South Wales, Australia
Elizabeth M. Hall, BSc, MSc, FRCPath
Principal Clinical Scientist
Department of Clinical Biochemistry
East Kent Hospitals University
NHS Trust
Kent and Canterbury Hospital
Canterbury, Kent, United Kingdom
Peter E. Hickman, MB BS, PhD, MPH,
MAACB, FRCPA
Associate Professor
Australian National University Medical
School;
Director of Chemical Pathology
The Canberra Hospital
Australian Capital Territory, Australia
Gregory A. Hobbs, PhD, DABCC
Clinical Laboratory Science
Bellarmine University
Louisville, Kentucky
David W. Holt, BSc, PhD, DSc(Med),
CSci, EurClin Chem, FESC, FRCPath
Professor of Bioanalytics
ASI, Ltd
London, United Kingdom
David G. Hughes, BAppSci, Grad Dip Sci
Scientist
Clinical Chemistry, ACT Pathology
The Canberra Hospital
Garran, Australian Capital Territory,
Australia
Mind Jin, PhD
Temple University
Philadelphia, Pennsylvania
Atholl Johnston, BSc, MSc, PhD,
FBPharmacolS, FRCPath
Professor of Clinical Pharmacology
William Harvey Research Institute
(School of Medicine and Dentistry)
Queen Mary
University of London
London, United Kingdom
Graham Jones, MBBS, DPhil, FRCPA, FAACB
Staff Specialist in Chemical Pathology
Department of Chemical Pathology
St. Vincents Hospital
Darlinghurst, New South Wales,
Australia
Saeed A. Jortani, PhD, DABCC, FACB
Associate Professor of Pathology and
Laboratory Medicine
University of Louisville
School of Medicine
Louisville, Kentucky
Lawrence A. Kaplan, PhD, DABCC
New York, New York
Steven C. Kazmierczak, PhD, DABCC
Professor of Pathology
Director of Clinical Chemistry and
Toxicology
Oregon Health and Science University
Department of Pathology
Portland, Oregon
Sandra Klingberg, B App Sci
Supervising Scientist, Protein
Laboratory
Pathology Queensland, Central
Laboratory
Royal Brisbane Hospital
Herston, Queensland, Australia
Gus Koerbin, BAppSci, AFAIM
Principal Scientist ACT Pathology
Adjunct Professional Associate
University of Canberra
ACT Pathology
The Canberra Hospital
Garran, Australian Capital Territory,
Australia
Magdalena Korecka, PhD
Senior Research Investigator
School of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
 641
William J. Korzun, PhD, DABCC, MT(ASCP)
Associate Professor
Department of Clinical Laboratory
Sciences
Virginia Commonwealth University
Richmond, Virginia
Edmund Lamb, PhD, FRCPath
Clinical Scientist (Biochemistry) and
Head of Department
Department of Clinical Biochemistry
East Kent Hospitals
NHS Trust
Kent and Canterbury Hospital
Canterbury, Kent, United Kingdom
Stanley S. Levinson, PhD, DABCC
Professor of Pathology and Laboratory
Medicine
University of Louisville
Director of Clinical Chemistry and
Immunochemistry
Department of Veteran Affairs Medical
Center
Louisville, Kentucky
Barry Lewis, MD, FRCPA, FHGSA
Head, Department of Clinical
Biochemistry
PathWest Laboratory Medicine WA
Princess Margaret Hospital
Perth, Western Australia, Australia
Jinong Li, PhD
Clinical Chemistry Fellow
Johns Hopkins Medical Institutions
Baltimore, Maryland
Greg Maine, PhD
Manager, Global Scientific Affairs
Associate Research Fellow
Abbott Laboratories
Abbott Park, Illinois
Christopher R. McCudden, PhD, DABCC,
NRCC
Assistant Professor
Department of Pathology and Laboratory
Medicine
School of Medicine
University of North Carolina
Chapel Hill, North Carolina
Denise A. McKeown, MSci, AMRSC
Senior Analyst
St Georges, University of London
Analytical Unit
Department of Cardiac and Vascular
Sciences
London, United Kingdom
Brett McWhinney, BSc, MSc, MBA, MPhil
Supervising Scientist
HPLC Section, Department of Chemical
Pathology
Royal Brisbane Hospital
Herston, Queensland, Australia
Danni L. Meany, PhD
Clinical Chemistry Fellow
Johns Hopkins Medical Institutions
Baltimore, Maryland
James J. Miller, PhD, DABCC, FACB
Professor
University of Louisville
School of Medicine
Department of Pathology and Laboratory
Medicine
Louisville, Kentucky
Michael Milone, MD, PhD
Assistant Professor of Pathology and
Laboratory Medicine
Associate Director, Toxicology
Laboratory
School of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
Gerald J. Mizejewski, BS, MS, PhD
Senior Research Scientist
Wadsworth Center
New York State Department of Health
Albany, New York
Scott A. Muerhoff, PhD
Volwiler Research Fellow
Infectious Diseases Research and
Development
Abbott Diagnostics
Abbott Laboratories
Abbott Park, Illinois
Anthony O. Okorodudu, PhD, MBA
Professor
Director, Clinical Chemistry Division
UTMB/CMC Outreach Laboratory Services
Department of Pathology
University of Texas Medical Branch
Galveston, Texas
 642
Peter OLeary, BSc, MAACB, AFACHSE,
ARCPA, PhD
Adjunct Professor, School of Public
Health (Curtin)
Adjunct Associate Professor
School of Womens & Infants Health
(UWA),
Director, Office of Population Health
Genomics
Public Health Division
Health Department of Western Australia
Perth, Western Australia, Australia
Matthew T. Olson, MD
House Officer, Department of Pathology
Johns Hopkins Medical Institutions
Baltimore, Maryland
Felix O. Omoruyi, PhD
Fellow
Department of Pathology
Clinical Chemistry
University of Texas Medical Branch
Galveston, Texas
Mauro Panteghini, MD
Professor of Clinical Biochemistry and
Clinical Molecular Biology
University of Milan Medical School
Laboratorio Analisi
Milan, Italy
Gerardo Perotta, MPA
Interim-Coordinator, Pathology
Education
Department of Pathology and Laboratory
Medicine
University of Cincinnati College of
Medicine
Cincinnati, Ohio
Michael A. Pesce, PhD
Professor Emeritus of Pathology and
Cell Biology
Columbia University Medical Center
New York Presbyterian Hospital
New York, New York
Julia M. Potter, B Med Sc(Hons), MB BS,
PhD, FRCPA
Professor of Pathology
Australian National University Medical
School
Executive Director, ACT Pathology
The Canberra Hospital
Garran, Australian Capital Territory,
Australia
Terry Pry, PhD
Retired-Manager Scientific Affairs,
Asia-Pacific
Abbott Diagnostic Division
Abbott Laboratories
Auckland, New Zealand
Kishor Raja, BSc(Hons), MSc, PhD, CSci
Principal Clinical Scientist/Honorary
Senior Lecturer
Clinical Biochemistry Department
Kings College Hospital
London, United Kingdom
Jordan Reynolds, MD
Resident Physician
University of Cincinnati
Department of Pathology and Laboratory
Medicine
Cincinnati, Ohio
Ken Robertson, BSc, AAIMS
Senior Scientist in Charge (Research)
PathWest Laboratory Medicine
Royal Perth Hospital
Wellington St. Perth, Western
Australia, Australia
Andrea M. Rose, PhD, MBA
Senior Clinical Support Consultant
Roche Diagnostics
Indianapolis, Indiana
Enrico Rossi, PhD, MAACB
Research Biochemist
PathWest Laboratory Medicine
Nedlands, Western Australia, Australia
Randal J. Schneider, MS, PhD
Director of Clinical Chemistry and
Toxicology
ProHealth Care Laboratories
Waukesha, Wisconsin
Les Shaw, BS, PhD
Professor
University of Pennsylvania
School of Medicine
Philadelphia, Pennsylvania
Run Zhang Shi, PhD
Instructor
Assistant Director of Clinical
Chemistry and Immunology
Department of Pathology
School of Medicine
Stanford University
Stanford, California
 643

Ravinder Jit Singh, PhD John G. Toffaletti, PhD

Co-Director, Endocrine Laboratory Professor in Pathology
Organization Clinical Laboratories and Department of
Mayo Clinic Pathology
Rochester, Minnesota Duke University Medical Center
Durham, North Carolina
Patricia Slev, PhD
Assistant Professor of Pathology Susan Vickery, MSc, PhD
(Clinical) Senior Clinical Scientist
University of Utah, East Kent Hospitals University
Medical Director NHS Trust
Serologic Hepatitis and Retrovirus Kent and Canterbury Hospital
Laboratory Canterbury, Kent, United Kingdom
ARUP Laboratories
Salt Lake City, Utah Ping Wang, PhD, DABCC
Medical Director of Clinical Chemistry
Ramasamyiyer Swaminathan, MBBS, MSc, The Methodist Hospital
PhD, FRCPath Houston, Texas
Professor and Head of Department of
Chemical Pathology Gregory Ward, BSc(Hons), MSc, MAACB,
St. Thomas Hospital FAACB
London, United Kingdom Head, Biochemistry and Endocrinology
Sullivan Nicolades Pathology (Sonic
Danyal B. Syed, BSc, MA, PhD, C(ASCP), Healthcare)
CC(NRCC), DABCC, FACB Brisbane, Queensland, Australia
Laboratory Director
William F. Ryan Community Health Center Alan H. B. Wu, PhD
New York, New York Professor, Laboratory Medicine
University of California, San Francisco
Danyel H. Tacker, PhD, FACB Clinical Chemistry and Toxicology
Clinical Chemist Laboratories
Ochsner Medical CenterNew Orleans San Francisco General Hospital
New Orleans, Louisiana San Francisco, California

Jillian R. Tate, BSc(Hons), MSc Odette Youdell, BAppSci(Hons), MAACB

Senior Scientist Senior Scientist
Pathology Queensland Clinical Biochemistry
Chemical Pathology Department Alfred Pathology Service
Royal Brisbane and Womens Hospital Melbourne, Victoria
Brisbane, Queensland, Australia Australia
 644

Foreword
Foreword for the 2009 edition of Methods in Clinical Chemistry

In the mid-1980s we perceived a need for an extensive, up-to-date, compilation of methods available for
use in clinical chemistry laboratories. To meet this need, we published in 1987 Methods in Clinical
Chemistry with C.V. Mosby. This volume provided not only a review of extant methodologies, but also a
critique of each method. This enabled the authors, when appropriate, to suggest one technique as a
recommended method. Since its initial publication, Methods in Clinical Chemistry has been repeatedly
updated and made available in electronic form (CD-ROM or Internet) by Pesce Kaplan Publishers.

Like the previous version, this edition is published in parallel with the current edition of our textbook,
Clinical Chemistry: Theory, Analysis and Correlation (5th edition; Elsevier, 2010). The editors of this
work, Peter Hickman and Gus Koerbin, have assembled an international group of expert clinical chemists
from the United State, Europe, and Australia/New Zealand. We have retained the scope of previous
editions, including:
144 revised method reviews of available technologies for the analysis of each analyte,
a critique of each methodology,
analytical quality goals (when available),
recent references,
a suggested procedure for manual methods.

This edition will be available in both electronic (CD-ROM) and printed (two volumes) formats. It is our
hope that this edition will be widely used and vigorously reviewed by its users. Using new software
technology, we will provide a mechanism for input from readers for future versions of this edition. We
have created an Internet site (http://www.pescekaplan.com/) where individuals can publicly post their
comments. Eventually, the Editors will redact the suggestions into changes incorporated into Methods in
Clinical Chemistry.
 645

Preface for the 2009 edition of Methods in Clinical Chemistry

It was certainly an honor and privilege to be invited by Larry Kaplan and Amadeo Pesce to undertake the
editorial role for the 2009 edition of Methods in Clinical Chemistry. The challenge for us was to provide a
text that was contemporary, detailed but readable, and of paramount importance, a useful resource and
laboratory tool for both students and professionals as we near the end of the first decade of this
millennium.

Much in clinical chemistry has changed since the first edition was published in 1987: the breadth and
depth of our knowledge base, the development of laboratory technology, and the widespread use of the
internet and digital technology. Review of each chapter and final publication for this edition of Methods
in Clinical Chemistry has been vastly different from the original compilation, with Internet and digital
technology significantly streamlining the process. In 2003, the methods portion of the text was removed
from the 4th edition of Kaplan and Pesces Clinical Chemistry Theory, Analysis, Correlation and provided
as a CD-ROM. This edition includes 131 new and revised methods plus 13 older methods that were not
revised. Each method contains a critique of alternate methodologies; contemporary and historical,
analytical quality goals, and performance data. Methods in Clinical Chemistry is not only offered in a CD-
ROM/DVD format, but also in a two volume printed format. In addition, the methods will be accessible to
purchasers of the 5th edition of Kaplan and Pesces Clinical Chemistry Theory, Analysis, Correlation via
the publishers (Elsevier) Internet site, Evolve.

We have been very fortunate to collaborate with an outstanding team of 76 authors from all over the
world. Our clinical chemistry experts reside in 16 states of the United States, most states of Australia, and
in New Zealand, Egypt, Italy, Belgium and England, making this a truly international project. Without the
contributions of these industry leaders, this volume could not have been completed and to the team we say
a heartfelt thank you. It is somewhat clich to say that it takes a cast of thousands to produce a body of
work such as this, but without these authors and the help of the following individuals, we would not have
succeeded: Professor Julia Potter, Executive Director of ACT Pathology, Canberra, Australia for
supporting our undertaking of this project, Priscilla Delatorre for her patience, along with her formatting
and word processing skills, Nataliya Polyakov of the College of American Pathologists Surveys team for
her assistance in accessing proficiency data (usually at very short notice), Elseviers Ellen Wurm-Cutter
for her valued advice and particularly for her patience and tenacity in keeping us on schedule and to her
editorial assistant, Jennifer Hermes, for her behind-the-scenes contribution. No list of acknowledgements
would be complete without a mention of the respected and appreciated guidance, praise, constructive
criticisms and most of all encouragement that Larry Kaplan and Amadeo Pesce have provided throughout
this process, which commenced in early 2007.

We trust that this edition will be widely used and reviewed by you the reader. We invite you to use the
internet site (http://www.pescekaplan.com/) created to accept your valued input, comments and
suggestions so that future editions of Methods in Clinical Chemistry can respond to the ever changing
needs of the laboratory professional into the second decade of this millennium and beyond.

We intend to continue with this project, progressively updating methods and releasing a new version as
dictated by progress in the field.
 646

We also intend to include more international contributors, with the hope that it would become the world
methods reference. Finding appropriate authors from diverse countries has proven to be a challenge and
we ask for recommendations from our colleagues.
.

Peter Hickman
Gus Koerbin
Australia, 2009
 647

Methods
1. 25-OH-Vitamin D 24. Barbiturates* Page 205
Ravinder Jit Singh Page 17 25. Bence Jones Protein
2. 1-Antitrypsin Stanley S. Levinson Page 219
John Beilby Page 28 26. Benzodiazepines* Page 232
3. Acetaminophen 27. Beta-hCG (beta-human chorionic
Gus Koerbin, gonadotropin)
David G. Hughes, James J. Miller Page 251
Julia M. Potter Page 33 28. Beta-2-Microglobulin
4. Adrenocorticotropic Hormone (ACTH) James J. Miller Page 257
Hassan M. E. Azzazy Page 43 29. Bilirubin
5. Alanine Aminotransferase R. Swaminathan Page 261
James J. Miller Page 46 30. Blood Gas Analysis and Oxygen
6. Albumin Saturation
Kee Cheung Page 52 Goce Dimeski Page 275
7. Albumin in Urine 31. C-Reactive Protein (CRP)
Graham Jones Page 61 Odette Youdell Page 288
8. Alcohol 32. Calcium
Ping Wang Page 67 Randal J. Schneider Page 296
9. Aldolase* Page 81 33. Cancer Antigen 125 (CA 125)
10. Aldosterone Hassan M. E. Azzazy Page 304
Hassan M. E. Azzazy Page 85 34. Carbamazepine
11. Alkaline PhosphataseTotal Gus Koerbin,
Danyal B. Syed Page 90 Julia M. Potter Page 307
12. Alpha-Fetoprotein 35. Carbohydrate Antigen 15-3 (CA 15-3)
Gerald J. Mizejewski Page 101 Gregory A. Hobbs Page 317
13. Aluminum (Aluminium) 36. Carbohydrate Antigen 19-9 (CA 19-9)
Tony Badrick Page 117 Hassan M. E. Azzazy Page 320
14. Amino Acid Screening 37. Carbon Dioxide and Bicarbonate
Kevin Carpenter Page 124 William J. Korzun Page 323
15. Ammonia 38. Carcinoembryonic Antigens (CEA)
Elizabeth M. Hall Page 128 Gregory A. Hobbs Page 327
16. Amniotic Fluid Phospholipids 39. CatecholaminesPlasma
(AFPL) LS Ratio and PG Brett McWhinney Page 337
Hassan M. E. Azzazy Page 132 40. CatecholaminesUrine
17. Amylase Brett McWhinney Page 344
Ming Jin Page 150 41. Cerebrospinal Fluid (CSF) Protein
18. Angiotensin Converting Enzyme Quantitation
(ACE) Danyel H. Tacker,
Hassan M. E. Azzazy Page 157 Anthony O. Okorodudu Page 363
19. Anion Gap 42. Ceruloplasmin
Tony Badrick Page 162 Ahmed Zakaria Page 372
20. Anticonvulsive Drugs* Page 165 43. Chloride
21. Apolipoproteins A-1 and B William J. Korzun Page 377
Jillian R. Tate Page 181 44. Cholesterol
22. Aspartate Aminotransferase John R. Burnett,
James J. Miller Page 190 Ken Robertson Page 382
23. B-Type Natriuretic Peptide, 45. Cholinesterase
Danyal B. Syed Page 393
NT-proBNP, and ProBNP
Alan H.B. Wu Page 197 46. CMV (Cytomegalovirus)
Terry Pry, Greg Maine Page 405
 648

47. Copper The following analytes are in Volume

K. Raja, R. Swaminathan Page 410 II
48. Cortisol
R. Swaminathan Page 420 70. Glucose
49. Creatine Kinase John Beilby Page 651
Mauro Panteghini Page 430 71. Glycated Hemoglobin
50. Creatine Kinase Isoenzymes Andrea M. Rose Page 662
Mauro Panteghini Page 436 72. Haptoglobin
51. Creatinine Joris R. Delanghe Page 670
Edmund J. Lamb Page 440 73. Hepatitis B
52. Cyclosporin (Cyclosporine A) Paul Coleman Page 674
David W. Holt, 74. Hepatitis C Virus
Denise A. McKeown, A. Scott Muerhoff Page 680
Atholl Johnston Page 452
75. High-Density Lipoprotein (HDL)
53. D-Xylose* Page 470 Cholesterol
54. Dehydroepiandrosterone and its John R. Burnett,
sulfate (DHEA and DHEA-S) Ken Robertson Page 686
Gus Koerbin Page 481 76. Holotranscobalamin
55. Digoxin and Digitoxin Marion Black Page 698
Randal J. Schneider Page 489 77. Homocysteine
56. Drug Screen Sheila Dawling Page 703
Christopher R. McCudden Page 495 78. Homovanillic Acid
57. Estradiol Lawrence A. Kaplan Page 718
Greg Ward Page 516 79. Human Immunodeficiency Virus (HIV)
58. Estriol* Page 520 Patricia Slev Page 728
59. Ethylene Glycol 80. Immunoelectrophoresis
Ping Wang Page 535 Stanley S. Levinson Page 740
60. Fecal Electrolytes and Osmolality 81. Immunoglobulin Quantitation
Felix O. Omoruyi, Karen Golemboski Page 753
Anthony O. Okorodudu Page 544 82. Insulin and C-Peptide
61. Fecal Fat and Fat Absorption Steven C. Kazmierczak Page 762
Lawrence A. Kaplan Page 547 83. Ionized Calcium
62. Fecal Occult Blood John G. Toffaletti Page 769
R. Swaminathan Page 559 84. Iron and Iron-Binding Capacity
63. Ferritin Gerardo Perrotta,
Hassan M. E. Azzazy Page 567 Jordan Reynolds Page 781
64. Folic Acid 85. Ketones
Sheila Dawling Page 573 Lawrence A. Kaplan Page 785
65. Follicle-Stimulating Hormone (FSH) 86. Lactate Dehydrogenase and Lactate
Angela Ferguson Page 586 Dehydrogenase Isoenzymes
66. Free Thyroxine and Free Mauro Panteghini Page 793
Triiodothyronine 87. Lactic Acid
Greg Ward Page 589 Steven C. Kazmierczak Page 797
67. Gamma-Glutamyl Transferase (GGT) 88. Lead
Danyal B. Syed Page 609 Gus Koerbin Page 803
68. Gastric Fluid Analysis* Page 618 89. Lipase
69. Gentamicin and Other Ming Jin Page 814
Aminoglycosides 90. Lipoprotein (a)
Danni L. Meany, Gregory A. Hobbs Page 820
William Clarke Page 625
91. Lithium
Danni L. Meany,
William Clarke Page 823
 649

92. Luteinizing Hormone 115. Pyruvic Acid

Gregory A. Hobbs Page 828 Steven C. Kazmierczak Page 1019
93. Lysozyme* Page 834 116. Renin
94. Magnesium Greg Ward Page 1025
Steven C. Kazmierczak Page 840 117. Rheumatoid Factor
95. Maternal Fetal Screening Terry Pry Page 1030
Peter OLeary, 118. Rubella
Barry Lewis Page 846 Terry Pry, Greg Maine Page 1035
96. MetanephrinesUrine 119. Salicylates
Brett McWhinney Page 856 Gus Koerbin,
97. Methotrexate Julia M. Potter Page 1043
Michael A. Pesce Page 865 120. Serum Protein Electrophoresis
98. Methylmalonic Acid Sandra Klingberg Page 1052
Kevin Carpenter, 121. Sirolimus
Kathryn Green Page 875 Magdalena Korecka,
99. Mycophenolic Acid Michael Milone,
Michal J. Figurski, Leslie M. Shaw Page 1062
Magdalena Korecka, 122. Sodium and Potassium
Leslie M. Shaw Page 880 William J. Korzun Page 1070
100. Myoglobin 123. Steroid Hormone
Alan H.B. Wu Page 892 Receptors* Page 1078
101. Opiates* Page 899 124. Sweat Electrolytes: The Sweat Test
102. Organic Acid Screening Ronda F. Greaves Page 1104
Kevin Carpenter Page 912 125. T3 Uptake
103. Osmolality Run Zhang Shi Page 1115
Goce Dimeski Page 916 126. Tacrolimus
104. Oximetry Michael C. Milone,
Goce Dimeski Page 921 Michal Figurski,
105. Parathyroid Hormone (Parathyrin) Magda Korecka,
(PTH) Leslie M. J. Shaw Page 1120
Susan Vickery, 127. Testosterone
Edmund J. Lamb Page 934 Greg Ward, Gus Koerbin,
106. Phenylalanine* Page 939 Peter E. Hickman Page 1127
107. Phenytoin 128. Theophylline and Caffeine
Gus Koerbin Page 946 Saeed A. Jortani Page 1140
108. Phosphorus and Phosphate 129. Thyroglobulin (Tg)
Steven C. Kazmierczak Page 957 Run Zhang Shi Page 1151
109. Plasma Free Metanephrines 130. Thyroid Autoantibodies
Brett McWhinney Page 964 Run Zhang Shi Page 1158
110. Porphobilinogen Screening and 131. Thyroid-Stimulating Hormone (TSH)
Quantitation Greg Ward Page 1168
Enrico Rossi Page 974 132. Thyroxine (Total)
111. Procainamide and N- Greg Ward Page 1175
Acetylprocainamide 133. Total Serum Protein
Matthew T. Olson, Kee Cheung Page 1182
William Clarke Page 982 134. Transferrin and Carbohydrate-
112. Progesterone Deficient Transferrin
John Galligan Page 991 Sandra Klingberg Page 1191
113. Prolactin 135. Transthyretin (Prealbumin)
Sheila Dawling Page 996 Danyel H. Tacker,
114. Prostate Specific Antigen (PSA) Anthony O. Okorodudu Page 1201
Hassan M. E. Azzazy Page 1013 136. Tricyclic Antidepressants
Jinong Li, William Clarke Page 1206
 650

137. Triglycerides 141. Urine Porphyrin Quantitation

John R. Burnett, Enrico Rossi Page 1258
Ken Robertson Page 1213 142. Urine Protein, Total
138. Troponins Susan Vickery,
Jillian R. Tate, Edmund J. Lamb Page 1264
Mauro Panteghini Page 1224 143. Vitamin B12
139. Urea Joe DAgostino Page 1270
Elizabeth M. Hall Page 1246 144. Zinc
140. Uric Acid Tony Badrick Page 1276
Elizabeth M. Hall Page 1252
*Updated in the last edition.
651
Glucose
Glucose
John Beilby
Name: Glucose,
Clinical significance: Refer to Chapter 38, Diabetes, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular formula: C6H12O6
Molecular mass: 180.16 D
Merck Index: 4319
Chemical class: Carbohydrate
Structure:
NRSCL Definitive and Reference Methods:
i commonly used to measure glucose in body fluids.
Principles of Analysis and Current Usage
Chemically, glucose is an aldohexose, with the
aldehyde form in equilibrium with the
glucopyranose form (which is shown above). The
latter is the favored structure at physiological pH.
The aldehyde/enediol equilibrium allows glucose to
be reduced and oxidized with facility.
All modern methods for glucose analysis employ
enzymes as reagents to increase analytical
specificity. The enzymes used are the hexokinase,
glucose oxidase, and glucose dehydrogenase
reactions. All method types have been automated,
with resulting high specificity and precision. A
specimen blank is used to correct for interfering
substances that absorb at 340 nm. There are many
suppliers of commercial reagents, and therefore the
general principles are described here.
Current Methods In Use Hexokinase- and
glucose oxidasebased methods are the two most
i
Glucose
Previous and current authors of this method:
First edition: Lawrence A. Kaplan
Methods edition: Lawrence A. Kaplan
Second edition: Lawrence A. Kaplan
Third edition: Steven C. Kazmierczak
Fourth edition: Lawrence A. Kaplan
Fifth edition: John Beilby
dextrose
NCCLS RS1-A
Survey data from the 2007 College of American
Pathologists (CAP) Participant Summary Report
showed that 58% of the participating laboratories
used hexokinase-based methods, with 92% of these
laboratories measuring the product at 340 nm.
Glucose oxidasebased methods were used by 42%
of laboratories. Of these laboratories, 60% used a
glucose oxidaseoxygen electrode combination.
Less than 1% of laboratories used glucose
dehydrogenase methods.
Whole blood samples, anticoagulated with lithium
heparin are frequently analyzed for glucose as part
of the profile on blood-gas instruments. These
instruments use a glucose oxidaseencased oxygen
electrode method. They can be laboratory-based or
used for point-of-care testing [1] in, for example,
emergency rooms and intensive care units.
Nonlaboratory-based methods are finger-prick
blood methods for monitoring glucose
concentrations in diabetic patients and urine
dipsticks for semi-quantitative measurement of
glucose.
Hexokinase Methods
The hexokinase method involves two coupled
reactions. The hexokinase reaction phosphorylates
hexoses with ATP and Mg2+. Glucose 6-phosphate
dehydrogenase (G6PD) reacts specifically with
glucose-6-phosphate produced by the hexokinase
reaction to yield 1 mole of nicotinamide adenine
652
Glucose
dinucleotide phosphate (NADP+ or NAD+) for each
mole of glucose that is oxidized. The amount of
NADPH produced is directly proportional to the
amount of glucose in the sample and is measured
by absorbance at 340 nm. G6PD derived from yeast
is used with the cofactor NADP+. Most assays use
an enzyme derived from bacteria that employs
NAD+ as a substrate rather than the NADP+ used
by mammalian enzymes. The advantage of using
the bacterial enzyme is that red blood cell G6PD
and 6-phosphogluconate dehydrogenase, which use
NADP as a substrate, do not interfere in the
analysis of glucose. This reduces the interference in
the assay resulting from hemolysis. Although other
hexoses can enter into the hexokinase reaction,
normal serum concentrations of these sugars do not
cause significant interference.
Glucose + ATP hexokinase
glucose-6-phosphate + ADP
glucose-6-
Glucose-6-P + NADP+ (NAD+) phosphate
dehydrogenase
6-phosphogluconate + NADPH (NADH) + H+
The hexokinase assay is not commonly performed
as a kinetic procedure because of the requirement
for a rapid initial reading. However, it is often
performed as an end-point reaction using
bichromatic measurements to help correct for
interferences.
Glucose Oxidase Methods
The glucose oxidase method uses two coupled
enzyme reactions. In this case, the initial reaction is
the specific one, and the indicator reaction is
nonspecific. The first reaction employs glucose
oxidase (GO) to oxidize glucose to glucuronic acid
and hydrogen peroxide. Glucose in solution is in
the ratio of 36% in the form and 64% in the
form. Since GO is highly specific for -D-glucose,
glucose requires mutarotation to the form, and
most preparations of GO contain the enzyme
mutarotase to catalyze the conversion of -D-
glucose to the form. The hydrogen peroxide from
the GO reaction can be measured by several
different techniques. In one method using dry
chemistry techniques (Vitros 250, Ortho Clinical
Diagnostic), the hydrogen peroxide reacts with a
non-colored dye precursor to produce a colored
dye. The intensity of the dye is monitored
photometrically at 540 nm using reflected light.
-D-glucose + O2 glucose oxidase
D-gluconic acid + H2O2
2H2O2 + 4-aminoantipyrine + 1,7-
dihydroxynaphthalene peroxidase
red dye
Many POCT analyzers, including blood-gas
instruments, measure glucose concentrations using
the GO method. In these instruments, a silver
cathode and a platinum anode are surrounded by an
electrolyte solution and a multi-layer membrane
mounted at the tip. The membrane consists of an
outer membrane permeable to glucose, a middle
enzyme layer which contains immobilized glucose
oxidase, and an inner membrane layer permeable to
H2O2. The H2O2 is transported across the inner
membrane to the platinum anode at a negative
potential of 675 mV and is oxidized. The electric
current produced is proportional to the amount of
H2O2, which in turn is directly related to the
amount of glucose in the sample. To complete the
electrical circuit, a reaction at the cathode converts
two Ag+ (from AgCl) to Ag for each molecule of
H2O2 oxidized.
Glucose
Glucose + O2 gluconic acid + H2O2
H2O2 2H + O2 + 2e-
+
Glucose Dehydrogenase Methods
In 1975, a relatively pure form of glucose
dehydrogenase (GDH) was isolated [2]. This
allowed the development of a glucose method
based solely on this highly specific enzymatic
reaction:
glucose dehydrogenase
-D-glucose + NAD+
+
D-glucono--lactone + NADH + H
Mutarotase is added to convert glucose from the
to the form of glucose. The reaction is monitored
at 340 nm as either a kinetic or an end-point
reaction. Glucose dehydrogenase isolated from
Bacillus cereus is highly specific for glucose, and
the results produced are in close agreement with
the hexokinase method. This GD method has been
adapted for use on several automated instruments
[3,4].
Measurement of Urine Glucose
Urine dipsticks are widely used to screen for
glucose in the urine. All strips use glucose oxidase
with a chromogenic assay. An example of a
reaction used in this method is to detect the H2O2
produced from the GO reaction, where potassium
iodide acts as a final oxygen acceptor, resulting in a
change of color.
653
Glucose
glucose oxidase
Glucose + 2H2O + O2
glucuronic acid + 2H2O2
H2O2 + 2KI peroxidase
I2 (green to brown) + KOH
Other compounds, such as 3,3,5,5-
tetramethylbenzidine and 1,7-
dihydroxynaphthalene, whose oxidized forms are
colored, are also used. However, testing for urine
glucose cannot be used for the diagnosis or
monitoring of diabetic patients for the following
reasons:
1. Urine dipstick methods are semi-quantitative
techniques that have a low sensitivity for the
diagnosis of diabetes and are not
recommended for routine care of patients with
diabetes or as a screening tool for diabetes
[5,6].
2. The renal threshold, the blood glucose level at
which glucose is detected in urine, averages
160 to 180 mg/dL (8.9 to 10.0 mmol/L) but
varies widely between individuals. It can be
lower in children and during pregnancy and
higher in longstanding diabetes.
3. A negative test does not distinguish between
euglycemia or hypoglycemia.
4. The method is not specific for glucose and
may suffer from interferences by fluid intake,
ascorbic acid, and urinary tract infections.
Reference and Preferred Methods
The definitive method for glucose determination is
isotope dilution mass spectrometry (ID-MS). This
technique measures the analyte concentration with
the greatest accuracy, so the systematic errors are
negligible, and there is a high level of precision
with the coefficient of variation (CV) is < 0.5%.
The technique for glucose is based on the addition
to the sample of [13C6]glucose, a stable isotope, as
the internal standard. The chemical behavior of the
internal standard and the analyte are the same. The
ratio between the unlabelled and labeled substances
remains unchanged during the overall procedure,
and no correction for sample recovery is needed
[7]. External quality assurance programs use this
technique to set the target values for their
lyophilized sera [8]. Manufacturers also use this
technique to accurately determine glucose
concentrations in calibrators for glucose assays.
A reference method for glucose based on the
hexokinase reaction has been developed and
validated [9]. This technique is accurate and
precise but is too time consuming for routine use.
Plasma or serum samples are deproteinized using
solutions of barium hydroxide and zinc sulfate. The
resulting supernatant is incubated at 25C with a
reagent containing hexokinase, G6PD, NAD+, and
ATP until the reaction is complete. The final step is
to measure the amount of NADH produced.
Blanks, quality control samples, and calibrators are
carried through the entire procedure.
Glucose concentrations are commonly used for the
diagnosis and monitoring of diabetes, and all the
methods need to be precise and accurate, especially
since cutoff values used for the diagnosis of
diabetes must apply to all methods.
All of the enzymatic methods discussed under
Current Methods (above) show adequate
performance.
Specimen Collection and Storage
The choice of specimen used for glucose
determination depends on the analytical method to
be used. Serum or plasma, free of hemolysis, is the
specimen of choice for automated enzymatic
methods. Heparinized whole blood specimens can
be used on instruments using ion-selective
electrodes. However, the glucose concentration in
whole blood is approximately 12% to 15% lower as
compared with plasma because of the higher water
content of plasma. Plasma is recommended for the
diagnosis of diabetes, since the diagnostic cutoff
points have been derived using plasma samples.
Glucose concentrations in heparinized plasma are
approximately 5% lower than serum [10]. Other
body fluids, such as cerebrospinal fluid (CSF),
pleural fluid, and urine can also be analyzed. CSF
may contain bacteria or excess numbers of cells
that metabolize glucose and should be analyzed
immediately after collection. If a delay is
unavoidable, the sample must be stored at either
4C or frozen at 20C or collected into a
fluoride/oxalate specimen tube. The CSF must be
clear for assay. Centrifugation may be necessary if
the sample is not clear.
A plasma sample drawn after an overnight fast of 8
to 16 hours is used for the diagnosis of diabetes.
The plasma should be separated from the cells
within 60 minutes of collection unless the tube
contains a glycolysis inhibitor [11]. Serum samples
are appropriate for glucose analysis, providing the
serum is not in contact with the cells for longer
than 90 minutes. Glucose in whole blood at room
temperature can undergo glycolysis at a rate of
approximately 5% to 7% (10 mg/dL or ~ 0.6
mmol/L per hour). The sample should be
centrifuged and removed from clot or cells as soon
as possible. In patients with leukocytosis or in
samples that are contaminated with bacteria, the
rate of glycolysis can be even higher. To preserve
blood that cannot be separated rapidly, samples
should contain the glycolysis inhibitor sodium
fluoride (which inhibits the enolase enzyme) at 2.5
mg fluoride/mL of blood. This reagent is used in
654
Glucose
combination with anticoagulants such as potassium
oxalate. The glucose concentrations are stable for
72 hours at room temperature with fluoride, but
there is a 10% drop in the concentration due to a
water shift from the cells. The glucose
concentration does not change in serum or plasma
samples collected in tubes that contain gel
separators. The glucose in these tubes is stable for
at least 1 week when stored at 4C.
Other fluids such as ascitic, pleural, peritoneal, and
drainage fluids can be analyzed for glucose. These
samples should be collected into tubes containing
fluoride/oxalate preservative. If the samples are
turbid, this suggests the presence of cells, and
samples must be centrifuged prior to analysis.
Capillary blood glucose measurement is widely
used to monitor glucose levels in diabetic patients.
Concentrations are approximately 2 to 5 mg/dL
(0.11 to 0.28 mmol/L) higher than venous blood
glucose concentrations in fasting patients.
However, following a glucose load, the difference
can be as great as 70 mg/dL (3.9 mmol/L).
Interferences
The disadvantage of the coupled glucose oxidase
assays is that many compounds present in serum or
urine (such as bilirubin, ascorbic acid, and uric
acid) can be oxidized by the hydrogen peroxide
produced by the glucose oxidase reaction, resulting
in a negative bias. The negative bias resulting from
the reaction between the hydrogen peroxide and
ascorbic acid can often lead to strikingly low
values in cerebrospinal fluid (CSF) [12]. This is
because glucose concentrations are relatively low
in CSF, and ascorbate concentrations in CSF are
higher than in serum [13]. Since the extent of the
interference is inversely related to the glucose
concentration, CSF samples from neonates are
particularly vulnerable to this form of interference.
The coupled glucose oxidase reactions performed
as a kinetic analysis are especially sensitive to
ascorbate interference [12]. Also, because
ascorbate is very unstable and breaks down rapidly,
the degree of interference will vary with time and
temperature of storage before analysis. This
interference can be avoided if all CSF specimens
are analyzed by an alternative technique that does
not use a glucose oxidasehydrogen peroxidase
generating method. These include the hexokinase,
rate oxygen consumption, and glucose
dehydrogenase methods [14].
In addition, there may be compounds that react by
oxidizing the indicator dye, resulting in positive
biases. The specificity and accuracy of the coupled
glucose oxidase procedures have been increased by
a choice of reaction conditions that minimize this
bias. The most common approach is to employ
kinetic analysis of the reaction. By selection of the
most appropriate initial and subsequent times for
spectrophotometric reading, a reduction in both
direct photometric and chemical interferences can
be made.
The semiquantitative dipsticks employing glucose
oxidase and peroxidase are highly specific for
glucose, with no other sugar producing a reaction.
Strong oxidizing substances such as hypochlorite
and chlorine bleach can produce a positive
reaction, and ascorbic acid at high levels can
interfere with the peroxidase step, reacting with the
hydrogen peroxide to give erroneously low glucose
concentrations.
Glucose Reference Intervals and Interpretation
There are many conditions that lead to low or
elevated blood glucose levels (see Chapter 38,
Diabetes, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation). However, the
primary reason to measure glucose is for the
diagnosis of diabetes.
Reference intervals are of use for neonates and
children, but the diagnosis of pre-diabetes and
diabetes is done using specific criteria published by
various Diabetes Associations. The new cut-points
recommended by the American Diabetes
Association (ADA) [15] are as follows:
Fasting blood glucose:
Normal glucose: < 100 mg/dL (<5.6 mmol/L)
Pre-diabetic: 100 to 125 mg/dL (5.6 to 6.9 mmol/L)
Diabetic: 126 mg/dL (7.0 mmol/L)
The ADA does not recommend the use of an oral
glucose tolerance test (OGTT) for the routine
diagnosis of type 1 or 2 diabetes. However, it does
recommend the use of the glucose challenge test
for the diagnosis of gestational diabetes mellitus
(GDM) [16]. Other expert bodies such as the World
Health Organization and the Australian Diabetes
Society recommend the use of an OGTT to screen
for type 2 diabetes [17,18]. Important limitations of
the OGTT are poor reproducibility, cost, and
inconvenience. However, it may have a higher
sensitivity than the fasting plasma glucose (FPG)
for detecting diabetes [19]. This is a controversial
area, and it is likely that further changes will be
made to the diagnostic criteria for diabetes as more
clinical studies are completed.
The Australian Diabetes Society criteria for
diagnosis of diabetes using a fasting sample are
[18]:
Normal glucose- 3.0-6.0 mmol/L
Impaired fasting glucose (IFG)- 6.1-6.9 mmol/L
Diabetic- 7.0 mmol/L
655
Glucose

A fasting blood glucose result 7.0 mmol/L in

subjects with typical symptoms is diagnostic of Performance Goals
diabetes. In subjects without symptoms, a Survey data from the 2007 CAP Participant
confirmatory test, preferably a fasting plasma Summary Report showed that 53% of laboratories
glucose concentration should be performed on a used hexokinase methods measured at 340 nm,
separate day. In addition, if a formal laboratory 25% used glucose oxidaseoxygen electrode
FPG measurement is between 5.5 and 6.9 mmol/L, methods, 17% glucose oxidase colorimetric
then a 75 g OGTT should be performed to exclude methods, 5% hexokinase colorimetric methods, and
diabetes [18]. less than 1% of laboratories use glucose
dehydrogenase (Table 2). The imprecision of these
Cord blood is a mixture of neonatal arterial and methods at a mean glucose concentration of 153.6
venous blood that is removed from the umbilical mg/dL (8.45 mmol/L) demonstrated that the
cord at the time of birth. Cord blood can be used glucose oxidase methods were the most precise,
for a variety of measurements, including blood with a mean CV of 2.0% to 2.4%, followed by the
gases, bilirubin, and glucose. The fetuss blood hexokinase UV methods, with a mean CV of 2.5%.
glucose concentration in cord blood is usually The glucose dehydrogenase methods have a mean
about 10% lower than the maternal blood at the CV of 2.7%.
time of birth [20].
Table 2. Methods Used for Glucose Analysis in 5259
Most new cases of type 1 (juvenile or insulin- Laboratories Enrolled in CAP 2007
dependent) diabetes are diagnosed in the acute Method Total Number of CV (%)
stage in the emergency room. In these cases, the (%) Laboratories
diabetic patient presents with hyperglycemia and Hexokinase,
ketoacidosis. Abnormalities of electrolytes and measured at 53% 2787 2.5% at mean
renal function tests are also common findings in 340 nm 153.9 mg/dL
type 1 diabetics acutely ill with ketoacidosis. (8.54 mmol/L)
The 95% confidence intervals in the glucose Hexokinase, 5% 250 2.8% at mean
concentrations of important cutoff points can be colorimetric 153.5 mg/dL
calculated by multiplying the analytical CV for the (8.52 mmol/L)
assay (CVa) by 1.96 [21]. If the CVa for the assay
were 2.0%, then for a true plasma glucose Glucose
concentration of 126 mg/dL (7.0 mmol/L), the 95% oxidase, 17% 878 2.4% at mean
confidence intervals (CI) for that result would be color 146.8 mg/dL
121 to 131 mg/dL (6.7 to 7.3 mmol/L). At a (8.15 mmol/L)
glucose concentration of 200 mg/dL (11.1 Glucose 25% 1316 2.0% at mean
mmol/L), the 95% confidence intervals would be oxidase 157.5 mg/dL
192 to 208 mg/dL (10.7 to 11.5 mmol/L). Table 1 oxygen (8.74 mmol/L)
lists the 95% CI around the major glucose cut- electrode
points for a CVa of 2.0% and 4.0%, which are the
approximate low and high CVa data for the 2007 Glucose <1% 17 2.7% at mean
CAP results. dehydroge 153.9 mg/dL
nase (8.54 mmol/L)
As can be seen from these results, the larger the
analytical CV, the greater uncertainty exists around CV, Coefficient of variation as published by the
the cutoff value. Therefore, it is important to keep 2007 College of American Pathologists Participant
the CVa as tight as possible. Summary Report.

Table 1
95% Confidence Intervals at Several Plasma Glucose Concentrations
Plasma 100 110 126 mg/dL 200 mg/dL (11.1 mmol/L)
glucose mg/dL mg/dL (7.0
(5.6 (6.1 mmol/L)
mmol/L) mmol/L)
Analytical 2.0 96-104 106-114 121-131 192-208
CV (%) (5.4-5.8) (5.9-6.3) (6.7-7.3) (10.7-11.5)
4.0 92-108 101-119 116-136 184-216
(5.2-6.0) (5.6-6.6) (6.5-7.5) (10.2-12.0)
656
Glucose
Acceptable Clinical Laboratory Improvement
Amendments performance criteria (CLIA-88) for
measurement of glucose require that laboratories be
accurate to within 6 mg/dL or 10% (greater) of
the peer-group mean. The intraindividual variation
of glucose in blood in healthy adults has been
determined to be approximately 6.5%. Desirable
specifications for analytical imprecision derived
from studies of biological variation indicate an
assay imprecision of no greater than 3.3% and a
total error of no greater than 7.9% [22]. All glucose
methods are within the desired performance criteria
as defined by CLIA-88 and the analytical
imprecision specification if biological variability
defines required performance.
Glucose Meters
Management of glycemia in diabetes is crucially
important for the prevention
of both acute and long-term complications. Self-
monitoring of blood glucose (SMBG) is effective
for patients with type 1 and type 2 diabetes using
insulin. The ADA recommends the use of SMBG
for all insulin-treated patients with diabetes, those
treated with sulfonylureas or other insulin
secretagogues, and in patients not achieving
glycemic goals [23]. However, there is debate
regarding the effectiveness of SMBG as a self-
management tool for type 2 diabetics who are not
using insulin. Welschen et al. reviewed the
literature and concluded that two of six studies
reported a significant lowering of HbA1c in
patients using SMBG [24]. It was concluded that
SMBG may be effective in improving glycemic
control in patients with type 2 diabetes who are not
on insulin. However, a large, well-conducted
random-controlled trial is required.
Individuals with diabetes use SMBG in an attempt
to maintain their glucose levels as close to those of
a nondiabetic as is possible. Due to the imprecision
of the meters and the difference in results between
different manufacturers of meters, they are not
recommended for the diagnosis of diabetes but only
for the monitoring of treatment.
The methodology used in the strips is the same as
described for laboratory glucose analysis. A drop
of blood is applied to the strip that contains all the
necessary reagents. Some strips use
electrochemistry to detect the flow of electrons
from the enzymatic reaction by an electrode
incorporated in the strip. Others use a dye that
reacts with the glucose oxidase-peroxidase
chromogenic reaction, and the color intensity is
proportional to the glucose concentration and is
quantified by reflectance photometry that measures
the rate of reaction or the final concentration of the
product. There are a wide variety of meters
available, and they all have slightly different
performance characteristics. All meters require
calibration. This can be automatic or done
manually with each lot change of reagents. The
manufacturers supply quality controls that must be
regularly checked to ensure the performance of the
meter. A number of the larger manufacturers also
provide external quality assurance programs. There
are multiple analytical goals proposed for the
performance of glucose meters.
The results from glucose meters are not as accurate
as those obtained using laboratory methods,
especially in the hypoglycemic range, where
accurate measurement is crucial and can
overestimate glucose concentrations in the higher
range [25-28]. Many analytical goals have been
proposed for the performance of glucose meters.
The ADA recommends the analytical error for
glucose results should be within 5% of the
reference values [29]. The Clinical and Laboratory
Standards Institute (CLSI) uses the ISO 15197
guidelines that recommend results for meters be
within 15 mg/dL (0.83 mmol/L) for glucose
concentrations less than 75 mg/dL (4.2 mmol/L)
and within 20% for higher blood glucose values
when compared with compared with the central
laboratory [19]. A meter is considered within the
guidelines if 95% of pairs meet these criteria in
both glucose ranges.
There are many preanalytical and analytical factors
that can reduce the accuracy of the meter results.
Preanalytical variables that may affect the
performance of SMBG are hematocrit, temperature,
hypoxia, low systolic blood pressure, elevated
triglycerides, and some drugs such as ascorbic acid
and acetaminophen. Other causes of poor results
include improper calibration and use of controls,
poor storage of test strips, lack of maintenance, and
the differences in the technology of the
instruments. Kost suggests a number of strategies
that may help to improve the performance of
meters [30]. These include standardized calibration,
proficiency testing based on the same global
standard, and improved technologies that eliminate
the effects of co-founding factors.
Whole blood glucose levels are 10% to 15% lower
than the values obtained from plasma samples. In
current clinical practice, plasma and blood glucose
are used interchangeably, with a consequent risk of
clinical misinterpretation. In human blood, glucose
is distributed (like water) between erythrocytes and
plasma. Many of the modern meters will
automatically adjust the glucose reading by
multiplying the whole blood glucose concentration
by a constant factor of 1.11 to convert the
concentration in whole blood to the equivalent
concentration in plasma [1]. The conversion will
provide harmonized results, facilitating the
657
Glucose
classification and care of patients, and lead to fewer
therapeutic misjudgments.
A recent study of glucose-meter accuracy was
reported comparing two of the newer generation
meters for the detection of subsequent nocturnal
hypoglycemia. Glucose concentrations were
measured throughout the day and night and every
15 to 20 min during a standardized exercise
protocol [27]. Reference samples were assayed in a
central laboratory, using a hexokinase enzymatic
method. Sensitivities for detection of hypoglycemia
were 96% and 100% for the two methods, and
corresponding false-positive rates were both 5%.
Thus in a controlled clinical setting using venous
blood samples, both meters had a high degree of
accuracy compared with the laboratory reference
over a broad range of glucose concentrations in
children with type 1 diabetes. However, a number
of other studies have not shown such good
correlation [30-32].
Minimally Invasive Glucose Monitoring
Continuous glucose monitoring system (CGMS)
technology is in a developmental phase, but in the
future will significantly change diabetes
management. The use of insulin-pump therapy
offers better diabetic control with reduced
postprandial hyperglycemia and reduced risk of
severe hypoglycemia, especially during the night
[33]. However, for this kind of therapy to be really
successful, a system that continuously monitors
glucose and is in real-time communication with the
infusion pump to provide the amount of insulin
needed for the maintenance of normal glucose
levels is required.
There are a number of CGMS on the market, and
more are under development [34]. These systems
measure glucose levels in subcutaneous interstitial
fluid. Recent reports describe the use of CGM in
clinical settings [35], but the monitors are not
easily used on a routine, clinical, long-term basis.
The CGMS developed by Medtronic, Medtronic
MiniMed (Northridge, California), was the first
continuous glucose monitoring device approved by
the U.S. Food and Drug Administration. The
CGMS sensor, consisting of glucose oxidase
plated platinum electrode is inserted through a
needle into the subcutaneous tissue of the anterior
abdominal wall and measures interstitial glucose
concentrations. Real-time glucose measurements
can also be continuously transmitted using wireless
technology to a glucose sensor.
Another FDA-approved glucose sensing system is
the GlucoWatch 2 Biographer (GW2B) (Cygnus,
Redwood City, California). It is worn on the
forearm, and glucose is pulled from the interstitial
fluid underneath the skin by reverse iontophoresis.
In a randomized clinical trial of 200 children with
type 1 diabetes mellitus, the use of the GW2B with
conventional glucose meter monitoring did not
lower HbA1c levels or the frequency of
hypoglycemia, compared with conventional
glucose monitoring alone [36,37].
A double-blind crossover study of insulin-treated
type 2 diabetes compared the use of the MiniMed
CGMS with SMBG in 160 individuals. Over the
16-week treatment period, it showed that the
CGMS was superior to SMBG in assessing daily
glucose fluctuations and in detecting unrecognized
hypoglycemic episodes, especially at night [38].
A study that compared CGMS with SMBG
techniques in patients with gestational diabetes
mellitus showed that CGMS detected a higher
proportion of mothers needing antihyperglycemic
medication, compared with self-monitoring of
plasma glucose [39].
CGMS techniques appear to hold considerable
promise, and once the technology becomes more
robust they are likely to be widely used.
Spectroscopy-based and fluorescence-based
noninvasive sensors are under development and
hold promise for the future, but the technology
needs further improvement before it can be used
clinically [40,41].
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18. Twigg SM, Kamp MC, Davis TM, Neylon
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20. Bossolan G, Trindade CE, Barreiros RC.
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24. Welschen LM, Bloemendal E, Nijpels G,
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26. Johnson RN, Baker JR. Error detection
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28. Wehmeier M, Arndt BT, Schumann G,
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38. McNally PG, Dean JD, Morris AD,
Wilkinson PD, Compion G, Heller SR.
Using continuous glucose monitoring to
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39. Kestila KK, Ekblad UU, Ronnemaa T.
Continuous glucose monitoring versus
self-monitoring of blood glucose in the
treatment of gestational diabetes mellitus.
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41. Pickup JC, Hussain F, Evans ND, Rolinski
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660
Glucose

Table 3: Methods of Glucose Analysis

Method 1: Hexokinase (HK); quantitative, spectrophotometric K or EP
Principle of analysis:
Glucose + ATP HK glucose 6-phosphate + ADP
Glucose 6-phosphate + NADP+ G6PD 6-phosphogluconate + NADPH + H+
Increased absorbance at 340 nm related to glucose concentration
Comments: Serum, CSF, urine; automated; most commonly used method; has been proposed as basis of
reference method; very good accuracy and precision
Method 2: Glucose oxidase coupled reaction (Trinder)
a. Quantitative, using various types of dyes as final O2 acceptor; K or EP
b. Quantitative or semiquantitative in dipstick screen, visual or reflectance photometry
Principle of analysis:
Glucose + O2 glucose oxidase gluconic acid + H2O2
H2O2 + reduced dye horseradish peroxidase oxidized dye + H O
2
(colored)
Peroxidase indicator reaction increased absorbance related to glucose concentration
Comments:
a. Serum, urine, CSF; easily and usually adapted to automated analysis; second indicator reaction
susceptible to false-positive interferences from a variety of compounds; good accuracy and precision
b. Used in all dipstick screens; serum, urine

Method 3: Glucose dehydrogenase (GDH); quantitative, EP, K

Principle of analysis:
GDH
Glucose + NAD+ D-gluconolactone + NADH + H
+

Increased absorbance at 340 nm is related to glucose concentration

Comments: Rare, serum, CSF; can be adapted to automated instruments

Method 4: Glucose oxidase (GO) oxygen consumption; quantitative, polarographic measurement using O2
electrode; K
Principle of analysis:
Glucose + O2 glucose oxidase gluconic acid + H2O2
H2O2 consumed in side reactions
O2 consumption measured polarographically by oxygen electrode
Comments: Serum, CSF; semiautomated and fully automated systems; correlates best with the proposed
reference method; very good accuracy and precision

EP, End-point analysis mode; CSF, cerebrospinal fluid; GDH, glucose dehydrogenase; K, kinetic analysis mode.

Historic Methods method used for glucose analysis. Although it is a

Most older methods for measurement of serum
glucose were based on the ability of glucose to
directly reduce cupric ions (Cu 2+) to monovalent
cuprous ions (Cu+). In the presence of heat, the
reduced cuprous ions can form cuprous oxide
(Cu2O), which can be detected by a variety of
methods. The most popular method was the
reduction of phosphomolybdate (FolinWu) or
arsenomolybdate (SomogyiNelson) to form blue
molybdenum compounds. Neocuproine (2,9-
dimethyl-1,10-phenanthroline) reduced by Cu+
ions to form a highly colored complex was a
more sensitive procedure than the other copper-
reduction methods, this method lacked specificity.
Benedicts modification of the copper-reduction
methods is still used today but only as a
semiquantitative method for estimation of urine
glucose. This procedure, sensitive to total reducing
compounds present in urine, yields red Cu2O and
yellow CuOH precipitates. The greater the
concentration of glucose, the redder the final color.
The alkaline ferricyanide reaction involves the
reduction of yellow ferricyanide, Fe(CN) 63, to
colorless ferrocyanide, Fe(CN) 64 , by glucose in
alkaline conditions. When this method was initially
661
Glucose

automated, the reaction was monitored by
measurement of the decrease in yellow color. Later
adaptations of this method in the 1960s and 1970s
to AutoAnalyzer equipment utilized indirect
measurement of ferrocyanide. The ferrocyanide
was reacted with excess ferric ions to form ferric
ferrocyanide (Prussian blue). The o-toluidine
reaction is based on the ability of many aromatic
amines in acid solutions to condense with the
aldehyde group of glucose to form glycosamines.
The most widely used aromatic amine, o-toluidine,
is believed to be a carcinogen and is no longer
used. These methods are no longer used and are
listed only for historic interest.
 662
Glycated Hemoglobin
Glycated Hemoglobin
Andrea M. Rose
Name: Glycated hemoglobin, glycohemoglobin, GHb
Clinical significance: Refer to Chapter 38, Diabetes, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular mass: 64,500 D
Merck Index: 4538 (hemoglobin)
Chemical class: Glycated protein
Reaction: See Figure 1 at end
Principles of Analysis and Current Usage
The IUPAC-IUB Joint Commission on Biochem i cal
Nomenclature suggests glycation as the term that should
be used to describe the nonenzymatic addition of glucose
or other sugars to the free amino groups of proteins [1].
Since the middle to late 1970s, the glycated protein used to
monitor glycemic control in patients with diabetes mellitus
has been glycated hemoglobin (GHb). The major
component of adult hemoglobin is HbA, a tetramer
consisting of two and two subunits. The best
characterized and most abundant adduct of HbA is termed
HbA1c and describes a hemoglobin chain with a glucose
bound to the N-terminal valine of this chain.
Sugars other than glucose have been shown to modify the
amino terminus of hemoglobin and share a nomenclature
with HbA1c based on the order of their elution from an
ion-exchange column. For example, HbA1a, A1b, A1c,
collectively termed HbA1, are also called the fast
hemoglobins, owing to their more negative charge and
earlier elution compared to HbA0. Additionally, lysine
residues located throughout the and chains can serve as
glycation sites. Collectively, glucose residues at the amino
terminus and/or the lysine residues of both and
subunits are referred to as total glycated hemoglobin
(TGlyHb) [2,3].
Measuring glycemic control in patients with diabetes
mellitus has improved in both methodology and
standardization since the previous edition of this chapter.
Current methods in routine use still fall into two broad
categories based on (1) charge differences between
modified and unmodified hemoglobin (ion-exchange
chromatography, electrophoresis), and (2) structural
i a semiautomated electrophoretic method certified in 2006.
Glycated hemoglobin
Previous and current authors of this method:
First edition: Not done
Methods edition: Mary Ellen King
Second edition: Mary Ellen King
Third edition: Andrea Rose
Fourth edition: Andrea Rose
Fifth edition: Andrea Rose
differences that recognize glucose-modified from
nonglucose-modified hemoglobin (boronate affinity,
immunoassay, enzymatic). Standardization effortswhich
have resulted in the formation of the National
Glycohemoglobin Standardization Program (NGSP)and
treatment goals based on outcomes from the Diabetes
Control and Complications Trial (DCCT) [4] and the
United Kingdom Prospective Diabetes Study (UKPDS) [5]
have led the American Diabetes Association (ADA) to
recommend that regardless of methodology or specific
analyte quantified, results should be reported in terms of %
HbA1c or % HbA1c equivalents [6].
Ion-exchange HPLC elutes hemoglobin species based on
differences in their charge. The sample is automatically or
manually injected into a column filled with resin or gel
having ionic functional groups of opposite charge from the
analyte ions. Buffers of increasing ionic strength pass
through the column, displacing the hemoglobin species.
The separated hemoglobin fractions pass through a flow
cell, where absorbance is measured spectrophotometrically.
Background noise can be reduced by using a secondary
wavelength. Software integrates the raw data, and time in
minutes from injection to the maximum point of the
elution peak is compared to an internal library of normal
hemoglobin and variant fractions. Current methods use
whole blood samples in their primary tubes and offer
automatic mixing, dilution, and injection. HPLC offers
excellent precision and the ability to detect the presence of
hemoglobin variants [7,8].
The 2007 College of American Pathologists (CAP)
participant summary, GH2-A, showed no participants
reporting GHb results by electrophoresis. The June 2007
NGSP list of certified methods lists one manufacturer with
Electrophoresis involves the separation of hemoglobin
species based on charge differences. The sample is applied
to an agarose gel, and the hemoglobins migrate across an
electric field. After electrophoresis, bands are scanned by a
densitometer and compared to a standard for quantification.
Boronate affinity chromatography employs a binding
ligand (boronic acid) immobilized to an inert, insoluble
 663
Glycated Hemoglobin
matrix that selectively interacts with cis-diol groups
present in sugars such as glucose to form a strong but
reversible covalent bond [9]. Glycated hemoglobin
remains bound, while nonglycated hemoglobin is eluted
with wash buffer. A soluble diol-containing agent such as
sorbital is then used to displace the glycated hemoglobin.
Both species are detected photometrically at 414 nm.
Boronate affinity chromatography detects total glycated
hemoglobin, meaning the binding ligand can attach to
glucoses at the amino terminus, as well as lysine residues
downstream of both the and hemoglobin chains. The
method, however, can be calibrated to reflect HbA1c
equivalent values. The ligand preferentially binds 1-
deoxyfructosyl derivatives [3].
Immunoassays use antibodies that recognize the glycated
amino terminus of the Hb chains including four or more
amino acids. Commercial methods use antibody-mediated
inhibition of latex agglutination or immunoturbidimetry to
obtain a HbA1c mass value [9]. HbA1c is expressed as a
percent after Hb is measured spectrophotometrically and
the ratio HbA1c/Hb is determined.
According to the NGSP website, one enzymatic procedure
was certified in April 2007 on an automated analyzer.
With this method, whole blood samples are first lysed, and
then the lysate is subjected to extensive protease digestion.
Glycated valines serve as substrate for the recombinant
enzyme, fructosyl valine oxidase. Hydrogen peroxide
formed in the initial reaction, plus an added chromagen,
are catalyzed by horseradish peroxidase to produce a
colored agent that is proportional to the concentration of
GHb. Both the color formed from the chromagen and Hb
are measured spectrophotometrically [10,11].
Reference and Preferred Methods
In 1993, a survey of CAP participants reporting GlyHb
results gave the following breakdown of methods in
routine use: affinity chromatography 35%, ion-exchange
HPLC 35%, electrophoresis 30%. The CAP 2007 GH2-A
participant summary gave the following breakdown of
survey participants by methodology for laboratories
reporting %HbA1c or %HbA1c equivalents: immunoassay
63.7%, HPLC 35.0%, boronate affinity chromatography
1.3%. In 2007, 99% of methods were NGSP certified at the
time of the survey. Only 25 laboratories were reporting
TGlyHb using either of two manufacturers.
The Diabetes Control and Complications Trial completed
in 1993, and subsequent studies such as the Epidemiology
of Diabetes Interventions and Complications (EDIC) and
the UKPDS, showed that the complications associated
with uncontrolled diabetes could be avoided in patients
who maintained strict glycemic control. However, lack of
standardization among laboratories performing GHb
testing led the American Association for Clinical
Chemistry (AACC) to form a subcommittee aimed at
developing a standardization program so that all
laboratories could report values traceable to the DCCT.
The NGSP, formed in 1996, still uses the DCCT reference
method (HPLC using Bio-Rex 70 resin) to provide the
anchor value for all primary and secondary reference labs
(PRL and SRL respectively) that are part of the NGSP
network [12]. This network provides the framework for
manufacturers and laboratories to obtain certification that
their methods are traceable to the DCCT. Bio-Rex 70
HPLC, used in the Central Primary Reference Laboratory
(CPRL) and PRLs, is considered a Designated Comparison
Method (DCM). Although the DCCT/EDIC Research
Group reports that the Bio-Rex 70 HPLC and other HPLC
methods used to support the DCCT and EDIC trials have
been stable and precise long term [13], there are known
interferences associated with this methodology [12,14].
This led the International Federation of Clinical Chemistry
to begin work in 1995 to develop a primary reference
material and reference method that specifically measures
HbA1c.
In 1994, in order to achieve uniform, international
standardization, the IFCC established a working group on
standardization of HbA1c [15]. The first step was to define
the analyte based on molecular structure rather than peak
(retention time) in an HPLC. The nomenclature
recommended by the IFCC/IUPAC is N-(1-deoxyfructos-
1-yl) hemoglobin chain [16]. HbA1c is defined as the
stable glucose adduct to the N-terminal group of one or
both of the -chains of hemoglobin A0, which may or may
not include glucoses attached at lysine residues further
downstream. Glucoses attached at lysine residues without
attachment at the N-terminal valine of the hemoglobin
chain are not defined as HbA1c [14]. The working group
was also charged with isolating pure HbA1c and HbA0,
developing a reference method specific for HbA1c,
establishing a Reference Laboratory Network, and
preparing secondary reference calibrators and controls to
aid manufacturers in standardizing their methods [15]. In
2001, the following final method was adopted by the
national member societies of the IFCC and published in
2002 [17]. Initially, hemoglobin isolated from lysed
erythrocytes is proteolytically digested with
endoproteinase Glu-C, which releases either a glycated or
nonglycated hexapeptide, C4,H9,O4-CO-CH2-NH-Val-His-
Leu-Thr-Pro-Glu-COOH or NH2-Val-His-Leu-Thr-Pro-
Glu-COOH, respectively. Note that cleavage occurs
between the sixth and seventh glutamic acid residues in the
hemoglobin chain. Therefore, the variant hemoglobins S
and C, with substitutions of valine and lysine respectively
at position six, are not cleaved by endoproteinase Glu-C
[17]. Post proteolytic cleavage, the glycated and
nonglycated hexapeptides are separated from other
digested proteins and quantified by HPLC followed by
either electrospray mass spectrometry or capillary
electrophoresis with UV detection. Both systems have
been shown to give identical results. In 2004, members of
the IFCC Working Group on HbA1c Standardization
published a study documenting the linear relationship
 664
Glycated Hemoglobin
between the IFCC method and each of three national
HbA1c standardization schemes from the United States
(NGSP), Japan (Japanese Diabetes Society/Japanese
Society of Clinical Chemistry), and Sweden. While each of
the DCMs differs in recovery from each other and the
IFCC Reference method (ion-exchange HPLC using Bio-
Rex 70 resin, high-resolution ion-exchange HPLC and
national calibrators, Mono S HPLC respectively), with the
linear relationships observed, the lower, more specific
IFCC values can be converted to the respective DCM
values via three distinct regression equations, allowing for
continued use of associated clinical decision points [18].
Specimen
Whole blood drawn in EDTA or other method-specific
anticoagulant may be used. Some methods require cell
lysis with hemolyzing reagent provided by the
manufacturer prior to analysis. Finger stick, as opposed to
venipuncture, may be used with point-of care devices.
Samples are generally stable for one week at 2C to 8C,
but stability may be method specific [19,20]. The literature
suggests that storage at 20C is generally not
recommended [18]; however, the reader should consider
manufacturers guidelines. Whole blood stored at 70C variants. However, some method-specific instances of
has been shown to be stable for one year [21].
Interferences
The accuracy of HbA1c measurements has been shown to
be affected by the following: (1) conditions that cause
anemia or shortened erythrocyte survival, (2) the presence
of hemoglobin variants, and (3) nonglucose adducts to the
hemoglobin chain via carbamylation; acetylation;
vitamin C, E, or alcohol; or elevated concentrations of HbF.
Red blood cells (RBCs) are freely permeable to glucose
and other substances. Since the average RBC life span is
120 days, the % HbA1c value reflects the nonenzymatic
addition of glucose adducts over the past 2 to 3 months [6].
Conditions that increase hemoglobin turnover will
decrease exposure of hemoglobin to glucose [22].
Additionally, hemodialysis patients treated with
erythropoietin to stimulate RBC production may have
HbA1c values that do not reflect glycemic control, owing
to an increased proportion of young erythrocytes [23].
Amino acid substitutions in the and chains of adult
hemoglobin give rise to hemoglobin variants that can
affect the accuracy of diabetes monitoring in a variant and
method-dependent manner. For example, immunoassay
methods use antibodies that recognize 4 to 10 N-terminal
amino acids plus the ketoamine linkage and glucose
[24,25]. Amino acid substitutions close to the amino
terminus might be expected to impact antibody recognition
[26-28]. Clinically significant bias compared to CLC330
boronate affinity, generally accepted to provide accurate
recovery with most hemoglobin variants [9,29], was
observed with both HbS (6Glu-Val) and HbC (6Glu-
Lys) variants, using the Roche COBAS Integra [30] and
the Metrika A1cNow [31] immunoassays. The Beckman
Synchron CX7 [28], Bayer DCA2000 [32], Roche Tina-
Quant immunoassay (Hitachi) [25,32], and COBAS
Integra [33-36] gave accurate recovery with these variants.
Immunoassay methods do not recognize nonglucose
adducts, which may also nonenzymatically attach to
hemoglobin as a result of uremia [37,38], high doses of
aspirin [39], or alcoholism [40].
Cation-exchange chromatography and electrophoresis
separate hemoglobin species based on charge differences.
Inaccurate results occur when a glycated or nonglycated
hemoglobin variant or chemical modification such as
acetylated or carbamylated hemoglobin cannot be
separated from HbA or HbA1c [9]. A seminal review by
Bry et al. [9], as well as other recent reports, describe
variants giving inaccurate results with HPLC [26,31,40-
47]. Interference is HPLC method and variant dependent.
Boronate affinity chromatography measures total glycated
hemoglobin via attachment of the cis-diol groups of
glucose to boronic acid. Because recognition is based on
structure and not charge, affinity chromatography does not
recognize nonglucose adducts and is impervious to most
inaccurate results with hemoglobin variants have been
described [48], including with the common variants S and
C [30,49]. A mutation that results in acetylation of the
amino terminus of the hemoglobin chain and a
consequent inability to glycate the amino terminus has
been described and would be expected to give decreased
reactivity with boronate affinity, as well as immunoassay
[26]. The CLC330 boronate affinity HPLC method from
Primus Corporation has been used in several studies, with
hemoglobin variants as the comparative method. Recently
this method was compared to the IFCC mass
spectrophotometric reference method in support of its
consistent relative recovery in patients with hemoglobin S
and C traits [29]. As noted under Reference and Preferred
Methods, hemoglobins with amino acid substitutions at
position 6, including HbS and HbC, are not recognized in
the IFCC reference method because of lack of enzymatic
cleavage in the initial step. Therefore, the consistent
recovery of the IFCC method (measures the ratio
HbA1c/total HbA) versus the CLC330 boronate affinity
method (measures total glycated Hb/total Hb) assumes no
difference in glycation rate for HbS and HbC [29]. While
this author is aware of no publications citing a difference
in glycation rate for S and C variants, the literature does
support such a difference with some variants [50,51], and
readers are cautioned that some methodologies may not be
suitable for monitoring select patients.
Substances such as ascorbic acid and other adducts that
compete for glucose binding to hemoglobin may result in
an inaccurate and conflicting reflection of the patients
mean blood glucose level as indicated by the HbA1c value,
which may differ with different methodologies [52].
Finally, both charge-based and structure-based methods for
 665
Glycated Hemoglobin
diabetes monitoring may underestimate the HbA1c value
in patients with persistent elevations in HbF and may not
accurately reflect the patients level of glycemic control
[53,54].
Reference Interval
Results from the DCCT and UKPDS clearly demonstrated
that lowering mean blood glucose values, and
consequently HbA1c, could reduce the complications
associated with uncontrolled diabetes. Based on these trials,
the American Diabetes Association currently recommends
that in general patients should achieve an A1c goal of <
7%. The individual goal recommended for each patient is
to achieve an A1c value as close to the high end of normal
(i.e., < 6 %) as possible without significant hypoglycemia
[55]. The high end of normal in this case refers to the
reference interval reported in NGSP values which is ~ 4%
to 6% HbA1c and is based on the outcomes and
instrumentation used in the DCCT and UKPDS outcomes
studies.
The development of the IFCC reference system and
subsequent joint effort to determine the relationship
between the DCM values of Japan, the United States, and
Sweden showed a strong correlation between each DCM
and the IFCC method. However, the increased specificity
of the IFCC reference method means that IFCC HbA1c
values are significantly lowerfor example, 1.5% to 2.0%
HbA1c compared to NGSP [56]. The IFCC method
reference interval is 2.8% to 3.8% HbA1c [57]. Currently,
manufacturers use the IFCC method to provide traceability
and an anchor to a higher-order reference method and
materials, as is required for manufacturers selling to
European Union countries. For patients and clinicians used
to NGSP values, a conversion formula is then used to
convert from IFCC to NGSP. This has led to debate in the
international clinical and scientific communities as to the
following: (1) Should the medical and scientific
communities adopt the lower IFCC values globally, or
should an IFCC to NGSP or other DCM conversion factor
continue to be used to anchor the values to major clinical
trials; (2) Should the name HbA1c be changed to the
metrologically correct name, with a shortened version for
general use, and the results reported in the standard
scientific unit of mmol/mol as supported by the IFCC; or
(3) If an international study currently underway proves
there is a linear relationship between HbA1c values and
mean blood glucose in both type 1 and 2 diabetics and
across ethnic groups, should laboratories report results of
diabetes monitoring in terms of mean blood glucose
[16,57-59]? On average, each 1% change in HbA1c has
traditionally equated to approximately a 2 mmol/L (35
mg/dL) change in mean plasma glucose [55]. A very
recent reassessment of the DCCT data indicates that the
relationship between MPG and HbA1c is not constant, as
previously thought. Moreover, the relationship appears
dependent on whether the patient was treated intensively
or monitored more conventionally, with the less closely
monitored group having a higher MPG at each HbA1c
level than the intensively treated group [60]. The picture is
further complicated by reports that more recent glucose
levels influence HbA1c levels more than earlier ones [61],
HbA1c levels may be partly genetically determined [62],
and that some individuals may be high versus low
glycators [62].
With pressure growing to eliminate differences in HbA1c
reporting globally, in May 2007, the Consensus Committee
of the American Diabetes Association, the European
Association for the Study of Diabetes, the International
Diabetes Federation, and the International Federation of
Clinical Chemistry and Laboratory Medicine reached
agreement and issued the following directives regarding
the worldwide standardization of the measurement of
HbA1c:
1 A1C test results should be standardized worldwide,
including the reference system and results reporting.
2 The new IFCC reference system for A1C represents
the only valid anchor to implement standardization of
the measurement.
3 A1C results are to be reported world-wide in IFCC
units (mmol/mol) and derived NGSP units (%) using
the IFCC-NGSP master equation.
4 If the ongoing average plasma glucose study fulfills
its a-priori-specified criteria, an A1C-derived average
glucose (ADAG) value calculated from the A1C result
will also be reported as an interpretation of the A1C
results.
5 Glycemic goals appearing in clinical guidelines should
be expressed in IFCC units, derived NGSP units, and
as ADAG.
Furthermore, the committee urges that these
recommendations be implemented globally as soon as
possible [64].
Interpretation
Glycated hemoglobins (hemoglobins with covalently
bound sugars) form nonenzymatically in red blood cells in
amounts mostly proportional (see above) to the average
blood glucose level over the 120-day lifespan of the
erythrocyte [65]. Hemoglobin A1, the major glycated
hemoglobin fraction, can be further separated into
subfractions, of which HbA1c is the predominant and best
characterized component. HbA1c differs from its parent
nonglycated hemoglobin, A0, in that it has a glucose
residue covalently linked to the amino group of the N-
terminal amino acid of the chain. An unstable Schiffs
base of glucose and hemoglobin (also termed the labile
component) forms initially and rearranges to the stable
ketoamine product, A1c [66]. Also present are smaller
amounts of glycated hemoglobins, which are products of
nonenzymatic glycation of the hemoglobin chain by
other sugars [67], as well as glucoses attached to lysine
residues of both and chains and at the -chain N-
terminal group [68].
 666
Glycated Hemoglobin
Data from the DCCT and UKPDS showed a reduction in
the microvascular and macrovascular consequences of
uncontrolled diabetes as % HbA1c values approached
nondiabetic levels in the intensively treated group.
However, previous to the formation of and continued
standardization efforts by the NGSP, lack of consensus on
a comparative method for assay standardization, or even
which analyte to measure (i.e., HbA1 [A1a, Alb, A1c],
HbA1c, or total glycated Hb) affected the clinicians
ability to effectively monitor patients and the ADAs
ability to implement recommendations for patient care. As
of the May 2007 survey, CAP proficiency data indicated
99% of laboratories participating were reporting results as
HbA1c or equivalent, and 99% of the methods used were
certified by the NGSP.
Performance Goals
It is generally accepted that U.S. laboratories should use
methods that are certified by the NGSP. Beginning
September 2007, NGSP certification requirements for
manufacturers and laboratories desiring level II
certification tightened from 1% HbA1c for the 95%
confidence interval of the differences to NGSP to 0.85%
HbA1c. The precision criteria will be eliminated. CAP has
in the past utilized a peer group grading system for HbA1c,
but starting with the 2007 GH2-A survey, proficiency
testing participants were required to recover assigned
NGSP target values within a 15% range limit [56]. For
example, acceptable performance at a target value of 6.0 %
HbA1c would be 5.1% to 6.9%. It is recommended that
laboratories achieve an intralaboratory CV of < 3% and an
interlaboratory CV of < 5% [69]. Recent CAP data
suggests that most but not all methods have sufficient
precision to achieve this goal.
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 669
Glycated Hemoglobin

Table 1: Glycated HemoglobinMethods of Analysis

Method 1: Boronate Affinity Column Chromatography

Principle of analysis: Spectral absorbance post separation of glycated and nonglycated hemoglobin. The
glycated component binds to boronate, while the nonglycated component passes through the column.
Comments: Measures total glycated hemoglobin (cis-diol groups present in sugars at the amino terminus,
as well as lysine residues downstream on both the and hemoglobin chains). Can be calibrated to reflect
HbA1c equivalent values. Preferentially binds 1-deoxyfructosyl derivatives.
Method 2: Electrophoresis
Principle of analysis: Spectral absorbance of separated hemoglobin components
Comments: Separation of hemoglobin species is based on charge differences. Any substance with the same
charge as HbA1c is measured as HbA1c.
Method 3: Enzymatic
Principle of analysis: Glycated valines serve as substrate for fructosyl valine oxidase in a coupled reaction
with horseradish peroxidase. The resulting colored agent, together with hemoglobin, is measured
spectrophotometrically.
Comments: New method.
Method 4: Immunoassay
Principle of analysis: Antibody-mediated inhibition of latex agglutination or immunoturbidimetry.
Comments: Measures HbA1c (four-plus N-terminal amino acids of hemoglobin and the ketoamine
linkage and glucose). Amino acid substitutions in this region may affect antibody recognition.
Method 5: Ion-Exchange HPLC
Principle of analysis: Spectral absorbance of separated hemoglobin components
Comments: Separation of hemoglobin species is based on charge differences. Any substance with the same
charge as HbA1c is measured as HbA1c.

Table 2 Comparison of Methods

Method *CLIA Complexity Type of Separation
Boronate Affinity Moderate (manual columns high) Structural differences
Automated HPLC
Electrophoresis High Charge differences
Enzymatic Moderate Structural differences
Immunoassay Moderate (POC waived) Structural differences
Ion-Exchange HPLC Moderate (manual columns high) Charge differences
*CLIA Complexity from www.accessdata.fda.gov

Figure 1. Formation of stable product.

670
Haptoglobin
Haptoglobin
Joris R. Delanghe
Name: Haptoglobin, Hp
Clinical significance: Refer to Chapter 40, Hemoglobin, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular mass: 85,000 D (type 1-1); other genetically determined types (2-1, 2-2)
form a series of higher-molecular-weight polymers
Chemical class: Glycoprotein
Principles of Analysis and Current Usage
i spectrophotometrically as reduced absorbance. The
Early methods for quantitation of serum haptoglobin
were based on formation of a complex between
haptoglobin (Hp) and hemoglobin (Hb). A known
amount of hemoglobin was added to serum. If no
complex was formed, no Hp was present; if all the Hb
formed Hb-Hp complexes, the concentration of Hp was
greater than the concentration of Hb; and if complexes
formed, leaving free Hb, the concentration of Hp was
less than the concentration of added Hb. The free Hb
was separated from the Hb-Hp complex by use of
electrophoresis or gel filtration, and the hemoglobin in
each component was measured by use of its peroxidase
activity (Table 1, Method 1). The amount of total Hb
complexed with Hp was the hemoglobin-binding
capacity and was taken as a measure of the Hp level.
Reference intervals for methods based on binding of Hb
were expressed as milligrams of Hb bound per liter.
Current methods for measuring haptoglobin are
immunological, including nephelometry (Table 1,
Method 2a), turbidity (Table 1, Method 2c), radial
immunodiffusion (RID) (Table 1, Method 2b), and
enzyme-linked immunosorbent assays (ELISA, Table 1,
Method 2d). In the nephelometric method, serum
containing an unknown amount of haptoglobin is mixed
with anti-haptoglobin antibody. The formation of an
antigen-antibody complex results in light scattering
when a beam of monochromatic light is passed through
the sample. The degree of light scattering is proportional
to the concentration of haptoglobin [1,2]. An alternative
nephelometric procedure that measures the rate of
formation of the antigen-antibody complex is available
[3]. This method has been shown to correlate closely
with a hemoglobin-binding assay [3].
Turbidimetric methods (Table 1, Method 2c) are similar
to nephelometric assays in that haptoglobin binds to an
anti-haptoglobin antibody forming a complex, which
causes turbidity. The turbidity is measured
i
Haptoglobin
Previous and current authors of this method:
First edition: Robert S. Franco
Methods edition: Robert S. Franco
Second edition: Not updated
Third edition: Steven C. Kazmierczak
Fourth edition: Harini Patel and Joris R. Delanghe
Fifth edition: Joris R. Delanghe
intensity of the incident light beam is reduced because of
the light-scattering effects (Rayleigh and Mie scattering)
of the immune complexes [2]. Addition of polyethylene
glycol is used to accelerate the formulation of the
complex. Readings usually occur at 340 nm. The
absorbance is plotted against Hp concentration on a
calibration curve; the absorbance is inversely
proportional to the concentration of the haptoglobin.
Enzyme-linked immunoadsorbent assays (ELISA, Table
1, Method 2d) are the most recent development in the
measurement of haptoglobin. In one of these techniques
[4], a specific monoclonal antibody (MoAb) to
haptoglobin was produced using pooled fecal extracts
containing the Hp antigen from patients with colorectal
cancer. The MoAb developed was coated on polyvinyl
chloride (PVC) microtiter plates. The extracts from feces
are added and incubated at 4C overnight, allowing
haptoglobin to bind to the MoAb. This complex is
reacted with a streptavidinhorseradish peroxidase
conjugate to form a sandwich. After washing, the plates
are developed by adding substrate (2,2-azino; 3-
bisethylbenzthiazoline-6-sulfonate [ABTS]) and citrate
buffer containing 0.02% hydrogen peroxide. The
absorbance at 405 nm is directly related to the amount of
Hp.
Radial immunodiffusion is no longer routinely used,
because the method is not very precise. Furthermore,
serum concentrations of the Hp2-2 phenotype are
underestimated because of the difficulty of migrating in
agar. In the RID method, an antibody to haptoglobin is
evenly dispersed in a thin, two-dimensional gel. Small
holes, or wells, are cut into the gel. A small amount of
serum (typically 5 L) is placed into the well and
allowed to diffuse radially into the gel surrounding the
well, forming a precipitin ring. The concentration of
haptoglobin in the sample is directly related to the
diameter or area of the ring.
Another ELISA technique [5,6] is based on the reaction
of the carbohydrate moiety of haptoglobin with a
mannosyl-specific lectin, concanavalin A (Con A). Con
A is immobilized to polystyrene microtiter plates.
Haptoglobin reacts quantitatively with the Con A,
forming a stable complex. Polyclonal or monoclonal Hp
antibodies [5,6] conjugated with horseradish peroxidase
are allowed to react with the haptoglobin complex,
forming a sandwich. Again, substrate is added to
produce a colored product measured
671
Haptoglobin
spectrophotometrically. The absorbance at 405 nm is
directly related to the amount of Hp. The range of Hp
binding to Con A is 25 to 300 g/L using polyclonal
antibodies, and 50 to 600 g/L using monoclonal anti-
haptoglobin antibody. Streptococcal cells with
haptoglobin binding receptors have also been used as
Hp-capture proteins in an ELISA system [7].
A horizontal electrophoresis method (Table 1, Method 3)
using a discontinuous polyacrylamide gel has been
described for measurement of haptoglobin polymorphs
from autopsy blood samples. This method has
advantages over the traditional vertical gel method,
including smaller sample requirement, easier gel
preparation, and shorter run time [8].
Reference and Preferred Methods
There is no reference method for measuring haptoglobin
concentration. Nephelometric and turbidimetric methods
are to be preferred because of their suitability for
automated analysis in combination with a good
analytical performance. Nephelometry is often used
when large numbers of samples must be analyzed. This
method is available on automated instruments.
Turbidimetry may be an attractive alternative if this is
automated on random-access chemistry analyzers.
Advantages of this method include a small sample size, a
fast turnaround time with results available the same day,
and relatively high precision. Disadvantages are
relatively high reagent costs and the need for a
specialized piece of equipment.
Radial immunodiffusion features a very small sample
size, extreme simplicity when commercially prepared
plates are used, and essentially no capital investment.
One disadvantage of RID is the relatively long time
before the precipitin rings can be measured, typically 18
to 48 hours.
The sensitivity of the concanavalin A ELISA assay is
higher than that of affinity immunoelectrophoresis by an
order of 1000 [9]. Binding of the Hp in the ConA ELISA
depends on the mannose-type carbohydrate moiety of
haptoglobin. Thus changes in the concentration of
haptoglobin in various pathological conditions could be
related to quantitative differences in hepatic haptoglobin
glycosylation [9]. The comparison of the reaction
conditions for RID, nephelometry, and turbidimetry of
haptoglobin is found in Table 2.
Specimen
Serum from an unhemolyzed blood specimen is used for
haptoglobin determination. The serum may be stored for
1 week at 2C to 8C or frozen at 20C for longer
storage [10]. Avoid repeated freeze/thaw cycles.
Interferences
Specimens should not be hemolyzed. Samples that are
grossly lipemic or turbid may interfere with some
nephelometric or turbidimetric methods.
Haptoglobin Reference Interval
An international reference preparation for plasma
proteins, lot CRM 470, certified by the Bureau of
Reference of the European Community (BCR), has been
adopted worldwide [2]. Calibrated against this standard,
the reference interval for haptoglobin in serums of adults
is between 0.3 and 2.0 g/L [11]. This reference interval
includes all three main subtypes of haptoglobin.
Newborns have little or no haptoglobin; their levels
approach those of adults by about 4 months [12]. Males
reportedly show haptoglobin concentrations that are 20%
to 25% greater than those found in females [13].
There are two allelic, autosomal genes (Hp1 and Hp2),
resulting in three major phenotypes (Hp1-1, Hp2-1, and
Hp2-2). Haptoglobin reference intervals are phenotype-
dependent: 0.57 to 2.27 g/L (Hp1-1), 0.44 to 1.83 g/L
(Hp2-1), and 0.38 to 1.5 g/L (Hp2-2) [2,14,15].
Expression of the Hp gene is absent in
anhaptoglobinemia (Hp0-0 phenotype), a condition
present in approximately 1 in 1000 Caucasians. In
blacks, especially of West African origin (Nigeria,
Cameroon), anhaptoglobinemia is more frequent
(>30%). In the United States, frequency of Hp0-0 in
blacks is considerably less: 4%. Hypohaptoglobinemia
has also been reported in a few nonblack families
carrying a silent allele with no gene product, Hp0
[2,15].
Interpretation
Haptoglobin is a genetically polymorphic serum protein
that binds free hemoglobin and thereby facilitates
hemoglobin removal by the reticuloendothelial system.
A decreased haptoglobin concentration is therefore
evidence of increased erythrocyte destruction.
Several studies indicate that for European and American
whites, type 1-1 occurs in 13% to 21% of the population,
type 2-1 in 47% to 59%, and type 2-2 in 28% to 36%.
Type 1-1 is more common in blacks, whereas type 2-2
predominates in Asian populations.
Haptoglobin is a positive acute-phase protein; that is, it
becomes elevated in blood after an acute disease or
shock to the body. It may be especially elevated in
seriously burned patients. In severe psychiatric
conditions, such as major depression, very high Hp
concentrations are found.
Haptoglobin is decreased in intravascular hemolysis as a
result of binding with hemoglobin, with subsequent
removal of the hemoglobin-haptoglobin complex by the
liver. The rate of removal of these complexes by the
liver is very high (serum Hb-Hp complexes show a half-
life of a few minutes). Serum Hb levels also decrease in
extravascular hemolysis if the capacity of removal by the
reticulo-endothelial system is insufficient and the free
hemoglobin enters the bloodstream. Note that
simultaneous hemolysis and inflammation may result in
a low, normal, or high serum haptoglobin level.
672
Haptoglobin
Because of the limited hemoglobin-binding capacity (1
mole of Hp binds 1 mole of Hb) [16], haptoglobin can
only be used as a parameter of mild or moderate
hemolysis. Hemopexin is a heme-binding plasma protein
which, after Hp, forms the second line of defense against
hemoglobin-mediated oxidative damage during
hemolysis. A decrease in plasma hemopexin
concentration reflects a recent release of heme
compounds in the extracellular compartment. Heme-
hemopexin complexes are cleared by receptor-mediated
endocytosis. Hemopexin determination in tandem with
Hp is useful in the assessment of hemolysis and allows
for the monitoring of the severity of hemolysis after
depletion of Hp. After saturation of the Hb binding
capacity, concentrations of serum hemopexin start to
decrease, whereas the Hp concentration remains low
(<0.3g/L). During intense hemolytic diseases,
monitoring of serum hemopexin should be preferred to
monitoring Hp. In contrast to other markers for
hemolysis (e.g., lactate dehydrogenase activity,
potassium), Hp and hemopexin concentrations are not
influenced by in vitro hemolysis [15].
One report describes the measurements of haptoglobin
concentration in the serum to differentiate between
malignant and nonmalignant tumors of the ovary [6].
Another report describes the detection of haptoglobin in
fecal matter by use of an ELISA technique [4]. The
authors argue that detection of haptoglobin has many
advantages compared to the current fecal occult blood
test for early detection of colorectal carcinoma. These
advantages include no requirement for dietary restriction
and the ability to differentiate between upper and lower
gastrointestinal bleeding with the same sensitivity and
specificity of the current test.
References
1 Van Lente F, Marchand A, Galen RS.
Evaluation of a nephelometric assay for
haptoglobin and its clinical usefulness. Clin
Chem 1979;25:2007.
2 Fink PC, Roemer M, Haeckel R, et al.
Measurements of proteins with the Behring
Nephelometer: a multicentre evaluation. J Clin
Chem Clin Biochem 1989;27:261-276.
3 Sternberg JC. A rate nephelometer for
measuring specific proteins by
immunoprecipitin reactions. Clin Chem
1977;23:1456.
4 Xing PX, Young GP, Ho D, Sinatra MA,
McKenzie IF. A new approach to fecal occult
blood testing based on the detection of
haptoglobin. Cancer 1996;78:48-56.
5 Katnik I, Dobryszcka W. Development of
concanavalin Aenzyme immunosorbant assay
for glycated haptoglobin using polyclonal and
monoclonal antibodies. J. Immunoassay
1992;13:145-162.
6 Katnik I, Judach J, Kmieciak K, Gerber J,
Dobryszcka W. Measurement of haptoglobin by
the reaction with concanavaline a in serum of
patients with ovarian tumors. Eur J Clin Chem
Clin Biochem 1995;33:727-732.
7 Katnik I, Lammeler C, Guszczynski T,
Dobryszycka W. Quantitation of human
haptoglobin by ELISA system based on
streptococcal haptoglobin receptors. Arch
Immunol Ther Exp 1993;41:105-109.
8 Quarino L, Samples M, San Pietro D, Shaler R,
Orta A, Jack D. Haptoglobin typing of
bloodstains using horizontal discontinuous
polyacrylamide gel electrophoresis. Sci Justice
1995;35:213-216.
9 Valette I, Pointis J, Rondeau, Y, et al. Is
immunochemical determination of haptoglobin
phenotype dependent? Clin Chim Acta
1979;99:1-6.
10 Dale JC, Pruett SK. Phlebotomy a minimalist
approach. Mayo Clin Proc 1993;68:249-255.
11 Dati F, Schumann G, Thomas L, et al. A new
international reference preparation for proteins
in human serum (RPPHS). Eur J Clin Chem
Clin Biochem 1996;34:517-520.
12 Davis ML, Austin C, Messmer BI, Nichols
WK, Bonin AP, Bennett MJ. IFCC-
standardized pediatric reference intervals for 10
serum proteins using the Beckman Array 360
system. Clin Biochem 1996;29:489-492.
13 Hashimoto S, Miwa M, Akasofu K, et al.
Changes in serum proteins of post-menopausal
women. Maturitas 1991;13:23-33.
14 Van Rijn HJ, Vander Wilt W, Stroes JW,
Schrijver J. Is turbidimetric immunoassay of
haptoglobin phenotype dependent? Clin
Biochem 1987;20:245-248.
15 Langlois MR, Delange JR. Biological and
clinical significance of haptoglobin
polymorphism in humans. Clin Chem
1996;42:1589-1600.
16 Nyman M. Serum haptoglobin: methodological
and clinical studies. Scand J Clin Lab Invest
1959;11:suppl 39.
673
Haptoglobin

Table 1: Methods of Haptoglobin Analysis

Method 1: Hemoglobin-binding
a. Electrophoresis
b. Gel filtration
Principle of analysis:
a. Hb separated from Hb-haptoglobin by difference in electrophoretic mobility; peroxidase stain for
hemoglobin sued to visualize bands
b. Hb separated from Hb-haptoglobin by molecular weight difference; quantitation by measurement of
A415 of hemoglobin
Comments:
a. Historical; semiquantitative
b. Not suitable for clinical use; quantitative
Method 2: Immunochemical
a. Nephelometry
b. Radial immunodiffusion
c. Turbidimetry
d. ELISA
Principle of analysis:
a. Antibody reacts with haptoglobin, forming complex that scatters light
b. Haptoglobin diffuses into gel containing anti-haptoglobin antibody; precipitin ring diameter
proportional to concentration
c. HpAb reacts with Hp to form complex which results in turbidity; the turbidity is proportional to the
concentration of Hp
d. Hp reacts with a binding agent (Ab, lectin) coated on a solid phase to form a complex; this reacts with
anti-HpAb on a second site on Hp to form a sandwich; this complex is proportional to the concentration of Hp
Comments:
a. Currently in clinical use; suitable for large number of samples
b. Currently in clinical use; suitable for small number of samples
c. Currently in clinical use; suitable for labs with automated equipment
d. Currently in clinical use; suitable for labs with automated equipment
Method 3: Electrophoretic
Principle of analysis:
Various Hp types are separated by charge
Comments:
Not currently in use; small sample volume

Table 2: Comparison of Conditions for Haptoglobin Analysis

Condition RID* Nephelometry Turbidimetry
Temperature 23 2C (RT) RT RT
pH 7.4
Time of reaction 18 h 6.0 min 20 sec
Sample Serum Serum Serum, plasma
Sample size 5 L 10 L 50 L, diluted
Reportable range 25-2760 mg/L 42-1300 mg/dL 20-1200 mg/dL
Precision: Mean (% CV) 143 mg/L (7.0%) 178 (3.9%) 86 (8.3%)

*Kallestad Endoplate, Sanofi Diagnostics Inc., Chaska, MN

Behring Nephelometer Analyzer (BNA), Dade Behring Diagnostics Inc., San Jose, CA
Behring Turbitimer (Turbitimer), Dade Behring Diagnostics Inc., Westwood, MA
674
Hepatits B Virus
Hepatitis B Virus
Paul Coleman i
Name: Hepatitis B virus (HBV)
Clinical significance: Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
The hepatitis B virus (HBV) was discovered in the early
1960s, using a blood sample from an Australian
aborigine (therefore, HBV was initially called Australia
antigen) [1]. The discovery of Australia antigen quickly
led to the development of diagnostic assays for hepatitis
B surface antigen (HBsAg), which continues to be a key
marker for HBV infection today. Forty years after its
discovery, HBV remains a global health care challenge,
with an estimated 350 million people currently infected
[2]. The worldwide prevalence of HBV infection is
about 10-fold higher than the global AIDS cases
estimated by the UN AIDS agency. As a consequence of
HBV infection, about 80 million chronic carriers have
progressed to hepatocellular carcinoma, and over
500,000 deaths occur each year. Infection can be
controlled by such measures as universal vaccination,
passive immunization, and more recently antiviral
therapy. Early diagnostic detection coupled with
infection-control measures has led to a significant
reduction in HBV infection rates in developed countries
[3].
HBV is classified in the genus Orthohepadnavirus of the
Hepadnaviridae family. It has a small, partially single-
stranded, circular DNA genome of approximately 3.2
kilobases that is unique in structure. The minus-strand
DNA is the size of the entire genome, whereas the plus-
strand DNA is about half this length [4]. The
encapsulated viral DNA contains a covalently linked
polymerase and an RNA oligomer to prime the
completion of plus-strand DNA [5]. The HBV genome
contains four genes with partially overlapping, open
reading frames (ORFs) that encode seven proteins: the
polymerase protein (Pol gene); core antigen and e
antigen (C gene); large, medium, and small surface
antigen proteins (S gene); and the X protein (X gene).
Surface antigen (HBsAg) forms the outer envelope of
the virion that surrounds the inner core (HBcAg),
protecting the single-copy genome. The X protein has
been implicated in the integration of HBV DNA into the
host genome and can also suppress viral replication; the
multifunctional polymerase protein directs the
replication of HBV DNA.
The HBV DNA replication cycle is unique as it proceeds
through an error-prone RNA reverse transcriptase
intermediary step where misincorporation of nucleotides
into the copy genome results in variants. Although gene
overlap constrains some viral variability, mutant or
variant forms have been identified for all four HBV
i
Hepatitis B
New method:
Fifth edition: Paul Coleman
genes. Examples are the recent emergence of vaccine
escape mutants with altered HBsAg [6] and antiviral-
resistance mutants with altered polymerase proteins [7].
Phylogenetic analysis of genome sequences has led to
the classification of HBV into eight genotypes (A to H)
based on a nucleotide divergence of approximately 25%
[8].
Acute infection with hepatitis B can result in a
distinctive yellow jaundice and may be accompanied by
fever, chills, fatigue, nausea, loss of appetite, and
abdominal pain. These symptoms usually subside after
several weeks. Acute infection outcomes can range from
asymptomatic infection to a fulminant hepatitis that is
rapidly fatal. Chronic infection is usually asymptomatic.
HBV transmitted vertically from mother to child usually
results in chronic infection 80% of the time, where the
child lacks a robust immune response to neutralize and
clear the virus. Chronic perinatal infection accounts for a
disproportionately large share of worldwide morbidity
and mortality. Chronic infection over a period of many
years often leads to cirrhosis, progression to liver cancer,
and death. In contrast, horizontal transmission to adults
through sexual contact, unsafe injections, household
contact, or other percutaneous transmission generally
results in about 90% of cases in an acute resolving
infection. An HBV vaccine is available (consisting of
recombinant HBsAg) which can be initiated at birth; it
provides long-term protection in more than 90% of
healthy people. These infection profiles can be further
complicated by co-infection with the hepatitis D virus
that requires HBV for its replication cycle.
The generalized appearance of HBV seromarkers in an
acutely infected patient is presented in Figure 1. Note
that a window period is present between the onset of
infection and the detection of HBV seromarkers. The
acute HBV infection-window period extends from 1 to 3
months (mean 59 days), as shown by an extensive
clinical study [9]. Improvements in diagnostic
technology continue to narrow this window period
during which an infectious, asymptomatic patient is
capable of spreading disease [10]. Closing the detection
window also allows earlier therapeutic intervention with
improved prognosis for the patient.
During the infection cycle, HBV secretes large amounts
of incomplete virions consisting of HBsAg only and also
secretes e antigen (HBeAg) in a process that is thought
to challenge the host immune response and permit
complete virions to continue the infection cycle.
675
Hepatits B Virus
Diagnostic monitoring of HBV antigens and the
subsequent host antibody response can differentiate both
the stage and severity of infection, as indicated in Table
1. The sentinel diagnostic markers for detecting HBV
infection are HBsAg and HBV DNA. A standardized
testing approach has recently been proposed [11].
Table 1: Description of HBV Seromarkers
and Their Diagnostic Outcome
HBV HBV infection outcome
Seromarker
HBsAg Persistence for longer than 6
months can indicate chronic
infection
Anti-HBs Seroconversion to anti-HBs
can indicate immunity
HBeAg Determines relative
infectivity
Anti-HBe Seroconversion to anti-HBe
indicates progression
towards infection resolution
Anti-HBc IgM Differentiates acute/recent
infection from chronic
carrier state or resolved
HBV infection
Anti-HBc Helps to establish the stage
of chronic infection
HBV DNA Persistence without
seromarkers can indicate
occult infection
Agar Gel Diffusion Test
The first screening assay for HBsAg was gel diffusion
based (Table 3, Method 1). This technology involved
adding high-titered guinea pig anti-HBs to a central well
that was ringed by test wells to which test serum samples
were added. The samples were allowed to diffuse
together. If HBsAg was present in the test serum sample,
the binding of antigen by the guinea pig anti-HBs caused
the formation of a visible aggregate called a precipitin
line. There are three characteristic precipitin line
reactions: a curved identity line, a crossed nonidentity
line, and partial identity when common and unique
antigenic determinants are present. These tests required
large amounts of antigen and antibody for proper
precipitin performance, making them relatively
insensitive compared to more modern tests. This test is
rarely used today, but it did set a precedent to
differentiate true-positive from false-positive results.
Radioimmunoassay
Radioimmunoassays (RIA) to detect HBsAg were
introduced in the early 1970s with the Ausria-125I test
(Abbott) (Table 3, Method 2) [12]. Sensitivity of the
RIA was 30 times that of the gel-diffusion assay. A
serum sample was incubated in polypropylene tubes
coated with guinea pig hyperimmune anti-HBs. Bound
antigen (if present) was detected in a second incubation
by forming a sandwich with 125I-labelled guinea pig
anti-HBs and then measured in a gamma counter. A
confirmatory assay was also introduced which utilized
high-titered human anti-HBs to reduce the RIA signal of
true positives while false-positive signal remained
unchanged. In this manner, the investigator could rapidly
distinguish true-positive results from false positives.
While RIA technology was the mainstay of HBV testing
for decades, the relatively short half-life of the
radioisotope and radioactive disposal concerns led to the
emergence of enzyme and chemiluminescent
immunoassays.
Immunoassay
Immunoassays to detect HBsAg primarily utilize one or
more monoclonal antibodies directed against the a
determinant of HBsAg to immobilize the antigen on a
solid phase (Table 3, Method 3). The a determinant is
a highly conformational, hydrophilic domain between
HBsAg amino-acid positions 100 to 160 that is stabilized
by a backbone of conserved, disulfide-bonded cysteine
residues. Bound HBsAg is detected by a second mono-
or polyclonal antibody to which either an enzyme
(horseradish peroxidase, alkaline phosphatase) or a
chemiluminescent reporter (acridinium, ruthenium) is
conjugated. Sensitivity of HBsAg assays is evaluated
using HBV seroconversion panels and antigen end-point
detection. The sensitivity of blood-screening HBsAg
assays is now less than 0.1 ng/mL and have been shown
to detect a wide range of viral variants [13]. Assay
sensitivity at this low sensitivity level closes the acute-
window period of infection by about 9 days [14].
Confirmatory assays are available to identify false-
positive test results. Recently there is an emerging
interest in quantitating the patient HBsAg level in order
to monitor response to antiviral therapy. The non-US
Architect HBsAg assay (Abbott) is currently the only
commercial immunoassay with an HBsAg quantitative
claim.
Nucleic Acid Testing
Nucleic acid testing (NAT) HBV assays amplify target
regions of circulating viral genomes in a patient blood
sample (Table 3, Method 4). The amplified viral product
can be detected (and in some cases quantified) by
hybridizing with specific oligonucleotide probes
conjugated with a visualization ligand. A colorimetric or
luminescent reaction is then read. Multiple NAT targets
can be amplified from one sample extraction. NAT
testing requires dedicated areas for sample extraction
versus detection to reduce the risk of contamination and
is generally regarded as technically more complex than
EIA technology. Two NAT technologies for HBV
detection are in use in blood bank settings: polymerase
chain reaction (PCR) and transcription-mediated
amplification (TMA).
The COBAS AmpliScreen HBV test (Roche) is an
example of a PCR qualitative in-vitro test for the direct
detection of HBV DNA in human plasma pools. PCR
produces DNA amplicons from a target template. The
COBAS test includes an internal control for extraction
and amplification and has a reagent to prevent
amplification of preexisting amplicons. An example of
TMA technology is the qualitative PROCLEIX ULTRIO
676
Hepatits B Virus
HBV assay (GenProbe) that produces RNA amplicons
from a target template. These NAT technologies have a
limit of HBV detection at approximately 30
genomes/mL in their most sensitive procedures, so
clinical sensitivity will vary depending on the specimen
pooling formatwhether the test serum pool is
composed of 24, 16, or 8 donor specimens. Individual
donor testing (ID NAT) is the most sensitive testing
format.
Closure of the HBV infection-window period by NAT
testing is tied to the size of the test specimen pool. Pools
of 24, 16, or 8 specimens show less, equivalent, or a
slight advantage (~3 days), respectively, in window-
period closure compared to best-in-class HBsAg blood
screening immunoassays, while ID NAT closes the
window period by 8 to 14 days [14,15]. Still, some HBV
infections are not detected by HBsAg or NAT assays in
blood samples but can be detected in liver biopsies [16].
Rapid Assays
Rapid immunochromatographic tests for HBsAg are
available (Table 3, Method 5). The advantages of these
assays are room-temperature storage and a visual readout
in 10 to 20 min. The relative insensitivity of this format
limits their application to health care settings in
developing countries [17].
HBV Antibody Assays
Monitoring the patients immune response to HBV
antigens is a key diagnostic tool in establishing disease
susceptibility and disease state. Two antibody responses
are routinely measured: antibody to HBV surface antigen
(anti-HBs) (Table 3, Method 6) and antibody to HBV
core antigen (anti-HBc) (Table 3, Method 7). Anti-HBs
can be induced by immunization (or past infection), and
antibody levels > 10 mIU/mL indicate that the patient is
protected from further HBV infection. Anti-HBc is a
marker of past HBV infection, and this response can
persist for years after infection. Antibody to HBV e
antigen (anti-HBe) is less often monitored and can be
used to establish the infection stage of chronic carriers.
Reference and Preferred Methods
There is no standardized reference method for HBV
detection, but there are standards for evaluating various
HBV assay formats. The World Health Organization
(WHO) offers standards for HBsAg immunoassay
testing: WHO International Standard for HBsAg
(00/588) and for NAT testing, WHO HBV DNA
International Standard (97/746) [18]. The WHO website
(http://www.who.int/biologicals) describes the available
reference standards. There are national standards from
regulatory agencies such as the U.S. Food and Drug
Administration (FDA), Center for Biologics Evaluation
and Research (CBER), HBsAg Lot-Release Panel#12.
Similar HBsAg panels for Germany are available from
the Paul-Ehrlich-Institut (http://www.pei.de), and French
HBsAg standards are available (http://afssaps.sante.fr).
Lastly, there are seroconversion panels available from
private firms to compare assay performance, such as the
HBsAg Sensitivity Panel (BBI Diagnostics), described
on their website (http://www.seracare.com).
Specimen
HBV test specimens can be serum, plasma, or cadaveric
samples. Tissue samples such as liver biopsies can also
be tested for HBV. Studies have shown NAT sensitivity
to be unaffected by eight short-term freeze/thaw cycles
[19]. HBsAg is an multimeric antigen that presents with
three-dimensional epitopes. The Abbott PRISM HBsAg
assay permits shipping at ambient temperature (30C or
colder) for a period not to exceed 7 days, then storage
for up to 14 days at 2C to 8C. If storage periods
greater than 14 days are anticipated, the serum or plasma
should be removed from the clot or red blood cells to
avoid hemolysis. Serum or plasma should be stored
frozen (20C or colder). For cadaveric specimens,
storage conditions are more restrictive, given the nature
of the sample. Previously frozen specimens must be
centrifuged to remove particulate material. Heparinized
blood tubes should be avoided because they can interfere
with both HBsAg and NAT assays.
Interferences
Blood is a variable sample, and manufactures describe
potentially interfering blood substances in their package
inserts. Specimens with rheumatoid factor or elevated
triglycerides, bilirubin, or hemoglobin are evaluated for
assay interference. Various microorganisms are also
evaluated. In addition, some specimens contain human
anti-mouse antibodies that can affect monoclonal
antibody assays by causing false-positive results.
Optimizing reagent diluents and washes can reduce
nonspecific assay interference. The package insert
specificity data for high-sensitivity blood-screening
HBsAg immunoassays is 99.99%, demonstrating that
specificity interferences have been reduced to very low
levels in certain assays.
Interpretation
The U.S. Centers for Disease Control and Prevention
(CDC) recommendations for interpreting hepatitis B
serological markers is shown in Table 2. Additional data
on marker interpretations is available on the CDC
website:
(http://www.cdc.gov/NCIDOD/DISEASES/HEPATITIS
/b/faqb.htm).
Table 2: Interpretation of Hepatitis
B Serological Test Results
Tests Results Interpretation
HBsAg
anti- negative
HBc negative Susceptible
anti- negative
HBs
HBsAg
anti- negative Immune due to
HBc positive natural
anti- positive infection
HBs
677
Hepatits B Virus

reviews are published so the user of these tests can make

Table 2: Interpretation of Hepatitis an informed decision as to which assay performance
B Serological Test Results goals best meet their needs. These performance goals
change (and will continue to change) in response to
HBsAg factors affecting infection-marker interpretation, such as
anti- negative Immune due to universal neonatal vaccination or co-infection with
HBc negative hepatitis B another infectious agent. Since diagnostic HBV testing
anti- positive vaccination can reduce but not totally eliminate the risk of
HBs transfusion-transmitted disease, future programs to
improve blood safety must consider strategies that
couple diagnostics with a second intervention such as
HBsAg
pathogen reduction [24].
anti-
HBc positive
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2 Kao JH, Chen DS. Global control of hepatitis B
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[21]. Improvements in NAT analysis will lead to new Echevarria J, Lee S, Mushahwar I et al. Genetic
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HBV Performance Goals HBsAg subtypes. Intervirology 2004;47:289-
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Laycock ME, Fiebig EW et al. Comparative
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Transfusion 2003;43:788-98.
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Multicenter performance evaluation of a
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19 Krajden M, Minor JM, Rifkin O, Comanor L.
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RNA quantification as measured with branched-
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20 Stramer SL. Current risks of transfusion-
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21 Valsamakis A. Molecular testing in the
diagnosis and management of chronic hepatitis
B. Clin Microbiol Rev 2007;20:426-39.
22 Cornberg M, Protzer U, Dollinger MM,
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Prophylaxis, diagnosis and therapy of hepatitis
B virus (HBV) infection: the German guidelines
for the management of HBV infection. Z
Gastroenterol 2007;45:1281-328.
23 Gish RG, Locarnini SA. Chronic hepatitis B:
current testing strategies. Clin Gastroenterol
Hepatol 2006;4:666-76.
24 Alter HJ. Pathogen reduction: a precautionary
principle paradigm. Transfusion Med Rev
2008;22:97-102.
679
Hepatits B Virus

Table 3: Methods for Hepatitis B Virus

Method 1. HBV Agar Gel Diffusion Test
Principle of analysis: Immunodetection of viral capsid
Usage: Infrequently used
Comments: Precipitin line formation, low sensitivity
Method 2. HBsAg Radioimmunoassay
Principle of analysis: Capsid antigen detected by antibody sandwich assay
Usage: Infrequently used
Comments: Easy to use but short shelf life
Method 3. HBsAg Immunoassay
Principle of analysis: Solid-phase antigen capture detected by antibody-enzyme conjugate
Usage: Frequently used
Comments: Detects all HBV genotypes, moderate to good sensitivity, less expensive, not overly complex to
run, confirmatory assay available
Method 4. HBV Nucleic Acid Testing
Principle of analysis: Amplified genome targets detected by probe hybridization
Usage: Frequently used in high-volume settings
Comments: High sensitivity in individual donor testing, complex, expensive
Method 5. HBsAg Rapid Assays
Principle of analysis: HBsAg wicks across support strip, conjugate binding causes stripe to appear
Usage: Infrequently used
Comments: Limited sensitivity, subjective visual read
Method 6. Anti-HBs Assay
Principle of analysis: Antibody to HBsAg detected by antigen sandwich assay
Usage: Frequently used to establish immune state
Comments: Antibody result of 10 mIU/mL or greater indicates protective status
Method 7. Anti-HBc Assay
Principle of analysis: Antibody to HBcAg detected by inhibition assay format
Usage: Frequently used
Comments: Indicates stage of infection, new assays are more specific

Figure 1: Appearance of HBV seromarkers in an acute infection profile.

 680
Hepatitis C Virus
Hepatitis C Virus
A. Scott Muerhoff
Name: Hepatitis C Virus (HCV)
Clinical significance: Refer to Chapter 31, Liver Function, and Chapter 32, Viral Hepatitis: Diagnosis and
Monitoring, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
i Currently, antibody screening test results are used in conjunction
Hepatitis C virus (HCV) is one of the five human viruses (known
as hepatitis A, B, C, D, and E) that infect the liver, causing
inflammation. All of these viruses cause acute disease, but
hepatitis B and C can result in chronic infection. It is estimated by
the World Health Organization that about 180 million people
worldwide are infected with HCV and that 130 million are
chronically infected. HCV is a leading cause of liver disease in
developing countries and is a significant risk factor for the
development of chronic liver disease (cirrhosis) and liver cancer.
In the United States, after chronic alcohol abuse, infection with
HCV is the leading indication for liver transplantation. Unlike
hepatitis B virus, there is no vaccine for HCV.
Hepatitis C virus was first described in 1989 as the causative
agent of non-A, non-B (NANB) post-transfusion hepatitis [1].
HCV is an enveloped, positive-strand RNA virus and is classified
as a member of the Flaviridae family. The single-strand RNA
genome is approximately 9400 nucleotides in length and encodes
a large polyprotein precursor that is cleaved by virus-encoded
proteases and the host cellular machinery into structural
components (nucleocapsid or core and envelope proteins 1 and 2)
and enzymes required for processing and replication (e.g.,
nonstructural region-3 serine protease and helicase and
nonstructural region-5b RNA polymerase). Identification of this
virus and the subsequent introduction of serological tests for
detecting circulating antibodies resulted in a dramatic reduction in
the rate of post-transfusion hepatitis in the United States and
worldwide [2].
Most new infections in the developed world are the result of
needle sharing. Diagnosis of acute infection is often difficult,
since as many as 60% to 70% of patients have no distinct
symptoms, while the remainder may exhibit jaundice, fatigue,
loss of appetite and abdominal pain. Antibodies to HCV are
detected by immunoassay in only 50% to 70% of patients at the
onset of symptoms, increasing to more than 90% after 3 months.
Viral RNA can be detected within 1 to 3 weeks post-exposure.
Individuals who report to the physician or hospital with
symptoms related to acute infection typically have elevated serum
bilirubin concentration and alanine aminotransferase (ALT)
activity. Only a minority of patients require hospitalization.
Fulminant hepatitis due to HCV infection occurs rarely but can be
present in patients with hepatitis B virus co-infection.
i
Hepatitis C virus
Previous and current authors of this method:
New method
Fifth edition: A. Scott Muerhoff
with other clinical parameters such as liver enzyme activity, in
particular ALT and AST, to aid in the diagnosis of HCV liver
disease. Since antibody tests do not distinguish active from
resolved infection, additional tests are used to confirm the
presence of HCV antibodies in serum or plasma or to detect
ongoing (i.e., persistent) infection. Among the latter are nucleic
acid tests (NAT), the recently developed core antigen assay, and
the even more recently developed HCV combination
antigen/antibody test. In addition, there are tests for the
determination of the HCV genotype. HCV exists as a collection
of six major genotypes that differ in their response to antiviral
treatment. It is therefore imperative that the infecting genotype be
determined prior to commencement of treatment. Genotyping
assays require isolation of viral RNA from serum or plasma,
followed by RT-PCR to amplify specific regions of the genome
for hybridization to genotype-specific probes or for direct
sequencing and subsequent comparison to a database of known
HCV genotypes.
Antibody Tests
HCV antibody tests utilize 2-4 recombinant antigens or peptides
derived from the core (c22 protein or peptide), serine
protease/helicase (NS3/4 region, C33c or C200 antigen), and
replicase (NS5) genes. Most commercial tests detect only IgG, the
exception being the ARCHITECT anti-HCV assay (Abbott
Laboratories) which detects both IgM and IgG. A prototype
research assay (Immunomatrix Inc., Gaithersburg, MD) for
detection of HCV antibodies in human oral fluid utilizes
conjugates for detection of IgA and IgM in addition to IgG [3];
however, this assay is not yet commercially available. Some
assays utilize an enzyme conjugate, whereas others tests use
chemiluminescent detection and therefore have greater dynamic
range and often greater sensitivity. The immunoassays are
validated for serum or plasma, although some manufacturers also
validate their assays for detection of antibodies in cadaveric
specimens, cord blood, or other bodily fluids.
Confirmation of HCV infection uses an FDA-approved
immunostrip blot assay produced by Chiron Corporation, known
as RIBA (recombinant immunoblot assay). RIBA results are
reported as positive (2 or more markers detected), indeterminate
(1 HCV marker detected), or negative (no HCV markers
detected). Current testing algorithms for determination of acute or
chronic infection utilize the primary antibody detection assay in
conjunction with the RIBA test and/or nucleic acid tests
(described below). The current Centers for Disease Control and
Prevention (CDC) recommendations [4] indicate that tests results
from third-generation screening enzyme immunoassays (EIAs)
with signal-to-cutoff (S/CO) ratios above 3.8 do not require
supplemental testing using RIBA, since the vast majority of these
individuals will confirm positive, and most are positive for
circulating RNA (see Interpretation section). Confirmation of
 681
Hepatitis C Virus
infection can also be achieved by use of nucleic acid tests which
detect viral genetic material (see below).
Core Antigen Test
An assay for detection of circulating HCV antigen has been
recently developed. The method utilizes monoclonal antibodies to
capture and detect the core antigen. Two versions of this test have
been reported. One version will detect antigen only during the
window period of infection, that is, prior to the detection of
antibodies [5-7]. Another version detects core antigen prior to and
after seroconversion to antibody positive status [8-10]. A
commercial total core antigen assay, the Ortho trak-CTM test,
was introduced in 2001 by Ortho Clinical Diagnostics, but this
test is not FDA-approved. An evaluation of this assay [11]
concluded that it has utility in viral load monitoring, including
determination of baseline viral load, the assessment of response to
treatment, and the examination of early viral kinetics during
therapy. However, the authors concluded that the total HCV core
Ag assay cannot be used as a marker of viral replication for HCV
RNA values below 20,000 IU/mL, thus limiting its use in
monitoring long-term response to treatment. The routine
application of the core antigen assay has not yet become
established in clinical practice; however, it may prove
useful/economical for detection of primary infection in blood
donors in low-incidence populations where RNA testing has not
been implemented and in groups at high risk for infection, such as
hospital personnel, laboratory workers, hemodialysis patients,
organ donors, and prison populations, where frequent
testing/monitoring would be beneficial and more cost effective
compared to nucleic acid tests. It may also be useful for
confirmation of infection, since there appears to be good
correlation between core antigenemia and viremia (i.e., RNA
titers).
Combination Antibody-Antigen Test
An assay for detection of both antibodies and core antigen is
available outside of the United States and Europe in a microtiter
assay format (Abbott-Murex HCV Ag/Ab Combination). Other
automated methods have been reported in the literature [12] but
are not yet commercially available. An antibody/antigen
combination assay will be useful for improvement of blood
supply safety in that it will detect seroconverters but, also a
portion of those who are infected but have yet to develop
antibodies. While not as sensitive at nucleic acid testing (NAT;
see below), the assay would be useful for screening blood in those
regions/countries where HCV endemicity is low. Clinical utility
of the automated combination assay has not been established.
Nucleic Acid Testing (NAT)
Infection with hepatitis C virus can be determined by detection of
viral RNA in serum or plasma [13]. Blood donations in the
United States and many other countries are tested for HCV RNA
by using nucleic acid amplification testsspecifically, reverse-
transcription polymerase chain reaction (RT-PCR) or an
isothermal method known as transcription-mediated
amplification (TMA). These tests are also used for confirmation
of HCV infection among individuals who are antibody positive.
In addition, they can be used to monitor treatment or disease
outcome in infected individuals. The assays have different
sensitivity end-points typically measured in International Units
per mL (IU/mL) and are either qualitative or quantitative.
Quantitative assays typically are less sensitive than qualitative
tests [14,15], but the former are very useful for monitoring
antiviral therapy outcomes. The lower-limit of detection of
qualitative assays range from 5 IU/mL (TMA-based method such
as Procleix HIV-1/HCV) to 50 IU/mL (AMPLICOR 2.0 or
Ampliscreen). Quantitative assays exhibit low-end quantitation
ranges of 10 IU/mL (TaqMan real-time PCR assays) to 615
IU/mL (Quantiplex bDNA 3.0) [16].
Genotyping
HCV exists as collection of genotypes designated 1 thru 6
[17,18]. Genotypes are defined as viral variants exhibiting more
than 30% sequence divergence from other strains. Individuals
infected with genotype 1 are typically more refractory to antiviral
treatment (combination therapy using Ribavirin and Pegylated-
interferon alpha). Individuals infected with genotypes 4, 5, and 6
exhibit better treatment outcomes but typically require more
aggressive therapy than individuals infected with genotype 2 or 3
[19]. Response to therapy is determined via quantitative
measurement of viremia levels before, during, and at the end of
treatment. The treatment goal is to reduce the circulating virus to
undetectable levels at the end of therapy.
Genotyping tests are specialized and are typically performed at
reference laboratories. Four types of tests are used: RFLP
(restriction fragment length polymorphism), RT-PCR with probe
hybridization, RT-PCR with sequencing, and the InnoGenetics
Line-Probe Assay, or INNO-LiPA HCV test [15]. The latter
involves the generation of PCR-amplified target sequences of the
viral genomic RNA (specifically, the 5-untranslated region) that
is then hybridized to type-specific probes immobilized onto solid
filter paper strips. The hybridized amplicons are detected via a
colorimetric system that produces banding patterns characteristic
of specific genotypes [20]. Because these methods require
extraction and amplification of nucleic acids, the use of
heparinized tubes for blood collection is not recommended.
Confirmatory Antibody Test
The only FDA-approved assay for confirmation of anti-HCV
status among EIA repeat-reactive donors is the Chiron RIBA strip
immunoblot assay [21]. This test utilizes three peptides, one from
core and two from the NS4 region, and two recombinant antigens,
one from NS3 and one from the NS5 region, immobilized onto a
paper membrane support (per Chiron RIBA3 Package Insert,
December 1998). The two NS4 region peptides are co-
immobilized into a single band; thus there are four HCV-specific
antigen bands on each strip. Visualization of antibody reactivity is
done using an anti-human IgG enzyme conjugate and colorimetric
substrate. Since the recombinant antigens are fused to human
superoxide dismutase (hSOD), a control band of hSOD is
included. Also included are two human IgG control bands, one at
each end of the strip and at different levels, to control for the
detection step and to determine assay background. For the test to
be valid, there must be reactivity to both human IgG control
bands, and the reactivity to the hSOD must be lower than the
Level 1 hIgG control. Interpretation of assay results are based
upon band intensity and banding pattern (see Table below). The
use of the RIBA confirmatory (also known as supplemental
testing) has diminished in recent years with the advent of nucleic
acid testing and with the recommendation by the CDC that
confirmatory testing need only be done when S/CO ratios from
screening or diagnostic tests are low (see algorithm in Figure 1).
 682
Hepatitis C Virus
Scoring and Interpretation of RIBA test results.
RIBA Band Intensity
Score
Absent
(minus)
Less than intensity of Level 1 IgG control band
+/
Equal to intensity of Level 1 IgG control band
1+
Greater than intensity of Level 1 IgG control band and
less than that of Level 2 IgG control band 2+
Equal to intensity of Level 2 IgG control band
3+
Greater than intensity of Level 2 IgG control band 4+
RIBA Antigen Band Pattern
Interpretation
No bands with 1+ or greater reactivity
Or
hSOD band alone with 1+ or greater reactivity
NEGATIVE
Any single HCV band with 1+ or greater reactivity
Or
hSOD band with 1+ intensity AND one or more HCV
bands with 1+ or greater intensity
INDETERMINATE
At least two HCV bands with 1+ or greater reactivity
POSITIVE
Reference and Preferred Methods
There are no currently accepted reference methods for
determination of HCV antibodies. HCV RNA detection assays
that are approved for routine clinical use are calibrated to the
World Health Organization International Standard. IgG antibodies
are detected within 3 months of onset of symptoms in more than
90 percent of cases. The average window period between RNA
and IgG detection is estimated at about 60 days [22]. Due to the
rapid ramp-up in viremia, HCV RNA is often detectable within 1
to 3 days of exposure. If acute infection is suspected, HCV RNA
should be checked within 4 to 6 weeks of exposure. HCV core
antigen is also detected within the window period but typically a
few days after RNA. In chronically infected individuals,
antibodies tend to persist, though viremia can fluctuate between
low, even undetectable, levels and very high titers (>800,000
IU/mL). For children born to HCV-positive mothers, presence of
antibodies should be checked at regular intervalsif still positive
at 18 months, qualitative NAT is preferred; if positive, infant is
chronically infected; if negative, NAT should be repeated in 6 to
12 months [16].
Interpretation of HCV markers
Anti-HCV HCV NAT Interpretation
- - No acute infection
- + Acute HCV infection
+ - Chronic or resolved
+ + Early seroconversion or
chronic
HCV NAT lower limit @ 50 IU/mL.
Specimen
Specimens for testing in HCV antibody assays should ideally be
collected as serum, citrated plasma, or EDTA plasma. Some
assays allow for use of lithium or sodium heparin plasma tubes,
but some do not (e.g., Abbott PRISM anti-HCV). Samples can
be stored at room temperature for 1 to 3 days and up to 7 days at
4C. For long-term storage, temperatures below 10C are
recommended. It is best to avoid repeated freeze/thaw cycles. For
core antigen tests, specimens can be stored at 2C to 8C for 3 to
5 days but should be stored frozen (<15C) if testing will be
done beyond 5 to 7 days. Repeated freeze/thaw cycles can reduce
core antigen detection sensitivity by up to 25% [23].
Manufacturers of the test kits provide detailed information
regarding sample collection, storage, and handling in their
package inserts. Heparinized tubes should be avoided if
specimens are to be used for nucleic acid testing.
Interferences
Analytical specificity among individuals infected with other
microorganisms is excellent (usually >98.5%), with most positive
results confirmed as true HCV antibody positives. Elevated levels
of some substances such as bilirubin, hemoglobin, serum lipids,
and red blood cells can interfere but only when present at very
high levels. Frozen samples should be centrifuged prior to testing.
Manufacturers of the test kits provide detailed information
regarding interfering substances in their package inserts.
Interpretation
An algorithm for hepatitis C virus antibody laboratory testing and
result reporting, as recommended by the CDC, is shown in Figure
1. This algorithm is applicable to the diagnostic and public health
setting and requires that RIBA testing be done for those who are
EIA-repeat reactive but NAT-nonreactive [4,24]. The FDA has
recently allowed blood banks variances to allow similar methods
for blood donors [25,26]. Results from third-generation antibody
screening tests are interpreted based upon S/CO ratios.
Individuals with S/CO ratios > 3.8 will return positive results via
confirmatory immunoblot assay > 95% of the time, hence
supplemental testing via RIBA is not required, and individuals are
reported as HCV infected. If S/CO ratios are < 3.8, supplemental
testing via RIBA3 and/or NAT is recommended. Interpretation of
results from the RIBA confirmatory test are as discussed above.
Nucleic acid test results are typically reported as negative (below
the limit of detection) or positive for qualitative tests.
Quantitative test results are reported in IU/mL, though they are
not typically used for diagnosis but rather therapeutic monitoring.
Interpretation of screening test results can include consideration
of supplemental test results (e.g., RIBA).
Diagnostic antibody test results are also reported as S/CO ratios.
Typically, values > 1.00 are considered positive, although some
manufacturers include a gray zone (e.g., 0.80 to 1.20) wherein
results are taken to be equivocal until the sample is retested in
duplicate. If only one of the duplicate tests is positive, the result is
reported as equivocal, and another specimen should be obtained
from the individual for further testing. Diagnosis of acute HCV
infection relies on a combination of antibody and NAT results
(see Table below) [14]. For diagnostic purposes, results of
serological tests should be used in conjunction with patient
history and other hepatitis markers for diagnosis of acute and
chronic infection.
 683
Hepatitis C Virus
HCV Performance Goals
The following recommendations for the performance and utility
of HCV tests were recently published in a report from the
National Academy of Clinical Biochemistry [27]. Performance
characteristics for each assay (i.e., sensitivity, specificity, percent
CV, etc.) are established by manufacturers of commercial assays
in accordance with the licensing body guidelines of the country in
which it is to be used.
1. EIA screening tests for HCV antibody are adequate for
diagnosis of past or current infection in a patient
population with a high prevalence of disease;
supplemental testing is not needed in such patients. If
confirmation is required, HCV RNA should be used.
2. Supplemental anti-HCV tests (e.g., RIBA) should be
used in populations with low prevalence of disease or to
confirm infection in a patient who is RNA negative.
3. HCV RNA detection and quantitation methods should be
calibrated to the World Health Organization
international standard.
4. Specimens for HCV RNA testing should either be
collected as EDTA or citrated plasma, or be promptly
centrifuged to prevent falsely low results.
5. Assays for HCV RNA detection should have a dynamic
range from < 1000 copies/mL to > 3.2 106 copies/mL.
6. Genotyping tests should reliably differentiate all six
major genotypes and also distinguish genotype 1a from
1b.
References
1 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW,
Houghton M. Isolation of a cDNA clone derived from a
blood-borne non-A, non-B viral hepatitis genome.
Science 1989;244:359-362.
2 Tobler LH, Busch MP. History of posttransfusion
hepatitis. Clin Chem 1997;43:1487-1493.
3 Zmuda JF, Wagoneer B, Liotta L, Whiteley G.
Recognition of multiple classes of Hepatitis C antibodies
increases detection sensitivity in oral fluid. Clin Diag
Lab Immunol 2001;8:1267-1270.
4 Alter MJ, Kuhnert WL, Finelli L. Guidelines for
laboratory testing and result reporting of antibody to
hepatitis C virus. MMWR Recomm Rep 2003;52:1-13.
5 Muerhoff AS, Jiang L, Shah DO, Gutierrez RA, Patel J,
Garolis C, et al. Detection of HCV core antigen in
human serum and plasma with an automated
chemiluminescent immunoassay. Transfusion
2002;42:349-356.
6 Leary TP, Gutierrez RA, Muerhoff AS, Birkenmeyer
LG, Desai SM, Dawson GJ. A chemiluminescent,
magnetic particle-based immunoassay for the detection
of hepatitis C virus core antigen in human serum or
plasma. J Med Virol 2006;78:1436-1440.
7 Courouce AM, Le Marrec N, Bouchardeau F, Razer A,
Maniez M, Laperche S, et al. Efficacy of HCV core
antigen detection during the preseroconversion period.
Transfusion 2000;40:1198-1202.
8 Tanaka T, Lau JY, Mizokami M, Orito E, Tanaka E,
Kiyosawa K, et al. Simple fluorescent enzyme
immunoassay for detection and quantification of
hepatitis C viremia. J Hepatol 1995;23:742-5.
9 Kashiwakuma T, Hasegawa A, Kajita T, Takata A, Mori
H, Ohta Y, et al. Detection of hepatitis C virus specific
core protein in serum of patients by a sensitive
fluorescence enzyme immunoassay. J Immunol Methods
1996;190:79-89.
10 Aoyagi K, Ohue C, Iida K, Kimura T, Tanaka E,
Kiyosawa K, et al. Development of a simple and highly
sensitive enzyme immunoassay for hepatitis C virus core
antigen. J Clin Microbiol 1999;37:1802-8.
11 Bouvier-Alias M, Patel K, Dahari H, Beaucourt S,
Larderie P, Blatt L, et al. Clinical utility of total HCV
core antigen quantification: a new indirect marker of
HCV replication. Hepatology 2002;36:211-218.
12 Shah DO, Chang CD, Jiang LX, Cheng KY, Muerhoff
AS, Gutierrez RA, et al. Combination HCV core antigen
and antibody assay on a fully automated
chemiluminescence analyzer. Transfusion
2003;43:1067-1074.
13 Scott JD, Gretch DR. Molecular diagnostics of hepatitis
C virus infection: a systematic review. JAMA
2007;297:724-732.
14 Chevaliez S, Pawlotsky JM. Use of virologic assays in
the diagnosis and management of hepatitis C virus
infection. Clin Liver Dis 2005;9:371-82.
15 Ferreira-Gonzalez A, Shiffman ML. Use of diagnostic
testing for managing hepatitis C virus infection. Semin
Liver Dis 2004;24 Suppl 2:9-18.
16 Scott JD, Gretch DR. Molecular diagnostics of hepatitis
C virus infection: a systematic review. JAMA
2007;297:724-732.
17 Simmonds P, Alberti A, Alter HJ, Bonino F, Bradley
DW, Brechot C, et al. A proposed system for the
nomenclature of hepatitis C viral genotypes. Hepatology
1994;19:1321-1324.
18 Simmonds P, Smith DB, McOmish F, Yap PL, Kolberg
J, Urdea MS, et al. Identification of genotypes of
hepatitis C virus by sequence comparisons in the core,
E1 and NS-5 regions. J Gen Virol 1994;75:1053-61.
19 Fried MW, Hadziyannis SJ. Treatment of chronic
hepatitis C infection with peginterferons plus ribavirin.
Semin Liver Dis 2004;24 Suppl 2:47-54.
20 Bouchardeau F, Cantaloube JF, Chevaliez S, Portal C,
Razer A, Lefrere JJ, et al. Improvement of hepatitis C
virus (HCV) genotype determination with the new
version of the INNO-LiPA HCV assay. J Clin Microbiol
2007;45:1140-1145.
21 Revised recommendations for testing whole blood, blood
components, source plasma and source leukocytes for
antibody to hepatitis C virus encoded antigen (anti-
HCV). FDA memorandum to all registered blood
establishments. Rockville, MD: U.S. Food and Drug
Administration; 1993 Aug 5.
22 Lynn SA, Wright DJ, Kleinman SH, Hirschkorn D, Tu
Y, Heldebrant C, et al. Dynamics of viremia in early
hepatitis C virus infection. Transfusion 2005;45:994-
1002.
23 Abbott/Murex HCV Ag/Ab Combination Assay Package
Insert, Version 2006.
24 Centers for Disease Control and Prevention.
Recommendations for prevention and control of hepatitis
C virus (HCV) infection and HCV-related chronic
disease. MMWR Recomm Rep 1998;47(RR-19):1-33.
 684
Hepatitis C Virus

25 Kleinman SH, Stramer SL, Brodsky JP, Caglioti S,
Busch MP. Integration of nucleic acid amplification test
results into hepatitis C virus supplemental serologic
testing algorithms: implications for donor counseling
and revision of existing algorithms. Transfusion
XXXX;46:695-702.
26 Variance request to use NAT results and be exempted
from HIV and HCV supplemental testing in specific
circumstances [monograph on the Internet]. Association
Bulletin 05-03. Bethesda: AABB; 2005. Available from:
http://www.aabb.org/content).
27 Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS,
Seeff LB. Diagnosis and monitoring of hepatic injury. I.
Performance characteristics of laboratory tests. Clin
Chem 2000;46:2027-2049.

Table 3: Methods for Hepatitis C Virus

Method 1. Antibody test

Principle: Immunoassay for antibody detection
Usage: Diagnosis or screening blood donations
Comments: Most tests detect IgG only, some IgG and IgM
Method 2. Nucleic acid tests
Principle: Amplify and detect viral RNA in serum and plasma
Usage: Qualitative for diagnostic confirmation, quantitative for
therapeutic monitoring
Comments: Detects evidence of ongoing viral replication
Method 3. Confirmatory antibody test
Principle: Western blot format, typically strip-blot
Usage: To confirm antibody positivity if S/CO is low
Comments: Detection of at least 2 markers is needed to confirm infection
Method 4. Genotyping test
Principle: Uses RT-PCR to amplify portion of virus genome, determine genotype
by hybridization to genotype-specific probes or by sequencing.
Usage: Identification of genotype in individuals known to be infected with
HCV
Comments: Genotype identification needed to determine treatment regime and
predict outcome.
Method 5. Core or nucleocapsid antigen test
Principle: Immunoassay (sandwich format) to detect circulating core antigen.
Usage: Detection of window period blood donations, as possible
confirmatory test, or quantitative assay can be used for
therapeutic monitoring.
Comments: Commercial tests for detection of core antigen are not yet available in
the USA.
Method 6. Combination antibody/antigen test
Principle: Simultaneous detection of core antigen and HCV antibodies
Usage: Detection of window period blood donations, routine and
frequent testing of individuals in high risk groups (injection
drug users, prison inmates, health care worker, hemodialysis
patients).
Comments: Commercial combination tests are not yet available in the USA.
 685
Hepatitis C Virus

Figure 1: Laboratory algorithm for antibody to hepatitis C virus (anti-HCV) testing and result reporting (3).
686
High-Density Lipoprotein (HDL) Cholesterol
High-Density Lipoprotein (HDL) Cholesterol
John R. Burnett and Ken Robertson
Name: High-density lipoprotein (HDL) cholesterol
Clinical significance: Refer to Chapter 37, Coronary Artery Disease: Lipid Metabolism, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass: 170,000 to 360,000 D (HDL)
Chemical class: Lipoprotein
Chemical structure:
i sphingomyelin and lysophosphatides [3]. Cholesterol is
Principles of Analysis and Current Usage
Evidence suggests that high-density lipoprotein (HDL)
cholesterol (C) is a primary and independent coronary
heart disease (CHD) risk factor [1,2]. The National
Institutes of Health (NIH) National Cholesterol
Education Program (NCEP) has periodically released
guidelines concerning the clinical importance of HDL-C
and CHD. These new guidelines place more attention on
HDL-C for (1) assessment of CHD risk, (2) inclusion
during initial total cholesterol screening, and (3)
consideration of choice of drug therapy. The widespread
use of HDL-C values in clinical medicine warrants a
critical analysis of the methods currently available for
cholesterol and apolipoprotein-lipoprotein
quantification.
By definition, HDL is the fraction of plasma lipoproteins
with a hydrated density of 1.063 to 1.21 g/mL in a
preparative ultracentrifuge. Electrophoretically, it has the
mobility of 1 globulins. The most dense of the
lipoproteins, it is composed of approximately 50%
protein, 25% phospholipid, 20% cholesterol, and 5%
triglyceride. The major lipid in HDL is phospholipid, of
which lecithin is the major fraction, followed by
i
High-Density Lipoprotein (HDL) Cholesterol
Previous and current authors of this method:
First edition: Herbert K. Naito
Methods edition: Herbert K. Naito
Second edition: Herbert K. Naito
Third edition: Herbert K. Naito
Fourth edition: Herbert K. Naito
Fifth edition: John R. Burnett, Ken Robertson
the next major lipid found in HDL. The ratio of
esterified to free cholesterol is about 3 to 1. HDL is a
heterogeneous group of compounds that have been
divided into two density classes: HDL2 (d = 1.063 to
1.125 g/mL) and HDL3 (d = 1.125 to 1.210 g/mL).
Using the techniques of density-gradient
ultracentrifugation and gradient polyacrylamide gel
electrophoresis, HDL2 has been further subdivided into
two subspecies (HDL2a and HDL2b), and HDL3 has
been subdivided into at least three subspecies [4,5].
These subspecies appear to be discrete molecular entities
with characteristic sizes, densities, and distribution.
Column chromatography [6,7,8], isoelectric focusing [9],
and high-performance liquid chromatography [11] have
been employed to further investigate the heterogeneity
within the HDL distribution.
There are two direct ways to measure HDL: (1) by
analytical ultracentrifugation and (2) by isolation of
HDL and measurement of the particles gravimetrically.
However, there are only a few clinical laboratories that
can afford an analytical ultracentrifuge and have the
expertise and time to perform the laborious and tedious
steps. To isolate the lipoproteins by column
chromatography, electrophoresis, preparative
ultracentrifugation, or a polyanion precipitation
technique followed by gravimetric determination of the
amount of HDL after the salts and water have been
removed is time consuming and requires expertise.
To circumvent some of these problems, the isolated
HDL fraction is not measured in its entirety. Instead,
either the protein moiety or the lipid moiety is measured
687
High-Density Lipoprotein (HDL) Cholesterol
as an indirect means of quantitating HDL. HDL is
composed of approximately 50% protein, of which
apolipoprotein (apo) A-I and apoA-II are the major
apoproteins, with apoA-I being quantitatively the more
important. The phospholipid component is the largest
lipid constituent by mass, but since cholesterol is simpler
to measure than phospholipids, the HDL-C has prevailed
as an indirect means of determining HDL concentration.
Numerous techniques are available for HDL-C
quantitation. They are based on two steps: (1) isolation
of the HDL and (2) quantitation of the cholesterol in the
isolated HDL. The various methods differ primarily on
how the HDL fraction is isolated, with the cholesterol
analysis usually being done by one of the many
acceptable cholesterol methods available. The method of
Albers et al. [10] can be recommended, though certain
compatible enzymatic procedures would be preferable.
The following HDL isolation procedures are used today
in the clinical laboratory [9,11-21].
Preparative Ultracentrifugation (Table 1, Method 1)
For the simultaneous separation of both very-low-
density lipoproteins (VLDL) and low-density
lipoproteins (LDL), the plasma or serum is adjusted to a
density of 1.063 g/mL by overlaying of the sample with
sodium or potassium bromide solution (415 mg of KBr
per 5 mL) and centrifugation of the sample at 105,000 g
for 24 hours at 16C. After the supernatant solution
containing the VLDL and LDL is removed, the
infranatant solution can be analyzed for the cholesterol
concentration. Technically, the infranatant solution
should be adjusted to a density of 1.210 g/mL so that one
can obtain a solution that truly reflects only HDL.
However, there are only minuscule amounts of other
lipoproteins besides HDL with a density greater than
1.063 g/mL. In addition, the extra step of adjusting the
solution with a density of 1.063 g/mL to a density of
1.210 g/mL introduces greater error than direct analysis
of the fraction with a density greater than 1.063 g/mL,
which for the most part represents all the HDL. This
method of isolating the HDL and measuring the
cholesterol content for HDL cholesterol estimation is a
classical procedure and is often regarded as a reference
procedure. However, it should be emphasized that
caution is warranted when the preparative ultracentrifuge
is used as a reference method for the isolation of
lipoprotein fractions. This method has been operationally
defined in terms of hydrated density and is generally
used to isolate the major lipoprotein fractions (VLDL,
LDL, and HDL). In more heterogeneous samples,
however, there is known to be an overlap of lipoproteins
within an operationally defined density range. For
example, occurrence of floating -lipoproteins (-
VLDL), HDL, and sinking pre--lipoprotein (Lp[a]),
respectively, in density ranges commonly used to
separate VLDL, LDL, and HDL is well documented [22-
25]. To make the preparative ultracentrifugation method
a standard method for HDL-C, one must correct for the
occurrence of apoB-containing lipoproteins in the
density range 1.063 to 1.21 g/mL. Warnick et al. [26]
have successfully improved the accuracy of HDL-C
values in the fractions with density d > 1.063 g/mL by
correcting for manipulative loss and cholesterol derived
from apoB-containing lipoproteins, including Lp(a).
Both Warnick et al. [26] and Srinivasan et al. [27]
reported that they found substantial amounts of apoB-
associated cholesterol in the fraction with d > 1.063
g/mL in samples drawn from a limited number of
children and adult men and women with normal serum
concentrations of lipids. Srinivasan et al. [27] found
contamination of the apoB-containing cholesterol in the
HDL fraction especially in hyperlipoproteinemic (types
IIa, IIb, and IV) persons. Hutt et al. [28] found that in
addition to Lp(a), apoE-containing HDL also contributed
significantly to the positive bias of the ultracentrifugal
method as compared to precipitation and column
chromatography techniques. In addition, recovery of
lipoproteins is often less than 90%. Thus both studies
[26,27] emphasize that without appropriate corrections,
the lipoprotein concentrations obtained by preparative
ultracentrifugation may not serve as appropriate
reference intervals.
Column Chromatography (Table 1, Method 2)
Ion-exchange chromatography and gel-permeation
chromatography have both been used in the isolation of
the major lipoprotein groups and the subpopulations
within each group. These procedures separate HDL
subclasses based on differences in charge or molecular
size, respectively. For instance, using hydroxyapatite
packing, the solution with a density of 1.063 to 1.210
g/mL can be subfractionated into 12 to 14 HDL
subclasses. The significance of each of these
subfractions is not well defined at the present time, but
as more information is gathered about the metabolic role
of each of these subclasses of lipoproteins, their
distinction will be more important. These methods are
not widely used today because of the critical care and
attention necessary in standardizing the columns, the
need to concentrate the eluent for cholesterol analysis,
and the need for large sample size. Similarly, the
preparative block electrophoresis methods are almost
never used for routine analysis. These methods also
require large samples and are very time consuming.
Their applications are more suited for the collection of
large amounts of lipoproteins.
Preparative Block Electrophoresis (Table 1, Method 3)
Both starch block and Geon-Pevikon block
electrophoresis methods are employed in the isolation of
major lipoprotein fractions and their subfractions. These
electrophoresis procedures separate the lipoprotein
classes on the basis of their net charge and size. The
smaller HDL molecules have the highest mobility
toward the anode. These methods are mainly used as
preparative techniques.
Agarose Gel Electrophoresis (Table 1, Method 4)
This method uses the standard lipoprotein
electrophoresis procedure on agarose medium, followed
by the overlaying of the electrophoresed sample with
enzymatic cholesterol reagent. The enzyme reagents
used include cholesterol esterase and oxidase with a
peroxidase indicator. More recently, cholesterol
688
High-Density Lipoprotein (HDL) Cholesterol
dehydrogenase rather than cholesterol oxidase has been
used with the dye nitroblue tetrazolium. This dye is
insoluble and stable following reduction. A densitometer
with an automated integrator is used to scan the agarose
strip after color development and quantitate each
lipoprotein fraction.
The agarose gel electrophoresis method is not often used
for routine analysis. The presence of -VLDL and Lp(a)
in the sample can introduce bias in the measurement of
pre- and lipoproteins, because the measurements are
based on electrophoretic distributions of and pre-
lipoproteins. If the calculations are based on the percent
distribution of the , pre-, and lipoproteins (derived
from the densitometric scans) multiplied by the total
cholesterol, it is conceivable that the presence of -
VLDL and Lp(a) would falsely elevate and pre-
lipoprotein cholesterols and falsely depress -lipoprotein
cholesterol.
Serfontein et al. [29] reported that HDL-C is selectively
underestimated by 10% when sodium phosphotungstate-
MgCl2 precipitation is used to visualize lipoprotein
bands separated by agarose electrophoresis. They
postulated that since the cholesterol is recovered from
the precipitation solutions used to visualize the
lipoprotein bands, the HDL, because of its relatively
smaller molecular mass and relative solubility in the
precipitation medium, diffuses out of the gel into the
precipitation medium.
Precipitation With Polyanion Solutions (Table 1,
Method 5)
Differential precipitation of lipoproteins with various
polyanion solutions is common practice and is suitable
for the clinical laboratory because of its simplicity,
elimination of expensive instrumentation, speed, and low
cost. These techniques are all based on the ability of
various agents to precipitate selectively the major
lipoprotein fractions, except HDL. HDL is left in the
supernatant solution for cholesterol quantitation. The
agents most frequently employed include heparin
manganese chloride (LRC method), dextran sulfate
magnesium chloride, sodium phosphotungstate, and
polyethylene glycol.
Dextran sulfate of various molecular mass with calcium
or magnesium salts has been used for HDL-C
quantitation. Most dextran sulfate procedures tend to
underestimate HDL-C, though this method does not
appear to be sensitive to variations in incubation and
centrifugation temperatures. Higher-molecular-mass
(50,000 D) dextran sulfates tend to produce HDL-C
values closer to those obtained using the heparin
magnesium chloride methods. Warnick et al. [30] have
described a dextran sulfatemagnesium chloride
procedure that is compatible with enzymatic cholesterol
determination. The method, when compared to the LRC
method [11], is considered simple, rapid, accurate, and
precise [31].
The use of sodium phosphotungstate is another method
of precipitating apoB-containing lipoproteins. This
method also underestimates HDL-C, but not so much as
the polyethylene glycol method does. This method is
particularly sensitive to temperature fluctuations and
reagent concentrations, and these can be a major source
of error. According to Assmanns group [32],
phosphotungstic acidMgCl2 can also be used for
complete precipitation of apoB-containing lipoproteins
in hypertriglyceridemic sera (up to 155 mg/dL; 4.0
mmol/L) with only marginal coprecipitation of HDL.
However, one disadvantage of this method is the
unfavorable ratio of sample to precipitation reagents
(10:1). Another disadvantage is the instability of the
reagent mixture containing phosphotungstic acidMgCl2
because of the formation of isopolytungstic acid after
prolonged storage [33].
Use of the modified reagent offers considerable
advantages over the use of the customary reagent: the
modified precipitation reagent remains fully functional
after a 12-month storage of the reagent at +50C [33],
whereas prolonged storage of the customary reagent
results in deposits of isophosphotungstic acid. Moreover,
when the modified precipitation reagent was used in sera
containing as much as 397 mg/dL (10.26 mmol/L) of
triglyceride, none of the precipitate floated to the surface
[22,23], whereas with the customary reagent, one can
expect floating of the precipitate in sera with triglyceride
concentrations greater than 220 mg/dL (5.7 mmol/L).
The proportional composition of sample to reagent of
1:2 allows for the addition of diluents to make the
precipitation procedure easier and improve the precision
of the analysis. Niedmann et al. [24] have suggested that
cholesterol oxidase-resistant 3,5-cholestadiene may
form under certain conditions with heparin-MnCl2 or
phosphotungstic acidMgCl2 precipitation methods for
HDL cholesterol quantification. With their highly
sensitive chromatographic techniques, they confirm that
definitely no inert 3,5-cholestadiene forms when the
present precipitation reagent is used.
Polyethylene glycol of various molecular weights has
been used occasionally for HDL-C determinations. Of
all the precipitation techniques, this method has the most
serious problems with accuracy and precision. Wiebe
and Smith [25], however, concluded that their method
was both accurate and precise.
There are a few studies that were extensive examinations
of different HDL-isolation methods. Warnick et al. [34]
evaluated improved, or second-generation, HDL-C
methods. The methods were compared on specimens
with a wide range of total cholesterol, triglyceride, and
HDL-C values. They also tested specimens to which
were added either sodium chloride, to increase the ionic
strength, or glucose, to approximate a specimen from a
diabetic patientfactors that reportedly affect
lipoprotein precipitation. Methods were compared for
completeness of lipoprotein sedimentation by assessing
the proportion of specimens with turbid supernatants.
Cholesterol was measured in all supernatants. They also
689
High-Density Lipoprotein (HDL) Cholesterol
measured total protein in a subset of supernatants and
precipitates to determine whether the effectiveness of
sedimentation of hypertriglyceridemic specimens might
be attributable to coprecipitation of other plasma
proteins.
The dextran sulfateMg2+ method was selected as the
comparison method for this report because previous
studies [30,35] demonstrated good specificity for HDL
separation. In the earlier works, dextran sulfateMg2+
supernatants of EDTA-treated plasma contained virtually
no apoB, indicating specific separation of LDL. On the
other hand, the dextran sulfateMg2+ precipitate
fractions contained little apoA-I, an indication that
precipitation of HDL may not have been excessive.
All the modified precipitation methods tested gave
similar results for HDL-C, an indication that
modifications of the methods may have led to improved
accuracy. Results by the heparin-Mn2+ (92 mmol/L) and
phosphotungstate-Mg2+ methods agreed best with those
by the dextran sulfateMg2+ procedure. The heparin-
Mn2+ (46 mmol/L) and the two polyethylene glycol
methods gave slightly higher results. Observed
differences were largest for specimens with high HDL-C
values. Addition of either NaCl or glucose within the
expected physiological range did not significantly affect
lipoprotein precipitation. In terms of sedimentation
effectiveness, the methods were ranged in the following
order: polyethylene glycol (100 g/L, pH 10) > dextran
sulfateMg2+ > heparin-Mn2+ (92 mmol/L) =
polyethylene glycol (75 g/L) > phosphotungstate-Mg2+
> heparin-Mn2+ (46 mmol/L).
Although no ideal method for measuring serum HDL-C
is available, it is important to recognize the limitations of
all procedures. Particular caution is urged for those
setting up this test for clinical evaluation of persons at
risk for CHD. Precision is also important; remember that
the mean difference in HDL-C concentration in the
Framingham Study populations with and without
myocardial infarction (MI) is only 40 mg/dL. As more
work is done using different polyanion solutions for the
selective precipitation of lipoproteins, a more sensitive,
accurate, and precise method will probably evolve.
Polyacrylamide Gel Electrophoresis (Table 1, Method
6)
Roche et al. [36] and Muiz [37] reported the use of
polyacrylamide gel electrophoresis (PAGE) for
quantitating HDL cholesterol. According to Roche et al.
[36], the PAGE method correlated well (r = 0.96) with
the ultracentrifugal method. This was a higher
correlation than that found by Muiz [37], who reported
a correlation coefficient of 0.84.
Magnetic Precipitation
Another HDL-isolation technique is based on the
isolation of HDL by selective precipitation of LDL and
VLDL using dextran sulfate (50,000 D) that has iron
attached to it [38]. Thus when the dextran sulfate
attaches to the LDL and VLDL, a magnet is used to
pull the lipoprotein complex to the bottom of the tube.
This approach eliminates the need for centrifugation to
obtain a supernatant solution containing the HDL. This
method also has the distinct advantage of being less
sensitive to lipemic specimens, which is a major
interferent that can cause incomplete precipitation of
LDL and VLDL (and chylomicrons) with other
precipitation methods. Triglyceride concentrations can
be several thousand mg/dL before problems arise. The
method appears to be precise and accurate, although a
small negative bias has been observed with conventional
dextran sulfate and a positive bias with phosphotungstate
precipitation.
Homogenous Assays
Recently, several so-called homogenous methods have
been developed that are capable of full automation
(Table 1, Method 7). These methods do not require off-
line pretreatment and separation and are considered to be
third-generation assays [39,40]. These assays may be
immunologic, making use of antibodies directed against
apoB and/or apoC-III, or they may involve reaction with
various precipitating or complexing agents to form
soluble or insoluble complexes of chylomicrons and
LDL and VLDL cholesterol. The majority of
laboratories have adopted the homogenous assays, even
though questions have been raised regarding the
specificity of these methods, especially in specimens
with unusual lipoprotein compositions.
Miscellaneous Method Considerations
Quantitation of HDL2- and HDL3-Cholesterol
Subfractions
The HDL2 subfraction may be better correlated with risk
of coronary heart disease than total HDL cholesterol.
The HDL2/HDL3 ratio was reported to be the best
biochemical marker of coronary heart disease risk of 22
that were tested [41]. Time-consuming zonal
ultracentrifugation, column chromatography, and
gradient-PAGE have all been used to isolate HDL2 and
HDL3. Selective precipitation of the HDL subfractions
has been reported. Double precipitation methods with
polyanions to separate HDL subfractions offer a more
practical procedure. The methods are relatively rapid,
economical, and simple. The method of Martini et al.
[42,43] uses heparin-MnCl2 to precipitate the VLDL and
LDL, followed by dextran sulfate (15,000 D) to
precipitate the HDL2. The supernatant solution contains
the HDL3 fraction used for cholesterol measurement in
the following relationship:
HDL2-C= Total HDL-C HDL3-C
The methods appear to be compatible with both
chemical and enzymatic cholesterol procedures [44].
Lundberg et al. [45] reported the use of polyethylene
glycol 6000 to precipitate the apoB-containing
lipoproteins, after which HDL2 was precipitated with
690
High-Density Lipoprotein (HDL) Cholesterol

dextran sulfateMgCl2. Estimation of total HDL-C and polymer mixture and acted upon to produce colorless
HDL2 and HDL3 fractions with dextran sulfateMgCl2 products. The second detergent reagent then releases the
cholesterol from LDL cholesterol, which then forms a
has been published and appears to be accurate and
colored product which is measured
precise [31].
spectrophotometrically.
Hirano et al. [47] have published a single-step
Reference Diagnostics distributes a product using Denka
precipitation procedure for the measurement of HDL-
Seiken reagents. This assay stabilizes the LDL
cholesterol subfractions. This technique involves
molecules by use of a surfactant. This means that the
precipitating apoB-containing lipoproteins and the HDL2
LDL cholesterol is nonreactive. The first reagent reacts
using a heparin/manganous chloride/dextran sulfate
with all non-LDL cholesterol to produce a colorless
reagent. The HDL3 in the supernatant is then measured
product. A second surfactant in the second reagent then
using a homogenous HDL-C assay. The HDL3 value is
releases the cholesterol from LDL cholesterol, which
then obtained by subtracting the HDL2 value (corrected
then is measured spectrophotometrically after formation
for volume) from the total HDL-C value (measured
of a colored compound.
using the same Denka Seiken reagent).
The Wako reagents are distributed in the United States
Quantitation of LDL Cholesterol by Sigma Diagnostics. In this technique, an enzymatic
Most clinical laboratories utilize the convenient method complex is used to stabilize the LDL molecules and so
of Friedewald to estimate LDL cholesterol. Since LDL is render the cholesterol nonreactive. Non-LDL cholesterol
known to be the critical atherogenic lipoprotein, it is is then reacted upon, again producing a colorless
natural that methods be developed for a convenient, product. The addition of the second reagent, which
direct, and rapid assay for LDL cholesterol. The majority contains a detergent, again releases the LDL cholesterol,
of the precipitation techniques involve the use of which is measured spectrophotometrically after
complex carbohydrates such as heparin, dextran sulfate, formation of a colored compound.
amylopectin, and polyvinyl sulfate to precipitate LDL
directly. The specificity of polyanion precipitation There are a number of other suppliers and manufacturers
depends on experimental variables such as polyanion of similar reagent kits who use a variety of other novel
concentration, polymer length, pH, ionic strength, and techniques to achieve the same end [48,49].
the concentration of divalent cations.
Reference and Preferred Methods
The introduction of immunologic-based LDL cholesterol
The Joint Committee for Traceability in Laboratory
procedures has led to the routine use of these methods
Medicine (JCTLM) accepts the CDC Reference Method
for LDL measurement. This method is based on
for HDL [50]. This technique involves
polyclonal antisera specific for HDL and VLDL; after
ultracentrifugation to remove everything of density less
centrifugation the supernatant solution contains the LDL
than 1.006, followed by use of a heparin/manganous
fraction, which is analyzed for cholesterol. Empirically,
chloride precipitation to remove non-HDL. The HDL-C
the direct LDL cholesterol procedure should be more
in the supernatant is then measured
precise and accurate than the estimated LDL cholesterol
spectrophotometrically. This technique is also the
by the Friedewald formula [46,48,49] because of three
reference method for LDL cholesterol. (LDL-C = Total
analytical variables (total cholesterol, triglyceride, and
Cholesterol post ultracentrifugation HDL-C).
HDL cholesterol measurements) compared to one (LDL
cholesterol).
There is no validated definitive method for HDL-C. The
accepted reference method has not been fully validated,
In recent years, a number of suppliers have released
as is the case for the cholesterol reference method.
direct assays for the measurement of LDL cholesterol.
Instead, the accepted accuracy target is a procedure used
These avoid the necessity for the precipitation step, so
at the CDC to assign HDL-C target values for human-
make them suitable for application as automated
based serum pools.
procedures. The LDL-C plus second-generation assay
distributed by Roche (using Kyowa reagents) utilizes the
The NCEP guidelines place more emphasis on the
process of selective micellary solubilization of LDL
measurement of HDL-C for the assessment of CHD risk.
cholesterol by a nonionic detergent. When this detergent
The medical decision values of less than 35 mg/dL and
is added to the standard cholesterol esterase/cholesterol
60 mg/dL or greater demand greater accuracy of
oxidase reagents, the relative reactivities of the
measurement to minimize the false positives or negatives
lipoproteins are altered so that HDL < chylomicrons <
[51].
VLDL < LDL. In the presence of Mg2+, a sugar
compound in the reagent further reduces the cholesterol
The direct gravimetric and ultracentrifugation techniques
enzymatic reaction of VLDL and chylomicrons, enabling
for HDL quantitation are rarely used for routine work.
the determination of LDL cholesterol.
These methods are too laborious and require highly
specialized techniques. Nevertheless, an
The Daiichi reagent, distributed in the United States by
ultracentrifugation-based method is considered the
Genzyme Diagnostics, uses a two-reagent method.
reference method.
During the first step, cholesterol from non-LDL
cholesterol components is released by a detergent
691
High-Density Lipoprotein (HDL) Cholesterol
Although the heparinmanganese chloride procedure is
not commonly used, it is considered one of the methods
of choice. The CDCs reference method is used to
standardize the participating laboratories in the Lipid
Standardization Program.
However, the heparinmanganese chloride method is not
without faults. Because of the presence of manganese
ions in the sample, this method is not compatible with
most cholesterol enzymatic systems, giving a consistent
false-positive bias. However, if a reliable and accurate
Liebermann-Burchard (L-B) method is used, this
becomes an excellent method. Hypertriglyceridemic
samples greater than 4000 mg/L (4.5 mmol/L) tend to
cause incomplete precipitation of the apoB-containing
lipoproteins, resulting in turbid solutions that contain
substantial quantities of VLDL and LDL. This problem
can be minimized by:
(1) Dilution of the samples with 0.9% saline and
reprecipitation with heparinmanganese chloride (92
mM of Mn2+)
(2) Centrifugation at a higher gravity force, such as
12,000 g for 10 min
(3) Removal of the chylomicron or VLDL fractions first,
using preparative ultracentrifugation at a density of
1.006 g/mL
(4) Ultrafiltration of the turbid supernatant solution with
a 0.22 m ultrafilter
(5) Use of undiluted samples but with twice the volume
of heparinmanganese chloride solution. In practice,
options 1 and 5 are preferred.
Another problem associated with the heparinmanganese
chloride method is the interval between sample
collection and precipitation. Because the HDL-C values
decrease with an increase in time interval, it is
recommended that the analysis be performed on the
sample soon after blood collection.
A detailed description of the heparinmanganese
chloride procedure is included (click here). Since the
mean difference in HDL-C concentration between
persons at normal risk for CHD and persons at high risk
is small (about 4 to 5 mg/dL), it is suggested that the
minimum precision for this method should be less than 3
mg/dL for within-day and day-to-day variance.
The homogenous methods for HDL-C quantitation are
steadily replacing the conventional precipitation
methods. These methods allow full automation of
previously tedious manual treatment of samples.
Specimen
In brief, the patient must be fasting for at least 12 hours
before the blood is drawn. It should be emphasized that
although nonfasting conditions do not appear to
influence the blood HDL-C levels in most people,
postprandial lipemia has the potential of interfering with
many of the analytical methods. To minimize this
analytical problem, it is always good laboratory practice
to request fasting specimens. Serum or plasma can be
used as the sample, but since plasma is usually preferred
for lipid analysis, HDL analysis will most frequently be
performed on the same plasma specimen. EDTA is again
the anticoagulant of choice, and the final concentration
should be 1 mg of EDTA per mL of blood. The sample
should be removed from the blood clot within 2 hours
and may be stored at 4C for up to 2 days. If specimens
are to be kept for longer than 48 hours, they should be
frozen.
HDL is relatively labile, and freeze/thaw cycles have
been shown to affect some of the precipitation methods.
However, if specimens are frozen, they should be kept at
temperatures below 50C, at which temperature they
are stable for up to 2 years. Once thawed, specimens
should be gently mixed prior to analysis. It is
recommended that tubes specifically designed for low-
temperature storage should be used.
Interferences
The homogenous assays for HDL-C differ somewhat in
the effect of various interfering substances. Triglycerides
less than 900 mg/dL generally do not interfere with any
of the homogenous methods. Hemoglobin < 2 g/L and
bilirubin < 100 mg/L also do not interfere with
homogenous assays, and some instrument manufacturers
claim even higher limits for TG, hemoglobin, and
bilirubin. Reports of interference in these assays from
specimens with atypical lipoprotein patterns indicate the
need for careful assessment of results from these types of
specimens. There have also been reports noting
unreliable results in samples from subjects with
Waldenstroms macroglobulinemia and in a case of
polyclonal gammopathy.
HDL Reference Interval
Just as population-based reference intervals for total
cholesterol were abandoned with the implementation of
the NCEP guidelines, HDL cholesterol population-based
reference intervals are being abandoned for
interpretation of patients risk for CHD. The medical
decision points selected by the NCEP Adult Treatment
Panel (ATP) were based on specific end-points in
clinical trials (heart attacks, deaths due to CHD,
abnormal EKG, etc.) and on simplicity. The NCEP
recommended only two medical decision points. In
2001, the NCEP increased the high-risk medical decision
level to < 40 mg/dL for HDL-C [51,52].
Interpretation
During the last 3 decades, an increasing amount of basic,
clinical, and epidemiological research has focused on the
role of plasma lipoprotein level as a primary risk factor
for CHD. Since 1965, when Fredrickson and Lees [54]
developed a system for phenotyping
hyperlipoproteinemia, the major emphasis has been on
chylomicrons, VLDL, and LDL. Although reports
linking cholesterol in the lipoprotein or HDL to lower
incidence of MI were available in the 1950s [53,55],
interest in and recognition of this information waned as
the emphasis was placed on the seemingly atherogenic
lipoprotein lipoprotein. There is no longer any doubt
that LDL plays an important role in the development and
progression of coronary atherosclerosis. Although
several investigators have clearly shown the deposition
of LDL and apoB in the intima and media of the vascular
692
High-Density Lipoprotein (HDL) Cholesterol
wall [56,57], the precise mechanism by which LDL is
involved with atherogenesis is not completely
understood.
Lipid and lipoprotein studies in the past have
emphasized the positive relationship among plasma total
cholesterol, LDL, and the increased risk for CHD.
However, other lipoproteinsthat is, chylomicrons,
VLDL (or VLDL-like particles), intermediate-density
lipoproteins (IDL), -VLDL, Lp(a), oxidized LDL,
homocysteine, and apoEhave now been implicated in
this multifactorial and complicated disease. It is now
well established that there is an inverse relationship
between hypertriglyceridemia and HDL (and HDL-C)
concentration. Thus although epidemiologists have not
shown a clear relationship between elevated triglycerides
and increased risk for CHD, a distinct relationship does
exist between HDL-C levels and CHD; that is, the lower
the HDL concentration, the greater the risk for CHD.
The relationship between elevated triglyceride-rich
particles (chylomicrons, VLDL, and IDL) and increased
risk for CHD is to a large extent probably attributable to
the depressed HDL (HDL-C) levels. Partitioning the
total cholesterol measurement into the atherogenic factor
(LDL cholesterol) and the antiatherogenic factor (HDL
cholesterol) provides more meaningful information that
shows a better statistical relationship with CHD than the
earlier method. There are numerous studies that have
clearly shown the inverse and independent relationship
between HDL or HDL cholesterol and risk for CHD; a
comprehensive review on this subject can be obtained
elsewhere [58-60].
HDL-C is only a reflection of HDL concentration. There
are other markers that might better reflect the HDL
concentration, such as HDL phospholipid [61] or
apoproteins [41]. Many investigators showed that MI
survivors have lower levels of apoA-I and apoA-II and
HDL-C than healthy adult males do. They postulated
that since hypertriglyceridemia is common in CHD
subjects, and since a reciprocal relationship between
HDL-C and triglyceride levels exists, the lower HDL-C
concentrations could in part reflect the higher
triglyceride levels in MI subjects, as suggested by
Carlson and Ericsson in 1975 [62]. In addition, they also
suggested that the small but statistically significant
difference in total apoprotein composition could reflect a
redistribution of HDL subclasses (HDL2 versus HDL3),
a compositional alteration of the HDL molecules, or both
[63].
Like other major classes of lipoproteins, HDLs are a
heterogeneous group of macromolecules with different
chemical and physical properties and, more than likely,
with different metabolic functions. Thus three subclasses
(HDL1, HDL2, and HDL3) of HDL have been
identified. It appears that the HDL2 but not the HDL3
has clinical significance from the standpoint of
association with CHD risk. Thus partitioning the major
lipoprotein classes into IDL and LDL or HDL2 and
HDL3 appears to increase their sensitivity, specificity,
and hence their predictive value for CHD risk [63,64].
Performance Goals for HDL-Cholesterol
Measurements
The NCEP Laboratory Standardization Panel
recommends that total error be within 13% of the true
value. The total error comprises imprecision (random
error) and inaccuracy or bias (systematic error). The
imprecision of any assay should be < 4%. The limits for
acceptable performance established by CLIA require that
laboratories be within 30% of the peer-group mean.
Survey data from the 2007 College of American
Pathologists (CAP) Participant Summary Report [65]
shows a wide range of imprecision values (% coefficient
of variation [CV]) for HDL cholesterol, ranging from
4% to 7% in most cases. There are a few methods,
however, which still show CVs of 8% to 12% or higher.
Overall, the results indicate good, acceptable
performance. Even techniques with very poor precision
appear to satisfy the Clinical Laboratory Improvement
Amendments (CLIA) criteria
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Pathologists; 2007.

Table

Table 1: Methods for Isolation of High-Density Lipoprotein (HDL)

Method 1: Ultracentrifugation
Principle of analysis: Plasma or serum adjusted to density of 1.063 g/mL with potassium bromide and centrifuged at high
speeds for 24 h; all lipoproteins separated by density, with HDL fraction in 1.063 to 1.21 g/mL range
Comments: Specialized research laboratories; reference method; laborious and time consuming
Method 2: Column chromatography
Principle of analysis: HDL isolated and separated into subclasses on basis of charge (ion-exchange) or molecular size (gel
permeation)
Comments: Specialized, research laboratories; rarely used; difficult to properly control chromatographic conditions
Method 3: Starch block electrophoresis
Principle of analysis: HDL separated from other lipoproteins on basis of charge and size
Comments: Rarely used; used for isolation of large amounts of HDL, not for quantitative purposes
Method 4: Agarose gel electrophoresis
Principle of analysis: (See Method 3)
Comments: Frequently used; precision not adequate for clinical use
Method 5: Precipitation
a. Heparinmanganese chloride
b. Dextran sulfate
c. Phosphotungstate
d. Polyethylene glycol
Principle of analysis: Polyanions (heparin, dextran sulfate), phosphotungstate, polyethylene glycol, in presence of divalent
cations, used to precipitate larger, less dense lipoproteins; HDL quantitated in supernatant as HDL-C
Comments:
a. Frequently used reagent; current method of choice; not compatible with all cholesterol procedures
b. Used occasionally; use of lower molecular mass dextran can produce biased results; higher molecular mass
dextrans produce excellent results; compatible with enzymatic procedures
c. Most commonly used; underestimates HDL; sensitive to temperature fluctuations
d. Used infrequently; poorest accuracy and precision; not recommended
Method 6: Polyacrylamide gel electrophoresis
Principle of analysis: (See Method 3)
Comments: Used infrequently; probably underestimates HDL levels
Method 7: Homogenous methods
Synthetic polymer/detergent
Polyethylene glycol
2+
Principle of analysis: Cyclodextrin and Mg or synthetic polymer with a polyanion selectively blocks, but does not
precipitate, non-HDL lipoproteins; HDL free to react with reagent enzymes; immunological
Principle of analysis: Antibody to human apoB in first reagent reacts with apoB-containing lipoproteins, chylomicrons, LDL
and VLDL; HDL free to react with enzymes added with second reagent
Comments: Most common methods in use today; better precision than manual precipitation methods
696
High-Density Lipoprotein (HDL) Cholesterol
Procedure: Isolations of High-Density Lipoproteins
by HeparinManganese Chloride
Principle
The larger, less dense apoB-containing lipoproteins
(those with low, very low, and intermediate density) are
precipitated overnight by heparinmanganese chloride.
After centrifugation to separate the precipitated
lipoproteins, HDL cholesterol in the supernatant solution
is quantitated.
Reagents
1. Manganese chloride solution, 1.0 mol/L.
Dissolve 19.791 g of MnCl24H2O in distilled water,
and bring volume to 100 mL. This is stable for 3 months
at 4C.
2. Heparin solution, 4000 U/mL. Dilute 1 mL of
heparin (A.H. Robins; 10,000 USP units/mL) with 1.5
mL of 0.9% saline solution. This is stable for 1 week at
4C.
Assay
Equipment: Refrigerated centrifuge capable of
maintaining temperature of 4C at 2000 g.
1. Pipet 1 mL of serum into a 13 100 mm
disposable culture tube.
2. Cap tubes with polyethylene stoppers, and place
in refrigerator for 30 min at 4C.
3. Add 50 mL of heparin solution; mix on a vortex
mixer for 1 min.
4. Add 50 mL of manganese chloride solution;
mix on a vortex mixer.
5. Cap tubes with polyethylene stoppers, and place
in refrigerator for 30 min at 4C.
6. Centrifuge the heparin-lipoprotein precipitate at
2000 g for 1 hr at 4C.
7. Using a transfer pipet, transfer supernatant
solution into a clean test tube. Store in a
refrigerator overnight to ensure complete
precipitation of all apoB-containing
lipoproteins.
8. The next morning, sediment any remaining
precipitate at 2000 g for 30 min at 4C.
9. Using a transfer pipet, transfer the clear
supernatant solution into a clean test tube for
measurement of cholesterol, using a standard
method compatible with heparinmanganese
chloride. The L-B method is recommended.
Notes
1. Biological sample. If one uses plasma samples,
the 92 mmol/L Mn2+ salt concentration should be
selected. However, if one uses serum, the 46 mmol/L
Mn2+ concentration is more ideally suited for HDL
cholesterol determinations. If EDTA plasma samples are
used with the heparin 46 mmol/L Mn2+ method, slightly
higher HDL cholesterol values will be obtained because
of incomplete precipitation of the apoB-containing
lipoproteins. If one uses the 92 mmol/L Mn2+ salt
preparation on serum samples, the HDL cholesterol
values will be slightly lower than results obtained using
the ultracentrifugal procedure (the reference procedure).
2. Purity check. If the sample shows an unusual
value (HDL cholesterol concentrations less than 20 or
greater than 80 mg/dL) or the -lipoprotein bands on the
electrophoresis medium do not agree with the serum or
plasma HDL cholesterol value, perform a purity check or
repeat the analysis or do both.
A purity check on the supernatant solution should be
done if there is the possibility of incomplete
precipitation of apoB-containing lipoproteins. To do this,
electrophorese the supernatant solution, and stain with a
lipid dye to show whether only one band exists. If there
is more than one lipid-staining band and the second band
is in the -globulin area or 2-globulin area, an apoB-
containing lipoprotein is probably present. One can
confirm this by doing immunological studies using apoB
antisera.
3. Double-concentration precipitation. If the
serum is turbid or hypertriglyceridemic (greater than 400
mg/dL), a double-strength solution should be used to
precipitate the beta-containing lipoproteins. To do this,
use 1 mL of serum, 100 L of heparin, and 100 L of
manganese chloride solution. Multiply the result by 1.2
to account for the dilution. A double-strength solution
should also be used for a serum sample if, after
conventional manganese chlorideheparin precipitation,
a floating lipid precipitate is present and the purity check
of the supernatant shows contamination.
4. Pooled human sera for quality control.
Reference materials are needed for calibration,
standardization, and quality control of HDL-cholesterol
analyses. The calibration and control materials must be
suitable for determining cholesterol in the HDL fraction
when specimens contain relatively low concentrations of
cholesterol and when nonspecific and interfering
substances may be present. Calibration materials
applicable to low cholesterol level analyses can be
standards containing about 25 and 60 mg/dL of
cholesterol. Serum control materials for these cholesterol
analyses can be sera diluted to contain about 25 and 60
mg/dL of cholesterol. These levels cover the range of
values of most HDL specimens. An alternative
procedure is to use a normal-range cholesterol serum
pool for calibration and to use both primary standard
solutions and serum pools of low concentration levels
for monitoring. Since a major cause of discrepancies
between HDL-C cholesterol results among laboratories
can be attributed to lack of appropriate standardization
of the total cholesterol method, an effort should be made
first to standardize the cholesterol method. Reference
materials, according to Cooper [66], that appear suitable
for calibrating and monitoring the determination of
cholesterol in HDL isolated fractions are: (1) primary
cholesterol standards, (2) serum calibrator pools, (3)
serum quality control pools, and (4) fractions of HDL
isolated by ultracentrifugation or precipitation methods
and labeled accurately with total cholesterol content.
697
High-Density Lipoprotein (HDL) Cholesterol
The calibration and control materials produced for use in
the first steppreparation of the HDL sample for
cholesterol analysisare often unsuitable because of an
unpredictable instability of HDL particles, varying
composition, and flux of lipids and apoproteins among
lipoproteins [2] or of apoB from HDL particles [51].
Compromises, therefore, have to be accepted. Under
these circumstances, it appears practical to seek HDL
reference materials that are homogeneous, are stable up
to 2 years, and possess a suitable similar-to-serum
matrix. The HDL reference materials that might meet
these criteria for control of the HDL isolation procedure
are (1) serum with low triglyceride or VLDL levels, (2)
bottom fraction 1.006 g/mL density prepared by
ultracentrifugation, and (3) bottom fraction 1.063 g/mL
density prepared by ultracentrifugation.
Studies by Cooper [66] suggest that for long-term
storage of HDL cholesterol pools, a temperature less
than 60C, rather than 20C or 5C, is necessary.
5. Calculation of VLDL and LDL cholesterol.
Although one can isolate VLDL (density less than
1.006 g/mL) and determine the cholesterol content,
VLDL cholesterol can be estimated with a fair
amount of accuracy by the following
calculation:[46]
Triglyceride (mg/dL) = VLDL-C (mg/dL)
5
This holds true only under the condition that serum
triglyceride concentration is less than 400 mg/dL.
Furthermore:
LDL-C = Total cholesterol (VLDL-C + HDL-C)
698
Holotranscobalamin
Holotranscobalamin
Marion Black i
Name: Holotranscobalamin, holoTC, active B12
Clinical significance: Refer to Chapter 43, Vitamins, in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Principles of Analysis and Current Usage
The diagnosis of Vitamin B12 deficiency is complex
because of the poor sensitivity and specificity of the
conventional assays [1,2].
Vitamin B12 (cobalamin, Cbl), the current biochemical
marker for investigation of vitamin B12 deficiency, has
been shown to have limited specificity, to be falsely
positive if the concentration of haptocorrin (HC), which
is a vitamin B12-binding protein, is reduced, and falsely
negative if haptocorrin is increased. The many different
methodologies available for measuring Cbl have resulted
in a range of method-dependent reference intervals
[2,3,4].
The concentrations of the metabolic markers
methylmalonic acid (MMA) and homocysteine (Hcy) are
considered to be more sensitive indicators of vitamin B12
status than total vitamin B12 (Cbl). Both MMA and Hcy
increase in vitamin B12 deficiency. However, Hcy has
been shown to have low specificity, being influenced by
lifestyle factors such as smoking and alcohol intake, and
increasing in patients with folate deficiency and renal
impairment. MMA, while considered a more sensitive
indicator of vitamin B12 status than Cbl, is an expensive
assay requiring specialized instrumentation not readily
available in most clinical laboratories.
Recently a new biochemical marker, holotranscobalamin
(holoTC), has been proposed as a sensitive early marker
for diagnosing vitamin B12 deficiency.
Vitamin B12 circulates in plasma bound to two proteins,
HC and transcobalamin (TC). HC, a glycoprotein with a
half-life of 240 hours, binds 70% to 90% of total Cbl and
is mostly saturated with Cbl [5,6]. HC has no known
functions other than its ability to bind metabolically inert
forms of vitamin B12 [7]. Vitamin B12 bound to TC,
referred to as holotranscobalamin (holoTC), has a half-
life of 1 to 2 hours and is the residual 10% to 30% of
total vitamin B12. HoloTC is the physiologically active
vitamin B12 fraction which promotes the specific uptake
of its vitamin B12 by all cells through receptor-mediated
endocytosis involving a specific TC receptor found in all
tissues.
The much shorter half-life of holoTC (1 to 2 hours) has
led to the view that measurement of holoTC would be
useful as a sensitive early marker of vitamin B12
i
Holotranscobalamin
New method
Fifth edition: Marion Black
deficiency. There have been a number of recent clinical
studies outlining the diagnostic value of holoTC in
predicting vitamin B12 status in various clinical settings,
in particular as an early marker of vitamin B12 deficiency
[4,8,9-13].
Serum holoTC has proved challenging to measure,
because it accounts for only 10% to 30% of the
circulating vitamin B12, and because the major part of
TC circulates unsaturated with vitamin B12 (apo-
transcobalamin [apoTC]) [14-16]. Early attempts to
measure holoTC involved separating TC from HC prior
to quantification of vitamin B12, allowing direct
measurement of the cobalamin attached to TC [17-19],
or an indirect calculation of holoTC concentration was
made by measurement of total plasma cobalamins and
the plasma cobalamins not attached to TC [20-22].
Nexo et al. [23] combined a sensitive enzyme-linked
immunosorbent assay (ELISA) for TC measurement
with a simple procedure for removal of apoTC and
developed an assay to determine both holoTC and total
TC in < 200 L of serum. They reported a method in
which magnetic beads coated with vitamin B12 (Cbl)
precipitated apoTC, then measurement of the holoTC
present in the supernatant was done by ELISA.
More recently, several specific and reproducible
commercial assays have become available for the direct
measurement of holoTC. The holoTC competitive-
binding radioimmunoassay (holoTC RIA) described by
Ulleland et al. [24] was the first commercially available
method for holoTC measurement. HoloTC is measured
as a two-step process. First, total TC is captured from the
sample by magnetic particles (microspheres) coated with
monoclonal anti-human TC antibodies. Then the Cbl
bound to holoTC is released and assayed by competitive-
binding RIA standardized with recombinant human
holoTC [24,25].
Following characterization of a monoclonal antibody
with > 100-fold specificity for holoTC over apoTC, a
direct immunoassay was developed in the ELISA format
[26].
Axis-Shield Diagnostics Ltd subsequently developed a
direct enzyme immunoassay utilizing the highly specific
mAb for holoTC. This assay was later adapted for use on
the Abbott AxSYM platform. The Abbott AxSYM
699
Holotranscobalamin
active-B12 (previously holoTC) assay [27] is a precise,
rapid, automated two-step, sandwich microparticle
enzyme immunoassay (MEIA) for the quantitative
determination of human holoTC in serum. The active-
B12 assay is based on two monoclonal antibodies. One is
a mouse monoclonal antibody specific for active B12
(holoTC), immobilized on latex microparticles. The
second binder is a mouse monoclonal antibody to TC.
The assay directly quantitates active B12 (holoTC),
avoids the pretreatment steps common to all vitamin B12
assays, and shows good correlation with the Axis-Shield
active-B12 (holoTC) RIA assay.
Since the commercial RIA assay for holoTC requires a
large sample volume (400 to 800L), Refsum et al.
developed a microbiological assay (MBA) requiring less
plasma volume (100L) that could measure both holoTC
and total TC [28].
In their MBA assay, holoTC is captured with magnetic
beads coated with TC antibodies, followed by a
conventional MBA for cobalamin measurement
(Lactobacillus leichmannii).The MBA assay showed
good precision and a strong correlation with holoTC by
RIA.
Reference and Preferred Methods
There is no reference method for the measurement of
holoTC. The methods currently in use are the ELISA
[23] and MBA [28] assays, the commercially available
RIA assay [29], and the automated MEIA assay [27] on
the Abbott AxSYM immunoassay analyzer. All assays
have been shown to have acceptable performance
[23,27-29]. Assay selection will depend on the
individual laboratorys needs. The automated MEIA
assaywith no preanalytical treatment required, a
turnaround time of 20 minutes, and an assay throughput
of 45 tests/houris suitable for testing large numbers of
samples in a clinical diagnostic setting [30].
Specimen
No significant circadian variation of serum holoTC has
been found in healthy, vitamin-replete subjects on a
standard, nonvegan nonvegetarian Danish normal diet.
This supports the view that holoTC is a marker of long-
term vitamin B12 status and that samples may be
collected from nonfasting subjects [7].
Only serum or lithium-heparin plasma is recommended
for the MEIA assay. Separated samples may be stored
for up to 28 days at 2C to 8C after collection. If testing
is delayed more than 28 days, specimens may be stored
for up to 6 months at 20C or colder [27].
Both serum and EDTA plasma may be used for RIA and
ELISA, although plasma values are a little higher (6% to
8%) than for serum [23,24].
The RIA assay [24,29] requires 400 L of sample (800
L if analyzed in duplicate). Samples may be stored for
up to 7 days at 2C to 8C and then frozen. Repeated
freeze/thaw cycles and extended exposure of specimens
to light should be avoided for both the MEIA and RIA
assays [27,29].
Both the RIA and ELISA methods determined that
holoTC concentrations were higher in EDTA plasma
than serum, but the MBA assay showed no differences
between plasma (EDTA) or serum, fasting or nonfasting,
specimens [28].
Interferences
Trace amounts of hemolysis (hemoglobin < 500 mg/dL),
icterus (bilirubin < 40 mg/dL), and lipemia (triglycerides
< 1500 mg/dL) do not generally interfere with holoTC
measurement in either the RIA or MEIA assays[27,29].
Holotranscobalamin Reference Interval
The holoTC reference interval is dependent on the
methodology. There is currently no consensus regarding
reference intervals for holoTC [31].
For the RIA assay, a study performed by Ulleland et al.
[24] involving 105 healthy volunteers (20 to 80 years of
age) reported a reference range of 24 to 157 pmol/L. A
later study by Loikas et al. [25] utilized a more carefully
selected reference group (n = 303 healthy adults, 22 to
88 years of age), in which they chose to exclude
individuals with any conditions or medications that
might cause Cbl deficiency. Loikas et al. determined the
95% central reference interval to be 37 to 171 pmol/L.
The MEIA assay quotes the 95% central reference
interval as 19 to 119 pmol/L, based on a study involving
281 healthy donors [27].
For ELISA, Nexo et al. determined the central 95%
reference interval to be 40 to 150 pmol/L (n = 137, 21 to
65 yrs of age, healthy blood donors) [23].
The MBA reference interval, 42 to 157 pmol/L, was
determined by Refsum et al. [28] in their study (n = 500,
18 to 69 yrs of age, healthy, nonfasting blood donors),
which was performed on serum samples. They also
proposed that separate reference intervals be considered
in younger women (45 years), holo TC being lower in
women than men in this age group.
A number of clinical studies have considered the
biological determinants of holoTC. Several studies have
reported a strong relationship between serum creatinine
and holoTC [8,32], but others have found no association
[4,33].
No major differences in holoTC concentrations between
males and females have been observed [23-25].
However, there may be hormonal regulation of holoTC;
younger women who were current users of oral
contraceptives (OC) were shown by Riedel et al. [34], to
have Cbl and holoTC levels 25% lower than non-users.
This was not associated with significantly higher
concentrations of the metabolic markers MMA and Hcy,
suggesting redistribution rather than depletion of
intracellular Cbl. Riedel et al. concluded that these
700
Holotranscobalamin
hormonal effects may lessen the diagnostic utility of
total Cbl and holoTC in OC users. Further studies are
required to determine if OC users with borderline Cbl
are likely to develop Cbl deficiency clinically [34]. This
study also found that hormone replacement therapy
(HRT) had no noticeable effect on circulating Cbl,
holoTC or the metabolic markers MMA and Hcy.
Interpretation
A number of studies have been published to support the
view that holoTC would be a better indicator of vitamin
B12 status than total serum Cbl:
HoloTC values have been shown to be low in
patients with biochemical signs of vitamin B12
deficiency [9]
Low values of holoTC have been reported in
both vegetarians and vegans and in populations
with a low intake of vitamin B12 [4,8,10]
Low holoTC (but not vitamin B12) has been
reported in patients with Alzheimers disease
[11]
HoloTC has also been shown to reflect vitamin
B12 absorption better than serum vitamin B12
[12]
Data from Hermann et al. [8] supported the view that
measurement of holoTC and MMA provide a better
indication of Cbl status than the measurement of total
vitamin B12, holoTC being a more sensitive marker than
the functional marker of vitamin B12 metabolism,
methylmalonic acid (MMA). They found that the use of
holoTC and MMA enabled them to differentiate between
storage depletion and functional vitamin B12 deficiency.
The effect of diminished renal function on holoTC
concentration is unclear. Hermann et al. [8] expressed a
cautionary note: because renal failure may impair
cellular holoTC uptake, renal patients will have a higher
requirement of circulating holoTC. They concluded that
holoTC could not be used as a marker of vitamin B12
status in patients with renal dysfunction [8].
Likewise, Hvas and Nexo [32] found that an increase in
holoTC and vitamin B12 were significantly associated
with plasma creatinine: the higher the plasma creatinine,
the higher the holoTC and vitamin B12 values.
Miller et al. [4] found no correlation between creatinine
and holoTC. More recently, Loikas et al. [33] examined
the effect of renal impairment on the vitamin B12
deficiency markers (vitamin B12, Hcy, MMA, holoTC) in
1011 aged subjects. Using serum cystatin C as the
indicator of renal impairment, they found that the
metabolic markers Hcy and MMA, but not vitamin B12
or holoTC, were affected by renal function impairment.
The effect of renal impairment on holoTC needs to be
further clarified, particularly since vitamin B12 deficiency
and renal impairment are common in the aged
population.
Since there is no gold standard method available to
diagnose vitamin B12 deficiency, owing to the decreased
availability of Schillings test (difficulties in obtaining
labeled vitamin B12 and native human intrinsic factor)
[35], it is difficult to assess the clinical usefulness
(sensitivity and specificity) of holoTC.
Hvas and Nexo [2] concluded that it is still debatable
how much additional value the measurement of holoTC
provides beyond the already existing tests for diagnosing
vitamin B12 deficiency. They suggested further studies
were required to decide whether holoTC can be used
alone or should be used in combination with one or the
other vitamin B12 markers.
Miller et al. [4], found that holoTC and vitamin B12 have
equal diagnostic accuracy in screening for metabolic
vitamin B12 deficiency and that measurement of both
holoTC and total vitamin B12 provides a better screen for
vitamin B12 deficiency than either assay alone.
A recent study by Clarke et al. [13] confirms that
holoTC has a modestly superior diagnostic accuracy
compared with conventional vitamin B12 for the
detection of vitamin B12 deficiency, but neither test can
be recommended to screen asymptomatic populations.
Holotranscobalamin Performance Goals
HoloTC is not currently regulated for proficiency
testing. Discussions are underway in Europe with a view
to introducing a holoTC proficiency testing scheme.
Clinical diagnostic laboratories tend to perform either
the automated (no pretreatment of sample) MEIA assay
[27] or the manual RIA assay [29]. The total precision
for the RIA assay (using manual pipetting steps) as
determined using the National Committee for Clinical
Laboratory Standards (NCCLS) Protocol EP5-T2 [36]
was shown to be
12% to 8%, across a range of holoTC concentrations
from 14 to 139 pmol/L [29].
The precision for the AxSYM active B12 automated
MEIA assay [27] was determined using the NCCLS
Protocol EP5-A2 [37]. The AxSYM active-B12 assay
[27] is designed to have a precision of less than 10%
total CV for holoTC (active B12) in the range of 23 to 50
pmol/L. This performance goal is achievable as
demonstrated by Brady et al. [30], who showed total
imprecision of 6.3% to 8.5%, and intraassay imprecision
of 3.4% to 5.1%. The ELISA holoTC assay has been
shown to have a total precision (CV) of 8% at 33 pmol/L
and 7% at 88 pmol/L [23]. For the MBA assay, Refsum
et al. [28] assessed between-assay imprecision for
holoTC using five human plasma pools (27 to 167
pmol/L). Between-day CVs for holoTC were 4% to 9%.
All of the above assays have demonstrated acceptable
holoTC assay performance.
References
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complementary or exclusive diagnostic
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Holotranscobalamin
2 Hvas AM, Nexo E. Diagnosis and treatment of
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3 Green R. Metabolite assays in cobalamin and
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4 Miller JW, Garrod MG, Rockwood AL,
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holotranscobalamin, singly and in combination,
in screening for metabolic vitamin B12
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5 Seetharam B, Li N. Transcobalamin II and its
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8 Hermann W, Obeid R, Schorr H, Geisel J.
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9 Obeid R, Journa M, Hermann W. Cobalamin
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of dietary vitamin B12 deficiency. Clin Chem
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11 Refsum H, Smith AD. Low vitamin B12 status
in confirmed Alzheimers disease as revealed
by serum holotranscobalamin. J Neurol
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transcobalamin concentration and
transcobalamin saturation reflect recent vitamin
B12 absorption better than does serum vitamin
B12. Clin Chem 2004;50:1043-1049.
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AM, Schneede J et al. Detection of vitamin B12
deficiency in older people by measuring
vitamin B12 or the active fraction of vitamin B12
, holotranscobalamin. Clin Chem 2007;53:963-
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14 Markle HV. Cobalamin. Crit Rev Clin Lab Sci
1996;33:247-356.
15 Herbert V. Staging vitamin B12 (cobalamin)
status in vegetarians. Am J Clin Nutr
1994;59(Suppl 5):1213S-22S.
16 Nexo E, Hansen M, Rasmussen K, Lindgren A,
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19 van Kapel J, Wouters NM, Lindemans J.
Application of heparin-conjugated sepharose
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20 Vu T, Amin J, Ramos M, Flener V, Van yo L,
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21 Herzlich B, Herbert V. Depletion of serum
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23 Nexo E, Christensen AL, Hvas AM, Petersen
TE, Fedosov SN. Quantitation of holo-
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24 Ulleland M, Eilertsen I, Quadros E, Rothenberg
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27 Abbott AxSYM Active-B12 Assay package
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holotranscobalamin (holoTC) assays in a
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32 Hvas AM, Nexo E. Holotranscobalamin: a first-
choice assay for diagnosing early vitamin B12
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33 Loikas S, Koskinen P Irjala K, Lopponen M,
Isoaho R, Kivela S-L, Pelliniemi T-T. Renal
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total vitamin B12 and holotranscobalamin in
screening for vitamin B12 deficiency in the
aged. Clin Chem Lab Med 2007;45:197-201.
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35 Schilling RF. Intrinsic factor studies II. The
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37 National Committee for Clinical Laboratory
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NCCLS Document EP5-A2. Wayne, PA:
NCCLS; 2004.

Tables
Holotranscobalamin Methods Summary Table
Method 1: RIA
Principle of analysis: Radioimmunoassay (competitive binding)

Comments: Serum, EDTA plasma, large sample volume required (400 L), sensitive, precise, time consuming,
multiple manual steps
Suitable for use in clinical laboratories

Method 2: ELISA
Principle of analysis: Magnetic beads coated with vitamin B12 (cobalamins) precipitate apo-transcobalamin
(apoTC), and the protein moiety of holoTC present in the supernatant is measured by ELISA.

Comments: Sensitive, precise, time consuming, requiring skill to pretreat the samples prior to running on an
automated platform. 100 L sample.

Method 3: MEIA
Principle of analysis: Microparticle enzyme immunoassay (MEIA). Two-step sandwich microparticle enzyme
immunoassay

Comments: Sensitive, precise, serum or lithium heparin plasma, automated, rapid assay suitable for use in clinical
laboratories

Method 4: MBA
Principle of analysis: Microbiological assay (MBA); holoTC captured with magnetic beads coated with TC
antibodies; followed by a conventional MBA for cobalamin measurement (Lactobacillus leichmannii)

Comments: Precise, time consuming, useful for studies with limited sample volume
703
Homocysteine

Homocysteine
Sheila Dawling
Name: Homocysteine or HcyH, total homocyst(e)ine or tHcy
Clinical significance: Refer to Chapter 37, Coronary Artery disease in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular mass: 135.2: convert molar to mass units: g/mL or mg/L = mol/L 0.1352
Chemical class: Amino acid
Structures:
O NH2 O O
HS HO S
OH S OH HS OH
NH2 O NH2 NH2

Homocysteine Homocystine Cysteine

(HcyH) (Hcy-Hcy) (Cys)

O O O
S S
H3C OH HO S OH O
NH2 NH2 NH2 H2N
S
Methionine Homocysteine Mixed Disulfide
(Met) (Hcy-Cys) Homocysteine thiolactone
i exists in a variety of oxidized forms, mostly through
Principles of Analysis and Current Usage
disulfide links with other sulfur-containing amino acids
Homocysteine (2-amino-4-mercaptobutyric acid [Hcy]) or those contained in proteins such as albumin and the
was first described in 1932. It is a nonessential, sulfur- globulin proteins IgG, 1-acid glycoprotein, and HDL. A
containing amino acid that is not required for protein smaller fraction becomes irreversibly bound through
synthesis, but its clinical relevance became apparent amide linkage via reaction with Hcy-thiolactone [2].
some 30 years later when McCully observed the Presence of an excess of some of these forms is
relationship between grossly elevated plasma associated with a variety of adverse effects. These
homocysteine and accelerated cardiovascular disease [1]. include but are not limited to folate and B12 deficiency
Its importance derives from two key factors. First is its [3,4], cardiovascular disease [5,6], birth defects [7],
position as an intermediary metabolic product that pregnancy complications [8], psychiatric disorders [9],
derives from the demethylation of the essential amino cognitive impairment in the elderly [10], and
acid methionine, making it a key player in the osteoporosis [11]. Whether homocysteine is directly
folate/vitamin B12 cycles. Once formed, homocysteine is responsible for these effects or is merely the tangible
either remethylated to methionine in a step involving evidence of perturbed one-carbon metabolism or other
vitamin B12 and folate, or it is metabolized to cysteine in thiol pathways of different etiologies is still highly
two vitamin B6dependent reactions (Figure 1). debated [12].
Defective homocysteine metabolism results in high Plasma Hcy Content Protein Distribution %
plasma concentrations of homocysteine, accompanied by Form mol/L % of Total Hcy Cys Cys-Gly
either elevated or decreased levels of methionine _____________________________________________
(cystathionine -synthase or methionine synthase Protein-bound 8 80
deficiency, respectively). Secondly, because of its strong forms
chemical reactivity as a reducing agent, its presence in Albumin
plasma in its native form is short-lived. In plasma, it 88 79 67
Globulins
12 21 33
i
Homocysteine Disulfides 2 20
Previous and current authors of this method: Hcy-Cys
First edition: Not done Hcy-Hcy
Methods edition: Not done Hcy 0.2 0.2
Second edition: Not done
Third edition: Not done tHcy 10 100
Fourth edition: Kirby Marsh, Gregory Rezinger
Fifth edition: Sheila Dawling Hcy, Free homocysteine; tHcy, Total homocysteine
704
Homocysteine
Accurate measurement of homocysteine poses
significant analytical problems, since the small
homocysteine molecule must be measured specifically
against a background of a 20- to 50-fold excess of
structurally similar molecules. Two approaches are taken
for the measurement of homocysteine in plasma: (1)
discrete analysis for the free homocysteine together with
some or all of the other oxidized species or (2) the
conversion of all the plasma forms to a single entity
(known as total homocysteine, or total homocyst(e)ine or
tHcy). Selection of the method depends on the clinical
application, although for routine screening, the second
approach is the one most usually taken, since this can
most often be accomplished with commercially available
reagents which run on automated chemistry platforms.
Initial methods for measuring homocysteine used an
amino-acid analyzer (Table 1, Method 1). In these
procedures, the heparinized plasma samples were
deproteinized and the free homocysteine (as well as
other amino acids) sequestered in the filtrate. These were
then separated by cation-exchange chromatography and
quantified by post-column derivatization with
Ninhydrin, using a dedicated amino acid analyzer [13].
An alternative approach is to prepare fluorescent
derivatives of the amino acids in the filtrate and then
separate these by HPLC with quantitation by
fluorimetric detection [14]. These methods require
specialized, expensive equipment and are extremely
slow, with analysis times of 2 to 3 hours per sample.
Until recently however, these were the only HPLC
methods available with sufficient sensitivity (1 mol/L)
to determine the free form of homocysteine in plasma.
These are still widely used for specialized monitoring of
patients with homocystinuria, where both plasma and
urine concentrations may be measured, together with
methionine and other essential amino acids, to monitor
the patients dietary status.
Reduction of all plasma species of oxidized
homocysteine to yield free, reduced homocysteine is
used by most procedures to yield total plasma
homocysteine (tHcy). Total Hcy is then either quantified
by HPLC (Table 1, Method 2) or is converted to S-
adenosyl-homocysteine (SAH) and subsequently
monitored traditionally with coupled enzymatic reactions
(Table 1, Method 4) or reacted with an SAH antibody
(Table 1, Method 3). These methods are described
further.
Stabler et al. [15], described a capillary gas
chromatography-mass spectrometry method using t-
butyldimethylsilyl derivatives and tritiated internal
standards to quantify plasma concentrations of total
homocysteine, cysteine, and methionine. They reduced
all plasma forms of the three compounds by reduction
with 2-mercaptoethanol and used selected ion
monitoring for identification and quantification of the
amino acids. This method has not received much
attention.
Three HPLC kits are currently available (Bio-Rad, Drew
Scientific, and ChromSystems) for total homocysteine.
These take the form of an initial supply of the
instrumentation, with purchase of additional reagents
and consumables as necessary. The Bio-Rad assay [16]
involves a 5-minute (50C) incubation of sample and
internal standard with trialkylphosphine reducing agent
and derivatizing reagent (4-aminosulfonyl-7-fluoro-
2,1,3-benzoxdiazole, ABD-F). After precipitation of
proteins with trichloroacetic acid, the supernate is
chromatographed by reverse phase, and compounds are
detected and quantified by fluorescence detection (385
nm excitation, 515 nm emission). Within less than 5
minutes, there is good separation of glutathione,
cysteine, homocysteine, and Cys-Gly. The Drew S30
system [17] is available on similar terms. This assay
involves a short incubation of sample and internal
standard (2-mercaptoethylamine) with TCEP (tris[2-
carboxyethyl]) phosphate reducing agent. After
precipitation of proteins with trichloroacetic acid, the
supernate is derivatized with ammonium SBDF (7-
fluorobenzo-2-oxa-1,3-diazole-4-silfonate) for 50
minutes (60C). The thiol derivatives are subsequently
chromatographed by reverse phase, and compounds are
detected and quantified by fluorescence detection. While
the Bio-Rad assay is a conventional HPLC system, the
Drew S30 takes the form of a small black-box
analyzer, producing a number rather than a
chromatogram printout. The instrument is configured to
identify cysteine and Cys-Gly but only reports the value
for homocysteine. Thus specimens that require dilution
must be diluted in a mixture of 200 mol/L cysteine and
30 mol/L Cys-Gly. Both assays compare well to the
CDC method, perform well, and are suited to high-
throughput analysis, with run times of 5 to 6 minutes per
sample. The ChromSystems kit has been the most recent
entry to the market. This is run on a conventional HPLC
system with fluorimetric detection. The assay range is
0.5 to 200 mol/L and compares well to other HPLC
methods [18].
Electrochemical detection for HPLC has the distinct
advantage over fluorimetric detection in that the thiols
can be detected at the same sensitivity without prior
derivatization, thus eliminating what can be a time-
consuming step. Martin et al. [19] described a method
that used penicillamine as an internal standard and
dithiothreitol as a reducing agent for 15 min at 37C.
Proteins were precipitated with sulfosalicylic acid and
the supernate injected directly onto a C18 column with a
mobile phase of 10 mM sodium dihydrogen phosphate,
pH 2.75 with phosphoric acid, 15% methanol, and 12
mM octane sulfonic acid modifier. The run time was
approximately 8 min. The electrochemical detector was
operated at a screening potential of +0.4 V and detection
at +1.0 V. ESA diagnostics promotes a method for use
on its eight-channel CoulArray detector. While not a
kit as such, the method is fully validated and U.S.
Food and Drug Administration (FDA)-approved. The
porous graphite electrodes require minimal maintenance
compared to other detectors of this type, and the assay,
705
Homocysteine
with a run time of about 15 minutes, gives results that
correlate well with the CDC reference method [20]. A
30-minute incubation with penicillamine internal
standard and TCEP (tris-2[carboxyethyl]-phosphine
hydrochloride) reducing agent is required. Proteins are
precipitated with perchloric acid, and the supernate is
applied to the C18 column. The mobile phase is 0.15 M
sodium dihydrogen phosphate with 1 mM sodium
dodecylphosphate modifier, pH 2.8 with 10%
acetonitrile. Four of the eight channels are used for this
application, at settings of 520, 750, 800 and 820 mV.
The assay is linear to 100 mol/L but measures only the
total homocysteine.
HPLC with tandem mass spectrometry has been applied
to the analysis of total homocysteine. Sample
preparation is as described above for electrochemical
detection, and the column effluent is monitored in SRM
(selected reaction monitoring mode) (136.1 90.0 amu
for homocysteine and 140.0 94.0 amu for d4-
homoysteine) by neutral loss of formic acid. Although
the method benefits from the use of deuterated internal
standards, it offers little advantage for the additional
expense incurred [21]. This procedure can be simplified
and adapted to 96-well plates for high throughput by
analyzing reduced specimens without derivatization and
monitored as above [22]. Tandem mass spectrometry is
used for high-throughput testing of dried blood spots for
the presence of homocystinuria in newborn screening
programs. However, for this specialist application, the
butylated derivatized specimen is aspirated directly into
the mass spectrometer without any chromatography, and
the amino acid methionine is monitored at mass 209.2 in
neutral loss mode.
Although now obsolete, the introduction of a
radioenzyme assay for homocysteine paved the way for
the chemical analysis of this compound [23]. Central to
this assay was the reversibility of the reaction catalyzed
by S-adenosyl-L-homocysteine hydrolase (SAHH),
which allowed both non-radiolabeled S-adenosyl-L-
homocysteine and L-homocysteine to act as L-
homocysteine donors in the synthesis of radiolabeled S-
[14C]-adenosyl- L-homocysteine from radiolabeled
[14C]-adenosine. The product was quantified by HPLC.
The first of the assays to be made available for a
commercial clinical chemistry platform was in the early
1990s, with the development of the fluorescence
polarization immunoassay (FPIA) for the Abbott IMx
[24] and subsequently the AxSYM platform [25]. In this
procedure (Table 1, Method 3a, and Figure 2) all
additions are performed automatically by the
instruments pipetting system. The homocystine, mixed
disulfide, and protein-bound forms of homocysteine in
the serum or plasma are reduced with dithiothreitol to
form the reduced, free form of homocysteine (tHcy).
This product is then converted to S-adenosyl-L-
homocysteine by the catalytic activity of the enzyme
SAHH in the presence of excess adenosine. The S-
adenosyl-L-homocysteine produced is quantified in a
competitive reaction between it and S-adenosyl-L-
homocysteinefluorescein tracer for sites on a
monoclonal antibody specific for the S-adenosyl-L-
homocysteine moiety. The amount of tracer bound is
quantified by measuring the fluorescence polarization of
the resultant mixture, with this being inversely
proportional to the free S-adenosyl-L-homocysteine
concentration and hence inversely to the tHcy in the
specimen. The IMx method is a batch procedure (20
specimens in 1 hour), whereas the AxSYM is a random-
access analyzer (35-minute assay). This method is still
widely used and compares very favorably with the CDC
reference HPLC method, and has excellent precision
(Table 2) [26]. The method is also available from Bio-
Rad as a microtiter plate assay (Axis) [27].
The Bayer ADVIA Centaur assay uses the same
chemistry as described for the FPIA assay. (Table 1,
Method 3b, and Figure 3). Dithiothreitol is used as a
reducing agent, and reduced forms of homocysteine are
converted enzymatically to SAH. The solid-phase
(paramagnetic particles covalently linked to SAH) is
added, followed shortly by the Lite Reagent (monoclonal
mouse antibodies to SAH labeled with acridinium ester).
Any SAH produced competes with SAHmagnetic
particle conjugates for acridinium-labeled antibodies.
After separation of the magnetic particles, free tracer is
liberated with the trigger reagent, and a
chemiluminescent signal is produced in inverse
proportion to the amount of homocysteine present in the
sample. Results are available in 18 minutes and
compared favorably with both FPIA and HPLC [28].
This is a widely used method, but precision is rather
disappointing (Table 2).
Neither of these immunoassays is susceptible to a hook
effect because of the relatively narrow range of
homocysteine concentrations determined, but both may
be the subject of HAMA or heterophilic interference.
Interference by heterophile antibodies should be
suspected when serial specimens from the same patient
or repeat analysis of the same sample produce wildly
different results, or when recovery on dilution is not
linear. These polyclonal autoantibodies, either IgG or
IgM, bind across a broad spectrum of different animal
antibodies used in kit manufacture and thus form
sandwiches between the capture and detection
antibodies, producing a spurious signal. Analysis by an
alternative method is usually sufficient to align the
measured result with the clinical picture. Should it be
necessary to prove that these antibodies exist, a broad
spectrum passive precipitant such as PEG can be used,
but it is preferable to use an active blocking agent such
as The Scantibodies Heterophilic Blocking Reagent
Tubes (HBRT). This proprietary mix of lyophilized
mouse anti-human IgM has high affinity for human anti-
animal antibodies commonly used in reagent
manufacture (mouse, goat, sheep, and rabbit) and to
rheumatoid factor. Each tube contains sufficient reagent
to inactivate 500 L of patient sera and is incubated for
1 hour at room temperature. Although FDA approved for
this purpose, the value obtained from a sample treated
with HBR should not be used as a reportable result.
706
Homocysteine
With recent renewed interest in homocysteine, enzymic
assays for chemistry platforms have been developed
(Table 1, Method 4). These are often dealt with as a
group, although as shown by the three examples below,
their chemistries are quite varied, and consequently they
often show poor precision when considered together
(Table 2).
Vitros MicroTip ECi Assay (Table 1, Method 4a).
This is a three-reagent homogeneous enzyme assay.
These same reagents are available as the Catch
Homogeneous Enzymatic Assay (Catch Inc, Bothell,
WA), which can be run on a number of automated
chemistry platforms [29]. Reagent 1 contains serine,
NADH, and lactate dehydrogenase (LD); Reagent 2
contains TCEP (tris-2[carboxyethyl]-phosphine
hydrochloride) reducing agent; Reagent 3 contains
cystathionine -synthase (CBS) and cystathionine -
lyase (CBL). The first step reduces all plasma forms of
homocysteine to free homocysteine using TCEP (tris-
2[carboxyethyl]-phosphine hydrochloride). This reacts
with added serine and the enzyme cystathionine -
synthase (CBS) to form L-cystathionine:
Serine + homocysteine CBS L-cystathionine
Cystathionine -lyase (CBL) is then added, which
catalyses the conversion of L-cystathionine to
homocysteine, pyruvate, and ammonia:
CBL homocysteine + pyruvate +
L-Cystathionine
NH3
The final reaction is the reduction of pyruvate to lactate
by added lactate dehydrogenase (LD):
Pyruvate + NADH LD lactate + NAD+
The NAD+ generated can be monitored by the rate of
change in absorbance at 340 nm, which is proportional
to the concentration of homocysteine in the plasma
sample.
Results compare favorably with both FPIA and the Bio-
Rad HPLC [29]. There is negative interference from
elevated triglycerides (>500 mg/dL). The assay also
measures endogenous cystathionine, but this is usually
an insignificant amount. The major drawback with this
assay is that it is not compatible with some of the other
tests typically run on chemistry platforms. Carryover on
reagent probes and reaction cuvettes from
hydroxylamine used in some colorimetric iron assays
can falsely lower homocysteine results. In addition, these
reagents contain a significant amount of lipase and
should not be used concurrently with plasma lipase
testing.
Diazyme Assay (Table 1, Method 4b).
This two- or three-reagent system can be run on a
number of automated chemistry platforms [30]. The first
step reduces all plasma forms of homocysteine to free
homocysteine using TCEP (tris-2[carboxyethyl]-
phosphine hydrochloride).
Reduced homocysteine is reacted with added S-
adenosyl-L-methionine and the enzyme homocysteine
methyltransferase, producing S-adenosyl-L-
homocysteine and methionine.
Homocysteine + SAM HMT SAH + methionine
Added SAHH then cleaves S-adenosyl-L-homocysteine
to adenosine and regenerates homocysteine, which
perpetuates the reaction.
SAH SAHH homocysteine + adenosine
This adenosine is enzymatically converted to inosine and
ammonia by added adenosine deaminase.
Adenosine ADA inosine + NH3
The final step in the reaction is to quantify the liberated
ammonia with the enzyme Glutamate Dehydrogenase in
the presence of added 2-oxoglutarate.
NH3 + 2-oxoglutarate + NADH GLDH glutamate +
NAD+ + H2O
The NAD+ generated can be monitored by the rate of
change in absorbance at 340 nm, which is proportional
to the concentration of homocysteine in the plasma
sample.
A third enzymatic approach (Table 1, Method 4c) is
based on a single, highly specific enzyme, homocysteine
,-lyase (rHCYase). As described by Tan et al. [31], the
reactions are performed in 96-well plates. In the first
step, all forms of homocysteine are reduced with
dithiothreitol, and the reduced homocysteine is
converted to 2-oxobutyrate, ammonia, and hydrogen
sulfide.
Homocysteine + SAM rHCYase 2-oxobutyrate + NH3
Vit B6 + H2S
In the second step, the reaction is terminated by the
addition of DBPDA (N,N-dibutyl phenylene diamine)
and oxidizing agent (potassium ferricyanide) in sulfuric
acid. DBPDA combines with hydrogen sulfide to form
3,7-bis(dibutyl amino)phenothiazine-5-ium chloride,
which is strongly fluorescent (665 nm excitation, 690 nm
emission).
H2S + DBPDA potassium 3,7-bis(dibutyl
amino)phenothiazine-5-ium
chloride ferricyanide
One 96-well plate can be processed in about 1 hour, but
this application has not yet been used commercially.
Results compared well with HPLC.
707
Homocysteine
Reference and Preferred Methods
The reference method is provided by the Centers for
Disease Control and Prevention (CDC) [32]. This HPLC
procedure uses reduction, precipitation, derivatization,
and fluorometric detection, as described at the end of
this chapter. This method is not applicable to routine
testing in the clinical setting because of time constraints
and the dedicated equipment required. However, it does
have value in the investigation of unusual or complicated
patients. Review of College of American Pathologists
(CAP) survey data (Table 2) shows almost 50% of labs
reporting results to CAP are using chemiluminescence
and 30% fluorescence polarization. Only a handful of
labs are using HPLC, although the incidence is rising
slightly as kit assays are becoming available. In contrast,
over half the participants in the United Kingdom
National External Quality Assessment Service (NEQAS)
are using FPIA and a quarter HPLC methods;
chemiluminescence is not widely used. FPIA
consistently outperforms other methods in terms of
variance. The comparative results are shown in Table 2
and discussed in the section on Performance Goals.
Specimen
After blood collection but before removal of the red
cells, there is a time- and temperature-dependent
increase in the serum/plasma homocysteine which is
attributed to its continued release from erythrocytes.
This increase can be as much as 100% in 8 hours at
room temperature [33,34]. Gel separators can help if
centrifugation is prompt. Therefore, most methods use
EDTA plasma as the specimen of choice; serum
produces slightly higher values because of the time
required for the clotting process. Heparin may be used if
EDTA interferes with the analytical method used. All
specimens should be cooled on ice and transported to the
lab for immediate centrifugation. Once plasma is
separated, homocysteine is relatively stable (>4 days at
room temperature, several weeks refrigerated, several
years at 20C). Homocysteine is stable to repeated
freeze/thaw cycles, but the matrix often degenerates,
making analysis difficult [33].
Adenosine analogues such as 3-deazaadenosine are
effective in preventing leakage of homocysteine from
red cells but are not compatible with assays that use
enzymatic reactions with SAHH [35]. In any event, the
use of a specialist blood collection tube is not practical
in a routine clinical setting.
Venous stasis does not affect the homocysteine content
of plasma, but samples collected in the supine position
are 10% lower than those with the patient upright,
presumably because of the decline in albumin, its main
serum binding protein. Plasma homocysteine
concentrations are usually measured fasting.
Homocysteine concentration may decrease 10% to 15%
over the first few hours following eating and then rise
again by 6 hours following eating, in proportion to the
methionine (protein) content of the meal [36]. A
methionine-loading test has been used to identify
persons heterozygous for CBS deficiency [37], which
involves a plasma homocysteine measurement 4 to 6
hours after intake of 100 mg methionine/kg body weight.
Interferences
Hemolysis has not been found to interfere with
homocysteine determinations. Homocysteine is stable to
repeated freeze/thaw cycles, but the matrix often
degenerates, making analysis difficult [33]. Adenosine
analogues such as 3-deazaadenosine are effective in
preventing leakage of homocysteine from red cells but
are not compatible with assays that use enzymatic
reactions with SAHH [35].
Heterophile antibody or HAMA interference with
immunoassay tests should be suspected when repeated
analysis produces poor replication, there is poor
recovery on dilution, or different methodologies provide
discrepant results, as discussed earlier.
Homocysteine Reference Interval
Rasmussen et al. proposed age- and gender-specific
reference intervals for total homocysteine in plasma
[38], but given the poor precision of some assays, these
differences are probably not reproducible in the clinical
setting. The following upper reference limits have been
proposed by an expert committee for different groups,
based on the major determinants: age, pregnancy, folate
supplementation, and following methionine challenge
[12]. Concentrations in newborns are approximately half
those seen in healthy adults.
Group Folate
Supplemented Not Supplemented
Fasting tHcy mol/L
Pregnancy 8 10
Children < 15 yr 8 10
Adults 15 65 yr 12 15
Elderly > 65 yr 16 20
Post-methionine-load 5 times fasting level or
4 6 hr 40 mol/L above fasting level
In patients with homocystinuria, concentrations of total
homocysteine are often > 100 mol/L on treatment and
> 250 mol/L untreated. Free, unbound homocysteine is
seen in the plasma of these patients, usually 1 to 10
mol/L, and larger amounts of homocysteine can be
detected in their urine.
Interpretation
Homocystinuria, a rare autosomal recessive disease, is a
consequence of the formation of an enzyme deficiency
(usually cystathionine -synthase, less frequently
methionine synthase or one of the other enzymes; see
Figure 1) involved in the homocysteine metabolic
pathway. Extremely high concentrations (20 to 50 times
normal) of homocysteine are present in the serum and
urine. Clinical sequelae include unexplained mental
retardation, thromboembolism, lens dislocation,
progressive myopia, osteoporosis, Marfan-like features,
psychiatric disorders, or megaloblastic anemia, as well
as affected siblings [39]. Because of the relationship
between high homocysteine concentrations and vascular
708
Homocysteine
disease, McCully, in 1969, suggested that mild to
moderate hyperhomocysteinemia might be a factor
involved in premature vascular disease [1]. At that time,
however, no methods were available to measure
homocysteine at the low concentrations normally present
in plasma. Recent improvements in methods, as
described earlier, have provided a means to detect
homocysteine at the concentrations present in normal
plasma.
It transpires that many factors influence plasma
homocysteine concentrations, more commonly an
elevation rather than a decline. These should be taken
into account when interpreting the result and can be
summarized in the following categories: genetic,
physiological, lifestyle-related, clinical conditions, and
drugs. There are excellent summaries of these in Tables
3, 4, and 5 of Refsum et al. [12] The most likely cause of
hyperhomocysteinemia depends on the age of the person
and magnitude of the elevation.
Folate and/or cobalamin deficiency and/or renal
impairment account for the majority of cases in a
population where food is not fortified with folic acid. In
folate-supplemented populations, renal impairment and
cobalamin deficiency prevail [12]. Homozygosity for
MTHFR (677C > T) polymorphism (see Figure 1) is the
most common genetic determinant. These individuals
usually have an elevation of some 2.5 mol/L, although
this may not be so relevant in persons with adequate
folate/B12 and riboflavin intake [40]. Mandatory folate
supplementation of grain products in the United States
since 1998 has resulted in a decrease of about 30% in
homocysteine concentrations in the population [41].
Studies that have sought to understand the role
hyperhomocysteinemia plays in the pathogenesis of both
arterial occlusive disease and recurrent venous
thrombosis have driven much of the analytical
improvement and molecular investigations [12]. Case-
controlled as well as prospective studies have
demonstrated that plasma total homocysteine is a strong,
graded, and independent risk factor for coronary heart
disease and stroke [6,43,44]. Evidence from Mendelian
randomization, demonstrating an association between
coronary heart disease (CHD) and the methylene
tetrahydrofolate reductase (MTHFR) polymorphism
(677C > T), provided additional support for a causal
relationship [45,46]. (NOTE: Mendelian randomization is
a method that allows one to test for, or in certain cases to
estimate, a causal effect from observational data in the
presence of confounding factors. It uses common genetic
polymorphisms with well-understood effects on
exposure patterns [e.g., propensity to drink alcohol] or
effects that mimic those produced by modifiable
exposures [e.g., raised blood cholesterol]. Importantly,
the genotype must only affect the disease status
indirectly via its effect on the exposure of interest.
Because genotypes are assigned randomly when passed
from parents to offspring during meiosis, the population
genotype distribution should be unrelated to the
confounders that typically plague observational
epidemiology studies. In this regard, Mendelian
randomization can be thought of as a natural RCT.
The method relies on getting good estimates from
genetic association studies. Misleading conclusions can
also be drawn in the presence of linkage disequilibrium,
genetic heterogeneity, pleiotropy, or population
stratification.) Plasma homocysteine can be lowered with
B vitamins and folic acid, and persons with high plasma
levels or dietary folate and B12 intake have a lower risk
of CHD [47-50]. In contrast to what was expected on the
basis of epidemiological evidence, large randomized
studies were unable to show that lowering homocysteine
with B vitamins prevented recurrent stroke, myocardial
infarction, or death in patients who had previously had a
stroke [51] or myocardial infarction [52]. Studies on
restenosis after percutaneous intervention also yielded
inconsistent results [19,20].
As expected, many pathological events appear to be
mediated by changing the activity or blood concentration
of the B vitamins, in particular folate and/or cobalamin,
since these vitamins are required for homocysteine
metabolism (Figure 1). Other mechanisms may arise
from altering renal function and more rarely by
influencing enzyme activity. An interesting suggestion is
that individual differences in the distribution of Hcy
amongst the plasma proteins may help explain some of
the inconsistency between tHcy and clinical
manifestations. This is because the Hcy bound to
globulins (such as HDL) is not as easily exchanged with
the aqueous phase as that bound to albumin and thus
represents a more permanent protein modification [53].
Homocysteine has been shown to have a number of
important actions at the cellular level, which can help
explain some of its pathological effects. It promotes
endothelial growth factors, stimulates platelet factor V
and XII activity, induces tissue factor expression,
reduces protein C activity, suppresses expression of
thrombomodulin and heparan sulfate, directly induces
vascular smooth muscle proliferation, inhibits nitric
oxide synthesis, and stimulates free radical production
and oxidation of LDL cholesterol [54]. Non-
cardiovascular effects include but are not limited to birth
defects [7], pregnancy complications [8], psychiatric
disorders [9], cognitive impairment in the elderly [10],
and osteoporosis [11]. Whether homocysteine is directly
responsible for these effects or is merely the tangible
evidence of perturbed one-carbon metabolism or other
thiol pathways of different etiologies is still highly
debated [12].
Treatment of hyperhomocysteinemia is usually
adequately achieved inexpensively through
supplementation with vitamins B6, B12, and/or folate.
Extreme cases, such as homocystinuria, may require a
methionine- and protein-restricted diet or betaine
supplementation.
Homocysteine Performance Goals
Desirable analytical specifications derived from studies
of biological variation indicate a desired assay bias of <
10% (0.375 interindividual coefficient of variation
[CV]) and an imprecision of < 5% (0.75
intraindividual CV) [55,56].
709
Homocysteine
The analytical range should cover the 5th to 95th
percentile of the general population (~3 to 40 mol/L)
without the need for dilution or concentration.
Acceptable performance criteria defined by the Clinical
Laboratory Improvement Amendments (1988) for
measurement of plasma total homocysteine requires that
laboratories be accurate to within 3 SD of the peer
group mean. Precision data from the CAP 2006-7
participant summary reports are shown in Table 2 for
approximately 750 and 33 laboratories returning results
for tHcy assays to CAP and NEQAS, respectively. Data
clearly show FPIA outperforms other methods in
precision, and there is a strong negative bias for
chemiluminescence compared to other methods, ranging
from 20% at 5 mol/L to 35% at 70 mol/L. This bias is
surprising, given that the assays correlate well with
patient specimens [28], and is most likely attributable to
a matrix effect introduced by the survey materials.
Certified reference material is lacking, and this has been
blamed for some of the poor correlation between
methods. A recently introduced calibration/linearity
verification survey at CAP has not been widely
subscribed.
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44 Nygard O, Nordrehaug JE, Refsum H, Ueland
PM, Farstad M, Vollset SE. Plasma
homocysteine levels and mortality in patients
with coronary artery disease. N Engl J Med
1997;337:230-236.
45 Wald DS, Law M, Morris JK. Homocysteine
and cardiovascular disease: evidence on
causality from a meta-analysis. BMJ 2003;32:1-
22
46 Casas JP, Bautista LE, Smeeth L, Sharma P,
Hingorani AD. Homocysteine and stroke:
evidence on a causal link from mendelian
randomisation. Lancet 2005;365:224-232.
47 Verhaar MC, Stroes E, Rabelink TJ, Folates and
cardiovascular disease. Arterioscler Thromb
Vasc Biol 2002;22:6-13.
48 Robinson K, Arheart K, Refsum H, Brattstrom
L, Boers G, Ueland P et al. Low circulating
folate and vitamin B6 concentrations: risk
factors for stroke, peripheral vascular disease,
and coronary artery disease. Circulation
1998;97:437-43; Erratum 1999;99:983.
49 Folsom AR, Nieto FJ, McGovern PG, Tsai MY,
Malinow MR, Eckfeldt JH, Davis CE.
Prospective study of coronary heart disease
incidence in relation to fasting total
711
Homocysteine
homocysteine, related genetic polymorphisms,
and B vitamins: the Atherosclerosis Risk in
Communities (ARIC) study. Circulation
1998;98:204-210.
50 Voutilainen S, Rissanaen TH, Virtanen J, Lakka
TA, Salomen JT. Low dietary folate intake is
associated with an excess incidence of coronary
events: the Kuopio Ischemic Heart Disease Risk
Factor Study. Circulation 2001;103:2674-2680.
51 Tool JF, Manilow MR, Chambless LE, Spence
JD, Pettigrew LC, Howard VJ et al. Lowering
homocysteine in patients with ischemic stroke
to prevent recurrent stroke, myocardial
infarction and death: the Vitamin Intervention
for Stoke Prevention (VISP) randomized
controlled trial. JAMA 2004;291:565-575.
52 Bonaa KH, Njolstad I, Ueland PM, Schirmer H,
Tverdal A, Steigen T et al. Homocysteine
lowering and cardiovascular events after acute
myocardial infarction. N Engl J Med
2006;354:1578-1588.
53 Hortin GL, Seam N, Hoehn GT. Bound
homocysteine, cysteine, and cysteinylglycine
distribution between albumin and globulins.
Clin Chem 2006;52:2258-2264.
54 Zinellu A, Zinellu E, Sotgia S, Formato M,
Cherchi GM, Deiana L, Carru C. Factors
affecting S-homocysteinylation of LDL
apoprotein B. Clin Chem 2006;52:2054-2059.
55 Clarke R, Woodhouse P, Ulvik A, Frost C,
Scherliker P, Refsum H et al. Variability and
determinants of total homocysteine
concentrations in plasma in an elderly
population. Clin Chem 1998;44:102-107.
56 McKinley MC, Strain JJ, McPartlin J, Scott JM,
McNulty H. Plasma homocysteine is not subject
to seasonal variation. Clin Chem 2001;47:1430-
1436.
712
Homocysteine

Table 1: Homocysteine Summary Methods

Method 1: Amino acid analyzer for free homocysteine
Principle of analysis: Plasma or serum is deproteinated, and free amino acids are sequestered in the filtrate.
Either: a. Amino acids are separated by ion-exchange chromatography and detected by post-column derivatization with
Ninhydrin, yielding a mauve color for monobasic amino acids proportional to their concentrations.
Or: b. Amino acids are derivatized with fluorescent probe and the products separated by ion-exchange
chromatography. Fluorescence detection of individual amino acids proportional to their concentrations.
Comments: Used for profiling patients with homocystinuria; expensive to operate and limited capacity.
Analytical times are on the order of 1 to 3 hours or more.
Method 2: High-performance liquid chromatography of total reduced homocysteine
Principle of analysis: Plasma is treated with reducing agent and deproteinated. Free, reduced homocysteine and
other thiols are sequestered in the filtrate.
Either: a. Reduced thiols are derivatized to fluorescent compounds which are separated chromatographically. The amount
of homocysteine present is proportional to the fluorimetric signal.
Or: b. The compounds are separated chromatographically without derivatization and detected by electrochemical
oxidation.
Or: c. The compounds are separated chromatographically with or without derivatization and detected by tandem mass
spectrometry in SRM mode.
Comments: HPLC methods require a great deal of technical skill. In addition, throughput is limited. Method 2a
is the CDC reference method. Direct aspiration LC/MS/MS of derivatized amino acids (without chromatography)
is the method of choice for high-throughput newborn screening programs
Method 3: Enzymic product immunoassays of tHcy (fluorescence polarization or chemiluminescence)
Principle of analysis: All plasma forms of homocystine and mixed disulfide are reduced with dithiothreitol to
form reduced free homocysteine. This is converted enzymatically to S-adenosyl-homocysteine by S-adenosyl-
homocysteine hydrolase. The S-adenosyl-homocysteine produced competes directly with added S-adenosyl-
homocysteine-tracer conjugate for sites on a monoclonal antibody specific for S-adenosyl-homocysteine. The
amount of tracer bound is quantified by measuring either:
a. The fluorescence polarization of the resultant mixture. The amount of polarization is inversely proportional to
the plasma homocysteine concentration, or
b. Chemiluminescence after addition of trigger reagents. The luminescence is inversely proportional to the plasma
homocysteine concentration.
Comments: This assay is fairly simple to perform, is automated, and takes 30 to 60 min.
Method 4: Coupled enzymatic assays of tHcy for automated chemistry platforms
Principle of analysis: Homocystine, mixed disulfide, and protein-bound forms of homocysteine in the serum or
plasma are reduced with TECP or dithiothreitol to form the reduced free form of homocysteine. The total free
homocysteine is reacted in a series of coupled enzymatic reactions:
a. With serine and CBS produces cystathionine; cystathionine with CBL produces HCY and pyruvate; pyruvate
with LDH; monitored at 340 nm
b. With SAM and HMT produces SAH and methionine; SAH with SAHH produces HCY and adenosine;
adenosine with ADA produces inosine and NH3; NH3 reacted with GLDH; monitored at 340 nm
c. rHCYase with Vit B6 produces H2S; H2S reacted with DBPDA and ferricyanide; fluorescence detection
Comments: Automated, simple to perform and less expensive than immunoassays; reaction times vary from
about 10 to 20 minutes. Methods a and b available for many platforms.
713
Homocysteine

Figure 1
Diagram of homocysteine metabolism showing the interrelationship of the methionine and folate cycles with
methylation

CBS, Cystathionine--synthase; MAT, methionine adenosyl transferase; MS, methionine synthase; MTHFR, methylene
tetrahydrofolate reductase; SAH, S-adenosyl homocysteine; SAHH, S-adenosyl homocysteine hydrolase; SAM, S-adenosyl
methionine.
714

Homocysteine

Table 2: Performance of Analytical Methods in External Quality Assurance Programs

CAP 2006 - 7 Spot Challenges
%
Method of Labs Individual Specimen Mean in mol/L and (CV %)
Specimen HMS-04 HMS-05 HMS-06 HMS-01 HMS-02 HMS-03 Mean Value CV %
Chemiluminescence 48.8 4.15 (16.3) 48.68 (18.3) 10.13 (15.5) 3.71 (14.7) 8.95 (10.2) 9.01 (10.1) 14.1 14.2
Enzymatic 16.5 5.19 (21.2) 68.18 (13.1) 13.16 (6.4) 4.27 (15.0) 11.16 (7.6) 11.14 (7.5) 18.9 11.8
FPIA 28.7 5.81 (7.4) 70.65 (7.5) 13.15 (5.9) 5.4 (6.8) 11.59 (5.0) 11.73 (4.6) 19.8 6.2
HPLC All 2.0 --- --- --- 4.89 (14.7) 11.05 (10.9) 11.09 (11.3) ---
Others 4.0
Assigned Value* 5.7 75.6 14.2 4.7 11.6 11.6 20.5

All Results %CV n = 740 21.0 22.4 16.9 20.7 14.8 14.5 --- ---

NEQAS 2006 - 4 Spot Challenges

Individual Specimen Median (Range) in
Method % of Labs mol/L
HO
Specimen HO 06:04 HO 07:01 07:02 HO 07:03
2.6 (2.2 -
Chemiluminescence 6.1 15.5 (15.0 - 15.9) --- 2.9) 9.8 (7.4 - 12.2)
Enzymatic 15.2 16 (15.2 - 19.8) 6.7 (5.8 - 8.0) --- 12.3 (6.4 - 18.1)
4.0 (2.4 -
FPIA 54.4 14.4 (12.1 - 15.7) 6.2 (5.3 - 7.9) 4.3) 15.5 (14.5 - 17.4)
7.3 (5.2 - 6.1 (3.0 -
HPLC All 24.2 14.3 (6.1 - 18.3) 11.7) 133) 15.5 (13.6 - 17.8)
Median Value 14.7 6.3 4.0 15.3

All Results %CV n = 33 15.0 18.5 69.8 18.1

* Based on reference labs performing CDC assay [32].
715
Homocysteine

Figure 2
Fluorescence polarization immunoassay (FPIA) (Method 3a in Table 1) for the Abbott IMx and
AxSYM platforms.

No SAH

YYY YYY
YYY YYY in sample

High P

SAH in
sample

Low P

All forms of homocysteine are reduced with dithiothreitol, which is then converted enzymatically to S-
adenosyl-L-homocysteine (SAH). This product is quantified in a competitive reaction between it and
SAHfluorescein tracer conjugate for sites on a monoclonal antibody specific for the SAH moiety. The
amount of tracer bound is quantified by measuring the fluorescence polarization of the resultant
mixture, with this being inversely proportional to the free SAH concentration and hence inversely to
the tHcy in the specimen. Direction of applied and emitted light is shown by the yellow arrows.

Figure 3
Chemiluminescent magnetic microparticle immunoassay (Method 3b in Table 1).

Capture Ab-
SAH in Acridinium label
patient sample
Y
YY

Incubate All forms of homocysteine are first

reduced with dithiothreitol, which is then
converted enzymatically to S-adenosyl-L-
homocysteine (SAH). A competitive
reaction is set up in which patient-derived
SAH and SAH covalently linked to
Magnetic micro-
particles coated
Y paramagnetic particles compete for binding
sites on a monoclonal antibody specific for
the SAH moiety. Pre-trigger (acid) solution
with SAH releases the complexed SAH-labeled
detection antibody from the microparticles,
Magnetic which are drawn towards the magnet.
Trigger solution (sodium hydroxide)
Capture
optimizes the pH for the release of
chemiluminescence, which is captured and
quantified in the photometer.

Y Pre-trigger
Addition
RLU
Trigger Measurement
Addition

Y Y
716
Homocysteine
Appendix
Detailed Method for Total Homocysteine and
Cysteine published from the Centers for
Disease Control (CDC) [32]
Principle
Homocysteine, a thio-containing amino acid
derived from the metabolism of methionine, is
readily oxidized in body fluids to the disulfides
homocystine and cysteine-homocysteine.
Reduction of homocystine and these disulfides,
as well as the release of protein-bound
homocysteine with tris(2-
carboxyethyl)phosphine (TCEP) followed by
trichloroacetic acid, allows this method to
measure total plasma homocysteine and
cysteine concentrations. After derivatization
with a thiospecific fluorogenic reagent,
ammonium 7-fluorbenzo-2-oxa-1,3-diazole-4-
sulphonate (SBD-F), the thiols are separated
by isocratic reversed-phase high-performance
liquid chromatography (HPLC) using
fluorescence detection. A calibration curve is
carried through the derivatization procedure
along with an internal standard, cysteamine.
Specimen
Whole blood collected into EDTA
anticoagulant is immediately placed in wet ice
for transport and centrifuged as soon as
possible at 3000 rpm for 10 min. The plasma is
separated from the cells and refrigerated (2C
to 8C) until analysis.
Reagents
HPLC Mobile Phase: 0.1 mol/L acetic acid-
acetate buffer, pH 5.0, containing 30 mL/L
methanol.
Mobile Phase Buffer (MPB):
Weigh out 13.6 g sodium acetate trihydrate and
QS to 1 L with H2O. Adjust the pH by addition
of glacial acetic acid to pH 5 (0.1). Stable for 1
month at room temperature.
Working Mobile Phase:
Combine 970 mL of this acetate buffer with 30
mL methanol. Mix well. Stable at room
temperature.
Phosphate Buffered Saline (PBS):
Sigma Chemical Co. Stable at room
temperature.
100 g/L TCEP (tris(2-
carboxyethyl)phosphine Hydrochloride:
Pierce Chemical Co.
Weigh 1 g of TCEP hydrochloride and QS to
10 mL with H2O. Stable at room temperature.
100g/L TCA (Trichloroacetic acid)
containing 1 mmol/L EDTA:
Weigh 1.0 g TCA and 3.72 mg EDTA-Na2
(FW=372.2). QS to 10 mL with H2O.
Ultrasonicate solution until EDTA is
dissolved. Stable at room temperature.
1.55 mol/L Sodium Hydroxide:
Weigh 6.2 g of NaOH and QS to 100 mL with
H2O. Stable at room temperature.
0.125 mol/L Sodium Borate Buffer (pH 9.5)
containing 4 mmol/L EDTA:
Weigh 4.767 g sodium borate decahydrate and
148 mg EDTA-Na2 into a beaker. Add 90 mL
H2O. Ultrasonicate solution until crystals are
dissolved. QS to 100 mL with H2O (pH should
be 9.5). Stable at room temperature.
1 g/L SBD-F (ammonium 7-fluorbenzo-2-
oxa-1,3-diazole-4-sulphonate) in Sodium
Borate Buffer:
Wako Chemicals.
Prepare sufficient SBD-F solution for the assay
tubes (50 L is used per tube, 20 tubes = 1
mL). Weigh appropriate amount (mg) of SBD-
F into a light-protected (amber) vial. Add
volume of sodium borate buffer needed to
make a 1 g/L (1 mg/mL) solution. Vortex.
Stock SBD-F is stored at 4C. Solution must
be prepared fresh for derivatization and can be
stored at room temperature until used.
Internal Standard 40 mmol/L Cysteamine:
Sigma Chemical Co.
Weigh 4.54 mg cysteamine hydrochloride (FW
113.61) into a vial. Add 1 mL MPB.
Refrigerate until used in assay. Prepare fresh
on day of use. Not stable overnight.
0.1 mmol/L L-Homocysteine Stock:
Sigma Chemical Co.
Weigh 1.352 mg homocysteine (FW 135.18)
into a vial. Add 10 mL of MPB. Refrigerate
until used in assay. Prepare fresh on day of
use. Not stable overnight.
0.8 mmol/L L-Cysteine Stock:
Sigma Chemical Co.
Weigh 12.6 mg cysteine hydrochloride (FW
157.62) into a vial. Add 10 mL of MPB.
Refrigerate until used in assay. Prepare fresh
on day of use. Not stable overnight.
Calibration Standards:
Prepare in thiol-free EDTA plasma, at
concentrations of 0, 5, 10, 25, and 50 mol/L
717
Homocysteine
homocysteine and 0, 80, 160, 400, and 800
mol/L cysteine using the dilutions below:
0, 0 mol/L MPB only
5, 80 1 in 200 dilution of each
stock solution
10, 160 1 in 100 dilution of each
stock solution
25, 400 1 in 40 dilution of each
stock solution
50, 800 1 in 20 dilution of each
stock solution
Add MPB to each standard so that the total
amount of MPB plus standard stock solution is
the same in each tube.
If a small mount of calibrator is needed, an
intermediate dilution of stock can be prepared
(e.g., 1 nmol/mL by a 100-fold dilution of the
0.1 mol/mL solution) in MPB
Control Samples:
Commercially available materials e.g., Utak,
Chromsystems, Bio-Rad. Refer to package
insert for reconstitution instructions and
stability.
Procedure:
Pipet 50 L patient, calibrator, or control
plasma into polypropylene tubes.
Add 25 L of internal standard and 25 L
PBS, followed by 10 L TCEP. Cap and
incubate for 30 min at room temperature.
Add 90 L trichloroacetic acid / EDTA, vortex
for 30 sec, and centrifuge for 10 min at 13000
g.
Transfer 50 L of the supernatant to an
autosampler vial containing 10 L NaOH, 125
L borate buffer/EDTA and 50 L SBD-F
solution. Cap and incubate for 60 min at 60C.
Derivatized samples may be stored at 20C
up to 1 week until analysis by HPLC.
HPLC Analysis:
Waters Alliance Model 2690 HPLC pump
Autosampler WISP 710B with 10 L injection
loop
Waters Model 474 Scanning Fluorescence
Detector
Altech Associates Adsorbosphere C18 pre-
column
Phenomenex Prodigy ODS2 analytical column
(5 m particle size, 3.2 mm 150 mm)
Millennium32 software
Column flow rate 0.7 mL/min
Column oven temperature 29C
Excitation wavelength 385 nm, emission 515
nm
Chromatogram showing separation of thiols
from a patient serum containing 12.5 mol/L
tHcy. (From Pfeiffer CM, Huff DL, Gunter E.
Clin Chem 1999;45:290-2, with permission).
Cys, Cysteine; Hcy, homocysteine; Cys-Gly,
cysteinylglycine; GSH, glutathione; CysNH2,
cysteamine internal standard.
Calculations:
On each patient chromatogram, five peaks of
interest can be found within a 6-min analysis
time, as shown in the specimen chromatogram.
Identify the peaks corresponding to
homocysteine and cysteamine. Their
concentrations in plasma samples are
calculated using the internal standard method.
Calculate the ratio of homocysteine and
cysteine peaks height (or area) to cysteamine
peak height (or area) for each standard. Plot
these ratios versus the concentration of the
standard, and calculate the linear least squares
regression equation of the relationship.
Calculate the same ratios for each patient
sample and control. Extrapolate from standard
curve to calculate the homocysteine and
cysteine concentration in each patient sample
and control.
Reference Interval:
Total homocysteine < 13.0 mol/L for adults
and < 10.0 mol/L for children
Critical high levels of homocysteine are >100
mol/L.
Total cysteine < 360 mol/L for adults.
Analytical Measurement Range:
Total homocysteine 1.6 to 50 mol/L; total
cysteine 1.6 to 800 mol/L
Patient samples exceeding the calibration
range are repeated using a smaller volume of
plasma diluted into PBS.
 718
Homovanillic Acid

Homovanillic Acid
Lawrence A. Kaplan
Name: Homovanillic acid, 3-methoxy-4-hydroxyphenylacetic acid, HVA
Clinical significance: Refer to Chapter 53, Neoplasia, in the 5th Edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Molecular formula: C9H10O4
Molecular mass: 182.17 D
Merck Index: 638
Chemical class: Substituted aromatic carboxylic acid (catecholamine metabolite)
Structure: see Figure 3

i With sensitivities around 0.5 mol/L, these assays could be

Principles of Analysis and Current Usage
High-performance liquid chromatography (HPLC) for the
measurement of urinary HVA (Table 1, Method 1) usually
requires extraction from the biological sample. Usually the
biogenic amines are first separated by anion-exchange
chromatography on XAD-X4, DEAE-cellulose, or an
equivalent resin, and then HPLC analysis is performed [1-
11]. In one report [12], diluted urine was employed as the
sample to measure hydroxyl-methoxy mandelic acid
(HMMA), also known as vanillylmandelic acid (VMA), 5-
hydroxyindoleacetic acid (5-HIAA), and homovanillic acid
(HVA) in an isocratic HPLC system with amperometric
detection. In all cases, final separation was accomplished
by reversed-phase, ion-paired (C18) chromatography, and
detection was accomplished by ultraviolet
spectrophotometry (see Figure 1) or amperometry. Many
procedures allow the simultaneous measurement of HVA
and VMA [2,3,5,6,7,9,10].
Capillary electrophoresis methods (Table 1, Method 2)
have been reported for HVA analysis [13-18]. These
procedures were used to measure HVA and VMA using a
fused-silica capillary and an ion-paired detergent.
Detection was by ultraviolet absorbance, either fixed at
245 nm [15-17] or with use of a diode array [13,19] or by
amperometry [18].
Competitive-binding enzyme immunoassays (EIA) for
HVA have been reported for measurement of urinary HVA
(Table 1, Method 3) [20,21]. These assays employ
monoclonal antibodies to achieve the desired specificity.
i
Homovanillic Acid
Previous and current authors of this method:
First edition: Not done
Methods edition: Steven J. Soldin, Lawrence A. Kaplan
Second edition: Not updated
Third edition: Not updated
Fourth edition: Steven J. Soldin, Lawrence A. Kaplan
Fifth edition: Lawrence A. Kaplan
used to detect neuroblastomas.
Although there are no reports or surveys comparing the
frequency of use of the methods employed for the
measurement of HVA, most recent literature citations
report using an HPLC method. The other historical
methods are reviewed below.
Historically, colorimetric analysis of HVA utilized the
reaction of 1-nitroso-2-naphthol with biogenic amines
(Table 1, Method 4). HVA formed a red chromogen,
which was measured at 500 nm [22]. For preparation of a
sample blank, potassium thiocyanate was added to remove
the color that resulted from the reaction of HVA [22]. An
improved colorimetric reaction was proposed by Knight
and Hammond, who substituted 1-nitroso-2-naphthol-4-
sulfonic acid for the previously used 1-nitroso-2-naphthol
[23]. The assay can also be performed after the partial
purification of HVA by anion-exchange chromatography.
Another approach to quantitation was the use of thin-layer
chromatography (TLC) performed on silica gel. The
biogenic amines were measured by scanning reflectometry
at 400 nm (Table 1, Method 5). The compounds, which
were allowed to photo-oxidize for 2 days, developed a red
color [24].
A gas chromatographic (GC) method was developed with
p-hydroxyphenyl acetic acid used as an internal standard
(Table 1, Method 6). The sample was extracted with ether
and derivatized with TRI-SII/TBT (Pierce Chemical Co.,
Rockford, IL) forming the silyl derivative. The compounds
were separated by gas chromatography with 3% OV-1 as
the stationary phase, and the HVA was detected with a
flame-ionization detector [25]. The HVA could also be
measured by electron-capture detection [26].
Gas chromatographymass spectrometry was also used for
quantitation of serum and urine levels (Table 1, Method 7)
[27]. The HVA in the samples was first separated by use of
Amberlite XAD-4 anion-exchange columns. The eluted
 719
Homovanillic Acid
compounds were converted to their trifluoroacetyl
hexafluoroisopropanol ester derivatives, the derivatives
were separated by GC, and the HVA derivative was
detected by a mass spectrophotometer. The limit of
detection was 2 mg/mL for plasma and 120 ng/mL for
urine.
Reference and Preferred Methods
There is no formal reference method for HVA analysis.
Because of its superior sensitivity and specificity, and
because of its ease of use, high-performance liquid
chromatography (HPLC) has become the method of choice
for the measurement of HVA. The HPLC methods allow
separation of HVA within 20 to 30 min. An HPLC
method, with either ultraviolet or amperometric detector, is
the preferred method for HVA analysis. The procedures
are flexible to run a few or many samples at a time.
However, because of its superior sensitivity, amperometric
detection is preferred to ultraviolet spectroscopy. The
HPLC-amperometric methods are similar to the HPLC
analysis of urinary catecholamines employing
amperometric detection [1,8,10,28].
The ability to quantitate simultaneously VMA and HVA
may not have much practical advantage. Urine HVA is
rarely elevated in pheochromocytomas, whereas urinary
VMA is almost always increased in neuroblastomas. A rise
in urinary HVA output is used to diagnose neuroblastomas.
By concentrating the HPLC assay on the analysis of either
VMA or HVA, one can increase the speed of analysis for
the specific tumor that is being investigated. Analyzing for
only VMA allows washout of the HPLC column as soon as
the VMA peak is eluted, whereas assaying for only HVA
allows elution to start with a stronger mobile phase and
HVA eluting more rapidly.
The capillary electrophoresis assays require equipment and
knowledge that may not be as commonly available as for
the HPLC technique. Since there have been few reports
using this technique, it is difficult to judge its efficacy.
Similarly, there have only been two reports of the use of
the competitive binding enzyme immunoassay, which
carries the burden of requiring a specialized monoclonal
antibody. The EIA does have the advantages of not
requiring any extraction of the urine sample and the ability
to perform many analyses at once, either in an automated
spectrophotometer or in a multi-well reaction plate.
However, unless research is being performed, the need for
the HVA assay is rather infrequent, obviating need for
analysis of many samples. In any case, neither assay seems
to have any advantage in terms of sensitivity or specificity
over the HPLC assays.
The colorimetric methods are not specific for HVA. In
addition, the reaction of 1-nitro-2-naphthol with HVA
results in the formation of an unstable chromogen. The
instability of this chromogen increases with increasing
HVA concentration, resulting in a rapid fading of the color
with elevated HVA concentrations [23]. The use of the 4-
sulfonic acid derivative improves the stability of the
chromogen formed upon reaction of HVA [23]. The 4-
sulfonate substrate does react with 5-HIAA and VMA.
Interference from 5-HIAA is minimized because its
reaction product has a significantly different absorbance
maximum. VMA interference is reduced by use of an
extraction solvent (CHCl3) that minimizes VMA recovery.
After partial purification of HVA by ion-exchange
chromatography, the accuracy of the method is much
improved, and small increases in HVA excretion can be
detected.
The TLC method is slow and requires specialized
equipment. The GC methods do work well for HVA, but
the need for derivatization and the relatively poor
sensitivity has made them less likely to be adopted.
Specimen
The preferred sample for HVA analysis is a 24-hour urine
collected with sufficient acid to maintain a pH of less than
3 during the collection period. While a complete 24-hour
urine collection is recommended, shorter collection times
may be acceptable if the result is expressed per milligram
of creatinine. Usually 15 mL of 6 M HCl added to the
collection vessel before collection of urine is sufficient to
maintain the proper pH. Specimens should be kept
refrigerated during collection. If analysis is delayed,
specimens may be frozen indefinitely. An investigation of
the diurnal variation of urinary HVA secretion indicates
that a random urine sample may be satisfactory for the
diagnosis of neuroblastomas, especially when the excretion
rate is normalized for creatinine excretion (mg of HVA/g
of creatinine) [29]. However, these results should be
validated in each laboratory.
The use of filter paper to collect and store urine samples
has been successfully demonstrated [30]. HVA was eluted
from the filter paper with tartrate buffer, allowing both
HVA and creatinine to be measured on the urine sample.
Interferences
Catecholamine metabolites, vanillin, vanillic acid, glyceryl
guaiacolate, and many other compounds are known to
interfere in colorimetric assays. The HPLC and capillary
electrophoresis methods show little interference from other
endogenous compounds or exogenous drugs. The
monoclonal EIA showed 0.5% cross-reactivity with VMA
and < 8% cross-reactivity with other structurally related
catecholamine metabolites.
Homovanillic Acid Reference Interval
The age-dependent percentile values for urinary HVA, as
mg of HVA/24 h or mg of HVA/g of creatinine, are
shown in Table 3. There appears to be no difference in
excretion rates between males and females.
Seasonal variations in urinary levels of VMA and HVA
have been reported using samples collected on filter paper
 720
Homovanillic Acid
and analyzed by HPLC [31]. Although statistically
detectable, the differences were not considered clinically
important. The fluctuations in the concentrations of VMA
and HVA were more apparent in a small study that used
random urine samples [32].
Interpretation
The most important endogenous catecholamines are
epinephrine, norepinephrine, and dopamine. The
catecholamines are produced primarily in the chromaffin
cells of the adrenal medulla, the brain, and the sympathetic
nervous system. Dopamine is primarily synthesized in the
central nervous system (CNS) and acts as a
neurotransmitter. This compound is metabolized to 3-
methoxy-4-hydroxyphenylacetic acid (homovanillic acid,
HVA) by monoamine oxidase (MAO) and catechol-O-
methyl transferase (see Figure 3). Work in rhesus monkeys
suggests that HVA is not substantially further metabolized
in primates and is excreted only by the kidney [33].
In 1865, Virchow described a highly malignant tumor
appearing in infancy and childhood, which has today been
termed neuroblastoma. Neuroblastoma is the most
common (approximately one quarter of all cancers) type of
cancer in the first year of life, having an overall incidence
of 5% to 7% in all childhood malignancies [34]. Males are
affected slightly more commonly than females [35]. As
many as 14 cases per million children from birth to 9 years
of age are reported annually in the United States [35,36].
Neuroblastomas are tumors that secrete mainly the
precursors of norepinephrine, that is, dopamine and its
metabolites. In recent studies [2,7] it has been shown that
urinary VMA and HVA were the analytes of choice for the
laboratory diagnosis of neuroblastoma, since urinary
dopamine is somewhat less reliable.
Measurement of HVA excretion was of no value in
identifying patients with pheochromocytoma but was of
great use in identifying patients with neuroblastoma (Table
4). The age-related 100th percentile value was exceeded in
14 out of 15 patients, and all neuroblastoma patients had
HVA values exceeding the age-related 95th percentile
[2,11].
An evaluation of 18 additional patients with neural crest
tumors (15 with a neuroblastoma and three with a
pheochromocytoma), not included in Table 4, showed an
elevation of VMA above the 100th percentile in 17 of 18
patients, whereas with 1 patient, a normal value was
recorded. In 13 of the 15 additional patients with
neuroblastoma, HVA was elevated above the 100th
percentile. In one case, the HVA value lay between the
95th and 100th percentile, and in the other a value less
than the 95th percentile was recorded. The 3 additional
patients with pheochromocytoma had normal HVA values.
Because the quantitation of urinary HVA afforded a
reasonable, simple, and inexpensive way to diagnose
neuroblastomas, several nations (Austria, Canada, France,
Germany, Japan, and the United Kingdom) proposed
general population screening for this disease in newborns.
The underlying belief was that once detected, the disease
could be eliminated. The screening programs were beset
with both false-positive and false-negative results, and it
was found that many neonatal neuroblastomas regress and
disappear. Statistical analysis of the results of these
screening programs could not support the continuation of
screening [27,38]. In July 2006 the National Screening
Committee policy on neuroblastoma screening reaffirmed
this conclusion [39].
Measurement of urinary HVA and VMA can be useful in
the diagnosis of familial dysautonomia (Riley-Day
syndrome). This neurological disorder, present with
highest frequency among Ashkenazic (Eastern European)
Jews, is noted for a defect in the metabolism of
catecholamines [40,41]. In this disease, the urinary
excretion of VMA (expressed as g of HVA or VMA per
mg of creatinine) is decreased, while the excretion of HVA
is increased. The ratio of HVA to VMA excretion rates is
thus greatly increased in children with familial
dysautonomia. The laboratory analysis of urinary HVA
and VMA can help to confirm the diagnosis of this
syndrome.
HVA analysis of cerebrospinal fluid (CSF) has been used
to assess a number of mental disorders, including drug
abuse [42], depression, and alcoholism [43], as well as
changes related to aging [44].
Homovanillic Acid Performance Goals
There are no published performance goals for the analysis
of HVA. However, data for the College of American
Pathologists (CAP) proficiency surveys provide some
guidance. For urine HVA measured by HPLC, the target
ranges (mean 3SDs) for acceptable proficiency results
were:
Mean (mg/L) SD, range (mg/L)
2.6 0.6, 2.42-2.78
105 13.3, 39.9-144.9
References
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AG. High-performance liquid chromatographic
analysis of urinary catecholamines employing
amperometric detection: reference values and use
in laboratory diagnosis of neural crest tumors.
Clin Biochem 1980;13:285-291.
2 Soldin SJ, Hill JG. Liquid-chromatographic
analysis for urinary 4-hydroxy-3-
methoxymandelic acid and 4-hydroxy-3-
methoxyphenylacetic acid and its use in the
investigation of neural crest tumors. Clin Chem
1981;27:502-503.
3 Yoshida A, Sakai T, Tamura Z. Simple method
for the determination of homovanillic acid and
vanillylmandelic acid in urine by high-
 721
Homovanillic Acid
performance liquid chromatography. J chromatography. J Chromatogr B 1997;693:463-
Chromatogr 1982;227:162-167.
4 Krstulovic AM, Zakaria M, Lohse L, Bertani- 16
Dziedzic L. Diagnosis of neural crest tumors by
reversed-phase high-performance liquid
chromatographic determination of urinary
catecholamine metabolites. J Chromatogr
1979;186:733-748.
5 Frattini P, Santagostino G, Schinelli S, Cucchi 17
ML, Corona GL. Assay of urinary vanilmandelic,
homovanillic and 5-hydroxyindole acetic acids by
liquid chromatography with electrochemical
detection. J Pharmacol Methods 1983;10:193-
198. 18
6 Yoshida A, Yoshioka M, Tanimura T, Tamura Z.
Determination of vanilmandelic acid and
homovanillic acid in urine by high speed liquid
chromatography. J Chromatogr 1976;116:204- 19
243.
7 Yoshida A, Yoshioka M, Yamazaki T, Sakai T,
Tamura Z. Urinary levels of vanilmandelic acid 20
and homovanillic acid determined by high-speed
liquid chromatography. Clin Chim Acta
1976;73:315-320.
8 Morrisey JL, Shihabi ZK. Assay of 4-hydroxy-3-
methoxyphenylacetic (homovanillic) acid by
liquid chromatography with electrochemical 21
detection. Clin Chem 1979;25:2045-2047.
9 Soldin SJ, Hill JG. Simultaneous liquid-
chromatographic analysis of 4-hydroxy-3-
methoxymandelic acid and 4-hydroxy-3-
methoxyphenylacetic acid in urine. Clin Chem
1980;26:291-294. 22
10 Baursfeld W, Diener U, Knoll E, Ratge D, Wisser
H. Determination of urinary vanilmandelic acid
and homovanillic acid by high performance liquid
chromatography with amperometric detection. J 23
Clin Chem Clin Biochem 1982;20:217-220.
11 Soldin SJ. High-performance liquid
chromatography: application in a childrens
hospital. Adv Chromatogr 1982;20:139-163. 24
12 Bonfigli AR, Coppa G, Testa R, Testa I, De Sio
G. Determination of vanillylmandelic, 5-
hydroxyindoleacetic and homovanillic acid in
urine by isocratic liquid chromatography. Eur J
Clin Chem Clin Biochem. 1997;35:57-61. 25
13 Caslavaka J, Gassmann E, Thormann W.
Modification of a tunable UV-visible capillary
electrophoresis detector for simultaneous
absorbance and fluorescence detection :profiling
of body fluids for drugs and endogenous
compounds. J Chromatogr A 1995;709:147-156.
14 Kati B, Elgstoen P, Jellum E. Capillary 26
electrophoresis for diagnosis of metabolic
disease. Electrophoresis 1997;18:1857-1860.
15 Jensen JS, Harding S, Roulund H, Rogers
IS, Emmett PM, Golding J et al. Analysis of
creatinine, vanilmandelic acid, homovanillic acid
and uric acid in urine by micellar electrokinetic 27
467.
Shirao MK, Suzuki S, Kobayashi J, Nakazawa H,
Mochizuki E. Analysis of creatinine,
vanilmandelic acid, homovanillic acid and uric
acid in urine by micellar electrokinetic
chromatography. J Chromatogr B 1997;693:463-
467.
Garcia A, Heinanen M, Jimenez LM, Barbas C.
Direct measurement of homovanillic,
vanillylmandelic and 5-hydroxyindoleacetic acids
in urine by capillary electrophoresis. J
Chromatogr A 2000;871:341-50.
Li X, Jin W, Weng Q. Separation and
determination of homovanillic acid and
vanillylmandelic acid by capillary electrophoresis
Analytica Chimica Acta 2002;461:123-130.
Kati B, Elgstoen P, Jellum E. Capillary
electrophoresis for diagnosis of metabolic
disease. Electrophoresis 1997;18:1857-1860.
Taran F, Frobert Y, Crminon C, Grassi J,
Olichon D, Mioskowski C, Pradelles P.
Competitive enzyme immunoassay with
monoclonal antibody for homovanillic acid
measurement in human urine samples. Clin Chem
1997;43:363-368.
Shi RZ, Ho YP, Yeung JH, Or PM, To KK, Lau
MW, Arumanayagam M. Development of an
enzyme-linked immunosorbent assay with
monoclonal antibody for quantification of
homovanillic acid in human urine samples. Clin
Chem 1998;44:1674-9.
Goldenberg H. Specificity of the nitrosonaphthol
reaction: detection of metanephrine and other
guaiacol derivatives [abstract]. Clin Chem
1967;13:698.
Knight JA, Hammond RE. Improved colorimetry
of urinary 3-methoxy-4-hydroxyphenylacetic acid
(homovanillic acid). Clin Chem 1977;23:2007-
2010.
Huck H, Dworzak E. Quantitative analysis of
catecholamine and serotonin metabolites on thin-
layer plates. II. reflectance measurements after a
specific photochemical reaction. J Chromatogr
1972;74:303-310.
Brewster MA, Berry DH, Moriarty M. Urinary 3-
methoxy-4-hydroxyphenylacetic (homovanillic)
and 3-methoxy-4-hydroxymandelic
(vanillylmandelic) acids: gas-liquid
chromatographic methods and experience with 13
cases of neuroblastoma. Clin Chem
1977;23:2247-2249.
Cahuhan J, Darbre A. Determination of
homovanillic, isohomovanillic and
vanillylmandelic acids in human urine by means
of glass capillary gas-liquid chromatography with
temperature programmed electron-capture
detection. J Chromatogr 1980;183:391-401.
Takahashi S, Yoshioka M, Yoshiue S, Tamura Z.
 722
Homovanillic Acid
Mass fragmentographic determination of
vanilmandelic acid, homovanillic acid and
isohomovanillic acid in human body fluids. J
Chromatogr 1978;145:1-9.
28 Frattini P, Santagostino G, Schinelli S, Cucchi
ML, Corona GL. Assay of urinary vanilmandelic,
homovanillic and 5-hydroxyindole acetic acids by
liquid chromatography with electrochemical
detection. J Pharmacol Methods 1983;10:193-
198.
29 Tuchman R, Robison LL, Maynard RC,
Ramnaraine ML, Krivit W. Assessment of the
diurnal variations in urinary homovanillic and
vanillylmandelic acid excretion for the diagnosis
and follow-up of patients with neuroblastoma.
Clin Biochem 1985;18:176-179.
30 Hanai J, Kawai T, Sato Y, Takasugi N, Nishi M,
Takeda T. Simple liquid-chromatographic
measurement of vanillylmandelic acid and
homovanillic acid in urine on filter paper for mass
screening of neuroblastoma in infants. Clin Chem
1987;33:2043-2046.
31 Nishi M, Miyake H, Takeda T, Takasugi N, Sato
Y, Hanai J. Seasonal variations in
vanillylmandelic acid and homovanillic acid in
infants urine. Jpn J Clin Oncol 1987;17:151-155.
32 Nishi M, Miyake H, Takeda T, Yamashiro K,
Takasugi N, Hanai J, Kawai T. Fluctuation in the
concentrations of vanillylmandelic acid and
homovanillic acid in mass screening for
neuroblastoma. Eur J Pediatr 1990;149:859-861.
33 Miller AL, Keenan RW, Maas JW, Asch RH.
Disposition of homovanillic acid in the primate.
Metab Brain Dis 1987;2:207-212.
34 Gurney JG, Severson RK, Davis S, Robinson LL.
Incidence of cancer in children in the United
States: sex-, race-, and 1-year age-specific rates
by histologic type. Cancer 1995;75:2186-2195.
35 National Cancer Institute. Neuroblastoma
Treatment PDQ. Available at
<http://www.cancer.gov/cancertopics/pdq/treatme
nt/neuroblastoma/healthprofessional>
36 Miller RW, Fraumeni JF, Hill J. A neuroblastoma
epidemiologic approach to its origin. Am J Dis
Child 1968;115:253-261.
37 Schilling FH, Spix C, Berthold F, Erttmann R,
Fehse N, Hero B et al. Neuroblastoma screening
at one year of age. N Engl J Med 2002;346:1047-
53.
38 Woods WG, Gao RN, Shuster JJ, Robison LL
Bernstein M, Weitzman S et al. Screening of
infants and mortality due to neuroblastoma. N
Engl J Med. 2002;346:1041-1046.
39 National Library for Health. National Screening
Committee Policy: Neuroblastoma Screening.
Available at
<http://www.library.nhs.uk/screening/ViewResou
rce.aspx?resID=57184>
40 Smith AA, Taylor T, Wortis SB. Abnormal
catecholamine metabolism in familial
dysautonomia. N Engl J Med 1963;268:705-707.
41 Geltzer AI, Gluck L, Talner NS, Polesky HF.
Familial dysautonomia. N Engl J Med
1964;271:436-440.
42 Roy A, Berman J, Williams R, Kuhn C. Higher
levels of CSF homovanillic acid in recently
abstinent cocaine-dependent patients. Am J
Psychiatry 2002;159:1053-1055.
43 Sher L, Oquendo MA, Li S, Huang Y-y,
Grunebaum MF, Burke AK et al. Lower CSF
homovanillic acid levels in depressed patients
with a history of alcoholism.
Neuropsychopharmacology 2003;28:1712 -
1719.
44 Rapoport SI, Schapiro MB, May C. Reduced
brain delivery of homovanillic acid to
cerebrospinal fluid during human aging. Arch
Neurol 2004;61:1721-4.
 723
Homovanillic Acid

Tables

Table 1: Methods of Homovanillic Acid (HVA) Analysis

Method 1: High-performance liquid chromatography (HPLC)
Principle of analysis: HVA is extracted with an ion-exchange resin and separated by reversed-phase
chromatography. Detection by amperometry.
Comments: Most common; preferred method
Method 2. Capillary electrophoresis
Principle of analysis: HVA is extracted with solvent and separated by electrophoresis. Detection by ultraviolet
spectrophotometry or amperometry.
Comments: Rarely reported method.
Method 3. Competitive-binding enzyme immunoassay (EIA)
Principle of analysis: Using monoclonal antibodies, EIA is performed directly on urine sample, with colorimetric
measurement of enzyme activity.
Comments: Rarely performed method.
Method 4. Colorimetric
Principle of analysis: Reaction of nitrosonaphthol to form blue compound with absorption maximum at 500 nm
Comments: Historical; lacks specificity
Method 5: Thin-layer chromatography (TLC)
Principle of analysis: HVA is extracted and separated on silica gel; color developed by photooxidation, quantified
by reflectance spectrophotometry
Comments: Historical; slow, not suitable for clinical laboratory
Method 6: Gas chromatography (GC)
Principle of analysis: HVA is extracted and the silyl derivative formed; detected by flame ionization
Comments: Rare; slow, research method
Method 7: Gas chromatographymass spectrometry (GC/MS)
Principle of analysis: HVA is extracted with an ion-exchange resin, converted to the trifluoroacetyl
hexafluoroisopropanol ester derivatives, and separated by gas chromatography with detection by mass spectrometry
Comments: Rare; slow, research method

Table 2: Assay Conditions for HPLC Analysis of HVA*

Parameter Values
Sample volume 4 mL
Fraction sample volume 0.1 (anion-exchange chromatography step)
0.2 (anion-exchange elution step)
Final concentration and reagents TrisHCl, pH 7.0, 9 mmol/L
EDTA, 0.09 mmol/L (anion-exchange
chromatography step)
NaCl, 0.2 mol/L (anion-exchange elution step)
Assay time Approximately 60 min
Sensitivity 0.02 g (at 0.05 AUFS)

AUFS, Absorbance units at full scale.

*For assay presented in this chapter; for simultaneous HPLC analysis of VMA and HVA.
 724
Homovanillic Acid

Table 3: Percentile Reference Values for Urinary HVA Excretion

(Using Method Described in VMA Chapter method review)
HVA
95th 100th
Age n mg/24 hr (mol/24 h)*
01 48 2.8 (15.4) 3.5 (19.2)
24 34 4.7 (25.8) 6.7 (36.8)
59 20 5.4 (29.6) 5.7 (31.3)
1019 40 7.2 (39.5) 8.5 (46.7)
>19 56 8.3 (45.6) 10.3 (56.5)
mg/g of creatinine (mmol/nmol of creatinine)
01 37 32.6 (20.3) 76.9 (49.4)
24 49 22.0 (13.7) 58.8 (36.5)
59 79 15.1 (9.4) 33.2 (20.6)
1019 55 12.8 (7.9) 44.2 (27.4)
>19 56 7.6 (4.7) 9.4 (5.8)
*Analyses performed on timed urine samples obtained from patients under investigation for hypertension.
Analyses performed on untimed urine specimens obtained from hospital patients not suspected of having a neural crest tumor.

Table 4: HVA Excretion by Patients with Neural Crest Tumors

(Using Method Described in VMA Chapter, click here)
HVA
(mmol/mol
Age mg/24 (mol/24 mg/g of of
(years) Sex hour hour) creatinine creatinine)
Neuroblastoma
<1 M 36.8 (202) 1421 (882)
<1 F 14.2 (78) 408 (253)
<1 F 14.1 (77) 190 (118)
<1 F 15.1 (83) 158 (198)
1 F 41.2 (226) 496 (308)
1 F 9.4 (52) 68 (42)
1 M 10.0 (55) 57 (35)
2 F 21.6 (118) 140 (87)
2 M 6.4 (35) 90 (56)
3 M 70 (384) 341 (212)
3 M 96.5 (530) 200 (124)
3 M 42.2 (232) 313 (194)
3 M 61.7 (339) 371 (230)
12 M 31.1 (171) 55 (34)
Pheochromocytoma
11 M 2.2 (12) 2.1 (1.3)
12 M 2.0 (11) 9.4 (58)
17 M <2.0 (<11) <10.0 (<6.2)
8 F <2.0 (<11) 2.7 (1.7)
30 F 17.1 (94) 8.1 (5.0)
28 M <2.0 (<11) 1.7 (1.1)
45 M 4.7 (26) 5.3 (3.3)
43 F 1.2 (<11) 1.2 (0.75)
22 F <2.0 (<11) 2.6 (1.6)
19 M 4.0 (22) 2.9 (1.8)
33 F 8.5 (47) 7.9 (4.9)
56 F <2.0 (<11) 2.3 (1.4)
70 M 4.0 (22) 3.9 (2.4)
43 F 3.2 (18) 7.4 (4.6)
62 M <2.0 (<11) <7.0 (<4.3)
34 F 6.4 (35) 7.5 (4.7)
 725
Homovanillic Acid

Figures

Homovanillic Acid: Figure 1

Absorption spectrum of homovanillic acid (HVA) in 1% acetic acid.

Homovanillic Acid: Figure 2

HPLC analysis of homovanillic acid (HVA) with ultraviolet spectrophotometric detection. A, 0.5 g HVA standard. B,
Unspiked patient sample. C, Patient sample spiked with 50 g of HVA.
 726
Homovanillic Acid
Homovanillic Acid: Figure 3
Metabolism of dopamine to homovanillic acid. MAO, Monoamine oxidase; COMT, catechol-O-methyltransferase.
Procedure: HPLC Methods for Urinary HVA Using
Electrochemical Detection
The procedure for VMA (vanillymandelic acid) can be
used for the quantitation of urinary HVA because this
method allows for the simultaneous measurement of VMA
and HVA in urine samples. The alternative method,
presented below, employs ultraviolet spectrometry for
detection and either isocratic or gradient elution of the
HVA (Homovanillic Acid Table: Assay Conditions for
HPLC Analysis).
Procedure: Homovanillic Acid Analysis by HPLC and
Spectrophotometric Detection
Lawrence A. Kaplan
Principle
This method involves a preliminary purification of the
urine by DEAE-Sephadex (G-25) column chromatography
and subsequent separation and quantitation by reversed-
phase HPLC with detection of the HVA peak by ultraviolet
spectrophotometry at 260 nm.
Reagents
Place 1.21 g of Tris and 37.2 mg of disodium
ethylenediaminetetraacetate in a 1-L volumetric flask.
Dissolve the salts in approximately 900 mL of distilled
water. Adjust the pH to 7.0 with HCl, and bring volume to
mark with distilled water. Stable for 3 months at 4C to
8C.
1. NaCl, 0.2 mol/L. Place 11.7 g of NaCl in a 1-L
volumetric flask. Dissolve salt in approximately 900 mL of
distilled water, and bring to mark with additional distilled
water. Stable 6 months at room temperature.
2. 1% acetic acid, 0.18 mol/L. Dilute 10 mL of
concentrated acetic acid to a final volume of 1 L with
distilled water. Stable 2 years at room temperature.
3. Methanol. HPLC grade, filtered through a 0.22
m filter.
4. DEAE-Sephadex G-25. Suspend dry resin in
excess Tris buffer, and decant fines. Store in refrigerator
when not in use.
5. DEAE-Sephadex columns. Polypropylene
columns consisting of a separate barrel, a plastic filter disk,
and a reservoir (total column volume, approximately 17
mL) can be purchased from BioRad Labs. The suspension
of DEAE-Sephadex is poured into the assembled column
until the lower barrel of the column (approximately 3 mL)
is filled with the beads. The columns should be washed
with 5 mL of Tris-EDTA buffer. These columns are
prepared fresh on the day of use.
5. Mobile phase. Add 200 mL of HPLC-grade
methanol to 800 mL of 1% acetic acid. Mix, and filter
through an aqueous solvent filter (0.45 m). Degas for 15
min, putting the mobile phase under negative pressure.
6. HVA stock standard, 1 g/L (5.49 mmol/L).
Place 50 mg of HVA in a 50-mL volumetric flask.
Dissolve HVA in approximately 40 mL of 1% acetic acid,
and bring volume to mark with additional 1% acetic acid.
Stable 1 year at 4C to 8C.
 727
Homovanillic Acid

7. HVA working standard, 50 mg/L (0.27 Calculation

mmol/L). Dilute 5 mL of HVA stock standard to a final % recovery: The extraction efficiency for each
volume of 100 mL with 1% acetic acid. Stable for 3 specimen is determined by calculation of the recovery of
months at 4C to 8C. the HVA spike. This equation can be used only when
Vsx = Vx.
Assay (see Table 2)
Equipment: HPLC system including an ultraviolet detector
Mstd (PHsx - PHx)1000 = % recovery
capable of reading at 254 or 260 nm, an injector (manual
or automated), two pumps, a recorder integrator, and a PHstd (Vx) Msp
solvent programmer. This method can be performed by use
where
of a C18 Bondapak from Waters, Inc. (Milford, MA), or
equivalent. 1000 = Factor to account for sample volume from
1. Add 4 mL of centrifuged urine to 36 mL of 0.01 extraction (10 mL) and for 100% recovery (10 100 =
M Tris0.1 mM EDTA solution. Adjust pH to 7.0
0.05 with 1 M NaOH. 1000)
2. Remove a 20-mL aliquot, and place in labeled test PHstd = Peak height of standard
tube (unspiked sample).
Mstd = Amount in micrograms of HVA standard injected
3. Add 50 L of HVA standard (1 mg/mL) to
remaining solution, and readjust pH to 7.0 (spiked PHx = Peak height of unknown
sample). Vx = Volume of unknown injected
4. Spiked and unspiked solutions are carefully PHsx = Peak height of spiked sample
applied to DEAE-Sephadex columns so as not to Vsx = Vx = Volume of spiked sample injected
disturb resin bed. (Use plastic columns with lower
Msp = Amount in micrograms of spike HVA added to
portion filled with resin. Before applying
samples, wash resin beds with a one-column urine
volume of Tris buffer.)
5. Allow samples to drain into column. Wash Urinary excretion
column with 10 mL of water, and discard eluate.
1. Mstd (PHx) x 10 x 100% = g of HVA/mL of urine
6. Place a clean test tube under column. Elute HVA
with 10 mL of 0.2 M NaCl, allowing eluate to PHstd (Vx) (% recovery) 2
drain into the clean test tube.
7. Inject 40 to 50 L of spiked and unspiked sample
into HPLC system. Homovanillic Acid: Figure 2 2. (g of HVA/mL of urine) TV (mL)
illustrates typical chromatograms for a standard = mg of HVA/TV, or
and a patient sample.
8. Chromatographic conditions: mobile phase (20% mg of HVA/24 hours
methanol in 1% acetic acid) is run through pump
A, 80% methanol in pump B. Run HPLC
isocratically at 100% pump A until peak HVA is 3. g of HVA/mL of urine = g of HVA/mg of creatinine
eluted, and then switch flow to the 80% methanol mg of creatinine/mL of urine
in pump B, and increase flow rate to 2.5 mL/min.
After approximately 5 more min, switch mobile where TV is total volume.
phase back to 20% methanol and reequilibrate for Note
5 min. Change flow rate to 1.0 mL/min, and insert
next sample. This procedure should quickly elute Alternatively, one could perform the analysis by running a
the more strongly retained compounds and linear gradient elution from 0% to 20% methanol in 1%
prevent interference during subsequent injections. acetic acid over 10 minutes, with 5 minutes for
Wavelength: 254 nm (absorbance units reequilibration at 1% acetic acid after washing the column
at full scale, 0.05) with 80% methanol to elute material retained on the
Flow rate: 1 mL/min column. This procedure is very useful to separate peaks
Chart speed: 0.5 cm/min that closely elute with the HVA peak and interfere in its
analysis.
 728
Human Immunodeficiency Virus (HIV)
Human Immunodeficiency Virus (HIV)
Patricia Slev
Name: Human Immunodeficiency Virus (HIV)
Clinical significance: Refer to Chapter 32, Viral Hepatitis: Diagnosis and Monitoring, in the 5th edition
of Clinical Chemistry: Theory, Analysis, Correlation.
Structure: See Figure below.
Courtesy of Bio-Rad Laboratories [1].
Principles of Analysis and Current Usage
Acquired Immunodeficiency Syndrome (AIDS) was first
recognized as a clinical entity in 1981. The syndrome was
associated with abnormalities in the immune response,
particularly immunodeficiency, neuropathology,
opportunistic infections, and rare forms of cancer.
Epidemiological studies suggested that the culprit was an
infectious agent that could be transmitted via intercourse
or blood products [2]. It was not until 1983 that the virus
responsible for AIDS was isolated from infected patients.
Originally named HTLV-III because it was a cytopathic T-
cell lymphotropic virus, further analysis of the biology of
the virus revealed that although HTLV-III was a member
of the lentivirus genus of the Retroviridae family of viruses
and therefore in the same family as HTLV-I and HTLV-II,
it was significantly different from the HTLV viral entities.
The virus was renamed Human Immunodeficiency Virus,
or HIV [3].
Today it is estimated that a total of 39.5 million people
worldwide are living with HIV, [4] and approximately 1
million of these HIV-infected individuals are in the United
i
Human Immunodeficiency Virus (HIV)
New method:
5th edition: Patricia Slev
States [5]. A significant proportion remain undiagnosed
and unaware they are infected [5,6]. This is unfortunate
because with the advent of extensive and effective
treatment options such as highly active antiretroviral
treatment (HAART), HIV has now become a treatable,
chronic disease. However, individuals who are unaware
they are infected cannot be treated promptly and
effectively; they continue to spread HIV infection
disproportionately and unknowingly. Therefore,
diagnosing, screening, and monitoring HIV infection is
more important than ever. Although technology has been
rapidly advancing to allow for earlier, improved detection
and diagnosis of HIV infection, understanding the
advantages, limitations and appropriate use of each of the
multitude of currently available assays is crucial.
The two broad methodological categories that have been
developed for donor screening, diagnosing, and monitoring
HIV infection are serological and nucleic acid testing
(NAT). Both serological and NAT assays are used to
screen for HIV infection in blood or organ donors.
However, serological methods have been primarily utilized
for diagnosing HIV infection, and molecular assays have
been reserved for monitoring HIV disease progression.
Two exceptions to this general approach are a suspected
acute HIV infection and HIV diagnosis in infants. In
 729
Human Immunodeficiency Virus (HIV)
suspected acute HIV infections due to recent exposure, a
serological screen may be followed by a viral-load assay.
In infants, guidelines suggest a combination of both RNA
and DNA HIV NAT for diagnosis [7].
Immunophenotyping or T-cell lymphocyte count assays
based on flow cytometry technology have also been
employed for monitoring HIV disease progression but will
not be discussed in detail in this section. HIV genotyping
and phenotyping for the purpose of monitoring
development of resistance to available antiretroviral
treatments are also available but will be only briefly
addressed in this chapter.
Regardless of the method employed, one problem that
must be taken into account is the ability of the test to
detect the diverse HIV strains that have been identified to
date. All assays, but particularly the serological screens,
are optimized and can be categorized according to the
types and groups of HIV detected. Based on molecular
analysis of the envelope gene, HIV can be classified
phylogenetically into two types: HIV-1 and HIV-2. Within
HIV-1 there are at least three notable groups: M (major), O
(outlier) and N (new). The majority of the HIV-1 strains
circulating in the United States are group M. Group M
strains can be further categorized into subtypes or clades,
A-J, based on sequence. Subtype B strains predominate in
the United States, Japan, Australia, and Western Europe.
Subtype A is prevalent in Russia, as well as West and
Central Africa, while subtype C is most common in
Southern and East Africa, India, and Nepal [8]. Group O
strains were first identified in the early 1990s and are low
in prevalence in the United States [9]. The N group was
discovered in the late 90s in Cameroon. HIV-2 is primarily
restricted to Western Africa and rare in the United States
[10]. In summary, nongroup M strains and HIV 2 are rare
in the United States and Europe. The ability to detect these
rare strains is limited and depends on the assay used.
Functionally, serological methods can be further classified
according to whether they are utilized as a screen or as a
confirmatory (supplemental) test for HIV diagnosis.
Screening assays include rapid immunoassays (results
available in less than 30 minutes) and the more traditional
laboratory enzyme immunoassay (EIA) or enzyme-linked
immunosorbent assay (ELISA). Screening assays are
designed to detect individuals potentially infected with
HIV. Although these assays are very sensitive, follow-up
testing must be performed to confirm that the antibodies
detected in the screening assay are indeed HIV-specific.
The most widely utilized confirmatory assay for HIV-1 is
the Western blot, although the indirect
immunofluorescence assay (IFA) is also U.S. Food and
Drug Administration (FDA) approved for this purpose.
Once HIV infection is confirmed in an individual, some
form of NAT is recommended to establish a baseline viral
load in the patient. This can be used to predict disease
progression and monitor response to antiretroviral
treatment.
As of October 2006, the FDA approved the APTIMA
HIV-1 RNA Qualitative Assay Gen-Probe Incorporated
(San Diego, CA) as the first viral-load assay for diagnosis
of HIV [11]. This represents a significant departure from
previous guidelines in that the APTIMA assay, a NAT
method, can now be used not only for monitoring but also
for confirming HIV infection.
HIV assays used in the United States must be licensed by
the FDA for the specific use, whether it is blood/organ
screening, diagnosing, or monitoring HIV infection. For
example, although there are over 40 available ELISA
assays worldwide, only 10 are approved for use in the
United States. There are many different algorithms for
diagnosing HIV infection, screening potential blood and
organ donors, and monitoring HIV disease progression
worldwide, depending on the local availability of assays.
This chapter will focus on FDA-approved methods
available in the Unites States, with an emphasis on
laboratory diagnosis and disease-progression monitoring,
rather than blood or organ-donor screening.
Antibody Tests
Antibody tests for HIV can be categorized by clinical
functionnamely, screening or confirmation. In terms of
screening for HIV infection, both rapid immunoassays and
traditional EIAs are available. Rapid immunoassays have
been developed in an effort to aid in the early
identification of HIV-infected individuals. Early
identification can reduce viral spread by individuals who
are not aware that they are infected and improve care for
those infected. As the name implies, rapid immunoassays
provide results quickly, usually less than 30 minutes, and
are relatively inexpensive and do not require
instrumentation, although most are still performed in a
laboratory. Rapid immunoassay testing is recommended
for screening high-risk individuals in high-prevalence
areas and women in labor and delivery who have not had
access to antenatal care. As such, rapid immunoassays
have been employed in a variety of clinical settings,
including emergency departments, labor and delivery, and
ambulatory clinical sites.
In the last few years, there has been a marked increase in
the number of rapid immunoassays available. The first
rapid immunoassay was approved by the FDA in 2002
[12]. Currently there are six FDA-approved screening
rapid immunoassays kits for diagnosis available in the
United States (Tables 2 and 3). Three of these are
approved for point-of-care testing and therefore use
unprocessed specimens such as whole blood and oral fluid
and have waived status under the Clinical Laboratory
Improvement Amendments of 1988 (CLIA-88) [13]. The
specifics of each of these tests are beyond the scope of this
discussion, but rapid immunoassays all have certain
characteristics in common. All require no instrumentation
and are interpreted visually. Periodic testing of both known
 730
Human Immunodeficiency Virus (HIV)
HIV-negative and HIV-positive specimens (controls) is
required under the following conditions: (1) new operator,
(2) new kit shipment, (3) new kit lots, and (4) specified
periodic intervals. The most common sample type for rapid
immunoassays is serum or plasma, although the
development of oral fluid as an acceptable sample type has
increased the popularity of rapid immunoassays. However,
oral fluid samples have slightly decreased sensitivity and
specificity when compared to whole blood samples using
the same kit [13]. The most common antigens tested are
HIV-1, gp 41, gp 120, and gp 160 (envelope proteins);
occasionally tested are HIV-1 p24 (core antigen) and HIV-
2 specific gp 36 (envelope protein) (see Anatomy of HIV
on page 1). Sensitivity and specificity are quite good for
group-M subtypes but vary for group O and HIV-2 [12].
Rapid immunoassays are screening tests that are growing
in popularity in both the United States and abroad.
Traditional EIA/ELISA still remains the most common
screening method used in the United States. Similar to
rapid immunoassays, EIAs can be categorized based on the
HIV strains they can detect. There are HIV-1- and HIV-2-
specific assays, as well as combination assays that detect
both (Tables 4, 5, and 6). Anti-HIV antibodies develop
approximately 2 to 12 weeks after HIV infection and
depending on the EIA assay used, may be detected as early
as 2 to 6 weeks post-infection.
Several generations of EIA or ELISA have been developed
since the original in 1985. First-generation assays relied on
viral lysates, whereas second-generation assays were
improved by the addition of synthetic peptide and
recombinant proteins as antigens but detected primarily
IgG. The third-generation assays, which are currently on
the market, developed the antigen sandwich technique.
Third-generation EIA are considered the most sensitive,
because they detect all immunoglobulin isotypes, including
IgG and IgM. There are several third-generation-plus
assays that detect HIV-1 group O as well.
The principle for an antigen sandwich EIA, which is the
most common third-generation assay format in the United
States is as follows. HIV-specific antigens are fixed onto a
solid support. The patients sample, possibly containing
the anti-HIV antibody, and then another HIV antigen
labeled with an enzyme are added in that order. The anti-
HIV antibody is sandwiched between two antigen
molecules, hence the name of the format. Subsequently, a
chromogen/substrate is added, and the intensity of the
color change is measured spectrophotometrically. The
optical density (OD) values obtained are directly
proportional to the amount of anti-HIV antibody present in
the patient sample, and an OD cutoff > 1.00 is considered
reactive (positive) [14]. A recent development has been the
FDA approval of the first automated HIV-1/O/2 EIA
offered by Bayer (Tarrytown, NY)) on the ADVIA
Centaur platform, using a microparticle
chemiluminometric immunoassay format (Table 5). All
HIV ELISA assays are qualitative (meaning
presence/absence) and run in triplicate if the initial result is
reactive.
Antigen Tests
EIA assays that detect HIV antigens, primarily p24, are
also available but are no longer used for diagnosis in the
United States; they are occasionally used for donor-
screening purposes.
Combination Antibody-Antigen Tests
Fourth-generation HIV tests that detect both HIV antigens
(p24) and anti-HIV antibody have been developed, but
none have been approved for use in the United States to
date.
All rapid immunoassays and traditional EIAs (antibody,
antigen) are considered to be screening tests. Because the
purpose of screening assays is to detect any potentially
infected individuals, these tests have very high sensitivity
and good specificity but low positive-predictive value in
populations with low HIV prevalence. Samples from
noninfected individuals have been known to be repeatedly
reactive in screening assays for a variety of reasons,
including reaction with HLA antigens that are present
owing to the manner in which the virus was propagated
and bacterial contamination due to generation of
recombinant proteins, both used in manufacturing the
antigens for the EIA. Recent reports suggest that flu
vaccination can also result in an occasional positive HIV
screen [15]. Therefore, algorithms for HIV testing in the
United States require supplemental testing with a more
specific test for confirmation of HIV-1 infection.
Confirmatory Antibody Tests
There are two commonly available methods in the United
States for confirmation of HIV-1 infection, namely
Western blot and Indirect Immunofluorescence Assay
(IFA) (Table 7). A Western blot is based on the principle
of electrophoresing proteins, which migrate according to
their molecular weight in a sodium dodecyl sulfate (SDS)
polyacrylamide gel, and then blotting the results onto a
nitrocellulose strip. In this case, the bands on the
nitrocellulose blot are discrete and correspond to different
HIV-1 proteins. From top to bottom, the following HIV-1
bands are present: gp 160 (env), gp 120 (env), p65 (pol),
p55 (pol), gp 41 (env), p 40 (gag), p3 (pol), p24 (gag) and
p18 (gag) (see Anatomy of HIV on page 1) [1]. The HIV
antigen strip is subsequently incubated with patient
sample, followed by the addition of an enzyme-labeled
(horseradish peroxidase or alkaline phosphatase)
antihuman immunoglobulin. A relevant colorless substrate
is subsequently added that produces an insoluble colored
band on the strip wherever an antigen-antibody complex is
present. The band pattern and intensity are reported for
each patient. Thus each patient has a relatively unique
Western-blot profile, depending on the type and amount of
antibody they have directed against different HIV antigens.
 731
Human Immunodeficiency Virus (HIV)
There are three possible interpretations based on the
Western-blot profile (band pattern and intensity): positive,
indeterminate, and negative. The cardinal bands, which are
of diagnostic significance and the first bands to become
positive, are HIV-1 gp 41, gp 120, and gp 160, as well as
p24. The Centers for Disease Control and
Prevention/Association of State and Territorial Public
Health Laboratory Directors (CDC/ASTPHLD) criteria
require that two of the three cardinal bands must be present
for the sample to be interpreted as positive. Presence of
any bands on the blot that do not meet positive criteria,
including nonviral bands alone, is interpreted as
indeterminate. For the blot to be considered negative, no
bands can be present [16]. Occasionally, Western blots
exhibit a nonspecific staining (NSS) pattern, which is
defined by a smear on the strip that obscures all or some of
the bands and is associated with heterophile antibodies
[17]. This pattern is uncommon and rarely associated with
HIV infection.
Indeterminate Western-blot results are clinically
challenging because of the multifactorial causes. An
indeterminate Western blot may be due to cross-reacting
antibodies (e.g., influenza vaccine), advanced HIV (low
antibody titers), orrarelyinfection with HIV-2. The
patient status and risk factors, as well as the specific
Western-blot profile, must be considered together. Any
indeterminate Western-blot result should be followed up
with a repeat blot 4 to 6 weeks later and another after 6
months to rule out any potential seroconverters. There are
a limited number of options for confirming HIV-2
infection, and none is FDA approved. There is, however, a
typical indeterminate pattern on an HIV-1 Western blot
that an HIV-2 infection can produce, and it consists of the
following: presence of gag (p55, p24, or p17) plus pol
(p66, p51, p32) in the absence of env (gp 160, gp 120, or
gp 41) [10].
IFA is a qualitative confirmatory method for HIV-1 that
requires HIV-1-infected T-lymphocytes and a fluorescent
microscope. First, uninfected (control) and HIV-1-infected
T-lymphocytes are fixed on a slide in two separate wells.
Dilute patient sample is then added to both wells.
Following a wash, the FITC (fluorescein isothiocyanate)
anti-human IgG antibody conjugate is added. Subsequent
to another wash, the results are visualized using a
fluorescent microscope [18]. This technique requires
interpretation expertise and an expensive fluorescent
microscope but can be used to resolve indeterminate
Western blots. For specifically confirming HIV-2
infection, research-use-only methods are available.
Nucleic Acid Testing (NAT)
Nucleic acid testing for HIV is available in a variety of
formats (Table 8). All forms of NAT testing rely on
detecting HIV-1 RNA in the sample, and although a few
are qualitative, the majority are quantitative and are
therefore generally referred to as viral-load tests. Viral-
load assays are critical for monitoring HIV infection in
response to therapy and predicting disease progression. As
stated previously, NAT was used almost exclusively for
monitoring HIV infection or screening donors for potential
HIV infection until 2006, when the FDA approved the
APTIMA HIV-1 RNA Qualitative Assay as the first viral-
load test that could be used to diagnose or confirm HIV
infection.
There are two basic formats for viral-load assays for HIV:
nucleic acid amplification and signal amplification. The
three nucleic acid amplification technologies are reverse-
transcriptase polymerase chain reaction (RT-PCR), nucleic
acid sequence-based amplification (NASBA) and
transcription-mediated amplification (TMA). Branched-
DNA amplification (b-DNA) is the only signal
amplification assay available. Although nucleic acid
amplification tests are the most complex and sensitive of
the nucleic acid technologies for viral detection, all NAT
technologies have wide and comparable dynamic ranges
(40 to > 1 million copies/mL). However, most formats
have a standard and an ultrasensitive test version, which
usually differ in their dynamic range. Generally, the
qualitative or ultrasensitive versions of a method are
geared towards the low-end range of viral copy numbers
and require initial centrifugation of the sample to
concentrate the virus. Because viral-load assays amplify
different regions of the HIV genome, like EIA assays, they
vary in their ability to detect different HIV strains,
including groups N, O, and HIV-2. Most are optimized for
HIV-1 group M, particularly subtype B detection, which
represents the most prevalent circulating strains in the
United States. Most importantly, although plasma viral
concentrations generally correlate between viral-load
assays, they can vary by at least twofold [19]. Therefore, it
is recommended that when patients are monitored, the
same viral-load assay be routinely employed [20].
PCR (Roche Molecular Systems, Pleasanton, CA) is the
most established and well known technology. Briefly, viral
nucleic acid is isolated, and a discrete nucleic-acid portion
is exponentially amplified using a pair of target-specific
primers and a thermostable polymerase, resulting in
million copies of the original sequence, or amplicons. In
the case of HIV, which is an RNA virus, complementary
or c-DNA template must first be generated using the
reverse transcriptase enzyme. In addition, for the HIV PCR
assays (nonreal time) biotinylated primers, as well as
AmpErase (uracil-N-glycosylase), which functions to
prevent amplicon contamination, are used in the PCR
reaction. Subsequent to the generation of multiple
amplicon copies, the amplicon is denatured to single-
strand copies and hybridized with target-specific
oligonucleotide probes. Unbound material is washed away,
and the Avidin enzymelabeled conjugate is allowed to
bind to the biotin-labeled amplicon, which has been
hybridized with the target-specific oligonucleotide probes,
 732
Human Immunodeficiency Virus (HIV)
followed by the addition of the substrate. Detection is
based on the color change that is read
spectrophotometrically [21].
There are at least three different versions of PCR-based
HIV assays, all offered by Roche, that are variations that
differ in the degree of automation, specific equipment
used, and sample preparation. The Amplicor HIV-1
Monitor Assay is the least automated; the most automated
platform is AmpliPrep/COBAS (comprehensive
bioanalytical system) in which both sample extraction and
the PCR reaction are automated [22].
In May 2007, the FDA approved the first real-time PCR
assays for HIV viral loads: the Roche Cobas
AmpliPrep/Cobas TaqMan HIV-1Test (Pleasanton, CA)
and Abbott RealTime HIV-1 assay (Abbott Park, Illinois).
Real-time PCR assay is a target amplification technique
that allows for real-time detection of accumulating
product, as opposed to the traditional end-point PCR
assays; it represents an important advance in current
technological methods.
NASBA and TMA are similar technologies. NASBA is
marketed as the Nuclisens assay by bioMerieux (Durham,
NC) and is an isothermal reaction useful for amplification
of RNA targets. It employs two analyte-specific primers
and three enzymes: reverse transcriptase, RNase H, and T7
RNA polymerase. In contrast, TMA offered by Gen-Probe
(San Diego, CA) is also an isothermal reaction that can
amplify RNA or DNA and uses two analyte-specific
primers and two enzymes, namely a T7 RNA polymerase
and reverse transcriptase. In both cases, a double stranded
c-DNA template that includes the T7-promoter sequence is
synthesized by the reverse transcriptase and the primer
pair. Secondly, the DNA template is transcribed by the
RNA polymerase, which recognizes the T7 promoter
sequence to generate 100 to 1000 RNA copies. The newly
transcribed RNA amplicons reenter the cycle as templates
for the original reverse transcriptase reaction. Detection of
amplicons is achieved by acridinium esterlabeled DNA
probes that are specific for the target and give off a
chemiluminescent signal when they hybridize to the
amplicon [22,23].
B-DNA technology is a signal amplification technology
that does not require RNA purification or nucleic acid
amplification. A plasma sample is first centrifuged to
concentrate the HIV virus, then the cells are lysed, and the
viral RNA is captured by HIV-specific probes coated onto
the microwell. Target probes that bind to different regions
of the viral RNA, as well as to the preamplifier probes, are
then allowed to hybridize. Multiple copies of the alkaline
phosphataselabeled amplifier probe are subsequently
added; upon binding and addition of the substrate, they
produce a chemiluminescent signal [24].
Genotyping and Phenotyping Assays
HIV drug resistance can be monitored by genotyping or
phenotyping assays. The genotyping assays are the most
common and usually based on sequencing of the HIV
reverse transcriptase and protease genes. Non-sequence-
based genotyping assays that depend on line probe
technology are also available. Phenotyping assays rely on
genetic engineering. First, a recombinant virus which
expresses the reverse transcriptase and protease genes
amplified from the patient sample (via a vector) is
generated and then tested in cell culture with varying
concentrations of the drug to be evaluated. Simultaneously,
wild-type virus is grown as a control. Resistance is
measured by determining the concentration of the drug that
inhibits replication of the virus by 50% as compared to the
wild-type. Assays that combine genotype and phenotype
database information (Virtual Phenotype Assay) are
another option [20].
Reference and Preferred Methods
There are no formal reference methods for HIV testing.
The preferred procedure
for HIV diagnostic testing has been screening a plasma or
serum sample with an EIA, followed by confirmatory
testing with a Western blot. With the advent of the rapid
immunoassays and particularly the ability to test oral fluid
and fingerstick specimens for HIV antibodies, rapid
immunoassays have gained popularity as screening assays.
It remains to be determined what impact the APTIMA HIV
assay will have on HIV diagnostics as a screening and/or
supplemental test option. For monitoring progression of
HIV disease, the preferred viral-load method is a PCR-
based assay. It is also likely that the recently approved
Roche and Abbott real-time PCR-based viral-load assays
may revolutionize viral-load tests because of their rapidity
and automation.
Specimen
Acceptable sample types approved by the FDA vary,
depending on the screening or confirmatory assay
employed. Acceptable sample types for rapid
immunoassays include plasma, serum, whole blood
(venous or fingerstick), and oral fluid. For traditional EIA,
acceptable sample types include serum (cadaveric is
acceptable for certain assays), plasma, dried blood spots,
urine, and oral fluid. IFA-acceptable specimens include
serum, plasma, and dried blood spots. For the Western-blot
method, all IFA-approved sample types, plus urine and
oral fluid, are acceptable (Tables 1-6).
Plasma is the preferred sample type for viral-load assays
(Table 8). With viral-load assays, the choice of
anticoagulant is important. In particular, heparin must not
be used for any PCR-based viral-load tests, although it is
acceptable for NASBA and bDNA technologies [25].
 733
Human Immunodeficiency Virus (HIV)
Interferences
As with any immunoassay, heterophile antibodies can be a
source of interference [26]. Heterophile antibodies may
interfere not only with EIA but also with Western-blot
interpretation by producing a nonspecific staining (NSS)
pattern [17]. In addition, flu vaccination has been
associated with false-positive EIA results (see above). In
regard to NAT, substances that can inhibit PCR reactions
can be an issue. This is why an internal or external control
for each sample, depending on the assay type, is always an
integral component of any viral-load assay. Therefore, a
result of none detected on a viral-load assay is not
synonymous with negative for HIV infection. In fact, the
goal of HIV antiretroviral therapy is to achieve a viral load
that is below the limit of detection (using < 50 copies/mL
as a cutoff) [27].
Replication-competent HIV-1 can persist in the resting T-
cells of these patients, although they have an
undetectable viral-load result. Nevertheless, untreated
individuals rarely have undetectable HIV viral loads.
Interpretation
Studies suggest that 25% to 45% of HIV-infected
individuals are unaware they are infected, and the majority
of these are not diagnosed with HIV infection until they
are within a year of clinical disease onset (AIDS) [5,6].
Therefore, HIV screening is now recommended by the
CDC for all individuals 13 to 64 years of age in all routine
health care settings [28].
Individuals are screened with either a rapid immunoassay
or EIA. Repeatedly reactive screening assay results must
be confirmed with one of the available confirmatory tests,
most commonly the Western blot [12,29]. Any repeatedly
reactive EIA (rapid or traditional) result that is not
confirmed by supplemental testing suggests a false-
positive screen, as long as recent exposure and the
seroconversion window can be ruled out. Indeterminate
confirmatory test results should be repeated. According to
CDC guidelines, individuals with two indeterminate
Western-blot results spanning at least 6 months are
considered negative for HIV infection, provided recent
exposure has been ruled out. Positive results for
confirmatory testing should be followed up with a viral-
load assay to establish baseline plasma HIV concentration.
Technically, the APTIMA HIV assay can now be used as a
confirmatory test, although it has not yet been widely
adopted for this purpose.
HIV diagnosis is particularly difficult in neonates that have
been exposed to the virus in utero. Passively acquired HIV
specific antibodies due to transplacental transmission can
be present in these infants until 18 months of age. HIV
infection should not be diagnosed in neonates based on
serological assays but rather by a combination of viral-load
assays (HIV RNA and HIV DNA) [7,12].
Once the diagnosis of HIV has been established, viral-load
assays are recommended for monitoring HIV disease
progression. The goal of successful HIV therapy is an
undetectable HIV viral load. Although viral-load assays
correlate well, it is recommended that only one method be
used for serial monitoring. A change in HIV viral load that
is > 0.5 log 10 copies/mL is clinically significant [20].
Performance Goals
In general, performance characteristics for each assay,
such as specificity and sensitivity, are dictated by the
manufacturers of the commercial assays in accordance
with recommended guidelines of the licensing bodies for
the country in which the assay is utilized. The CLIA-88
require that laboratories performing non-waived HIV
antibody testing participate in a proficiency testing (PT)
program. Since most of the HIV antibody testing methods
are deemed at least moderately complex, with a only a
couple of rapid immunoassay exceptions, most hospital
and reference laboratories participate in a PT program.
Currently there are a number of active programs for HIV
antibody testing, including the one offered by the College
of American Pathologists (CAP), which has the highest
enrollment. Challenge samples are sent out periodically,
and a score of 80% must be achieved to receive a passing
grade, as is typical with all analytes and the CAP PT
program. In addition, many of these programs have
expanded to include rapid immunoassays, although a
proportion of the currently available rapid immunoassays
are CLIA waived.
A recent survey evaluating HIV-1 antibody testing
performance results showed that overall performance or
efficiency (TP+TN/all results) for HIV-1 EIA was 99%,
HIV-1 Western blot 99.5%, and HIV-1 IFA 98.7%. Only
4.3% utilized IFA for confirmation of HIV infection, and
approximately 1.2% used the Gen-Probe APTIMA HIV-1
RNA qualitative assay as a form of confirmation for
repeatedly reactive EIA results [31]. The same program is
no longer being offered for HIV-1 RNA determinations,
but the results from the February 2006 report indicate that
the performance for quantitative and qualitative tests
combined was 96.5%; more specifically, the performance
for quantitative was 96.3% and 100% for the qualitative
tests [32].
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York (NY): New York State Department of
Health; 2005 Feb. 13p.
31 Centers for Disease Control and Prevention
Model Performance Evaluation Program (MPEP).
Human Immunodeficiency Virus Type 1 (HIV-1)
Antibody Testing. Report of Results for the
Performance Evaluation Survey Conducted
During January 2007.
32 Centers for Disease Control and Prevention
Model Performance Evaluation Program
Human Immunodeficiency Virus Type 1
ribonucleic Acid (RNA) Determinations. Report
Results for the Performance Evaluation Survey
Conducted during February 2006 ).
33 http://www.fda.gov/cber - TABLES (accessed
June 2007)
 735
Human Immunodeficiency Virus (HIV)

Table 1: HIV methods summary

Method 1. Rapid Immunoassay
Principle: Detects antibodies against HIV
Usage: Screening for HIV infection
Comments: Many kits approved in the US for various sample types including whole
blood & oral fluid; increasing in popularity
Method 2. Enzyme Immunoassay / Enzyme Linked Immunosorbent Assay
( EIA/ELISA)
Principle: Detects antibodies against HIV or HIV antigens on asolid support
Usage: Screening for HIV infection
Comments: Many kits available, differ in the ability to detect HIV types, groups and subtypes
Method 3. Western Blot
Principle: Detect antibodies against HIV antigens electrophoresed on a strip
Usage: Confirming HIV infection
Comments: Most common confirmatory test for HIV infection
Method 4. Indirect Immunofluorescence Assay (IFA)
Principle: Detect antibodies against HIV antigens on infected cells
Usage: Confirming HIV infection
Comments: Less common confirmatory test for HIV infection
Method 5. Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Principle: Determine HIV plasma concentration based on PCR nucleic acid amplification
Usage: Monitoring HIV infection
Comments: Common method for monitoring HIV infection
Method 6. Branched DNA (b-DNA)
Principle: Determine HIV plasma concentration based on nucleic acid signal amplification
Usage: Monitoring HIV infection
Comments: Does not require nucleic acid extraction, purification
Method 7. Nucleic Amplication (NASBA)
Transcription Mediated Amplification (TMA)
Principle: Determine HIV plasma concentration based on non-PCR based nucleic acid amplification
Usage: Monitoring HIV infection
Comments: Both technologies are similar in principle but use different enzymes

Table 2 - HIV-1 Rapid Immunoassays (Antibody)

Trade Name Format Sample Use Manufacturer Approval
Date
Murex SUDS HIV-1 Test Rapid Serum / Plasma Donor Murex Diagnostics, 5/22/1992
Immunoassay Screen Inc
Norcross, GA
US License 1152

Reveal Rapid HIV-1 Rapid Serum / Plasma Diagnostic MedMira 4/16/2003

Antibody Test Immunoassay Laboratories, Inc
Halifax, Nova Scotia
Canada B3S 1B3

Uni-Gold Recombigen Rapid Serum / Plasma / Diagnostic Trinity Biotech, plc 12/23/2003
HIV Immunoassay Whole Blood Bray Co., Wicklow
(venipuncture and Ireland
fingerstick)
 736
Human Immunodeficiency Virus (HIV)

Table 3 - HIV-1,2 Rapid Immunoassays (Antibody)

Trade Name Format Sample Use Manufacturer Approval
Date
Multispot HIV-1/HIV-2 Rapid Test Rapid Plasma / Serum Diagnostic Bio-Rad Laboratories 11/12/2004
Immunoassay Redmond, WA

OraQuick ADVANCE Rapid HIV- Rapid Whole Blood, Diagnostic OraSure Technologies 6/22/2004
1/2 Antibody Test Immunoassay Plasma, Oral Fluid Bethlehem, PA
18015-1360

HIV 1/2 STAT-PAK ASSAY Rapid Fingerstick & Diagnostic Chembio Diagnostic 5/25/2006
SURE CHECK HIV 1/2 ASSAY Immunoassay venous whole blood, Systems, Inc
serum, plasma Medford, NY

Table 4 - HIV-1 EIA Antibody Assays

Trade Name Format Sample Use Manufacturer Approval Date

HIVAB HIV-1 EIA EIA Serum / Donor Screen Abbott Laboratories 3/1/1985
Plasma Abbott Park, IL
US License 0043

Genetic Systems EIA Serum / Donor Screen Bio-Rad Laboratories Blood 6/29/1998
rLAV EIA Plasma Virus Division
Redmond, WA
US License 1109

Vironostika HIV-1 EIA Serum / Donor Screen bioMerieux, Inc 12/18/1987

Microelisa System Plasma Durham, NC 27712
US License 1624

Vironostika HIV-1 EIA Plasma / Diagnostic bioMerieux, Inc 6/6/2003

Plus O Microelisa Serum / Durham, NC 27712
System Dried Blood US License 1624
Spots

HIVAB HIV-1 EIA EIA Dried Blood Diagnostic Abbott Laboratories 4/22/1992
Spot

HIV-1 Urine EIA EIA Urine Screen Diagnostic Calypte Biomedical Corp 8/6/1996

Genetic Systems EIA Dried Blood Diagnostic Bio-Rad Laboratories Blood 6/29/1998
rLAV EIA Spot Virus Division

Vironostika HIV-1 EIA Dried Blood Diagnostic bioMerieux, Inc 4/11/1990

Microelisa System Spot Durham, NC 27712

Oral Fluid EIA Oral Fluid Diagnostic bioMerieux, Inc 12/23/1994

Vironostika HIV-1 Durham, NC 27712
Microelisa System
737
Human Immunodeficiency Virus (HIV)

Table 5 - HIV-1,2 EIA Antibody Assays

Trade Format Sample Use Manufacturer Approval
Name Date
Abbott EIA Serum / Plasma / Donor Screen Abbott 2/14/1992
HIVAB Cadaveric Serum Laboratories
HIV-1/HIV- Abbott Park, IL
2 (rDNA) US License 0043
EIA

Genetic EIA Serum / Plasma / Donor Screen Bio-Rad 12/9/2000

Systems Cadaveric Serum Laboratories
HIV-1/HIV- Blood Virus
2 Peptide Division
EIA Redmond, WA
US License 1109

Genetic EIA Serum / Plasma / Donor Screen Bio-Rad 8/5/2003

Systems Cadaveric Serum Laboratories Inc
HIV-1/HIV- Redmond, WA
2 Plus O US License 1109
EIA

ADVIA Microparticle Plasma/Serum Diagnostic for Bayer Healthcare, 5/18/2006

Centaur Chemilumin- qualitative LLC
HIV 1/O/2 ometric determination of Tarrytown, NY
Enhanced Immunoassay antibodies to the
ReadyPack human
Reagents immunodeficiency
virus type 1,
including Group O,
and/or type 2 in
serum or plasma

Table 6 - HIV-2 EIA Antibody Assays

Trade Name Format Sample Use Manufacturer Approval
Date
Genetic Systems EIA Serum / Donor Bio-Rad Laboratories 4/25/1990
HIV-2 EIA Plasma Screen Blood
Virus Division
Redmond, WA
US License 1109
 738
Human Immunodeficiency Virus (HIV)

Table 7 - Confirmatory Assays

Trade Name Format Sample Use Manufacturer Approval
Date
Cambridge Biotech HIV-1 WB Serum / Donor Calypte Biomedical Corp 1/3/1991
Western Blot Kit Plasma Supplemental Berkeley, CA
US License 1207

Genetic Systems HIV-1 WB Serum / Donor Bio-Rad Laboratories 11/13/1998

Western Blot Plasma Supplemental Blood Virus Division

Fluorognost HIV-1 IFA IFA Serum / Donor Waldheim Pharmazeutika 2/5/1992

Plasma Supplemental GmbH
Vienna, Austria
US License 1150

Cambridge Biotech HIV-1 WB Urine Diagnostic Calypte Biomedical Corp 5/28/1998

Western Blot Kit Supplemental

Genetic Systems HIV-1 WB Dried Diagnostic Bio-Rad Laboratories 11/13/1998

Western Blot Blood Spot Supplemental Blood Virus Division

OraSure HIV-1 Western WB Oral Fluid Diagnostic Epitope, Inc 6/3/1996

Blot Kit Supplemental

Fluorognost HIV-1 IFA IFA Dried Diagnostic Waldheim Pharmazeutika 5/14/1996

Blood Spot Supplemental GmbH
 739
Human Immunodeficiency Virus (HIV)

Table 8 - HIV Nucleic Acid Testing (HIV-1 NAT)

Trade Format Sample Use Manufacturer Approval
Name(s) Date
Roche PCR Plasma Prognosis / Roche Molecular 3/2/1999
Amplicor Patient Systems, Inc
HIV-1 Management Branchburg
Monitor HIV-1 Viral Township, NJ
Test Load Assay

NucliSens NASBA Plasma Prognosis / bioMerieux, Inc 11/19/2001

HIV-1 QT Patient Durham, NC
Management 27712
HIV-1 Viral
Load Assay

COBAS PCR Plasma Donor Screen Roche Molecular 12/20/2002

Ampliscreen Expanded Systems, Inc
HIV-1 Test Indications For Pleasanton, CA
Use: Source 94566-0990
Plasma donors,
other living
donors, and
organ donors

Procleix HIV-1/HCV Plasma Donor Screen Gen-Probe 2/8/2002

Nucleic Acid Expanded San Diego, CA
Test (TMA) Indications For 92121
Use: Source
Plasma donors,
living organ
donors and
cadaveric
samples

UltraQual PCR Plasma Donor Screen National Genetics 9/18/2001

HIV-1 Institute
RT-PCR Los Angeles, CA
Assay 92121

Versant Signal Plasma Patient Bayer Corp 9/11/2002

HIV-1 amplification Monitoring Berkeley, CA
RNA 3.0 nucleic acid probe 94702
(bDNA)

All tables were adapted from the FDA CBER website [33].
740
Immunoelectrophoresis
Immunoelectrophoresis
Stanley S. Levinson
Name: Immunoelectrophoresis
Clinical significance: Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
i applications for immunoelectrophoresis, the greatest
Electrophoresis
Protein electrophoresis is based on the principle that
proteins that are composed of amino acids show
charges that depend on the properties of the amino
acids, the configuration of the protein, and the pH of
the buffer and medium. The migration of proteins in
an electric field is largely determined by the charge
on the molecule and the pH of the electrophoretic
medium. Different proteins will separate from one
another in accordance with the molecular charge. In
gels containing a cross-linked matrix, such as
acrylamide or starch, the separation may be
substantially influenced by the size of the molecules.
Immunoelectrophoresis is usually conducted in
agarose gel that contains minimal cross-linking, so
the charge on the molecule largely determines the
migration distance.
In clinical laboratories, agarose electrophoresis is
optimized for the identification of monoclonal
immunoglobulins. This is called high resolution
protein electrophoresis (HRPE). All kits presently
available for the clinical laboratory use such an
optimized approach.
Immunoprecipitation
After electrophoretic separation, the proteins are
treated with specific antibodies. Except for capillary
zone electrophoresis, where there is no gel, the
resultant antigen-antibody complex(es) become
insoluble and precipitate in the gel. The rate of
precipitation depends on the proportions of the
reactants, temperature, salt concentration, and pH of
the solution. Proteins that have not reacted with the
specific antibodies are removed from the gel by
washing. The antigen-antibody complex is best
visualized after staining. Although there are other
i nonspecific proteins that have not precipitated in the
Immunoelectrophoresis
Previous and current authors of this method:
First edition: Gayle Birkbeck
Methods edition: Gayle B. Jackson
Second edition: Gayle B. Jackson
Third edition: Steven C. Kazmierczak
Fourth edition: Gayle B. Jackson
Fifth edition: Stanley S. Levinson
demand exists in the medical laboratory for the
detection and identification of monoclonal
immunoglobulin gammopathies in serum and urine.
Agarose is chosen because it is sufficiently porous to
allow antibodies to enter the gel but sufficiently
dense to trap precipitated immune complexes within
the gel. Antibodies cannot enter more tightly cross-
linked media such as acrylamide. As a result, specific
antibody identification of proteins after separation on
less porous media requires removal of the proteins
from the gel onto membranes (Western blotting),
which is more tedious but widely used in research
laboratories.
Precipitation of the complexes after reaction with
specific antibodies is dependent on the antigens
(proteins that are themselves most often antibodies)
reacting with antigen-specific antibodies (also
proteins), which combine to form a lattice (immune
complex). According to the Heidelberg principle, if
the antibody concentration is held constant, the
amount of precipitate will increase as the antigen
concentration increases until a zone of equivalency is
reached. As the antigen concentration decreases
below equivalency, there will be less precipitate
(antigen and antibody will become increasingly
soluble). This phenomenon is called prezoning. Also,
as the antigen increases above equivalency, solubility
will also increase (prozoning). As will be discussed
below, these processes may become important when
interpreting the results of the immunoelectrophoresis,
since prozoning may cause fading of the precipitant
that must be recognized by the interpreter. Except for
capillary zone electrophoresis (immunosubtraction
where the electrophoresis is performed in free
solution without a solid gel) (Table 1, Method 4),
after precipitation, the gels are washed to remove the
agarose, then the agarose gel is stained with a stain
that binds proteins. The stain allows the specifically
precipitated protein to be better visualized.
Although immunoelectrophoretic techniques can be
used to identify any protein, the most common use is
for typing monoclonal immunoglobulins. Generally,
741
Immunoelectrophoresis
in clinical laboratories, four types of
immunoelectrophoresis have been used:
Electroimmunoassay
Electroimmunoassay (EIA) has also been called the
rocket or Laurell rocket [1,2] (Table 1, Method
1) technique. In this method, the antibody is mixed
into the gel, and the specific protein migrates to the
point of equivalency, where it precipitates. The
precipitate appears as an arrowhead or rocket.
Known calibrators with known amounts of protein
are run in some lanes. Unknown amounts of protein
run in others. This technique quantifies the amount of
unknown protein by comparing the length of the
rocket from the origin with the calibrators that are
graphed on a curve. Generally, this technique has
been supplanted by more accurate and
methodologically sensitive two-site solid-phase
immunoassay techniques. Some kits are available for
quantifying the coagulation factors Protein C and
Protein S by this technique.
Classical Immunoelectrophoresis
Immunoelectrophoresis (IEP) was first described by
Grabar and Williams [3]. (Table 1, Method 2).
Electrophoretic separation of replicate samples in
agarose gels is conducted in multiple lanes in the first
step. In the second step, immunoglobulin-specific
antiserum is placed in preformed troughs cut parallel
to each lane. The gels are allowed to incubate in a
closed chamber for 24 to 48 hours, while the antisera
and antigen diffuses into the gel. The proteins from
the patients serum which were separated by
electrophoresis in the prior step migrate by diffusion
towards the antisera. A precipitate develops, fixing
the immunoglobulins in the form of an arc. The gel is
then washed and stained. Examples of this technique
can be found in Ritzmann and Lawrence [4] and Penn
[5], as well as Gayle Jacksons description in
previous editions of this textbook.
Considerable skill is needed to interpret the results,
which are difficult to relate to routine protein
electrophoresis. A shorter, narrow arc more distant
from the trough and close to the antigen source
indicates a decrease in antigen, while a longer,
thicker band close to the trough indicates an
increased antigen concentration. However, if too high
a concentration of protein is present, the precipitate
may form very close to the trough or within the
trough. Also, prozoning may occur when the antigen
is present in high concentration. The presence of an
abnormal immunoglobulin is inferred from
comparing the size, shape, and distortions of the arc
with the control. The identification of small abnormal
components is particularly difficult.
Immunofixation Electrophoresis (IFE)
A brief history of this technique (Table 1, Method 3)
can be found in Levinson and Keren [6]. The
technique was first described for clinical laboratory
use in 1976 by Ritchie and Smith [7] and Cawley and
associates [8] for identifying monoclonal
immunoglobulins in serum, urine, and cerebrospinal
fluid. These papers illustrate exact methods well. The
major change in currently used methods is the
availability of the complete procedure as a kit.
As illustrated in Figure 1, replicate electrophoresis of
samples is performed in multiple lanes. Following
this, the fractionated proteins in the electrophoretic
lanes are precipitated (fixed) into the agarose by
overlaying with antisera specific for each type of
immunoglobulin to be identified. After a wash step to
remove unfixed proteins, the strips are stained with a
protein stain and washed to remove the excess stain.
The separated proteins precipitate in a pattern that is
easily related to the pattern on routine HRPEshown
on the strip on the left in Figure 1, designated serum
protein electrophoresis (SPE). IFE shows three to
five times more sensitivity than routine HRPE,
because the antibodies that are bound to the selected
proteins increase the overall protein mass that is
stained [9]. In clinical laboratories, IFE is currently
the predominant technique used for identifying
proteins of interest and is the recommended method
for the identification of monoclonal proteins (M
proteins) [10].
The advantages of IFE over IEP are several-fold,
particularly with regard to interpretation.
With IEP, interpretation depends largely on the
skill of the examiner, and subtle distortions of the
arcs may be overlooked, whereas with IFE, the
abnormal proteins migrate as restricted bands that
line up with the bands seen on routine SPE,
allowing for simple direct comparisons, as seen in
Figure 1 for the IgG kappa paraprotein.
IgM interference is eliminated. Monoclonal IgM
is particularly difficult to identify with IEP due to
an umbrella effect in which polyclonal IgG
interferes with the identification of monoclonal
IgM paraproteins. This effect occurs because the
smaller 7S IgG molecules diffuse faster than the
larger 19 S IgM molecules towards the trough. As
a result, the light chain antisera is entirely
complexed by the intact light chain bound to the
polyclonal IgG prior to the arrival of the larger
monoclonal IgM. The light chain associated with
the slower moving IgM does not react with the
depleted light chain antisera, and it is not
precipitated. As a result, the monoclonal IgM
742
Immunoelectrophoresis
appears to be polyclonal. When a monoclonal
IgM abnormality is suspected, it is necessary to
reduce the IgM by treating the serum with a
reducing agent such as mercaptoethanol prior to
electrophoresis, which breaks the 19S molecule
down to 7S. This allows it to migrate more
rapidly and compete equally with the IgG. IFE is
not affected by this phenomenon.
Biclonal gammopathies are identified with IFE as
two bands, one staining for kappa and the other
lambda. With IEP, an increase in the density of
both lambda and kappa arcs may be
misinterpreted as a polyclonal increase.
IEP is insensitive for picking up very small
monoclonal proteins, those less than 1 g/L,
because diffusion of the antigen causes the
concentration to be reduced. With IFE, the
antigen is concentrated into a narrow band. IFE is
very sensitive, detecting proteins to
concentrations as low as 100 mg/L. This
sensitivity can be substantially increased with
cerebrospinal fluid (CSF) and urine by
mechanically concentrating the fluid.
Serum samples run in antigen excess are usually
still interpretable with IFE, whereas with IEP,
prozoning may occur in the region of antibody
excess, causing a failure of arc formation. The
reason for this is because comparison of the SPE
with the IFE pattern provides a simple check for
identifying prozoning, as will be discussed below
under Interpretation.
The turn around time for IFE is much more rapid
(2 to 4 hours) than IEP, which requires a 24- to
48-hour incubation for the diffusion step.
Capillary Zone Electrophoresis
Capillary Zone Electrophoresis (CZE) (Table 1,
Method 4) measures proteins by absorbance [11]. No
protein stains are needed for this technique.
Molecules are separated in free buffer in thin, fused-
silica capillaries, generally of 20 to 100 mm internal
diameter, by the application of a very high voltage.
Generally, CZE gives results similar to SPE by
HRPE. The major advantage of this technique is that
many samples can be run rapidly, so for high-volume
laboratories, it offers a quick alternative compared to
agarose electrophoresis. However, the sensitivity is
somewhat greater so that fibrinogen that migrates in
the beta-region of the gel, and is present in some
serum samples at very low concentrations, sometimes
appears to be a monoclonal protein (about 3% to 5%
of the time), which requires rerunning the sample by
HRPE. Nevertheless, with high volume, there are
great savings in being able to run so many samples
more rapidly.
Following electrophoresis, immunotyping can be
accomplished by use of immunosubtraction that is
performed by incubating the serum sample with
beads coupled to anti-gamma, alpha, mu, kappa, and
lambda antisera. Following incubation with each of
the heavy- and light-chain antisera solid phase
reagents, the supernatants are reanalyzed by CZE to
determine which of the immunoglobulin fractions has
been removed.
Although CZE is useful for high volume SPE, it is
not recommended for paraprotein determinations on
urine or CSF analysis [11], and even paraproteins that
are identified by CZE in serum are generally typed by
IFE.
Reference and Preferred Methods and Specimens
Although there is no definitive reference method, IFE
(Table 1, Method 3) has been defined by the College
of American Pathologists (CAP) Expert Panel as the
preferred method for definitive identification and
typing of monoclonal proteins [10]. This expert panel
was selected and sponsored by the American Society
of Hematology, the American College of
Rheumatology, the Clinical Center of the National
Institutes of Health, and the Clinical Immunology
Society. The panel discouraged the use of the older
IEP method (Table 1, Method 2). The panel noted
that because of its greater sensitivity, IFE may be
useful for identifying a very small monoclonal
protein, even when agarose gel HREP is negative,
especially for identifying monoclonal kappa and
lambda free light chains [10]. An International
Federation of Clinical Chemistry Committee on
plasma proteins and SIbioC Study Group on proteins
also recommended IFE as the choice for identifying
monoclonal free light chains in urine [13].
Specimen
Serum: Serum samples should be screened by HRPE
and those with paraproteins typed by IFE.
CSF: IFE is also very sensitive for identifying
oligoclonal banding in CSF (see Interpretation
below), although many laboratories now use
isoelectric focusing techniques. CSF should be
concentrated 100 for initial examination, although
repeats with dilutions of the concentrate may be
necessary.
Urine: Urine samples may be screened by HRPE, but
IFE is more sensitive for identifying monoclonal free
light chains in urine (Bence Jones Proteins), and IFE
should be applied to all suspect samples by screening
with kappa and lambda antisera for free and bound
immunoglobulins (see Interpretation below) [6,10]. It
743
Immunoelectrophoresis
is important to concentrate the urine 100 to 150
prior to HRPE and IFE so that very low concentration
Bence Jones protein (BJP) can be identified (see
Interpretations below). It is also important to perform
the testing on 24-hour collections. Studies have
shown that testing on 2 or 3 consecutive 24-hour
urines essentially rules out Bence Jones proteinuria
(BJPuria) [12].
Two types of antisera are available for fixation of
light chains: antisera that react with free light chains
only (FLC-only) and antisera against both free light
chains and intact immunoglobulins (light chains
bound to heavy chains), often called antisera against
free and bound (F & B). The former is produced by
affinity purification, adsorbing out light chain
specific antibodies that react with intact
immunoglobulins, leaving only those that react with
FLC in solution. It usually has less avidity than
antisera against F & B. As such, it is more
susceptible to prozoning (see Interpretation below).
Some preparations of antisera against FLC-only may
also show somewhat diffuse band patterns on IFE,
but in recent years, most preparations produce
distinct patterns. Due to its lower avidity, antisera
against free should not be used for initial screening
but only in conjunction with antisera against F & B
as an adjunct for better definition when the BJP is
migrating close to an intact monoclonal protein
containing the same light chain type [13].
Reference Intervals
See Interpretation below.
Interpretation
Serum Electrophoresis
For serum samples, current guidelines suggest screening
for monoclonal proteins using HRPE followed by IFE for
definitive identification [10]. Figure 1 illustrates a
classical picture for multiple myeloma, where SPE by
HRPE show a paraprotein band (restriction migrating as a
band) in the gamma region of the gel, and IFE indicates
that it is an IgG- monoclonal protein (M-protein). The
SPE indicates there is very little immunoglobulin except
for the monoclonal band (clear gel on both sides of the
band). The IFE also shows little or no polyclonal
immunoglobulin (little diffuse staining on the IFE lanes).
In many cases of multiple myeloma, the uninvolved
immunoglobulins are very low because the normal plasma
cells are decreased, while the abnormal clone is
substantially increased (see the chapter on Bence Jones
Proteins).
Classical Profile versus Monoclonal Gammopathies of
Unknown Significance
The classical profile in Figure 1 can be contrasted with
the appearance of low-concentration paraproteins shown
in Figure 2. Notice that in Figure 2, both SPE and IFE
show substantial polyclonal (diffuse) staining near the
monoclonal IgG proteins. This indicates that there are
substantial amounts of polyclonal Ig. Most commonly,
these low concentrations of paraproteins are not
associated with disease but are monoclonal gammopathies
of unknown significance (MGUS). Visual inspection of
the SPE gels in Figure 2 indicates that the monoclonal
proteins are less than 10% of the total staining in the
gamma region or 1000 mg/L. Kyle and associate have
found that monoclonal proteins < 5 g/L will develop into
myeloma only about 6% of the time, < 10 g/L about 7%,
< 15 g/L about 11%, < 20 g/L about 20%, < 25 g/L about
24%, and < 30 g/L about 34% of the time [14]. In some
cases, it has been shown that these MGUS disappear with
time [14,15]. In other cases, low concentration
monoclonal bands turn out to be due to myeloma upon
bone marrow biopsy, especially when they are > 20 g/L,
and there is little diffuse polyclonal staining associated
with them (hypogammaglobulinemia of the normal
immunoglobulins). Nevertheless, there is generally no
advantage in using HRPE that identifies very tiny
monoclonal proteins in serum as compared to HRPE
methods that are not quite as sensitive, since the great
majority of tiny bands will be MGUS.
Position of Migration and Multiple Monoclonal Bands
The IFE in Figure 2 indicates that on the average, the
polyclonal IgA migrates most rapidly toward the anode,
since IgA is the most negative of the three major
immunoglobulins, whereas the polyclonal 19S IgM
tetramers that are too large tend to stay near the origin. As
seen in Figure 2, of the three major immunoglobulin
classes, polyclonal IgG migrates most towards the
cathode. Because of this more rapid migration, IgA M
proteins are often found in the beta region, at about the
beta-2 position, and low level IgA monoclonals are
sometimes masked by beta-2-migrating serum proteins
such as transferrin. Nevertheless, because of the wide
charge variability in the Fab region of the molecule, there
is substantial overlap for the migration position of any
single immunoglobulin. As a result, IgA M proteins may
be found in the more cathodal gamma region and IgG M
proteins in the beta region. Some laboratories test SPE
gels that show an elevation in the beta-2 fraction by
testing for / ratios by immunonephelometry or
immunoturbidimetry; others test by running IFE using
trivalent antisera against IgA, IgM, and IgG. Figure 3
shows a biclonal pattern with such a rapidly migrating
monoclonal IgG- in the beta region of the gel, as well as
an IgG- running cathodally.
As shown in Figure 3, in some cases two or more
monoclonal bands are seen. IFE may show the bands are
an intact monoclonal immunoglobulin and a free light
744
Immunoelectrophoresis
chain. Generally, the free light chain will not be seen on
SPE, either because it is too small to show up on the less
sensitive SPE or because it is migrating in the beta region
and masked by normal beta-migrating proteins. In other
cases, the bands turn out to represent two or more intact
monoclonal proteins (so-called biclonal or triclonal
gammopathies). Occasionally, one band turns out to be a
heavy chain without a light chain.
When two intact M proteins are seen, and one band is
kappa and the other lambda, as shown in Figure 3, the
pattern is a true biclonal gammopathy and indicates that
the M proteins originated from two different clones of B
cells. If the two bands are both IgG- or one is IgG- and
the other IgA-, or similarly both are lambda, it is unclear
that there are two abnormal clones, since it is more likely
that cells from a single clone are class switching, or that
an abnormal clone has given rise to a progeny that is
producing a different class of immunoglobulin or a
fragment, in which case both monoclonal proteins may
not be from different clones. In these cases, the pattern
may not represent a true biclonal gammopathy.
Multiple monoclonal bands may also be seen in cases of
IgA and IgM monoclonal gammopathies. Secretory IgA is
a dimer, and serum IgA is a monomer. A dysplastic cell
may produce both, so that two bands may be seen, both
typing for the same light chain type. The faster migrating
band is the monomer. IgM myeloma is very rare. Usually
IgM monoclonal proteins are associated with
lymphoproliferative diseases such as Waldenstroms
syndrome. Normally serum IgM is a pentamer, but
abnormal cells may produce monomers or even dimers, so
that two or three IgM M-protein (always the same light-
chain type) bands may be seen.
IgD and IgE
If a monoclonal protein is identified on serum HRPE
and stains for kappa or lambda but not for IgG, IgA,
or IgM with IFE, it may be a monoclonal free light
chain, or it may be an intact IgD or IgE. Under this
circumstance, testing for IgD and IgE should be
performed. It is very unusual for a monoclonal free
light chain to reach high enough concentrations in
serum to be identified by SPE, because most of the
free light chain spills over into the urine. Moreover, if
urine from the same patient is analyzed and a BJP is
identified, this does rule out an IgD or IgE, since IgD
paraproteins frequently have an associated BJP.
Prozoning
One difficulty with IFE is that when the M proteins
are at high concentration, they exhibit prozoning.
With serum proteins, this rarely causes a problem
because of the limited range of concentrations
encountered (about 1 g/L to about 6 g/L). It may
cause the band to appear as a donut with a faded
center, as shown in Figure 4, that will appear as a
solid band with a 3 to 5 dilution, as shown.
Prozoning may be a particular problem with CSF and
especially urine, because concentration of the sample
(approximately 100) prior to electrophoresis may
result in concentrations of the M protein of the order
of 60 g/L. In these cases, multiple dilutions with
repeat analysis may be necessary. These problems
will be discussed below.
Proteins Other Than Immunoglobulins
IFE is useful for identifying other proteins besides
monoclonals. For example, in some cases, samples
that are thought to be serum may contain some
fibrinogen [16]. Figure 5 illustrates the identification
of fibrinogen by IFE. We have found IFE useful for
identifying lysozyme in urine as well (see the chapter
on Bence Jones proteinuria) [9]. If desired, exact
identification can be made by IFE for the specific
proteins, as shown in Figure 5, for fibrinogen. If
necessary, highly purified antisera for many proteins
are available in laboratories performing
immunonephelometry or turbidimetry for protein
quantification, and these antisera can be used for IFE.
Urine Protein Electrophoresis
Figure 6 illustrates extreme prozoning that may occur
when examining very concentrated urine when a high
level BJP is present. HRPE of urine (UPE) shows a
very large monoclonal is present. The initial IFE was
performed on the undiluted 100 concentrate. A BJP
is clearly apparent from analysis of the initial 100
concentrate, since the dense area on UPE combined
with extensive prozoning when fixed with F & B
kappa, but not lambda, implicates a -BJP [17]. For
demonstration, the IFE was repeated with dilutions.
With dilution, a discrete monoclonal band is seen.
Also, notice that antibody against kappa FLC-only
does not show a reaction with the 100 concentrate.
This occurs because of its lower avidity. These
problems will be discussed in more detail in the
chapter dealing with Bence Jones proteins.
CSF Protein Electrophoresis
Multiple sclerosis (MS) is a chronic, inflammatory,
demyelinating disease that affects the CNS. Magnetic
resonance imaging (MRI) of the brain and spine is
often used to evaluate individuals with suspected MS.
MRI shows areas of demyelination as bright signal
on T2-weighted images or FLAIR (fluid attenuated
inversion recovery) distributed in the white matter of
the corpus callosum, optic nerves, brainstem, and
spinal cord [18]. Because MRI can reveal lesions that
occurred previously but produced no clinical
symptoms, it can provide the evidence of chronicity
745
Immunoelectrophoresis
needed for a definite diagnosis of MS. When
oligoclonal banding is found in CSF but not serum,
this indicates intrathecal synthesis (within the spinal
theca) of immunoglobulins. This may provide
supporting evidence of MS when the diagnosis is
uncertain. Oligoclonal banding is also associated with
diseases such as neurosyphilis, acute bacterial or viral
meningitis, progressive multifocal
leukoencephalopathy, subacute sclerosing
panencephalitis, progressive rubella panencephalitis,
polyneuritis, optic neuritis, trypanosomiasis, and
other infectious and autoimmune diseases.
Nevertheless, in the presence of MRI and clinical
evidence, it is especially helpful in identifying active
MS.
CSF samples are initially concentrated 80 to 100 for
analysis by HRPE and IFE. Oligoclonal banding
implies multiple bands (often only two) seen in
serum and CSF which are from a few clones
containing both kappa and lambda Ig, but they are
not monoclonal. Generally, oligoclonal banding is
superimposed on a diffuse polyclonal pattern, as
shown in the HRPE of CSF in Figure 7. Notice that
in this case, the bands are more distinct on CSF
electrophoresis than on IFE using IgG antibody,
because the IFE, which shows greater sensitivity, is
overloaded. If the sample used for IFE were diluted,
the bands would show up better. But in the case
shown, the gel was purposely overloaded for
demonstration purposes to show that the bands are
not monoclonal but exhibit characteristics of both
kappa and lambda (notice the bands stain for kappa
and lambda). In general practice, it is not necessary to
stain CSF for kappa and lambda but only for IgG. In
the sample shown in Figure 7, it would not be
necessary to run the IFE, because oligoclonal
banding is seen on HRPE of CSF. In Figure 8,
oligoclonal banding is not seen on the HRPE of CSF,
but they are seen on the more sensitive IFE after 10
dilution of the 100 concentrate. The labor associated
with dilution and re-assay is probably the main
reason many laboratories have switched to isoelectric
focusing for identifying oligoclonal bands. Because
of its expense and because most laboratories analyze
very few CSF samples for oligoclonal banding,
isoelectric focusing is only run in specialized
laboratories.
Immune Complex Interference
In some rare cases, circulating immune complexes
(CIC) may appear as paraproteins on agarose
electrophoresis. The CIC consist of IgM or IgG
rheumatoid factors and very rarely IgA that are
bound to the Fc portion of polyclonal IgG. The
rheumatoid factors may be monoclonal IgG or IgM
or polyclonal IgM. Since the CIC are very large, they
are trapped in the gel and do not wash out. They
appear to be a paraprotein on SPE. With IFE, they
remain trapped in the gel and may appear as shown in
Figure 9, where each strip shows a band regardless of
the fixation antibody. In some cases, the paraprotein
appears as a band on the strips treated with IgG, IgM,
kappa, and lambda but not IgA. This latter type of
profile is usually due to polyclonal IgM RF binding
IgG, whereas bands in all lanes are more often due to
monoclonal RF.
Components of the CIC are best determined by
treating the serum with 2-mercaptoethanol (see the
Methods Section, G. Mercaptoethanol treatment).
This breaks the sulfhydryl bonds so that the RF loses
its affinity for the polyclonal IgG. Treatment with
mercaptoethanol may be for 5 hours or 24 hours at
37C. The IFE is repeated after treatment. The
sample shown in Figure 9 required 24 hours of
treatment because a small band was seen in all lanes
after an initial 5-hour treatment. Sometimes 12-hour
treatment will break the immunoglobulins into
fragments, so it is best to treat first for 5 hours, and
then repeat with a 12-hour treatment if the pattern is
not clear. Notice after treatment, it can be seen that
there is a small monoclonal IgG- and polyclonal
IgG. The best interpretation is IgG- monoclonal
with RF activity complexed with polyclonal IgG.
Figure 10 shows a broad restriction on SPE that with
IFE stains mildly for IgG and lambda but more
strongly for IgM and kappa. After a 5-hour treatment
with mercaptoethanol, a monoclonal IgM- band can
be seen, while the IgG and lambda appear diffuse.
The best interpretation would be monoclonal IgM-
RF that was complexed with polyclonal IgG. Notice
after treatment, there is a slight precipitate at the
origin. This could have been removed by centrifuging
the sample before IFE.
Immunofixation Performance Goals and Quality
Control.
The single most important characteristic of correct
daily performance is that a sample with a monoclonal
protein should show a sharp band when fixed with an
appropriate specific antisera and that there is little
nonspecific staining. Appropriate banding patterns
are shown in Figures 1 through 4. Diffuse staining
(Figures 2 through 4) due to polyclonal
immunoglobulins migrating near the monoclonal is
appropriate.
As with most testing, performance goals are
periodically assessed by proficiency testing. The
CAP provides testing assessments for monoclonal
746
Immunoelectrophoresis
gammopathies in serum and urine (Bence Jones
Protein specimens) and for oligoclonal banding in
CSF. These require identification as to whether or not
there is a monoclonal immunoglobulin and whether
or not there is oligoclonal banding. The type of
monoclonal must also be determined.
Since the results of IFE are interpretive, exact control
limits for various applications are not defined. Most
electrophoretic plates contain wells at the bottom of
the plates for control sera that qualitatively indicates
whether the reaction has occurred: a precipitant ring
indicates a positive reaction (Figure 1).
New lots of antisera should be tested against patient
samples with known monoclonal gammopathies or
controls containing monoclonal proteins. The best
negative control is to test new lots against normal
sera, where only diffuse polyclonal patterns should
be seen.
References
1 Laurell CB. Quantitative estimation of
proteins by electrophoresis in agarose gel
containing antibodies. Anal Biochem 1966;
15: 45-52.
2 Laurell CB. Composition and variation of
the gel electrophoretic fractions of plasma,
cerebrospinal fluid and urine. J Clin Lab
Invest 1972; 29 suppl 124: 71-82.
3 Grabar P, Williams LA Jr: Mthode
permettant ltude conjugue des proprits
lectrophortiques et immunochemiques
dun mlange de protines: application au 15
serum sanguine. Biochim Biophys Acta
1953; 10:193-94.
4 Ritzmann SE, Lawrence M. Qualitative
Immunoelectrophoresis. In: Ritzmann SE,
Daniels JC, eds. Serum protein
abnormalities. Boston, MA: Little Brown,
1975:44.
5 Penn G, Batya J. In: Interpretation of
immunoelectrophoretic patterns. Chicago,IL:
Am Soc Clin Pathol, 1978.
6 Levinson SS, Keren DF. Free Light Chains
of Immunoglobulins: Clinical Laboratory
Analysis: Crit Rev Clin Chem 1994; 40:
1869-1878.
7 Ritchie RF, Smith R. Immunofixation. III.
Application to the study of monoclonal
proteins. Clin Chem 1976;22:1982-1985.
8 Cawley LP, Minard BJ, Tourtellotte, M,
Chelle C. Immunofixation electrophoretic
techniques applied to identification of
proteins in serum and cerebrospinal fluid.
Clin Chem 1976; 22:1262-1268.
9 Levinson SS, Elin RJ, Yam L. Light chain
proteinuria and lysozymuria in a patient with
acute monocytic leukemia. Clin Chem 2002;
48: 1131-1132.
10 Keren DF, Alexanian R, Goeken JA,
Gorevic PD, Kyle RA, MD, Tomar RH.
Guidelines for Clinical and Laboratory
Evaluation of Patients With Monoclonal
Gammopathies. Arch Pathol Lab Med 1999;
123: 106-107.
11 Sheldon J, Riches P. Capillary
electrophoresis for investigation of proteins
in biological fluids. J Clin Ligand Assay
2004; 27: 227-233.
12 Bridgden ML, Neal ED, McNeely MD,
Hoag GN. The optimum urine collections
and monitoring of Bence-Jones proteinuria.
Am J Clin Pathol 1990; 93: 689-693.
13 Graziani M, Merlini G, Petrini C; IFCC
Committee on Plasma Proteins; SIBioC
Study Group on Proteins. Guidelines for the
analysis of Bence Jones protein. Clin Chem
Lab Med. 2003; 41:338-346.
14 Kyle RA, Therneau TM, Rajkumar SV,
Larson DR, Plevak MF, Offord JR et al.
Prevalence of monoclonal gammopathy of
undetermined significance.N Engl J Med
2002; 346: 564-569.
Strobel SL.Transient paraproteinemia: an
intriguing immunological anomaly. Ann
Clin Lab Sci 2003; 33: 265-270.
16 Qiu LL, Keeling KL, Levinson SS, Elin RJ.
A Convenient and Effective Method for
Removing Fibrinogen from Contaminated
Serum Specimens for Protein
Electrophoresis. Clin Chem 2003; 49: 868-
872.
17 Levinson SS. Urine protein electroporesis
and immunofixation electrophoresis
supplement one another in characterizing
proteinuria. Ann Clin Lab Sci 2000; 30: 79-
84.
18 Rudick RA. A 29-year old man with
multiple sclerosis. JAMA 1998; 280: 1432-
1439.
747
Immunoelectrophoresis

Table 1: Immunoelectrophoresis Methods Summary

Method 1: Electroimmunodiffusion (EID) (Laurell rocket); quantification by size of immunoprecipitate in gel.

Principle of analysis: Electrophoresis of antigens into agarose gel containing monospecific antibodies for
the protein of interest; the length of gel pattern (rocket) from the origin is proportioned to antigen
concentration.
Comments: Used for special tests such as Protein C and Protein S.
Method 2: Immunoelectrophoresis (IEP); identification of monoclonal proteins through precipitin arc formation.
Principle of analysis: Proteins separated in agarose by zone electrophoresis; After electrophoresis,
antiserum is placed in a trough horizontal to the electrophoretogram and allowed to diffuse for 24 hours;
precipitin arcs form where antigen and antibody meet in the zone of equivalency; monoclonality can be
determined from shape and mobility of arc
Comments: No longer recommended for use in clinical laboratories.
Method 3: Immunofixation; identification of protein bands on zone electrophoresis by formation of precipitin bands
after overlaying the gel with highly purified antisera.
Principle of analysis: Proteins are separated by zone electrophoresis; area containing the protein to be
identified is overlain with a monospecific antibody, and its corresponding antigen precipitates.
Comments: Serum, urine, and CSF; can be used with the IFE to identify monoclonal proteins and
oligoclonal banding. Because of its greater speed of completion, parallel similarity of banding to SPE,
UPE, and CSF electrophoresis, this method lends itself to simplicity in interpretations and great sensitivity.
This is now the recommended method for SPE and UPE identification.
Method 4: Capillary electrophoresis and immunosubtraction
Principle of analysis: Protein fractions measured by absorbance following electrophoretic separation in a
small-bore capillary tube in free solution; following electrophoresis, serum sample is incubated with beads
coupled with anti-IgG, IgA, IgM, kappa, and lambda antibodies. The proteins of interest are captured by the
bead, and CZE is repeated. Disappearance of a peak is evidence of its type.
Comments: Although useful in laboratories that analyze very large quantities of samples for SPE, it has
not been perfected for immunoglobulin typing in either serum or urine, nor is there good evidence that it is
useful for analysis of BJP in urine. Typing after CZE is routinely performed by IFE.

Methods Overview
(generally obtained as a kit from a commercial
source).
A. Ingredients
1. Agarose gel, usually in buffer (tris-barbital
solution with preservatives (sodium azide).
2. Buffer pH 8.4 to 8.8. Often barbital and
sodium barbital, with 0.1%sodium azide as
preservative
3. Stain: Usually amido black, Coomassie blue,
or acid blue stain in 5% glacial acetic acid.
4. Protein fixative (precipitates all proteins)
10% sulfosalicylic acid and 10% acetic acid.
5. Highly purified monospecific antisera to IgG,
IgA, IgM, F & B kappa and lambda, and FLC-only
when needed for identification of BJP (see chapter on
BJP). These antisera are especially prepared for IFE,
lesser grade antisera will show artefacts and non-
specific staining that may confuse the interpretation.
B. Urine specimen concentration
1. For the detection of BJP, concentrate the
urine to 100 to 150 using stationary concentrators
such as Minicon with pore size that does not pass >
20,000 kD proteins. Most mechanical concentrators
concentrate to about 100. This usually takes 2 to 6
hours. Try to avoid the sample becoming dry, since
much protein may be lost on the containers walls.
To achieve 150 concentration, the concentrator
would have to be refilled with sample. In some cases,
the urine may be very dense, so that even 100
concentration cannot be easily achieved. Centrifugal
concentrators are more rapid but will not concentrate
to 100 and are therefore not adequate. Dilution of
the concentrate with saline may be necessary if the
initial pattern is confusing (see Interpretations
above).
C. CSF specimen preparation
1. Concentrate CSF to 80 to 100 for typing
oligoclonal bands in CSF. Always perform IFE on a
serum specimen from the same patient for
comparison with the CSF IFE. Dilution of the
concentrate with saline and repeat assay may be
necessary if the pattern appears overloaded (see
Interpretations above)
748
Immunoelectrophoresis
D. Electrophoresis step
1. Samples are usually in the 1 to 10 L range
and applied into wells or into the manufacturers
template on HRPE.
2. Prior to sample application, concentrate urine
and CSF appropriately.
3. Perform electrophoresis.
E. Immunofixation
1. After the gel has been removed from the
electrophoresis chamber, align the antisera template
on the gel so that the slits in the template are aligned
over the application area of the gel, ensuring good
contact between the template and the agarose. In
some cases, the antisera may be applied on filter
paper by laying it on the gel. Most kits now use
templates, since less antisera is used, and there is no
diffusion onto adjacent strips.
2. Many gels contain control wells near the
bottom (see Figure 1). Apply an appropriate sample,
usually 1 to 10 L, into the control wells (or as
specified by the manufacturer). The
immunoglobulins in the control will react with the
antisera applied in Step 4 so that a control reaction
should occur.
3. Usually there is a blotting step at this point.
4. With the gel slanted, apply onto the gel 1 or 2
drops of the Protein Fixative to the appropriate lanes
and 1 or 2 drops of appropriate antisera to the
appropriate lanes so that the drops flow quickly and
evenly down each lane. The protein fixative
precipitates all of the proteins in the SPE, UPE, or
CSF protein lane, whereas the antisera fixes only the
specific immunoglobulin or other specific protein
(see Figures 1, 2, and 5).
5. Incubate the gel in a closed incubation
chamber.
6. At this point, the gel is usually washed with
saline.
7. Staining is now performed, after which
excess stain is removed from the gel with glacial
acetic acid washes.
F. Quality control
1. For serum samples, the control wells at the
bottom of the strip found with many kits help insure
that the antisera is effective; a circular precipitate is
seen, indicating the reaction was appropriate (see
Figure 2). Although these controls contain intact light
chain, they usually do not contain FLC that are the
most important monoclonal protein assayed in urine.
3. Therefore, before being put into use, it is
important to test each new batch of FLC-only
antisera with urines containing known kappa and
lambda BJP that have been saved
frozen in aliquots. It is also worthwhile to test
new FLC-only antisera with a normal serum sample
or control to be sure FLC-only antisera is not reacting
with intact immunoglobulins (no reaction should be
seen, since there is so little FLC in normal serum).
G. Mercaptoethanol treatment
In some cases, after IFE, band restrictions will
be seen in the same position in multiple lanes. This
indicates that rheumatoid factor (RF) is present and
has bound IgG into immune complexes. The RF is
usually IgM, but may be IgG or very rarely IgA. RF
is an endogenous antibody that binds to the Fc
portion of endogenous IgG and forms immune
complexes. These are so large that they are fixed in
the gel prior to immunofixation, so they do not wash
out during the wash procedure. Bands may appear in
all lanes or the IgG and IgM lanes and very rarely in
the IgA and IgG lanes. The tip-off is restricted
staining at the same position in both the kappa and
lambda lanes.
To prove an immune complex is present and to
identify which immunoglobulins are involved, it is
necessary to break the sulfide bonds binding the
immunoglobulins together. This causes the RF to
loose its antibody activity and migrate separately.
The bonds are broken by treatment with the reducing
agent mercaptoethanol, and the IFE is repeated.
1. Make a 1:10 dilution of 2-mercaptoethanol with
isotonic saline. Make a mixture by adding 10 L of
the 1:10 dilution to 100 L of the patients serum.
2. Allow the mixture to incubate in a tightly capped
tube for 5 hours or overnight at 37C. Use the
resulting serum directly for IFE. If a precipitate
appears, centrifuge and use the supernatant for IFE. If
further storage is necessary, keep the mixture at
refrigerated temperature, but be sure to warm it well
and shake it before electrophoresis. (Usually an
overnight incubation is preferred, but in some cases,
the immunoglobulins are destroyed, and a 5-hour
incubation is required).
3. If a precipitate appears after mercaptoethanol
treatment, centrifuge the tube to collect the
precipitate, and use the supernatant for
electrophoresis. The immunoglobulins are still in
solution.
749
Immunoelectrophoresis

Figures

Figure 1. Serum protein electrophoresis (SPE) and

IFE. SPE (channel to the far left) indicates the lane
containing the SPE. This lane was fixed to
precipitate all proteins. The remainder of the
channels to the right were fixed with specific
antisera for IgG, IgA, IgM, kappa, and lambda
serum proteins as indicated. Migration is towards the
anode. In the SPE lane, albumin is the fastest
migrating band. Bands in order of migration are:
albumin, alpha-1-globulin, beta-1-globulin, beta-2-
globulin, and gamma-globulin. Normal polyclonal
gamma globulin shows diffuse staining, as shown in
Figure 2. In Figure 1, a band restriction is shown.
This indicates a paraprotein. IFE shows the paraprotein is an IgG-kappa monoclonal protein. Note that very little or
no staining is seen in the IgA, IgM, and lambda lanes, indicating that these proteins are decreased (compare with the
polyclonal staining in Figure 2). Wells at the bottom of the gel are control wells; a precipitate in these wells ensures
that a reaction has occurred. Arrow pointing right (O) indicates the origin (placement of the sample). Arrow pointing
left indicates the position of the monoclonal band.

Figure 2. Low concentration paraproteins on

SPE and IFE. Arrows indicate the position of
the M proteins. The monoclonal protein in the
gel above is an IgG-kappa, whereas that
below is an IgG-lambda. Note the darker,
diffuse, polyclonal staining about the band, as
compared to Figure 1, indicating that there
are substantial amounts of normal
immunoglobulins, as well as monoclonals, in
the gels shown in Figure 2. For other details
see Figure 1.

Figure 3. Biclonal gammopathy. Arrows point to the two different monoclonal bands. Otherwise, details are similar
to Figure 1.
750
Immunoelectrophoresis

Figure 4. Prozoning in serum. The band may be shaped

like a donut. Note how the band becomes solid with a 5
dilution. Otherwise, details are similar to Figure 1.

Figure 5. IFE of fibrinogen. In some cases, fibrinogen is not completely

removed, or a plasma sample is thought to be serum (often a sample with
anticoagulant is pored into a red top [serum] tube). Contamination by
fibrinogen can be mistaken for a monoclonal protein on SPE. Fibrinogen
or other serum proteins can be identified by IFE. In the case shown, the
direction of migration is up, toward the anode. Arrow indicates the
fibrinogen band.

Figure 6. Extreme prozoning with urine electrophoresis. 100 concentrated urine was diluted as indicated for IFE. F
& B indicates antisera against free and bound light chain, and F indicates antisera against FLC-only. UPE was fixed
with total protein fixative. The number 100 along the bottom indicates the amount of mechanical concentration.
After concentration, samples were diluted with saline to lesser concentrates as indicated. Zero indicates
unconcentrated urine and 1:10 indicates a 10-fold dilution of the unconcentrated urine. For explanation, see the text.
For other details of the Figure, see Figure 1.
751
Immunoelectrophoresis

Figure 7. Oligoclonal banding in CSF is both kappa

and lambda. HRPE of serum and CSF with IFE is
shown. Oligoclonal banding is seen in CSF protein
electrophoresis and on the IFE fixed with IgG,
kappa, and lambda but not in the serum protein
electrophoresis. In this case, oligoclonal banding
was apparent from the CSF electrophoresis without
IFE. The IFE was performed to demonstrate the
bands are both kappa and lambda. Details of the gels
are the same as in Figure 1.

Figure 8. Oligoclonal banding in IFE but not

CSF protein electrophoresis. In this case, the
oligoclonal banding is not seen on routine CSF
electrophoresis but is seen on the more
sensitive IFE after diluting the 100
concentrate to 10. Details of the gels are
similar to Figure 1.

Figure 9. Interference by IgG RF circulating

immune complexes. Above, SPE and IFE before
mercaptoethanol treatment and below, after
treatment. Notice (above) that the SPE (gel to
the far left) shows a paraprotein in the beta-
gamma region. IFE shows bands in all lanes.
After treatment, below, a rapidly migrating IgG-
kappa monoclonal protein (arrow) is seen in the
lane fixed for IgG, along with diffuse staining.
The paraprotein in the other lanes is no longer
visible. The interpretation is IgG monoclonal
protein with rheumatoid factor activity forming
an immune complex by binding to polyclonal
IgG. Details of the gel are the same as in Figure
1.
752
Immunoelectrophoresis

Figure 10. Interference by IgM RF circulating immune complexes. Above, SPE and IFE before mercaptoethanol
treatment and below, after treatment. The arrows point to the location of the IC that appears as a broad band before
treatment. After treatment (below) an IgM-kappa monoclonal protein is seen, along with diffuse staining on the lane
stained for IgG. Other details are similar to the legend in Figure 1.
753
Immunoglobulin Quantitation
Immunoglobulin Quantitation
Karen Golemboski
Name: Immunoglobulin quantitation
Clinical significance: Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular formula and weight: See Immunoglobulin Table: Physical Properties
Chemical class: Protein
i
Principles of Analysis and Current Usage
Historically, measurement of immunoglobulins (Ig) was
included as part of the total globulins present in human
serum (Table 1, Methods 1 and 2). Globulin isolation
was achieved by salt or gold precipitation and
quantitation by the biuret assay. The introduction of
electrophoretic separation techniques allowed
fractionation of the globulins into alpha, beta, and
gamma components, the gamma fraction consisting
almost entirely of immunoglobulins. Qualitative and
semiquantitative estimates of total immunoglobulin
levels could be obtained from immunoelectrophoresis
and protein electrophoresis, respectively (Table 1,
Method 3).
Knowledge of immunoglobulin structure and the
availability of monoclonal antibodies to each
immunoglobulin class (that is, specific for the constant
regions of the heavy and light chains) has made it
possible to quantitate each immunoglobulin class. The
resulting complex of immunoglobulin (as antigen) and
antibody can be detected by physical means (either by
precipitation of immune complexes or by
immunonephelometry, which detects the light-scattering
properties of immune complexes) or by measurement of
a label attached to the anti-immunoglobulin antibody
(e.g., fluorescent, enzymatic, or chemiluminescent label)
(Table 1, Methods 4 through 9).
Differences in structure and relative predominance in
serum and other body fluids (for example, there is a
millionfold difference between the serum concentrations
of IgG and IgE) necessitate different methods of
quantitation for the different classes of
immunoglobulins. Igs are generally assayed as separate
classes, and not all Ig classes are measured in every
clinical situation. Serum levels of IgD generally are not
of clinical importance unless a plasma-cell malignancy is
producing a monoclonal IgD protein. Quantitation of IgE
is usually performed as part of an evaluation of allergic
conditions.
i
Immunoglobulin Quantitation
Previous and current authors of this method:
First edition: Gayle Birkbeck
Methods edition: Gayle B. Jackson
Second edition: Gayle B. Jackson
Third edition: Gayle B. Jackson
Fourth edition: Gayle B. Jackson
Fifth edition: Karen Golemboski
IgM, IgG, IgA
Single radial immunodiffusion (RID; Table 1, Method 4)
was an early method of Ig quantitation used in clinical
settings. It consists of an agar plate with antibody
incorporated throughout the agar. Test samples, placed
in antigen wells, diffuse into the agar and form a
precipitin ring of antigen-antibody (Iganti-Ig)
complexes around the well. The diameter of the
precipitin ring is directly proportional to the amount of
antigen placed in the well. This procedure is used today
in laboratories performing low volumes of IgD or IgG
subclass determinations, and the method is described in
detail at the end of this chapter. A variation of the RID
technique, which shortened the lengthy incubation time,
was electro-immunodiffusion (also known as Laurell
rocket electrophoresis). Automated methods of
immunoglobulin quantitation using nephelometry (Table
1, Method 5) or immunoturbidimetry (Table 1, Method
6) have replaced RID techniques for measurement of
IgM, IgG, and IgA, providing faster results. The
nephelometric method measures light scattering
produced by the antigen-antibody complexes formed in
the test sample tube. The relative light-scattering values
are transformed into reportable units (that is, mg/L or
International Units [IU]/L). Both end-point and kinetic
methods of measurement are used. The end-point
method measures the light scattered by the complexes
formed after a steady state has been achieved. Thus
antibody is added to the patient sample, and the sample
is incubated for 15 min to 1 hour before quantitation.
The kinetic or rate method measures the maximum rate
of increase of forward light scatter produced by the
formation of antibody-antigen complexes. With this
method, a lengthy incubation of the sample is not
required before quantitation. Automated nephelometric
methods are available (Beckman Instruments, Brea CA;
Siemens Healthcare Diagnostics/Dade Behring, Inc.,
Deerfield, IL). Turbidimetric methods measure the
increase in absorbance resulting from the presence of the
precipitate. The absorbance reading can be taken at
equilibrium or on a kinetic basis (at selected time
intervals during the reaction). Automated turbidimetric
methods include those from Abbott Laboratories (Irving,
TX), Beckman Instruments, Olympus America
(Melville, NY), Roche Diagnostics (Indianapolis, IN),
and Siemens Healthcare Diagnostics/Dade Behring.
Results in both methodologies are compared with a
standard curve.
754
Immunoglobulin Quantitation
IgD
IgD, whose physiological function is still unknown, is
present in the serum at a concentration less than
IgM/A/G but higher than that of IgE. Serum levels of
IgD are usually measured when production of a
monoclonal IgD protein is detected by immunofixation.
Methods of IgD quantitation include radial
immunodiffusion (particularly, the Mancini end-point
method), which is of relatively poor sensitivity, and the
more sensitive but less accessible methods of labeled
immunoassay. In addition, reagents for nephelometric
IgD quantitation are available for use on the Behring
BNII Analyzer (Siemens/Dade-Behring, Deerfield, IL).
IgE
Of the five antibody classes, IgE is present in serum in
the smallest quantity. Sensitive methods such as
immunonephelometry, using an antibody specific for the
heavy chain, are required to detect these low levels of
protein. One example is the Dimension (Dade-Behring,
Deerfield, IL), which measures light scattered by
immune complexes (nephelometry), the intensity of light
scatter being proportional to the concentration of IgE
antibodies in the sample. Another automated method is
the Elecsys II IgE system (Hoffman-La Roche, Nutley,
NJ), a sandwich immunoassay in which the anti-IgE
antibody is bound to streptavidin. A biotin-labeled
enzyme binds to the streptavidin-IgE complex, allowing
chemiluminescent detection of IgE in the patient
specimen (Table 1, Method 9). The ImmuneTech Total
IgE system (ImmuneTech Corporation, Menlo Park, CA)
is also biotin-avidin based, with the specimen being
combined with anti-IgE-coated microspheres. A biotin-
labeled anti-human IgE antibody is followed with
fluorescent-labeled streptavidin, and the fluorescent
signal from the resulting complex is directly proportional
to the amount of IgE in the sample. Fluorescence is
measured with a flow cytometer. Biokit S.A. (Barcelona,
Spain) markets the Quantia IgE, an immunoturbidimetric
assay enhanced with latex particles. The UniCAP Total
IgE method from Pharmacia/Pfizer (New York, NY) is a
fluoroimmunoassay (Table 1, Method 7).
Quantitation of total serum IgE is usually performed to
diagnose or confirm hypersensitivity (allergy). However,
since many patients with allergy or atopic disease may
have normal or only slightly elevated total IgE, this
measurement may or may not be sufficient. Much of the
development in immunoglobulin testing over the last 2
decades has been directed at measurement of specific
IgE antibody levels for individual allergens, as an
adjunct to skin testing for allergy. Specific IgE
antibodies are assayed by coating a solid surface with the
antigen in question, allowing patient antibodies to bind
and detecting IgE with a labeled anti-IgE antibody. The
Immulite 2000 system (Diagnostic Products
Corporation, Los Angeles, CA) uses allergens bound to
beads; IgE is detected with alkaline phosphataselabeled
anti-IgE. A chemiluminescent substrate emits light in
direct proportion to the amount of allergen-specific IgE
in the sample. The AP 1800 CLA (Hitachi Chemical
Diagnostics, Inc., Mountain View, CA) employs
cellulose threads as the solid surface and
chemiluminescent detection of an enzyme-labeled
second antibody. Multiple antigen-coated threads may be
combined in the test chamber to allow simultaneous
testing for IgE antibodies to multiple antigens. Rolling-
circle amplification, a highly sensitive detection
technique, utilizes labeled DNA for detection, with a
DNA primer being attached to the reagent antibody (in
this case, anti-human IgE). When circular DNA, DNA
polymerase, and nucleotides are added, linked copies of
circular DNA are produced, which can then be
hybridized to labeled complementary oligonucleotide
probes (Qiagen, Valencia, CA).
Current in-vitro allergy testing methods are much
improved compared with original radiolabeled methods
(radiolabeled allergosorbent testing [RAST]; Table 1,
Method 8). Many methods now calibrate results to
World Health Organization (WHO) reference material,
and preparation of antigens has become more
standardized, although reference antigen preparations are
not yet available. Despite advances in allergen-specific
IgE quantitation, assays are still less sensitive than
traditional skin testing [1].
Light-Chain Quantitation
In certain lymphoproliferative disorders, unbalanced
synthesis of immunoglobulin subunits results in excess
production of or light chains. These free light chains
may circulate in serum or be excreted in urine.
Quantitation of free light chains in serum is more
sensitive than urine immunoglobulin quantitation
methods and also allows detection and monitoring of
monoclonal gammopathies which are non-secretory or
which secrete only light chains. Immunonephelometric
and immunoturbidimetric assays use antibodies specific
for light chains not associated with heavy chains (The
Binding Site, San Diego, CA) [2].
Standardization and Quantitation
The immunoglobulins are divided into five major classes
with four immunologically characterized subclasses of
IgG and two of IgA, differing in heavy-chain structure
and glycosylation. The ratio of these subclasses may
vary with some infectious and autoimmune diseases and
will certainly vary with monoclonal gammopathies.
Immunoglobulins also vary in size: IgM is usually
present as the stable 19S pentamer of 7S subunits but
occasionally may be found in free 7S form. IgA is 7S in
the monomeric form but 11S in the dimeric form (see
Table 2).
Reference Materials
The WHO first introduced a reference preparation for
IgG, IgA, and IgM in 1970 (NIBSC 67/86), allowing
conversion of mg/dL levels to IU/L. The CAP and the
European Community Bureau of References (BCR)
jointly produced a subsequent preparation in 1993, CRM
470 (BCR)/ RPPHS (CAP) [3]. IgE methods are
currently standardized against the 2nd WHO
International Reference preparation, NIBSC 75/502.
Standard reference material for IgD is also available
(British Research Standard 67/37).
755
Immunoglobulin Quantitation
Reference and Preferred Methods
The International Federation of Clinical Chemistry and
Laboratory Medicines Committee on Plasma Proteins
has designated both immunonephelometry and
immunoturbidimetry as reference methods for
quantitation of immunoglobulins IgG, IgA, and IgM in
serum or plasma [4]. Automated versions of both
methods are readily available, and according to 2007
CAP survey data, approximately equal numbers of each
are in use, and no respondents indicated use of any other
methods for quantitation of these immunoglobulins.
Both methods performed consistently within acceptable
tolerances on the survey.
No methods for quantitation of IgE have been designated
as reference methods to date. Respondents to 2007 CAP
surveys indicated a wide variety of methods in use; the
two most commonly reported were nephelometry (65)
and chemiluminescence (175). The remaining
respondents used labeled immunoassay (fluorescent-,
enzyme-, or radiolabeled).
High-resolution serum (and urine) protein
electrophoresis is the recommended method for
detection of a monoclonal (M) protein. Once detected,
densitometry should be used to quantitate M proteins,
and the heavy- and light-chain classes of the M protein
should be defined by immunofixation (the use of
immunoelectrophoresis is discouraged). If the level of
secreted monoclonal protein is low, such that the
densitometer peak may be obscured by normal levels of
other immunoglobulins (e.g., monoclonal IgA < 2.0
g/dL), then quantitation by nephelometry is indicated.
For high levels of M protein, however, values
determined by nephelometry (especially IgM and IgA)
are often falsely elevated [5,6].
Specimen
Immunoglobulin quantitation, for routine diagnostic
purposes, is performed most commonly on serum
specimens. Samples for quantitation may be stored for
up to 5 days at 2C to 8C if they are protected from
contamination and evaporation. For sample shipment or
longer storage periods, samples should be frozen at
20C or colder. Once thawed, samples generally should
not be refrozen, because repeated freezing and thawing
may cause deterioration of proteins.
Immunoglobulins in Other Body Fluids
Urine
Under normal conditions, immunoglobulins are not
found in urine. The presence of Igs in urine generally
indicates an overload proteinuria resulting from excess
production, which may be due to a monoclonal
gammopathy or in some cases polyclonal Ig production
during an infectious disease. Urine specimens generally
are concentrated 20 to 50 times before they are evaluated
by electrophoresis or by immunofixation. Detection of
IgD in urine, even after concentration, has not been
reported [7].
Free light chains are small enough to pass through the
glomerular membrane and therefore may be excreted in
urine (Bence-Jones proteins), although concentration
may be required.
CSF
An increased level of intrathecal immunoglobulin
production is associated with demyelinating diseases
such as multiple sclerosis (MS). IgG levels are compared
with CSF albumin to control for increased plasma levels
or damage to the blood-brain barrier. Measurement of
CSF IgM or IgA may be also be indicated if conditions
such as neurotuberculosis, mumps meningitis, or
adrenoleukodystrophy are suspected. Automated
nephelometric methods (Dade Behring BNII System and
Beckman Coulter Immage) have demonstrated sufficient
sensitivity and acceptable linearity for immunoglobulins
in CSF [8].
Tears
Detection of elevated levels of IgE in tears indicates an
ocular allergic response. A solid-phase
immunochromatographic test, Lacrytest, is designed as a
strip which is placed in the eye until sufficient tears are
absorbed (Adiatec SA; Loire-Atlantique, France). The
test detects IgE above 2.5 KIU/L (3 ng/mL), which is
considered the upper limit of normal levels.
Other
Normal levels for immunoglobulin in thoracentesis fluid,
synovial fluid, and other serous exudates are not well
defined, and the clinical usefulness of these fluid
quantitations is limited.
Interferences
Since light scattering produced by contaminating
particles in the antiserum can interfere with quantitation,
antiserum that is free of contamination must be used.
Turbid patient samples may have to be filtered before
use on the nephelometer. The presence of rheumatoid
factor or heterophilic antibodies which bind to reagent
antibodies may interfere with immunoassay. Prozone, or
hook, effects in the presence of high levels of
immunoglobulin (e.g., in multiple myeloma) may lead to
falsely low results and require dilution of sample for
accurate quantitation [9].
Immunoglobulin Quantitation Reference Interval
Immunoglobulin concentrations decrease during
pregnancy and reach lowest levels in the early
postpartum period. Levels vary widely in children and
adults; immunoglobulins present in infants as a result of
transplacental transfer decline until about 6 months of
age. Normal levels in children increase through
adolescence to adult levels (Table 2). Declining levels of
IgG and IgM in aging individuals have been reported
[10,11].
Interpretation
Immunoglobulin abnormalities can be divided into three
groups: (1) increased levels of several different
immunoglobulins (polyclonal gammopathy), (2)
increased levels of a single immunoglobulin type or of
proteins related to immunoglobulins (monoclonal
756
Immunoglobulin Quantitation

gammopathy), and (3) decreased levels of one or more lymphomas, monoclonal gammopathy of undetermined
immunoglobulins. significance (MGUS), and old age.

Human Serum Immunoglobulin Reference Intervals (mg/mL)

Immunoglobulin
class IgG IgM IgA IgD IgE
Cord specimen 7.6616.93 0.040.26 0.00040.09
0.53 months 2.998.52 0.151.49 0.030.66
36 months 1.429.88 0.181.18 0.040.90
612 months 4.1811.42 0.432.23 0.0140.95
12 years 3.5612.04 0.372.39 0.131.18 116122 ng/mL
23 years 4.9212.69 0.492.04 0.231.37 80122 ng/mL
36 years 5.6413.81 0.512.14 0.352.09
47 years 140442 ng/mL
69 years 6.5815.35 0.502.28 0.293.84
1014 years 374674 ng/mL
1216 years 6.8015.48 0.452.56 0.812.52
Adult 8.0016.00 0.502.00 1.403.50 00.14 22000 ng/mL

Data from:
Meites S, editor. Pediatric clinical chemistry; 2nd edition. Chicago: American Association for Clinical Chemistry;
1981.

A normal human serum immunoglobulin class is

composed of a heterogeneous mixture because of
Serum protein electrophoresis, followed by
subclass differences and the presence of numerous,
immunofixation electrophoresis, may be used to classify
different, heavy- and light-chain variable regions. Many
the increase as polyclonal or monoclonal and to identify
different clones of plasma cells synthesize and secrete
the monoclonal protein, if present. Monoclonal proteins
any one of the different class, subclass, and type
may be produced in a variety of diseases classified as
combinations, such as IgA, kappa. However, only one
monoclonal gammopathies, including multiple myeloma
clone synthesizes an IgA, kappa protein with a given
(producing monoclonal immunoglobulins which may be
heavy- and light-chain variable regionamino acid
of any class or may secrete only light chains),
sequence (antigen specificity).When many different
Waldenstrms macroglobulinemia (monoclonal IgM),
families or clones of plasma cells are responsible for an
heavy-chain disease (monoclonal production of
increased production of immunoglobulins, the increase is
immunoglobulin heavy chains only), and amyloidosis, in
termed polyclonal. When only one clone is responsible
which the monoclonal immunoglobulin deposits fibrils
for the increase, the increase is homogeneous and is
in multiple organs [12].
termed monoclonal. Immunoglobulin quantitation alone
cannot determine whether an immunoglobulin elevation
Quantitation of a monoclonal protein is important both
is monoclonal or polyclonal. For this purpose, protein
in the initial diagnosis and staging of disease and in
electrophoresis and/or immunofixation should be used in
following the disease course. In multiple myeloma, the
conjunction with immunoglobulin quantitation. A
amount of monoclonal protein is directly related to the
monoclonal protein will appear as a narrow band on the
size of the tumor burden, and quantitation of the
protein electrophoresis. Immunofixation will
monoclonal protein is one factor used in following the
demonstrate an increase in just one heavy chain and one
therapy. Malignant monoclonal gammopathies are often
light chain class. In a polyclonal increase, the band will
accompanied by decreases in the levels of normal
be diffuse on protein electrophoresis, and
polyclonal immunoglobulins.
immunofixation will demonstrate increases in more than
one antibody class.
Immunoglobulin deficiencies are best demonstrated by
Increased immunoglobulins may indicate a normal
quantitation of the immunoglobulin classes. Protein
polyclonal response to infection, chronic inflammation, a
electrophoresis can be used as a screen for
hypersensitivity response (allergic disease), or
hypogammaglobulinemia, in which B-cell activity is
production of an abnormal monoclonal immunoglobulin.
deficient or absent, and there is virtually no IgG detected
Polyclonal increases are nonspecific and may be found
in serum. However, the other immunoglobulin classes
with infections, autoimmune diseases, advanced
contribute so little to total levels that the densitometer
cirrhosis, chronic active hepatitis, biliary cirrhosis,
curve shows little change if they are decreased or absent.
sarcoidosis, narcotics addiction, amyloidosis, and many
parasitic diseases. Monoclonal gammopathies are found
Decreased total or individual immunoglobulin levels
with multiple myeloma, Waldenstrms
suggest an immunodeficiency disorder, which may be
macroglobulinemia, heavy-chain disease, essential
congenital or acquired. Most immunodeficiencies
cryoglobulinemia, chronic lymphocytic leukemia,
emerge in childhood, usually after maternal antibodies
are lost at about 6 to 8 months of age, but some may first
757
Immunoglobulin Quantitation
become evident in adulthood. Altered immunoglobulin
levels may be the result of a defect in B cells, T cells, or
both, since responses of B cells are not independent of T
cells.
About half of primary immunodeficiencies are the
consequence of B-cell defects and result in decreased
immunoglobulin levels. Individuals with these disorders
generally suffer repeated infections with encapsulated
bacteria such as Streptococcus pneumoniae or
Haemophilus influenzae. A defect in B-cell maturation
results in Brutons (X-linked) agammaglobulinemia,
resulting in markedly decreased or absent B cells and
immunoglobulins. Common variable immunodeficiency
(CVI) represents a group of disorders, all of which result
in hypogammaglobulinemia; these patients may present
in childhood, but the peak age of onset is in the third
decade of life.
Alterations may also occur in specific immunoglobulin
classes. Selective IgA deficiency is the most common
immunodeficiency disorder, with an incidence of about 1
in 500-700 individuals, although many are
asymptomatic. These individuals have IgG and IgM
levels within normal limits and respond well to vaccines,
but they may suffer increased respiratory and
gastrointestinal infections. IgA-deficient individuals may
also be deficient in one or more subclasses of IgG,
especially IgG2. Since antibodies to polysaccharide
antigens (such as bacterial capsular antigens) are usually
of the IgG2 subclass, individuals deficient in IgG2 may
show increased susceptibility to bacterial infection, even
though total IgG levels may be within normal limits. A
decrease in IgG and IgA with increased IgM
concentration is seen in hyper-IgM syndrome, in which
T cells are unable to produce the cytokines necessary to
induce B cells to switch from IgM to production of other
immunoglobulin classes.
Combined immunodeficiency disorders, affecting both B
and T cells, may also result in abnormal immunoglobulin
levels. These include severe combined
immunodeficiency syndrome (SCIDS; markedly reduced
IgG/A/M), Wiskott-Aldrich syndrome (decreased IgM,
elevated IgA and IgE), and ataxia-telangiectasia
(decreased or absent IgA; decreased IgE, IgG subclasses,
or a combination) [13].
Acquired immunodeficiency may be secondary to
concomitant malignancy or other diseases or may result
from protein-calorie malnutrition or from infection with
human immunodeficiency virus (HIV) [14].
Immunoglobulin Performance Goals
Survey data from the 2007 College of American
Pathologists (CAP) Participant Summary Report show
imprecision values (% coefficient of variation [%CV])
for immunoglobulins G, A, and M to range from
approximately 2% to 8% in samples with
immunoglobulin concentrations at the upper end of the
normal reference interval [5]. Acceptable performance
criteria (Clinical Laboratory Improvement Amendments
88) for measurement of immunoglobulins A, E, and M
require that laboratories be accurate to within 3SD of
the peer-group mean, and for immunoglobulin G,
laboratories must obtain results that are within 25% of
the peer-group mean. Maximum SD values for
immunoglobulins at medically significant decisions
levels are as follows [15]:
Immunoglobulin Decision level Maximum SD
IgA 400 mg/L 17 mg/dL
IgE 200 IU/mL 38 IU/mL
IgG 500 mg/dL 13 mg/dL
IgG 2000 mg/dL 125 mg/dL
IgM 300 IU/mL 9 IU/mL
The intraindividual variability of immunoglobulins G, A,
and M, measured in healthy adults over a 3- to 5-month
period, were found to range from 4% to 6% [15]. Ricos
et al. established desirable specifications for analytical
imprecision derived from studies of biological variation,
finding an assay imprecision for IgA, IgG, and IgM of
no greater than 2.7%, 2.3%, and 3.0% and total error of
no greater than approximately 14%, 8%, and 17%,
respectively [16].
Current automated methods for IgG, IgA, and IgM
quantitation appear to perform well within established
performance ranges; CAP data indicated only one IgA
high value result that exceeded the CLIA +3SD limit
(out of nearly 3000 reports). Quantitation methods for
IgE are also consistent, with CV values averaging
around 7.5%.
References
1 Hamilton RG, Adkinson NF Jr. In-vitro assays
for the diagnosis of IgE-mediated disorders. J
Allergy Clin Immunol 2004;114:213-115.
2 Katzmann JA, Clark RJ, Abraham RS, Bryant
S, Lymp JF, Bradwell AR, Kyle RA. Serum
reference intervals and diagnostic ranges for
free and free immunoglobulin light
chains: relative sensitivity for detection of
monoclonal light chains. Clin Chem
2002;48:1437-1444.
3 Johnson AM. Effect of a new international
reference preparation for proteins in human
serum (CRM 470) on results of the College of
American Pathologists surveys for plasma
proteins. Arch Pathol Lab Med 2000;124:1496-
1501.
4 Blirup-Jensen S. Protein standardization III:
method optimization. Basic principles for
quantitative determination of human serum
proteins on automated instruments based on
turbidimetry or nephelometry. Clin Chem Lab
Med 2001;11:1098-1109.
5 Keren DF, Alexanian MD, Goeken JA, Gorevic
PD, Kyle RA, Tomar RH. Guidelines for
clinical and laboratory evaluation of patients
with monoclonal gammopathies. Arch Pathol
Lab Med 1999;123:106-107.
758
Immunoglobulin Quantitation
6 Alexanian R, Weber D, Liu F. Differential
diagnosis of monoclonal gammopathies. Arch
Pathol Lab Med 1999;123:108-113.
7 Vladutiu AO. Immunoglobulin D: properties,
measurement, and clinical relevance. Clin
Diagn Lab Immunol 2000;7:131-140.
8 Owen WE, Roberts WL. Performance
characteristics of four immunonephelometric
assays for the quantitative determination of IgA
and IgM cerebrospinal fluid. Am J Clin Pathol
2003;119:689-693.
9 Butch AW. Dilution protocols for detection of
hook effects/prozone phenomenon. Clin Chem
2000;46:1719-1720.
10 Warren JS. Immunoglobulin quantification and
viscosity measurement. In: Detrick B, Hamilton
G, Folds J, eds. Manual of Molecular and
Clinical Laboratory Immunology. 7th ed.
Washington, DC: ASM Press; 2006:69-74.
11 Lock RJ. Immunoglobulins and
immunoglobulin subclasses in the elderly. Ann
Clin Biochem 2003;40:143-148.
12 Katzmann JA, Kyle R. Immunochemical
characterization of immunoglobulins in serum,
urine, and cerebrospinal fluid. In: Detrick B,
Hamilton G, Folds J, eds. Manual of Molecular
and Clinical Laboratory Immunology. 7th ed.
Washington, DC: ASM Press; 2006:88-100.
13 Conley ME. Primary antibody deficiency
diseases. In: Detrick B, Hamilton G, Folds J,
eds. Manual of Molecular and Clinical
Laboratory Immunology. 7th ed. Washington,
DC: ASM Press; 2006:906-913.
14 Stevens RA, Lempicki RA, Natarajan V,
Higgins JH, Adelsberger JW, Metcalf JA.
General immunologic evaluation of patients
with human immunodeficiency virus infection.
In: Detrick B, Hamilton G, Folds J, eds. Manual
of Molecular and Clinical Laboratory
Immunology. 7th ed. Washington, DC: ASM
Press; 2006:847-861.
15 Ford RP, Mitchell PEG, Fraser CG. Desirable
performance characteristics and clinical utility
of immunoglobulin and light chain assays
derived from data on biological variation. Clin
Chem 1988;34:1733-1736.
16 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biologic variation: pros, cons and
progress. Scand J Clin Lab Invest 1999;59:491-
500.
17 Ritzman SE. Immunoglobulin abnormalities. In:
Ritzmann SE, Daniels JC, eds. Serum protein
abnormalities, diagnostic and clinical aspects.
Boston: Little, Brown; 1975:351-485.
18 Berne BH. Differing methodology and
equations used in quantitating immunoglobulins
by radial immunodiffusion in a comparative
evaluation of reported and commercial
techniques. Clin Chem 1974;20:61-68.
759
Immunoglobulin Quantitation

Table 1: Methods of Immunoglobulin Quantitation

Method 1: Salt precipitation; isolation of total immunoglobulin by salt precipitation
Principle of analysis: Immunoglobulins are less soluble in certain salt solutions and precipitate; amount of
immunoglobulins quantified by total protein assay (such as biuret)
Comments: Serum; historical, nonspecific
Method 2: Gold precipitation; isolation of total immunoglobulin by gold precipitation
Principle of analysis: Similar to salt precipitation; gold used as precipitant
Comments: Serum; historical, nonspecific
Method 3: Electrophoresis; estimation of total immunoglobulin by physical separation
Principle of analysis: Proteins separate based on class (albumin, gamma globulin), calculation of each class as
percentage of total protein
Comments: Serum; good screening method for detection of monoclonal immunoglobulins
Method 4: Radial immunodiffusion (RID); quantitation by immunoprecipitation in gel
Principle of analysis: Immunoglobulin diffuses into gel containing antibody, forming a ring-shaped
immunoprecipitate; diameter of ring proportional to concentration
Comments: Serum, body fluids; accurate, slow, expensive; used for IgG subclass and IgD determination.
Variation: Electroimmunodiffusion (Laurell rocket); immunoglobulin electrophoresis into antibody-containing
agarose gel; height of gel pattern (rocket) proportional to immunoglobulin concentration.
Method 5: Nephelometry; quantitation by immunoprecipitation in solution
Principle of analysis: Reaction of immunoglobulin with its specific antibody results in immunoprecipitate,
which has light-scattering properties; amount of light scatter proportional to immunoglobulin concentration
Comments: Serum, body fluids; accurate, rapid, adaptable to automation; most commonly used
Method 6: Turbidimetry; quantitation by size of immunoprecipitate in gel
Principle of analysis: Immunoglobulin reacts with antibody or other precipitation agent; turbidity proportional to
immunoglobulin concentration
Comments: Serum, body fluids; accurate, rapid, adaptable to automation; commonly used
Method 7: Immunofluorometry; quantitation by competition for labeled antibody on a solid phase
Principle of analysis: Immunoglobulin adsorbed onto solid surface competes for fluorescent-labeled antibody
with immunoglobulin sample; fluorescence signal is inversely proportional to immunoglobulin concentration
Comments: Serum, some body fluids; precision slightly less than nephelometry; adaptable to automation
Method 8: Labeled immunoassay, competitive
Principle of analysis: Immunoglobulin reaction with antibody displaces labeled immunoglobulin. Label may be
detected by colorimetric, fluorometric, or chemiluminescent method. Amount of label detected is inversely
proportional to immunoglobulin concentration in specimen.
Comments: Any body fluid; sensitive
Method 9: Labeled immunoassay, noncompetitive
Principle of analysis: Immunoglobulin reacts with antibody on solid surface; reaction quantitated by second,
labeled antibody. Label may be detected by colorimetric, fluorometric, or chemiluminescent method. Amount of
label detected is proportional to immunoglobulin concentration in specimen.
Comments: Any body fluid; sensitive
760
Immunoglobulin Quantitation

Table 2: Physical Properties of Human Immunoglobulins

Immunoglobulin
class IgG IgM IgA IgD IgE
Molecular 150,000 900,000 160,000 (monomer) 185,000 200,000
mass (Daltons) 320,000 (dimer)
Sedimentation 6.6 18.019.0 6.2510.9 6.27.0 7.867.92
coefficient, S
Heavy chains
Heavy-chain 1 , 2 , 3 , 4 1, 2 1, 2
subclasses
Light chains or or or or or
Molecular IgG()22 IgM()(22 )5 IgA()(22 )1-3 IgD()22 IgE()22
formula IgG()22 IgM()(22)5 IgA()(22)1-3 IgD()22 IgE()22
Reference Intervals Serum Concentrations (mg/mL) (by age)
Cord specimen 7.6616.93 0.040.26 0.00040.09
0.53 months 2.998.52 0.151.49 0.030.66
36 months 1.429.88 0.181.18 0.040.90
612 months 4.1811.42 0.432.23 0.0140.95
12 years 3.5612.04 0.372.39 0.131.18 116122 ng/mL
23 years 4.9212.69 0.492.04 0.231.37 80122 ng/mL
36 years 5.6413.81 0.512.14 0.352.09
47 years 140442 ng/mL
69 years 6.5815.35 0.502.28 0.293.84
1014 years 374674 ng/mL
1216 years 6.8015.48 0.452.56 0.812.52
Adult 8.0016.00 0.502.00 1.403.50 00.14 22000 ng/mL
Data from Seligson O, ed. Immunology. In: Handbook Series in Clinical Laboratory Science. Vol 1. Section F. Part I. Boca
Raton, FL: CRC Press; 1978; and Meites S, ed. Pediatric Clinical Chemistry. 2nd ed. Chicago: American Association for
Clinical Chemistry; 1981.

IgG Subclass Quantitation by Radial
Immunodiffusion
Introduction
For laboratories performing few IgG subclass
quantitations, the technique of radial immunodiffusion
yields appropriate quantitative information. Commercial
kits such as those from the Binding Site are available.
There are several methods of obtaining the quantitative
result by this technique. The method described in this
procedure uses an end-point calibration curve in which
standards and unknowns are allowed to diffuse to end-
point before a calibration curve is constructed.
Two radial immunodiffusion methods have been used in
clinical laboratories. The Mancini method is based on an
end-point (equivalence-zone) reading. The plates are
incubated for about 48 hr (72 hr for IgM). The diameter
of the precipitin ring is measured, and then the antigen
level is determined from a standard curve. A standard
curve is prepared by plotting on linear graph paper the
concentrations of three or four reference sera against the
ring diameter squared (mm2). The Fahey-McKelvey
method measures the ring diameter before equivalence
(such as 18 2 hr). One constructs the standard curve by
plotting ring diameter (mm) against the concentration of
the reference standards on two-cycle semilogarithmic
graph paper. The Fahey technique has the advantage of
producing results more rapidly; however, the accuracy
and precision are somewhat less than those obtained
with the end-point technique [17,18]. Commercial plates
are available for radial immunodiffusion quantitation.
Many plates can be used with either the Fahey or
Mancini technique.
Reagents and Materials Required
Kits (includes calibrators and 7% BSA)
IgG subclass 1
IgG subclass 2
IgG subclass 3
IgG subclass 4
Reagent Preparation, Storage, and Stability
Plates: If plates are kept closed and properly stored (4C
to 6C), they may be used until expiration date. They
must not be frozen.
Calibrators: Each kit contains four calibrators (one for
each subclass) and 0.01% thimerosal as preservative.
The concentrations of the calibrators are indicated on the
labels. Note that this is expressed in mg/liter. Medium
and low-level dilutions of the calibrators are required to
provide calibration curves. These can be made by
diluting the calibrators to 60% and 10%. It is
recommended that 120 L of calibrator be mixed with
761
Immunoglobulin Quantitation

80 L of BSA for the 60% dilution and that 25 L of quoted on the control is 6100 mg/L, this is equivalent to
calibrator be mixed with 225 L of BSA for the 10% a ring diameter of 6.4 mm (from the RID reference
dilution. table). The control serum range should be the values
Human Serum Control: Store at 4C. obtained for 6.1 to 6.7 mm, which reads as 5370 to 6880
mg/L. Control values must be within stated ranges
Specimens before patient results can be reported. Notify supervisor
Use serum collected by normal means. Perform IgG if controls are out of range.
quantitation before setting up subclasses. This is to be
used as a guideline for making sample dilutions. Reporting Results
Compare patient values with expected values based on
Procedure previous IgG quantitation and the reference interval for
1. Dilute control and patient samples in 7% BSA. that age. If these correlate, answer test in computer. If no
a. Each subclass requires a different ring was detected, answer NONE. If ring was smaller
sample dilution. The standard than lowest standard, repeat patient at a lower dilution or
dilutions are: neat. If ring at neat dilution is still smaller than lowest
standard, answer < _____ (lowest standard mg/dL
IgG1 1:10* value).
25 L sample + 225 L of 7% BSA If a sample must be repeated for any subgroup, also
IgG2 1:10* repeat it for the other subgroups to keep the use of the
25 L sample + 225 L of 7% BSA wells consistent on all plates.
IgG3 Neat Expected Values
IgG4 Neat
IgG Subclass Reference Intervals
* For control, a smaller volume may IgG1 IgG2 IgG3 IgG4
be needed for 1:10 dilution. Age (years) mg/dL mg/dL mg/dL mg/dL
b. Because of the wide variation in less than 1 190620 30140 962 613
normal values based on patient age, it 1 230710 30170 1198 443
may be appropriate to use other 2 80830 40240 6130 3120
dilutions.
3 350790 50260 998 5180
c. Dilute Human Serum Control for use
45 360810 60320 9160 9160
as a control, using 1:10 dilution for
IgG 1 and IgG 2; neat control for IgG 67 2801120 30630 4250 11620
3 and IgG 4. 89 2801740 80550 22320 10170
2. Label worksheets to indicate the proper wells 1012 2701290 110550 13250 7530
and dilutions for each sample. 1315 2801020 60790 14240 1330
3. Fill appropriate wells with 5 L of samples 16 to adult
(neat or diluted, as appropriate). 327855 52556 4117 1114
4. Tightly close the plates, and incubate at room
temperature on a flat surface until end-point is In healthy adults, IgG constitutes approximately 75% of
reached (72 hr). the total serum immunoglobulins. Within the IgG class,
5. Using the RID viewer, read and record ring the concentrations of the four subclasses are
diameters to 0.1 mm. approximately as follows: IgG1, 60%; IgG2, 30%; IgG3,
6. To construct the standard curve, plot the square 5%; and IgG4, 4% (see table above). These figures vary
of the diameters against the concentration for somewhat from individual to individual, and this may be
each standard. At end-point, this should result under genetic control.
in a straight line. Construct a best-fit straight
line through adjacent points. IgG is the only class of immunoglobulins that can pass
7. Determine the concentration of the patient the placenta in humans and is responsible for protection
sample from the standard curve. Multiply by of the newborn during the first few months of life. IgG2
the appropriate dilution factor. is transferred more slowly than the other subclasses. The
IgG subclasses are also capable of fixing complement in
Quality Control the following order of descending efficiency: IgG3 >
Record control values for each subclass on QC sheet. IgG1 > IgG2 > IgG4. IgG4 is unable to fix complement
The ranges for the Human Serum Control are based on by the classic pathway but may be active in the alternate
the RID reference table enclosed in the kit. The pathway.
concentration mean of the control is listed on the control
bottle. Take this mean and determine the concentration Macrophages bear surface receptors which bind IgG1
on the table closest to the mean value. Then determine and IgG3 and their Fc fragments which are responsible
the ring diameter of this concentration as listed in the for arming the macrophages. Many infections in infants
reference Table. The range of the control should be may be due to failure to produce the normal proportions
within +0.3 mm; for example, if the IgG1 concentration of the subclasses, particularly IgG2.
 762
Insulin and C-Peptide
Insulin and C-Peptide
Steven C. Kazmierczak
Name: Insulin and C-peptide
Clinical significance: Refer to Chapter 38, Diabetes, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular mass: Insulin, 6000 D
C-peptide, 3000 D
Proinsulin, 9000 D
Merck Index: 4866
Chemical class: Protein (hormone)
Principles of Analysis and Current Usage i
Insulin and C-peptide are synthesized in the rough
endoplasmic reticulum of the beta cells of the pancreatic
islets of Langerhans. Like other polypeptide hormones and
related proteins that enter the endoplasmic reticulum,
insulin and C-peptide are synthesized as part of a larger
prohormone. Insulin is stored in secretory granules as an
inactive 9000-D precursor molecule known as proinsulin.
Proinsulin contains the insulin molecule covalently linked
to a single polypeptide chain of 3000 D known as C-
peptide. Proteolytic cleavage of the proinsulin polypeptide
releases one molecule of insulin and one molecule of the
C-peptide. Normally, 90% to 97% of the product released
from the beta cells is C-peptide and insulin in
equimolecular amounts. The remainder is primarily
proinsulin [1]. C-peptide is not biologically active. The
half-life of C-peptide is significantly longer than that of
insulin, resulting in fasting concentrations of C-peptide
that are 5- to 10-fold higher than those of insulin.
Active insulin is a 51amino acid protein of 6000 D. It is
composed of two chains (A and B) that are joined by
disulfide bridges. The insulin molecules of humans,
horses, cattle, sheep, pigs, and rabbits are biologically and
(to a lesser extent) immunologically similar.
The analysis of insulin holds a position of singular
importance in clinical chemistry, since it was this
substance that was measured in the first radioimmunoassay
(Table 1, Method 1) [2]. In the modern clinical laboratory,
measurement of insulin is accomplished using a variety of
iInsulin
Previous and current authors of this method:
First edition: Michael D.D. McNeely
Methods edition: Michael D.D. McNeely
Second edition: Not updated
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Steven C. Kazmierczak
immunoassays. Enzyme immunoassay (Table 1, Method 2)
is one of the most popular techniques. Other assays include
insulin radio-receptor methods using cultured leukocytes,
and bioassays that measure the stimulation of glucose
oxidation.
The quality of the various assays used for measurement of
insulin is a matter of concern. Problems include lack of
standardization, nonspecificity of the antibodies used, and
the presence of anti-insulin antibodies and insulin auto-
antibodies in patient samples [3]. Problems due to widely
disparate results obtained with different insulin
immunoassays have been recently confirmed, and these
problems hinder efforts towards achieving consistent
measures for treatment guidelines [4].
Many persons who have received diabetic therapy have
circulating endogenous antibodies to insulin. These
antibodies may increase or decrease the apparent insulin
concentration because of an effect on the binding antibody
or on the separation step [5]. The endogenous antibodies
can be measured and then removed by precipitation (see
following discussion).
For assay purposes, antibodies to insulin are usually raised
in guinea pigs, using purified porcine insulin. Because
human and rabbit insulin are very similar, antisera
developed in rabbits generally do not have a high affinity
for human insulin. Insulin is sufficiently immunogenic to
initiate an antibody response without being covalently
linked to a carrier. However, the hypoglycemia caused by
injection of pure insulin is a limiting factor when one is
designing an immunization schedule. A good insulin assay
will allow the detection of 10 to 30 pg (24 to 72 U) of
insulin per assay tube. Antibody cross-reactivity with
human, porcine, and bovine insulin; human growth
hormone (HGH); C-peptide; and proinsulin should be
determined for all assays. Cross-reactivity will be nearly
100% with various animal insulins, up to 60% with
proinsulin, but less than 1% with C-peptide and HGH.
Immunometric assays that are specific for insulin are
commercially available for a large number of instrument
 763
Insulin and C-Peptide
platforms. Unfortunately, these assays may not recognize
insulin analogs used for treatment of diabetes [6]. A recent
study that compared 11 different insulin assays found that
measured values varied by a factor of two. These
commercial insulin assays were standardized against the
first International Reference Preparation 66/304 (IRP
66/304) [7].
Insulin is readily labeled with radioactive iodine (125I)
using the chloramine-T procedure. Separation of bound
and free labeled insulin has been carried out by use of most
available techniques. These include dextran-coated
charcoal, double-antibody precipitation, and polyethylene
glycol. Endogenous insulin antibodies influence the
separation.
The original enzyme immunoassays (EIAs) for insulin
showed good correlation with RIA procedures [8,9] but
were not so easily performed. The EIAs employ a
sandwich technique. In this technique, antibodies to insulin
are adsorbed onto a solid phase (such as disks or beads).
Insulin from the sample remains bound to the solid-phase
antibodies after a washing step. Anti-insulin antibodies
directed to a second site on the insulin molecule and
conjugated to an enzyme are added to the solid phase.
After washing, the amount of bound antibodyenzyme is
determined by incubation of the solid phase with enzyme
substrate. The enzyme most often used is horseradish
peroxidase, which reacts with substrates such as 5-
aminosalicylic acid[9] and p-hydroxyphenylacetic acid
[10]. The products of the enzyme reaction are measured
spectrophotometrically (at 500 nm) [9] or fluorometrically
[10] (Dexcitation, 317 or 325 nm; Demission, 414 nm). The
amount of product formed is directly proportional to the
amount of insulin present in the sample. A homogeneous
EIA for insulin has not been developed.
A number of non-isotopic immunoassays for insulin have
also been developed. In these competitive insulin
immunoassays, enzyme labels employing fluorometric or
luminometric measurements of activity, as well as
fluorescence labels for use in fluorescence polarization
immunoassays, have been employed [11-13].
The non-isotopic immunoassays provide good sensitivity
and specificity, and the reagents used are typically stable
for longer periods of time when compared to RIA
techniques [14].
One common non-isotopic technique used for measuring
insulin is a two-site immunoenzymometric assay (Table 1,
Method 3). Insulin is bound with mouse monoclonal
antibody that has been immobilized on a magnetic solid-
phase bead. A second enzyme-labeled mouse monoclonal
antibody is added, which also binds to the insulin, forming
an antibody-insulin-labeled antibody sandwich. The solid
phase beads are used to remove any unbound enzyme-
labeled antibody and then incubated with a fluorogenic
substrate. The amount of fluorescence produced is directly
proportional to the insulin concentration within the sample.
Another nonisotopic immunoassay procedure for insulin is
that based on microparticle enzyme immunoassay (MEIA)
technology (Table 1, Method 4). In this procedure, insulin
is incubated with monoclonal (mouse) anti-insulin-coated
microparticles, producing an antibody-insulin complex. An
aliquot of this reaction mixture containing insulin bound to
the anti-insulin-coated microparticles is transferred to a
glass fiber matrix. The matrix is washed to remove
unbound materials and a second anti-insulin antibody
conjugated with alkaline phosphatase is dispensed onto the
glass fiber matrix, where it binds to the antibody-antigen
complex. Unbound materials are removed from the matrix
using a second wash step, and the substrate, 4-
methylumbelliferyl phosphate, is added to the matrix. The
fluorescence produced is directly related to insulin
concentrations within the specimen.
A variety of high-performance liquid chromatography
(HPLC)-based procedures for insulin have been described,
but these are primarily used in research settings. Recently,
mass spectrophotometric methods for insulin have been
described [15].
Assays for C-peptide are used for detecting self-induced
hyperinsulinism, since pharmaceutical preparations of
insulin do not contain C-peptide. In addition, measurement
of C-peptide can be used to monitor pancreatic surgery.
Levels should be undetectable following radical
pancreatectomy and should increase following successful
islet-cell or pancreas transplantation. C-peptide RIA
development is difficult, since the molecule does not
contain tyrosine, and synthetic or tyrosylated molecules
must be used. Standardization of the C-peptide assay is a
source of interassay variability. It is generally agreed
[10,16] that prior precipitation of endogenous antibodies is
mandatory before a C-peptide assay is performed.
Although measurement of C-peptide offers several
advantages over insulin determinations, several problems
need to be resolved. The issue of standardization must be
addressed, as various antisera used are known to differ in
their antigenic domains, and different materials have been
used as standards [17,18]. In addition, even minor cross-
reactivity of C-peptide antisera with human proinsulin can
significantly interfere in C-peptide immunoassays if
proinsulin secretion still persists in a diabetic patient who
has circulating insulin antibodies induced by insulin
therapy [17,19].
Routine analysis of C-peptide in the clinical laboratory can
be performed using a variety of commercial
chemiluminescence and enzyme-linked immunoassays [3].
Chromatographic methods have been described but are
used primarily in the pharmaceutical industry and in
research. Procedures based on HPLC-MS following solid-
phase extraction or immunoaffinity chromatography have
been described [20,21].
 764
Insulin and C-Peptide
Reference and Preferred Methods
There are no internationally recognized reference methods
for insulin or C-peptide.
The gold standard method for insulin is a bioassay, since
this measures biological activity and thus is of greater
physiological significance. Such methods are used to
standardize pharmaceutical insulin preparations and are
only available in specialized laboratories.
For routine insulin assay, non-isotopic immunoassays are
most commonly used. The selected method should not
employ an antibody that cross-reacts more than 1% with
C-peptide or growth hormone. With the isotopic assays, if
dextran-coated charcoal is used to separate bound from
free insulin, endogenous antibodies bind the tracer, falsely
lowering the values. Double-antibody separation
techniques may give falsely increased values in the
presence of endogenous antibodies [22]. For this reason,
an appropriate method for removal of endogenous
antibodies should be available. The sensitivity of the assay
should be equivalent to at least 10 U/mL (416.7 pg/mL)
of sample. For C-peptide measurements, a technique with
a preliminary antibody precipitation is the method of
choice. Before selecting a method for routine use, consult
van Rijn et al. [16]
A method for the detection of insulin antibodies is
sometimes required. For this purpose, the method of
Gerbitz and Kemmier [23] is recommended. The 2007
College of American Pathologists (CAP) Special Ligand
quality-assurance survey showed that essentially all
laboratories utilized a non-isotopic method for insulin
analysis. Non-isotopic assays provide good precision and
accuracy for determination of insulin and C-peptide.
Specimen
Insulin concentrations are identical in serum and
heparinized plasma. The hormone is stable for at least 12
hours at room temperature, for a week at 4C, and for a
month at 10C. Proinsulin and its cleaved metabolites
have been reported to have limited stability in human
serum. Use of protease inhibitors to promote stability of
the proinsulin-related peptides has been recommended [4].
Increases of up to 75% have been seen in frozen samples
following long-term storage (>2 years) [24]. Random
insulin assays are not useful. Specimens for insulin
analysis should always be collected as part of a planned
clinical protocol.
Interferences
The presence of an insulin-degrading enzyme in
erythrocytes may result in falsely decreased insulin
concentrations in specimens that are hemolyzed [25].
Falsely increased results caused by heterophile antibodies
or rheumatoid factors have been observed in
immunoassays which use a sandwich technique [26,27].
Serum for insulin determination should be separated from
the red blood cells within 5 hours following collection
[28].
Insulin Reference Interval
There is no single range for insulin in serum. All values
must be considered in relation to the clinical state of the
patient, particularly the dynamics of the plasma glucose.
Insulin concentrations in fasting individuals are usually
less than 25 U/mL (1042 pg/mL, 0.17 pmol/mL). During
a glucose tolerance test, the insulin value usually peaks
between 50 and 100 U/mL (2083 to 4167 pg/mL, 0.35 to
0.7 pmol/L) and follows the glucose curve by about 15
min.
When the serum glucose concentration falls below 500
mg/L (2.8 mmol/L), insulin concentration should be
suppressed below 20 U/mL (833.4 pg/mL). During a
hypoglycemic episode, the glucose/insulin ratio in normal
persons is usually greater than 25 U/mL. If the ratio is
higher, an insulin-secreting tumor should be suspected.
Interpretation
Serum insulin concentrations can be increased in patients
with noninsulin dependent diabetes mellitus (NIDDM),
hyperthyroidism, type IV hyperlipoproteinemia, renal
failure, liver cirrhosis, and generalized acute illnesses.
Decreased serum insulin concentrations are found in
patients with insulin-dependent diabetes mellitus (IDDM)
and can be associated with pheochromocytoma,
malnutrition, and cystic fibrosis. However, the findings of
increased or decreased insulin levels in these pathological
states are incidental findings and rarely of diagnostic use.
The primary reason for the measurement of serum insulin
is to evaluate fasting hypoglycemia. Neurological and
behavioral symptoms are frequently seen in patients with
insulin-induced hypoglycemia. The neurological
symptoms (excessive sweating, palpitation, tachycardia,
tremor, and weakness) are the result of increased
sympathetic nervous system activity with increased
epinephrine levels. Patients with severe hypoglycemia may
present in a coma or experience convulsions.
The most important cause of repetitive episodes of
hypoglycemia is an insulin-secreting tumor (insulinoma)
or pancreatic hyperplasia. Because surgical procedures can
easily correct this life-threatening condition, it is important
to make the diagnosis quickly. Another frequently seen
cause of insulin-induced hypoglycemia is factitious
hypoglycemia, that is, a self-induced hypoglycemia. In this
situation, the patients are either purposefully or
accidentally overtreating themselves with pharmaceutical
preparations of insulin.
Since insulin release from insulinomas may be episodic in
nature, the most accurate procedure for evaluation of
suspected insulin-induced hypoglycemia is to perform
multiple serum insulin and glucose determinations while
the patient is fasting. Fasting periods as long as 72 hours
have been used. The immunoreactive insulin/glucose
 765
Insulin and C-Peptide
(II/G) ratio is then calculated. In healthy, nonobese
persons, this ratio is normally 0.3 (insulin expressed as
U/mL, and glucose in mg/100 mL). If the ratio is greater
than 0.3, or in obese persons greater than 0.3 and a serum
glucose less than 600 mg/L, the insulin level is
inappropriately elevated for the corresponding glucose
level, and an insulinoma should be considered. After a 48-
hour fast, the II/G ratio has a sensitivity of 85% for
insulinomas. After a 72-hour fast, the sensitivity of the
ratio rises to 95% [29].
There are multiple clinical indications for measurement of
C-peptide. Although C-peptide is not used for the routine
monitoring of diabetes, it can be an important tool in
certain clinical situations. In some countries, C-peptide
measurements are used to qualify medical insurance
coverage for insulin-pump therapy. Measurement of C-
peptide can aid in the differential diagnosis of factitious
hypoglycemia and hypoglycemia caused by
hyperinsulinism. Since pharmaceutical insulin preparations
do not contain C-peptide, an increased serum insulin
concentration accompanied by a normal C-peptide level
should raise the suspicion of self-induced hypoglycemia.
C-peptide measurements can help assess the success of
surgical removal of all or part of the pancreas and islet
transplantation. Decreased C-peptide concentrations have
been observed in starvation and in Addisons disease.
Insulin Performance Goals
The Clinical Laboratory Improvement Amendments of
1988 (CLIA-88) evaluation criteria for acceptable
performance for insulin measurements require that
laboratories achieve results that are within 3 SD of the
mean value of all reporting laboratories. The precision of
the various methods commercially available can be
strikingly different; the interlaboratory coefficient of
variation (CV) ranges from approximately 3% to 25%
[30].
The within-individual biological variability for insulin has
been reported as 21% CV, and the within-group variability
for healthy individuals has been reported as 58% CV [31].
Based on these values for biological variability, one can
derive a desirable allowable measurement bias of 15.5%,
imprecision of 10.6%, and total analytical error of 32.0%
for a single reportable result at insulin concentrations
within or near the normal reference interval. The precision
of commercially available insulin methods meet the
criteria for acceptable performance.
References
1 Wahren J, Ekberg K, Jornvall H. C-peptide is a
bioactive peptide. Diabetologia 2007;50:503-509.
2 Yalow RS, Berson SA. Immunoassay of
endogenous plasma insulin in man, J Clin Invest
1960; 39: 1157-1175.
3 Clark PM. Assays for insulin, proinsulin(s) and
C-peptide. Ann Clin Biochem 1999; 36: 541-564.
4 Marcovina S, Bowsher RR, Miller WG, Staten M,
Myers G. Caudill SP, et al. Standardization of
insulin Immunoassays: Report of the American
Diabetes Association workgroup. Clin Chem
2007;53:711-716.
5 Nisselbaum JS, Fleisher M, Schwartz MK.
Discrepancies between serum insulin RIA
methods in the presence of endogenous
antibodies. Clin Chem 1977; 23: 1167. (abstract).
6 Heald AH, Bhattacharya B, Cooper H, Ullah A,
McCulloch A, Smellie S, et al. Most commercial
insulin assays fail to detect recombinant insulin
analogues. Ann Clin Biochem 2006; 43; 306-308.
7 Manley SE, Stratton IM, Clark PM, Luzio SD.
Comparison of 11 human insulin assays:
implications of clinical investigation and research.
Clin Chem 2007; 53: 922-932.
8 Hinsberg WD III, Milby KH, Zare RN.
Determination of insulin in serum by enzyme
immunoassay with fluorimetric detection. Anal
Chem 1981; 53: 1509-1512.
9 Yoshioka M, Taniguchi H, Kawaguchi A,
Kobayashi T. Murakami K. Seki M, et al.
Evaluation of a commercial enzyme immunoassay
for insulin in human serum, and its clinical
application. Clin Chem 1979; 25: 35-38.
10 Meistas MT, Kumar MS, Schumacher OP.
Diagnosis of self-induced hyperinsulinism in an
insulin-dependent diabetic patient by
radioimmunoassay of free C-peptide. Clin Chem
1981; 27: 184-186.
11 Tsuji A, Maeda M, Arakawa H, Shimizu T,
Ikegami Y, Sudo, H, et al. Enzyme immunoassay
of hormones and drugs by using fluorescence and
chemiluminescence reaction. In: Dal SB, ed.
Enzyme labelled immunoassay of hormones and
drugs. Berlin-New York: Walter de Gruyter &
Co., 327-39, 1978.
12 Yamajuchi Y, Hayaski C, Miyai K: Fluorescence
polarization immunoassay for insulin
preparations. Anal Letts 1982; 15: 731-737.
13 Toivonen E, Hemmila I, Marniemi J, Jorgensen
PH, Zeuthen J and Lovgren T. Two-side time-
resolved immunofluorometric assay of human
insulin. Clin Chem 1986; 32: 637-640.
14 Andersen L, Dinesen B, Jorgensen PN, Poulsen F,
Roder ME. Enzyme immunoassay for intact
human insulin in serum or plasma. Clin Chem
1993; 39: 578-582.
15 Van Uytfanghe K, Rodriguez-Cabaleiro D, Stockl
D, Thienpont LM. New liquid chromatography-
electrospray ionization-tandem mass spectrometry
measurement procedure for quantitative analysis
of human insulin in serum. Rapid Commun Mass
Spectrom 2007; 21: 1-3.
16 van Rijn, H.J.M., Hoekstra, J.B.L., and Thijssen,
J.H.H.: Evaluation of three commercially
available C-peptide kits, Ann. Clin. Biochem.
19:368-373, 1982.
17 Koskinen P. Nontransferability of C-peptide
measurements with various commercial
 766
Insulin and C-Peptide

radioimmunoassay reagents. Clin Chem 1988; 34: 25 Duckworth WC, Hamel FG, Bennett R, Ryan MP,
1575-1578. Roth RA. Human red blood cell insulin
18 Faber OK, Binder C, Markussen J, Heding LG, degrading enzyme and rat skeletal muscle insulin
Naithani VK, Kuzuya H, et al. Characterization protease share antigenic sites and generate
of seven C-peptide antisera. Diabetes 1978; identical products from insulin. J Biol Chem
27(Suppl 1): 170-177. 1990; 265: 2984-2987.
19 Myrick JE, Gunter EW, Maggio VL, Miller DT, 26 Boscato LM, Stuart C. Heterophilic antibodies: a
Hannon WH. An improved radioimmunoassay of problem for all immunoassays. Clin Chem 1988;
C-peptide and its application in a multi-year 34: 27-33.
study. Clin Chem 1989; 35: 37-42. 27 Highton J, Hessian PA. A solid-phase enzyme
20 Darby SM, Miller ML, Allen RO, LeBeau M. A immunoassay for C-reactive protein: clinical
mass spectrophotometric method for quantitation value and the effect of rheumatoid factor. J
of intact insulin in blood samples. J Anal Toxicol Immunol Methods 1986; 68: 185-192.
2001; 25: 8-14. 28 Walters E, Henley R, Barnes I. Stability of
21 Rogatsky E, Balent B, Goswami G, Tomuta V, insulin in normal whole blood. Clin Chem 1986;
Jayatillake H, Cruikshank G et al. Sensitive 32: 224 (letter).
quantitative analysis of C-peptide in human 29 Ravel, R. Clinical laboratory medicine, ed. 3,
plasma by two-dimensional liquid Chicago, 1978, Year Book Medical Publishers, p.
chromatography-mass spectrometry isotope- 330.
dilution assay. Clin Chem 2006; 52: 872-879. 30 College of American Pathologists Special Ligand
22 Feldkamp CS, Chapin E, Shearer G. Blanking in Participant Summary Report Y-B, Washington,
insulin radioimmunoassay. Clin Chem 1977; 23: DC, 2001, CAP.
1167 (abstract). 31 Fraser CE. Biological variation: From principles
23 Gerbitz KD, Kemmier W. Method for rapid to practice. Washington, DC: AACC Press, 2001.
quantitation and characterization of insulin 32 Greenwood FC, Hunter WM, Glover JS. The
antibodies. Clin Chem 1978; 24: 890-894. preparation of I-131-labeled human growth
24 Feldman JM, Chapman BA. Radioimmunoassay hormone of high specific radioactivity. Biochem J
of insulin in serum and plasma. Clin Chem 1973; 1963; 89: 114-123.
19: 1250-4.
Tables
Table 1: Methods for Insulin Analysis
Method 1: Radioimmunoassay (RIA)
Principle of analysis: Radioactive iodine (125I)-labeled insulin competes with sample insulin for binding sites on
anti-insulin antibodies. Separation of bound from free ligand can be accomplished by double-antibody precipitation,
solid phase, and so on.
Comments: Most frequently used; can be affected by endogenous anti-insulin antibodies
Method 2: Enzyme immunoassay (EIA)
Principle of analysis: Employs sandwich technique, in which second antibody is coupled to horseradish
peroxidase. Reaction products can be measured spectrophotometrically (500 nm) or fluorometrically (excitation, 317
nm; emission, 414 nm)
Comments: Research; as sensitive as RIA
Method 3: Immunoenzymometric assay (IEMA)
Principle of analysis: Insulin bound to antibody immobilized to magnetic solid phase support. Addition of second
antibody labeled with ALP added to form sandwich. Fluorogenic substrate added following wash step to remove
unbound labeled antibody
Comments: Frequently used; interference from endogenous anti-insulin antibodies and human anti-mouse antibodies
possible
Method 4: Microparticle enzyme immunoassay (MEIA)
Principle of analysis: Similar to Method 2, except insulin bound to anti-insulin-coated microparticles transferred to
glass fiber matrix where anti-insulin - ALP conjugate is added to form sandwich complex. 4-methylumbelliferyl
added as substrate for ALP
Comments: Frequently used; same as for Method 3
 767
Insulin and C-Peptide

Table 2: Assay Conditions for Radioimmunoassay of Insulin (Ref 16)

Condition RIA
Reaction temperature 4C
Sample volume 100 L
Fraction of sample volume 0.2
Assay time Approximately 20 h
Final concentration of reagents 125I-insulin: 0.2 ng/mL (40,000 cpm)

Phosphate buffer (pH 7.5): 40 mol/mL

Albumin: 2 mg/mL
Sensitivity Approximately 10 U/mL
Linearity 10288 U/mL
Interferences Anti-insulin antibodies
periodically, and adjust if necessary.
Procedure: Radioimmunoassay for Insulin Dissolve 1195 g of NaH2PO42H2O in about 200 mL of
Antibody Precipitation
deionized water and bring to a volume of 250 mL. Use this
Principle solution to adjust the pH of the phosphate-BSA solution to
Polyethylene glycol is added to serum or plasma in a exactly 7.50.
concentration that causes precipitation of IgG (and 2. Dextran-coated charcoal. Click here for
therefore endogenous antibodies). description.
3. Insulin antiserum. Antiserum to porcine insulin,
produced in guinea pigs, is available from New England
Reagent Nuclear (549 Albany St., Boston, MA 02118). For use,
1. Polyethylene glycol (250 g/L). Dissolve 2.5 g of dilute the antiserum in phosphate-BSA buffer to produce
PEG 8000 (7000 to 9000 D) (Fisher Scientific Carbowax 40% to 60% binding of tracer.
PEG 8000), and bring to 10 mL with deionized water. 4. Stock standard (10 g/mL; 0.24 U/mL, 1.67
nmol/mL). Dissolve 10.0 mg of crystalline porcine insulin
(Sigma 13505, 24 U/mg) in 0.01 mol/L of HCl, and dilute
Assay to 10 mL with phosphate-albumin buffer. Remove 1.00
1. Combine 1.0 mL of serum (or plasma) with 1.0 mL of this solution, and bring to 100 mL with phosphate-
mL of PEG solution. albumin buffer. This solution is stable for 1 month stored
2. Mix on a vortex mixer. at 70C in 1 mL aliquots and for 7 days at 4C after
3. Centrifuge at 3000 rpm (approximately 1000 g) at thawing.
4C for 15 min. 5. Working standards. Each day, prepare a series of
standards by diluting 250 L of stock standard to 50 mL
4. Remove supernatant, which may now be used as a
with phosphate-BSA to produce a 1200 U/mL (50
sample in the insulin assay. The insulin level
ng/mL) standard. The 1200 U/mL standard is then diluted
obtained on the supernatant must be multiplied by
with phosphate-BSA according to the following values:
2.

Insulin Assay [10,14,16,17] Concentration mL of 1200 Final Volume

(U/mL) U/mL standard (mL)
Principle 9.6 0.200 25
A guinea pig antiserum to porcine insulin is incubated with 19.2 0.400 25
the test serum and 125I-labeled human insulin. Separation 28.8 0.600 25
is achieved with dextran-coated charcoal. 48.0 0.400 10
72.0 0.600 10
96.0 0.800 10
Reagents 144.0 1.200 10
1. PhosphateBSA buffer, 0.05 mol/L, pH 7.5. 192.0 1.600 10
Dissolve 8.90 g of Na2HPO42H2O in about 500 mL of 288.0 2.400 10
deionized water using mild heating. Add 2.5 g of bovine
serum albumin (BSA), and slowly mix to dissolve. Adjust 6. Tracer (125I-labeled insulin). Obtain iodinated
pH to 7.5 using NaH2PO4 solution described next. Bring insulin from New England Nuclear (Boston, MA) (NEX-
volume to 1000 mL with deionized water. Stable for 3 104), or label porcine insulin using the method of
months at refrigerated temperatures. Check pH Greenwood et al. [32]. Dilute the tracer in phosphate-BSA
to produce a minimum of 20,000 cpm (counts per min) in
 768
Insulin and C-Peptide
200 L with approximately 0.1 ng of insulin. This is stable
for 48 h at 4C when diluted.
Assay
Equipment: A gamma-ray counter that will record 125I
and a refrigerated centrifuge capable of generating 1000 g
force.
1. Combine 200 L of antiserum and 200 L of
labeled insulin tracer in a series of 12 100 mm tubes
sufficient for standards, controls, unknown samples, and
background counts.
2. Add 100 L of standards, controls, or unknown
sera.
3. Prepare total-count (TC) and nonspecific binding
(NSB) tubes by combining 200 L of tracer and 300 L of
phosphate-BSA. Save TC tube for measurement of total
counts, and process NSB tube with samples.
4. A blank tube may be prepared for any sample by
substitution of 200 L of phosphate-BSA for antiserum.
5. Incubate overnight at 4C (18 h).
6. Add 2.0 mL of well-mixed dextran-coated
charcoal. Mix, and centrifuge at 1000 g for 15 min at 4C.
Decant, and count the supernatant.
Calculations
Subtract the NSB counts from all tubes. Compute for each
specimen and standard as follows:
__Counts NSB_ 100% = B/T
Total count NSB
where B = antibody-bound counts
T = total counts nonspecific binding
Plot % B/T versus concentration.
Note: If the value of the blank tubes is significantly greater
than that of the NSB tubes, an endogenous insulin
antibody should be suspected.
 769
Ionized Calcium
John G. Toffaletti
Name: Ionized calcium
Clinical significance: Refer to Chapter 33, Bone Disease, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Atomic symbol: Ca2+
Atomic mass: 40.08 D
Merck Index: 1613
Chemical class: Alkaline earth element, ionic form
Principles of Analysis and Current Usage
Calcium in serum or plasma exists in a variety of forms
as a result of the binding equilibria between calcium ions
and both high- and low-molecular-mass anions. The
term complex-bound calcium describes the calcium
bound to low-molecular-mass anions such as
bicarbonate, citrate, and phosphate [1]. Complex-bound
calcium constitutes from 10% to 15% of the total serum
calcium. The protein-bound calcium exists
predominately as calcium-albumin and constitutes
approximately 40% of the total calcium [2]. The
proportions of each form of calcium detected by ionized,
ultrafiltrable, and total calcium methods is shown in
Table 1.
The unassociated calcium ions, hereafter referred to as
ionized calcium, represent 45% to 50% of the total
calcium. The equilibria between these forms are affected
by pH, temperature, ionic strength, and magnesium-ion
concentration, as well as by the addition or removal of
calcium from blood. The pioneering work of McLean
and Hastings on the effect of calcium on frog heart
muscle contraction [3], established that the ionized
calcium in body fluids is the most physiologically active
form of calcium [4]. The concentration of ionized
calcium in blood affects many functions, including
excitation and contraction of cardiac and skeletal muscle
[5,6], neurotransmission, and secretion of parathyroid
hormone (PTH) [7-9].
Although methods based on bioassay or ultrafiltration
have been developed in the past to measure ionized or
ultrafiltrable calcium, ion-selective electrodes (Table 2,
Method 2) are virtually the only routine method in use
today. Current commercially-available electrodes are
relatively durable, easily serviced, suitable for use with
serum and blood, and automated for precision,
convenience, and rapid results in clinical work.
i membranes may increase the response time of the
Ionized Calcium
Previous and current authors of this method:
First edition: Not done
Methods edition: John G. Toffaletti
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: John G. Toffaletti
All modern calcium-selective electrodes are based on
similar designs and principles. Differences between
instruments are found mostly in the flow systems,
method of calibration, control materials, and types of
ion-selective membranes used. A basic diagram of such
an electrode is shown in Figure 1.
The ion-selective membrane is critical to the specificity
of the electrode. It must selectively interact with calcium
ions in the presence of other ions such as magnesium,
potassium, hydrogen, and sodium. These membranes
consist of a calcium-ion sensor and a solvent mediator.
The sensor is a molecule with high selectivity for
calcium that is dissolved in the solvent mediator. The
mediator functions to disperse and align the calcium
sensor to optimize the affinity and selectivity of the
sensor for calcium ions. The sensor-solvent mediator is
often incorporated into a polyvinyl chloride (PVC)
matrix that gives structural support to the membrane.
The sensor and solvent mediator must be insoluble in
aqueous solutions.
The calcium sensors are usually either ion exchangers or
neutral carriers. Ion exchangers are calcium salts, usually
of an organophosphate. Neutral carriers are organic
molecules without net charge that form a molecular
pocket with a favorable steric and electrostatic
environment for binding calcium ions. These sensors are
illustrated in Figure 2.
The ion-selective membrane functions by allowing
calcium ions to bind to the sensors in the membrane. If
calcium ions are unequally distributed on the internal
and external surfaces of the membrane, an electric
potential will develop across the membrane. Since the
internal electrode solution has a constant ionized calcium
concentration, variations in the membrane potential are
related to the ionized calcium concentration (activity) of
the sample. An inert cellophane membrane has been
used to prolong electrode life by preventing direct
contact between protein and lipids in the sample and the
calcium sensor in the electrode. However, such
electrode.
A potential develops at the junction of the reference-
electrode solution and the sample, called the liquid-
junction potential, as shown in Figure 1. If all samples
and calibrators produced the same junction potential, it
would be of no consequence. However, whole blood
 770
samples can develop a liquid-junction potential that is
different from that developed by serum or blood.
Manufacturers of ionized calcium analyzers have
designed their analyzers to minimize this effect.
Examples include: (1) allowing time for the sample to
2+
stabilize while in contact with the Ca electrode before
electrical contact is made with the reference electrode,
then reading the potential immediately, before the
junction potential is fully developed; and (2) using a
high concentration of anions and cations with similar
ionic mobilities in the reference-electrode solution.
Reference Methods
Although not designated as an official reference method
for ionized calcium, a designated comparison method
was published and later adopted by the National
Committee on Clinical Laboratory Standards (NCCLS)
as a means to achieve comparability of analytical results
among the commercially available calcium ion-selective
electrodes [10].
Specimen
For measurement of ionized calcium in clinical samples,
the use of heparinized whole blood collected
anaerobically is preferable and is far more prevalent than
use of serum. Heparinized whole blood may be analyzed
immediately (no clotting and centrifugation is needed)
and provides nearly double the usable sample compared
to serum, an especially important factor when analyzing
blood from neonates. A concern with ordinary sodium
heparin is that the heparin anion binds to calcium ions,
which alters the ionized calcium concentration.
However, modern syringes are readily available that
contain either a calcium-balanced heparin or a very low
amount of heparin dispersed in a polysaccharide web,
either of which has minimal effect on the ionized
calcium concentration. Although serum contains no
anticoagulant to bind calcium, the clotting process has
variable effects on the pH and ionized calcium [11,12].
Perhaps the only remaining advantage of serum is that it
is more stable in storage if analysis has to be delayed.
With modern blood-gas/electrolyte/metabolite analyzers
that analyze whole blood, the need for a rapid ionized
calcium result as part of this panel of tests has also made
whole blood the predominant sample for clinical use.
Ideally, samples should be analyzed as soon as possible,
preferably within 1 hour after collection. If this is not
possible, the ionized calcium concentration in either
clotted or heparinized blood stored in sealed tubes is
stable for 6 hours or more at 4C if in vitro hemolysis is
not evident [13]. This stability is fortuitous, however,
because the effects of lactate and acid production during
storage apparently offset the effects of each other on the
ionized calcium concentration: the hydrogen ion releases
calcium ions from albumin while lactate anions chelate
about the same amount of free calcium ions [14].
Therefore, although it is desirable to analyze samples as
soon as possible, whole blood stored at 4C for several
hours may still be acceptable. Serum kept in tightly
sealed syringes, with no air bubbles present, is stable for
1 to 2 hours at room temperature and over 24 hours at
4C.
Specimens should be collected anaerobically, because
CO2 is lost when blood is exposed to atmospheric air,
which causes pH to increase. As mentioned, a lower pH
(more H+ ion) will increase the concentration of ionized
calcium in serum, and a higher pH will have the opposite
effect [15]. If strictly anaerobic sampling or handling is
not possible, reporting the calculated ionized calcium at
pH 7.4 is an alternative that gives results within 3% to
4% of the uncorrected result on fresh sample if the pH
correction is not too large.
The patient should be seated or supine and relaxed when
blood is drawn, tourniquet application should be as brief
as possible, and the tourniquet should remain on the arm
until all blood is withdrawn. Although posture influences
total calcium more than ionized calcium, standing can
increase ionized calcium concentration by 1% to 2%
[16]. Prolonged venous stasis can also increase ionized
calcium by about 2%, and a few minutes of forearm
exercise with stasis can elevate ionized calcium by 8%
because of localized acid production [17]. Bedrest for 3
to 12 days has been shown to elevate ionized calcium
into the abnormal range [18]. This should be considered
when one is interpreting results on patients who are
completely bedridden.
Interferences
Anticoagulants that strongly bind calcium ions, such as
citrate, oxalate, or EDTA, significantly lower the ionized
calcium concentration. Heparin, the most widely-used
anticoagulant for ionized calcium measurements, is a
weaker binder of calcium ions. Ordinary sodium or
lithium heparin at a concentration of 30 IU/mL in plasma
lowers ionized calcium by about 3% to 5%. To minimize
the binding effect of heparin, a variety of modified
heparin salts have been used. For many years, calcium-
balanced or electrolyte-balanced heparins have been
available that virtually eliminate any effect on ionized
calcium in either plasma or whole blood. This type of
heparin has small amounts of calcium and other
electrolytes added that effectively occupy the binding
sites on heparin and prevent further binding in the blood
sample. Although this type of anticoagulant virtually
eliminates the effect of heparin over a wide range of
ionized calcium concentrations, samples with very low
ionized calcium concentrations will have a slight
positive bias, and samples with very high ionized
calcium concentrations will have a slight negative bias
[12]. Another approach has been to use a very low
amount of heparin dispersed in a soluble polysaccharide
web which appears as a puff of anticoagulant in the
syringe. The final concentration of heparin is so low
(~2.5 IU/L) that the interference to ionized calcium is
effectively eliminated at all concentrations. The
polysaccharide web dissolves very rapidly to quickly
disperse the heparin in the blood sample.
Heparin affects ionized calcium slightly less in whole
blood than in plasma because red cells can artifactually
increase the ionized calcium concentration by their effect
on the liquid junction potential. Syringes and evacuated
collection tubes of whatever size should be filled as
completely as possible and kept tightly sealed to
 771
minimize loss of CO2, which increases pH, and to
prevent an undesirably high concentration of heparin
anticoagulant.
It should be noted again that serum is less than ideal for
collection of ionized calcium samples. Serum has no
anticoagulant to bind calcium ions, but the clotting
process apparently has variable and unpredictable effects
on the pH and ionized calcium when compared to whole
blood collected without any anticoagulant and analyzed
immediately [11,12].
Ionized Calcium Reference Intervals
Depending on the type of anticoagulant used, the
population studied, and to some extent the analyzer used,
the lower reference limit for ionized calcium may range
from 1.13 to 1.16 mmol/L and the upper reference range
from 1.30 to 1.32 mmol/L. The above values are for
adults. Values for children appear to be about 0.05
mmol/L higher.
The above intervals are intended as guidelines that
should be confirmed by data for the specific instrument
and population served by the hospital. Variables that
may influence reference intervals are the concentration
and type of anticoagulant used, sample handling
practices, and the design and calibration system used by
the manufacturer of the electrode. Values for serum,
plasma, and whole blood differ; whole blood is the usual
sample collected and analyzed.
Interpretation
Changes in blood electrolyte concentrations are common
during major surgical operations involving the heart,
liver, and abdomen. Ionized calcium is often measured
during these procedures to monitor and control blood
levels, with decisions about calcium supplementation
based on these measurements. Administration of
heparin, citrate, bicarbonate, drugs, protein solutions,
along with alterations in blood pH and body temperature,
can significantly affect the calcium concentration in
blood. Because of alterations in blood pH and protein
concentration, total calcium measurements are of little
use in these circumstances [19]. Several recent studies
emphasize the importance of monitoring ionized calcium
instead of total calcium during surgery and recovery [20-
24]. With no supplemental calcium or magnesium given
during the procedure, ionized calcium was found to
decrease by up to 20% during cardiac surgery, and both
ionized and total magnesium decreased by
approximately 25% [25].
Hypocalcemia appears to be a frequent complication in
trauma patients, with contributing factors being colloid-
induced hemodilution, severe shock, pH disturbances
and elevated lactate [26]. More importantly, an ionized
calcium on admission of less than 1.00 mmol/L was
associated with greater injury severity, shock, longer
ICU stays, greater need for ventilation, and higher
mortality [27].
Perhaps the most prevalent use of ionized calcium
measurements is for monitoring critical care patients in
the ICU. Hypocalcemia is common in ICU patients who
develop sepsis or renal, cardiac, or pulmonary failure or
in patients with thermal burns. Total calcium
measurements frequently have little meaning, because
serum protein concentrations are reduced, acid-base
disturbances are present, and citrated blood products
have been given. If calcium is administered,
measurement of ionized calcium provides a guide to
proper dosage in these patients [28].
Critically ill patients, such as those with infections and
sepsis, pancreatitis, burns, and major trauma are all
susceptible to hypocalcemia. Because of its importance
in maintaining cardiac output, arterial pressure, and
systemic vascular resistance, adequate calcium
concentrations are especially important, and guidelines
have been reported on administering calcium [29]. A low
ionized calcium concentration has been associated with
increased mortality in patients admitted to the ICU
[30,31] and in patients with pancreatitis [32]. Patients
with sepsis commonly have decreased blood ionized
calcium concentrations [33], and the mechanism of
hypocalcemia may be related to the production of
various cytokines. These patients, especially if an
infection is present, also have low or suppressed
parathyroid hormone (PTH) levels. In sepsis, factors
such as TNF, IL-6, and CRP were all markedly elevated
on the first day of admission to the ICU [34]. It should
also be noted that yet another study reported that
albumin-corrected total calcium measurements are not
suitable for diagnosis of calcium disorders in the
critically ill [35]. Interestingly, one report claims that
ionized magnesium can be inferred from total
magnesium, but that ionized calcium must be measured
directly [28].
In neonates, ionized calcium measurements are
especially important for following their calcium status.
Neonatal hypocalcemia appears to be a normal
occurrence during the first week of life and may act as a
stimulus to induce the infants parathyroid glands to
become functional [36]. Typically, no calcium
supplementation is necessary or even beneficial [37].
However, in preterm infants or neonates with severe or
prolonged hypocalcemia, calcium supplementation may
be beneficial [38,39]. In neonates with seizures,
hypocalcemia and hypomagnesemia should be
considered as causes [40]. In critically ill children,
ionized hypocalcemia was both common and associated
with a higher mortality [41]. Ionized calcium measured
by point-of-care analyzers may be of special importance
in critically ill children when lab results are not available
rapidly [42].
In renal disease, after renal transplantation, or in patients
on hemodialysis, calcium metabolism is often altered,
sometimes profoundly. During hemodialysis,
maintaining a slight positive calcium balance is
important for maintaining good cardiac contractility and
blood pressure [43,44], and ionized calcium
measurements are probably the best means to monitor
 772
this, since total or corrected total calcium values do not
agree with directly measured ionized calcium in many
patients with renal disease [45]. The levels of PTH and
ionized calcium may be important determinants in long-
term survival of patients on dialysis [46,47].
For the differential diagnosis of altered calcium
metabolism in patients with hyperparathyroidism,
hypoparathyroidism, malignancy, or renal disease,
ionized calcium measurements may be paired with PTH
measurements to provide an important differential for
the cause of the calcium abnormality [48-51].
Ionized calcium measurements can be useful in acute
pancreatitis, during which ionized calcium typically
decreases during the first 24 hours of an attack and
return to normal by 48 hours [52]. Because both total
calcium and albumin also decrease, hypocalcemia may
be missed by measurements of either the total or
corrected total calcium. This is significant because
persistently low ionized calcium has been associated
with severe pancreatitis [32].
Ionized calcium has been reported to be increased in a
higher percentage of patients with malignancy than total
calcium [53,54]. In general, whenever the cause of
hypercalcemia cannot be readily determined, malignancy
must be considered. In this case, additional measurement
for PTH, cyclic AMP, or phosphate may be useful.
Ionized Calcium Performance Goals
Kost has published critical limits for ionized
hypocalcemia of 0.82 +0.14 mmol/L and for ionized
hypercalcemia of 1.55 +0.19 mmol/L [55].
Consequently, performance goals for ionized calcium
would be standard deviations (SD) of 0.03 to 0.05
mmol/L in the low and normal range and within 0.08
mmol/L in the upper range. Review of recent CAP
quarterly summaries for ionized calcium indicates that
each major brand of analyzer meets these goals. For four
control materials (AQ-01, AQ-02, AQ-11, and AQ-12)
with means of 0.96, 0.73, 1.90, and 1.30 mmol/L,
respectively, the SDs among the major brands of
analyzers ranged from 0.027 to 0.052, and the CVs
ranged from 1.9% to 4.3%. As a specific example, for
the AQ-12 material with a mean of 1.30 mmol/L, the
SDs ranged from 0.026 to ,0.035 and the CVs ranged
from 1.9% to 2.6%. Thus even for the analyzer with the
highest CV, 99% of the results for this control material
would range from 1.20 to 1.40 mmol/L.
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ionized calcium measurements in randomly
selected samples compared with clinical
diagnoses. Ann Clin Biochem 1983;20:349-
352.
54 Shemerdiak WP, Kukreja SC, Lad TE, York
PAJ, Henderson WJ. Evaluation of routine
ionized calcium determinations in cancer
patients. Clin Chem 1981;27:1621-1622.
55 Kost GJ. The significance of ionized calcium in
cardiac and critical care. Arch Pathol Lab Med
1993;117:890-896.
 775

Table 1: Detection of Calcium Components by Various Methods

Calcium Component Ion-Selective Electrode: Ultrafiltrable Total
Ionized Calcium (%) Calcium (%) Calcium (%)
Calcium ions 100 100 100
Calcium phosphate 0 100 100
Calcium bicarbonate 0 100 100
Calcium citrate 0 100 100
Calcium albumin 0 0 100

Table 2: Methods for Free Calcium Measurements

Serum, plasma, and whole blood:
Method 1: Biological assay; stimulation of muscle contraction
Principle of analysis: A heart is perfused with solutions containing Ca2+. The amplitude of
contraction is directly related to the concentration of Ca2+.
Comments: Historically important but an extremely tedious method
Method 2: Ion-selective electrode; potentiometry
Principle of analysis: Calcium ion-selective membrane specifically binds calcium ions, creating an
electrochemical potential across the membrane that is proportional to Ca2+ concentration.
Comments: A rapid and durable method for both stat and routine analyses; widely used in
commercially available instruments; some give pH corrected result
Serum or plasma:
Method 3: Ultrafiltrable; ultrafiltration with analysis by atomic absorption or other means
Principle of analysis: Pressure (mechanical or centrifugal) causes protein-free filtrate to pass through
ultrafilter; calcium measured in ultrafiltrate.
Comments: A time-consuming method that may be suited for laboratories without specialized
equipment for measuring ionized calcium; detects both calcium ions and all calcium complexes.
 776

Figure 1: Basic design of calcium ion-specific electrode system.

The calcium-ion sensor develops a potential proportional to the ionized calcium concentration, while the reference sensor
maintains a stable potential independent of the sample composition. Both electrodes maintain a stable internal filling
solution so that the only variable read by the voltmeter is the potential across the ion-selective membrane in contact with
the blood sample. (Courtesy of Nova Biomedical.)

Figure 2: Calcium-ion sensors.

 777

Figure 3: Typical series arrangement of analyte-specific electrodes in a modern blood gaselectrolyte analyzer.
Peristaltic pumps and valves automatically flow the blood sample to the electrodes while measurements are taken, pump
the blood out to waste, then clean the electrodes with a rinse solution. A reference electrode is used to measure the
potentials between it and each analyte-specific electrode.

Figure 4

Figure 4. Radiometer ionized calcium electrode design

(cross-sectional diagram)

Ag rod coated with AgCl

- provides electrical contact to buffer solution
- Ag/Ag+ equilibrium maintains a stable
potential

Electrolyte solution

Porous pin
- absorbs electrolyte solution
- holds PVC membrane

PVC membrane
- contains a Ca++ ion exchanger

Cellophane membrane
- protects PVC membrane
- prevents protein build-up
- held in place by white plastic gasket

Figure 4: Diagram of the Radiometer ionized calcium electrode.

A chloridized silver wire provides a stable potential within this half-cell. Note that this electrode design uses a cellophane
membrane to protect the ion-selective membrane (PVC with ion exchanger). (Courtesy of Radiometer America.)
 778

Figure 5. IL GEM ionized calcium sensor

(cross-sectional diagram)

Card
Reference
Electrode

Figure 5: Diagram of the IL GEM ionized calcium electrode.

This electrode system uses the ionophore ETH 1001 in a PVC membrane deposited on a plastic card which contains
miniature sensors for other analytes in a multi-use disposable cartridge. A hydrogel electrolyte matrix is deposited over an
Ag/AgCl wire, followed by a coat of ion-selective membrane. (Courtesy of Instrumentation Laboratory.)

Figure 6a. i-STAT ionized calcium sensor and reference electrode

(side-view diagram)

iCa SENSOR
ION SELECTIVE MEMBRANE
INSULATION LAYER
SOLID ELECTROLYTE
AgCl
Ag
SILICON DIOXIDE

SILICON BASE
REFERENCE ELECTRODE
H2O VAPOR PERMEABLE LAYER INSULATION LAYER
SOLID ELECTROLYTE + KCl
AgCl AgCl
Ag
SILICON DIOXIDE

SILICON BASE

Ion-selective membrane
Solid Electrolyte
AgCl
SiO2 Layer
Insulating Layer
Ag

Ag contact
pad

Figure 6: Diagram and image of the i-STAT ionized calcium sensor on a single-use card.
A calcium ion-selective film is deposited over a solid electrolyte gel that covers the Ag/AgCl wire. The reference electrode
has a thin layer of KCl solution covered by a water-permeable, electrolyte-impermeable silicone layer. A small opening in
the silicone layer forms a liquid junction. The image shows a top view of these components as they are arranged on a
miniature test card. (Courtesy of i-STAT Corporation.)
 779

Analysis of Ionized Calcium by Ion-Selective Electrode

Principle of Standard Laboratory Analyzers

The ion-selective electrodes, reagents, and flow systems of modern ionized calcium analyzers have many similarities. Most
measure ionized calcium within a panel of critical care tests that include blood gases, pH, Na, K, glucose, and lactate.
Some analyzers also include ionized magnesium and creatinine in this panel. A series of analyte-specific electrodes are in
solution contact with a reference electrode, and a potentiometer measures the specific potential that develops at each
analyte electrode, including the ionized calcium electrode, with a typical example shown in Figure 3. While the potential in
the ionized calcium (analyte) electrode changes in proportion to the ionized calcium concentration in the sample, the
reference electrode provides a stable, fixed potential that is not altered by the sample composition.

The calcium-ion electrode has an ionophore (a molecule that specifically binds calcium ions) imbedded within a stronger
polyvinyl chloride (PVC) or other type membrane. This ion-selective membrane may be covered with a cellophane-type
membrane that prevents contamination by proteins and other constituents in the blood samples being analyzed. An
electrolyte solution with a constant concentration of ionized calcium is contained within the electrode and is in contact with
the inner layer of the ion-selective membrane. As a blood sample contacts the outer layer of the ion-selective/cellophane
membrane, an electric potential develops across the membrane. A potentiometer or voltmeter reads the potential between
this ion-selective electrode and a constant-potential reference electrode, with this potential related to the analyte
concentration. Standard laboratory blood gaselectrolyte analyzers include those made by Bayer (Siemens),
Instrumentation Laboratory, Nova Biomedical, and Radiometer. Diagrams showing the design of ionized calcium
electrodes are shown for Radiometer (Figure 4) and Instrumentation Laboratory (Figure 5).

In most modern analyzers, once the sample is injected or aspirated into the analyzer, the remaining analysis is automated:
flowing the sample to the various analytical and reference electrodes, taking stable measurements and calculating
concentrations, then removing the sample to waste and rinsing for the next sample. A peristaltic pump and a series of flow
selector valves pull the sample through the analyzer, allow the rinse solution to flow, and open/close the injection and exit
ports. These actions are automatically coordinated once a sample is injected. The results are displayed approximately 60
sec later, and another sample may be analyzed 180 sec after injecting the previous sample.

In addition to calibrator fluids, ionized calcium analyzers use detergent rinse solutions between each sample to remove the
blood sample and clean the electrodes and flow system. Periodically, or when a clog is detected in the system, a stronger
cleaning solution (either bleach or alkaline solution) may be sent through the system.

Principle of Single-Use Test Cartridges for Ionized Calcium

Disposable, single-use cartridges are now in widespread use that analyze ionized calcium along with other blood gas and
electrolyte tests as part of a test panel. Although in a physically different form, the principles of analysis of these single-use
cartridges are very similar to those in standard laboratory analyzers. These cartridges are inserted and read in a small
(hand-held) test device and are typically used near the patient in point-of-care settings. The test system is largely within
the disposable cartridge itself and contains the sample injection port, the ion-selective and reference electrodes, a calibrant,
and the flow system.

One such system (i-STAT) has a calcium ionselective film of dioctylphenyl phosphonate plasticized polyvinyl chloride
containing the calcium salt of an organic phosphate compound that is coated over the active area of the electrode. The
reference electrode is an Ag/AgCl electrode covered by a thin KCl electrolyte layer, which in turn is covered with a water-
permeable, electrolyte-impermeable silicone layer. A small opening in the silicone layer allows formation of a liquid
junction and measurement of the electrochemical cell voltage between the sample and calibrant, which is related to the
concentration of the ionized calcium in the blood sample. Both a cross-section diagram and image of this single-use
electrode is shown in Figure 6.

Calibration
A typical calibration consists of analyzing one or more levels of calibrants or standards. Typically this is done initially
when an electrode or test pack is changed, then may be done manually or automatically at times necessary to satisfy
regulatory requirements.

Quality Control
Until relatively recently, quality of results were maintained by analysis of two or three external quality-control samples
provided by manufacturers in sealed glass ampules or other containers. Ionized calcium concentrations in these controls
should cover a range of concentrations from low to high levels. CAP and/or CLIA still require analysis of control materials
at least every 8 hours. However, over the past 10 to 15 years, quality-control procedures have undergone significant
evolution. In basic terms, these quality-control procedures are aimed at minimizing the need to analyze external control
material at frequent intervals, which are especially troublesome with near-patient use by caregivers. These new control
 780
Ionized Calcium

procedures also depend on systems that are inherently stable over an extended period of time, from several hours to several
weeks.

Electronic quality-control devices. These devices are often used with point-of-care test systems. The electronic control
devices are inserted into the cartridge reading system to check the electronic and physical integrity of the system. Although
these devices should determine if the cartridge-reading system is functioning properly, they have no relevance to the
integrity of the analytical cartridge itself.

Internal calibrant/control solutions. After an initial calibration of the cartridge lot, to help ensure that the cartridge and
electrode systems are functioning properly during the life of the test pack lot, an internal calibrant/control solution, sealed
within the cartridge, is analyzed with each blood sample. If this internal solution gives a reading within established limits,
the clinical analysis is considered valid, and the results are reported. The single-use i-STAT cartridge incorporates a
calibrant solution within each disposable test cartridge. Instrumentation Laboratory uses a control system based on analysis
of several solutions sealed within gas-impermeable multi-use test packs to verify calibration stability. The responses of
these control fluids and the blood samples are also monitored in multiple ways to help identify failures of the instrument,
flow system, or electrodes, and also identify problems with specific samples. When problems are detected, corrective
actions by rinsing and recalibration may be automatically initiated.
781
Iron and Iron-Binding Capacity
Iron and Iron-Binding Capacity
Gerardo Perrotta and Jordan Reynolds
Name: Iron and Iron-Binding Capacity
Clinical significance: Refer to Chapter 39, Iron, Porphyrin, and Bilirubin Metabolism, in the 5th
edition of Clinical Chemistry: Theory, Analysis, Correlation.
Atomic symbol: Fe2+ (ferrous), Fe3+ (ferric)
Atomic mass: 56 D
Merck Index: 4942
Chemical class: Element, metal
i
Principles of Analysis and Current Usage
There are many methods available for determining
serum iron. All colorimetric procedures have in common
a reaction in which ferric iron (Fe3+) is reduced to the
ferrous state (Fe2+) by the addition of strong reducing
agents such as hydrazine, ascorbic acid, thioglycolic
acid, or hydroxylamine [1-3].
Once the reduction has taken place, colorimetric
reactions use an iron-complexing chromogenic agent to
bind the ferrous iron [2-4]. The more commonly used
complexing agents, according to one of the recent 2007
College of American Pathologists (CAP) surveys, are
5,5(3-(2-pyridyl)-1,2,4-triazine-5,6 diyl)-bis-2-
furansulfonic acid (Ferene Iron Reagent), with or
without prior protein removal, and 3-(2-pyridyl)-5,6-
bis(4-phenylsulfonic acid)-1,2,4-triazine (FerroZine Iron
Reagent).
In summary, all colorimetric procedures employ the
following reaction (Table 1, Methods 1 and 2):
The use of coulometry for measurement of serum iron
has also been developed (Table 1, Method 3). The
method first removes iron from transferrin by the
addition of an alcoholic HCl solution. The freed iron is
exposed to different specific potentials in a multi-
electrode sensor. The transfer of electrons between the
2+ 3+
Fe and Fe generates a current. The instrument detects
the electrons in transition and relates the total electron
flow to iron concentration [5].
3+ 2+
Fe + e- Fe
i
Iron and Iron-Binding Capacity
Previous and current authors of this method:
First edition: Gerado Perotta
Methods edition: Gerado Perotta
Second edition: Gerado Perotta
Third edition: Gerado Perotta
Fourth edition: Gerado Perotta
Fifth edition: Gerado Perotta, Jordan Reynolds
Other methods, such as radiometry and atomic
absorption spectrophotometry, complete the list of
methods available for assaying serum iron. Most recent
CAP survey data [4] indicate that virtually all
laboratories utilize a colorimetric procedure, and none
are listed as using the atomic absorption method.
Serum iron analysis is almost always accompanied by a
measurement of the total iron-binding capacity (TIBC).
As indicated in Table 1, serum iron is bound to
transferrin, but only a portion of the transferrin molecule
is saturated with iron. The unsaturated iron-binding
capacity of transferrin (UIBC) denotes the available
iron-binding sites of serum. On the other hand, the
amount of iron that serum transferrin can bind when
completely saturated is the total iron-binding capacity
(TIBC).
In most colorimetric methods, TIBC is measured by first
saturating the transferrin with excess ferric iron. The
remaining iron that cannot be bound to transferrin is
removed by a chelator, such as magnesium carbonate,
which forms an insoluble complex with the excess ferric
iron. The total amount of iron saturating the transferrin is
then measured as described for total serum iron analysis.
Non-transferrin-bound iron (NTBI) and iron bound to
ferritin can cause apparent higher transferrin saturation
levels [6].
NTBI can also occur in patients in whom the binding
capacity of transferrin is overloaded, such as those
patients in an iron overloaded state (thalassemia and
hemochromatosis). Not all of the iron is associated with
transferrin.
An ion-exchange technique for measurement of serum
iron may also be used. A sample of serum is added to an
aliquot of ion-exchange resin preloaded with iron. After
a short period of equilibration, the serum sample
(transferrin) is saturated with iron, since the affinity of
iron for the transferrin is greater than its affinity for the
resin. An aliquot of this iron-saturated serum is then
analyzed the same way as for total iron analysis.
It is also possible to measure TIBC by an additive
procedure. A known iron standard (5000 g/L) is
incubated with the serum sample at a pH of 8.5, and the
transferrin becomes saturated. The remaining unbound
782
Iron and Iron-Binding Capacity
(free) iron is measured by the regular iron procedure.
The 5000 g/L standard minus the excess Fe not bound
to transferrin is the UIBC, or unbound iron-binding
capacity.
UIBC + Serum iron = TIBC
Reference and Preferred Methods
In 1978, the International Committee for Standardization
in Hematology (ICSH) proposed a reference method
with bathophenanthroline and revised it in 1990 by
replacing the chromogen with ferrozine or ferene [7].
The Clinical and Laboratory Standards Institute (CLSI),
formerly NCCLS, proposed its own method that differed
from the ICSH in terms of reagent concentration [8].
Tietz et al. compared the ICSH methods to five
commercial methods and found many discrepancies at
low concentrations. Effort towards a more reliable and
perhaps definitive reference method will hopefully be
available in the future [9].
Current methods using either FerroZine or TPZ as the
iron complexing reagent yield consistently higher values
than the bathophenanthroline method. The most common
comparison is between colorimetric methods that use
prior protein removal and those without prior protein
removal. Most laboratories reporting in a recent CAP
survey [4] used methods that fall into the category of
analysis without prior protein removal. Itano reviewed
the results of a questionnaire that was used to determine
the cause of the differences reported in the CAP survey
between the various methods listed. His findings
suggested that significantly lower values were common
for the FerroZine and TPZ procedures not using prior
protein removal [4,10]. However, there appeared to be
no significant bias between these two chromogenic
reagents used for either category. His study reinforced
the need for some reference method.
Specimen
No significant differences in apparent concentration
have been reported between serum and plasma [11].
Phlebotomy should be accomplished with minimal stasis
to allow the free flow of blood into the collection device.
A fasting, early-morning sample is optimal for analysis,
because a diurnal variation may decrease the iron
concentration by 30% during the course of a day. Peak
iron concentrations occur in the early- to late-morning
hours. The specimen should be centrifuged as soon as
possible after collection. Storage of whole blood without
separation of serum or plasma renders specimens
unsuitable for analysis. Serum iron is stable up to 1 week
at 4C to 8C and is stable indefinitely when frozen at
20C. Transferrin is stable for at least a week at
refrigerated temperatures and at least a month when
frozen.
Interferences
Hemolyzed samples must be rejected, since erythrocytes
contain iron and therefore falsely increase apparent iron
concentration. Response to hemolysis is both
heterogeneous and unpredictable, and therefore no
reliable corrective measures can be made [12]. Copper
has been found to interfere with ferrozine-based
methods. Administration of oral contraceptives will
elevate iron and TIBC levels. Drugs containing azo
groups may interfere with the coulometric analysis.
Iron and Iron-Binding Capacity Reference Interval
The range for serum iron in healthy persons is very
method dependent. Reference intervals range from 40 to
160 g/dL, to 20 to 250 g/dL in male adults and 20 to
200 g/dL in female adults. Iron concentration in
premenopausal women may be 10% to 15% lower than
for men, though product information for most methods
cite one range for both sexes. Reports indicate that
postmenopausal women have serum iron concentrations
that are not significantly different from those of men of
similar age. Neonates have serum iron concentrations
approximately twice that of healthy adults.
In healthy persons, transferrin is usually approximately
30% saturated with serum iron. TIBC reference values in
healthy adults are approximately 225 to 425 g/dL.
Interpretation
Measurement of serum iron alone is insufficient. It is
usual to make simultaneous measurement of iron, TIBC,
and ferritin as an index of iron stores. The ratio of iron to
TIBC is usually expressed as the transferrin saturation.
Iron deficiency is common and is characterized by low
serum iron, low transferrin saturation, and low ferritin.
Iron deficiency is more common in women. Women
during childbearing years lose about 600 to 800 mg of
iron during each pregnancy. This loss must be balanced
by the absorption of an equivalent amount of dietary iron
[13]. There is also significant blood loss at parturition,
and iron is lost during lactation.
Iron excess may be as a result of ingestion of iron or
combination tablets, or it may occur in the condition of
hemochromatosis, where there is increased absorption of
iron from the GI tract.
Iron toxicity occurs more frequently in children, though
it may occur in adults as well. Ingestion of therapeutic
iron tablets is the most common cause. Toxicity is
usually defined as a serum iron result that exceeds the
TIBC. In normal individuals, the transferrin is one-third
saturated, but in patients with iron overload, the
transferrin is completely saturated [14]. Common
symptoms of iron toxicity include vomiting, upper
abdominal pain, headaches, and diarrhea.
Iron poisoning most commonly occurs in patients
younger than 19 years of age. Serum iron concentration
greater than 350 g/dL is considered to be indicative of
systemic toxicity; however, lower serum levels may also
be present in toxic patients [15]. Serum iron levels reach
a maximum 2 to 6 hours after ingestion, and
measurement at about 4 hours is recommended.
However, some methods of measurement in the
overdose situation may be inaccurate because of the high
concentrations observed [16].
783
Iron and Iron-Binding Capacity
Iron overload occurs in conditions such as
hemochromatosis, ineffective erythropoiesis, and
transfusional iron overload, for example in -thalassemia
major [17].
Iron is rarely measured in urine specimens; rather the
urine is used for hemosiderin analysis. This iron-binding
protein may appear in urine as yellow-brown granules or
in cells or casts. Hemosiderinuria is useful for detection
of hemochromatins, hemolytic anemias, and paroxysmal
nocturnal hemoglobinuria.
Iron and Iron-Binding Capacity Performance Goals
Survey data from the 2007 CAP Summary Report shows
imprecision values (% coefficient of variation) for
measurement of iron at a concentration of 167 g/dL to
range from 1.7% for ferene methods without
precipitation to 4.8% for the nitroso PSAP methods [4].
Desirable specifications for analytical imprecision
derived from studies of biological variation indicate an
assay imprecision of 13.2% (0.5 within individual
biological variation [CVw]) [18]. The acceptable
performance criteria according to the Clinical
Laboratory Improvement Amendments of 1988 (CLIA-
88) require that laboratories be within 20% of their
respective peer group.
References
1 Peters T, Giovanniello T. Apt L, Ross JF. A
simple, improved method for the determination
of serum iron II. J Lab Clin Med. 1956;48:280-
288.
2 Levy A, Vitacce P. Direct determination and
binding capacity of serum iron. Clin Chem.
1961;7:241-248.
3 Goodwin J, Murphy B, Guillemette M. Direct
measurement of serum iron and binding
capacity. Clin Chem. 1966;12:47-57.
4 College of American Pathologists. CAP
Surveys 2007 Participant Summary, Chemistry
(C-A). Washington, DC: CAP, Chemistry
Resource Committee; 2007.
5 FerroChem Model 3050, Serum Iron and Total
Iron Binding Capacity Analyzer Instruction
Manual. 2nd ed. Bedford, MA: Environmental
Sciences Associates; 1979. Revised February
1981.
6 Al-Refaie FN, De Silva CE, Wonke B,
Hoffbrand AV. Changes in transferrin
saturation after treatment with the oral iron
chelator deferiprone in patients with iron
overload. J Clin Pathol. 1995;48:110-114.
7 Lewis SM. International Committee for
Standardization in Hematology proposed
recommendation for measurement of serum
iron in human blood. Am J Clin Pathol.
1971;56:543-545.
8 Valcour AA, Krzymowski G, Onoroski M,
Bowers GN Jr, McComb RB. Proposed
reference method for iron in serum used to
evaluate two automated iron methods. Clin
Chem. 1990;36:1789-1792.
9 Tietz N, Rinker A, Morrison S. When is a
serum iron really a serum iron? The status of
serum iron measurements. Clin Chem.
1994;40:546-551.
10 Itano M. CAP comprehensive chemistry, serum
iron survey. Am J Clin Pathol. 1978;70:516-
522.
11 Romeo R, Boedeker M, Duncan C.
Comparisons between serum and plasma
analytes measured on the Baxter Paramex. Clin
Chem. 1991;37:967.
12 Lippe G, Salvagno GL, Montagnana M,
BroccoG, Guidi GC. Influence of hemolysis on
routine clinical chemistry testing. Clin Chem
Lab Med. 2006;44:311-316.
13 Sonnenwirth AC, Jarett L. Gradwohls clinical
laboratory methods and diagnosis. 8th ed. St
Louis: Mosby; 1980.
14 Breuer W, Cabantchik Z. A fluorescence-based
one-step assay for serum non-transferrin-bound
iron. Anal Biochem. 2001;299:194-202.
15 Mills KC, Curry SC. Acute iron poisoning.
Emerg Med Clinics North Am. 1994;12;397-
411.
16 Curry SC. Iron. In: Reisdorff EJ, Roberts MR,
Wiegenstein JG. Pediatric emergency medicine.
Philadelphia: Saunders; 1993:673-679.
17 Olivieri NF, Brittenham GM. Iron-chelation
therapy and the treatment of thalassemia.
Blood. 1997;89:739-761.
18 White GH, Farrance I. Uncertainty of
measurement in quantitative medical testing: a
laboratory implementation guide. Clin Biochem
Rev. 2004;25:S1-4.
784
Iron and Iron-Binding Capacity

Tables

Table 1: Iron Methods Summary

Method 1: FerroZine iron reagent; colorimetric
Principle of analysis:
Transferrin (Fe3+) buffer 2Fe2+ + transferrin
2
reducing agent
Fe2+ + 3 FerroZine Fe (FerroZine)2+(magenta color complex, 560 nm)
Fe2+ + 3 NSPAP Fe (NSPAP)2+ (tense green complex, 750 nm)
Comments: Good coefficient of variation; no prior protein removal is necessary; manual or automated
assay
Method 2: Du Pont aca; colorimetric
Principle of analysis:
Transferrin (Fe3+) pH 4.2 Fe3+ + Transferrin
Fe3+ + Hydroxylamine Fe2+
Fe2+ + Bathophenanthroline Red complex (540 nm)
Comments: Good for stat tests; automated
Method 3: Electrochemical; coulometric
Principle of analysis: Fe3+ + e- Fe2+; sum of electrons in this transition is between two possible
valence states of Fe
Comments: Correlates well with FerroZine method; has very good accuracy and precision; excellent
for microsamples and stat tests; automated
Method 4: Spectroscopy; atomic absorption
Principle of analysis: Iron is concentrated by chelation with bathophenanthroline and is extracted into
methyl isobutyl ketone (MIBK); extract is analyzed by atomic absorption at 248.3 nm
Comments: Impractical and low sensitivity; mainly for research
Reaction between acetone and the sodium nitroferricyanide (nitroprusside) formulation found in Ketostix
(Ames, Division of Miles Laboratories, Elkhart, IN).

Figure
Iron: Figure 1

Absorption spectra of FerroZine [3-(2-pyridyl)-5,6-bis(4-phenylsylfonic acid)-1,2,4-triazine] reagent. Dashed

line, Reagent blank; solid line, reagent plus iron.
 785
Ketones

Ketones
Lawrence A. Kaplan
Name: Ketones
Clinical significance: Refer to Chapter 38, Diabetes, in the 5th Edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Common name: Acetoacetic acid Acetone -Hydroxybutyric acid
Molecular formula: C4H6O3 C3H6O C4H8O3
Molecular weight: 102.09 D 58.08 D 104.10 D
Merck Index: 51 58 4727
Chemical class: Ketocarboxylic acid Ketone Hydroxycarboxylic acid
Structure: See Figure 1 See Figure 1 See Figure 1
Metabolome sites:
Acetoacetic acid: http://www.hmdb.ca/scripts/show_card.cgi?METABOCARD=HMDB00060.txt
Acetone: http://www.hmdb.ca/scripts/show_card.cgi?METABOCARD=HMDB01659.txt
-Hydroxybutyric acid: http://www.hmdb.ca/scripts/show_card.cgi?METABOCARD=HMDB00011.txt

Principles of Analysis and Current Usage i
Ketones are biochemicals that have structural moieties of
ketones (that is, ; see structures).
Commonly found serum ketones include pyruvate,
acetoacetic acid, and acetone. Their reduced forms of the
first two ketones are lactic acid and -hydroxybutyric acid,
respectivel
Another term used to describe these biochemicals is
ketone bodies, which are acetoacetic acid, acetone, and -
hydroxybutyric acid. This discussion will only review the
analysis of these ketone bodies, which are associated with
diabetes, the clinical context in which ketones are most
frequently measured. Less commonly, other ketoacids can
be produced in certain diseases such as maple syrup urine
disease (see Chapter 52 and Reference 1). The discussion
of methodologies for pyruvate and lactic acids and the
complex ketoacids are presented elsewhere.
ketone bodies. A clinically meaningful assay that
specifically measures one of these biochemicals will lack
the ability to detect the others or to detect all the ketone
bodies. In addition, any clinically useful test for ketones
should be able to be performed rapidly, simply, 24 hours a
day.
Total Ketones; Urine and Serum
Total ketones are the sum of the measurable ketones found
in serum of diabetics, that is, acetoacetic acid and acetone.
The reaction between ketones and nitroprusside (sodium
nitroferricyanide) under alkaline conditions is the most
widely used procedure for the detection of total ketones in
diabetes care (Table 1, Method 1) [2,3]. Acetoacetic acid
and acetone both form a purple color in this reaction in the
presence of glycine and buffered by disodium phosphate
(Figure 1). This method is used most frequently as a
semiquantitative measure of total ketones in serum and
urine to screen for diabetic ketoacidosis. This assay does
not detect -hydroxybutyric acid.

The analysis for ketones has been limited by the fact that
the proportions of specific ketone bodies present in
diabetes can vary considerably and by the fact that no
single chemical or enzymatic method measures these
i Ketones
Previous and current authors of this method:
First edition: Lawrence A. Kaplan
Methods edition: Lawrence A. Kaplan and Nancy Gau
Second edition: Lawrence A. Kaplan
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Lawrence A. Kaplan
A modification of the nitroprusside test is the
incorporation of the reagents into a reagent strip (Ketostix)
or a tablet (Acetest). This technique is also incorporated
into the multitest urine chemistry dipsticks that are used
for routine urinalysis. The dipsticks are most accurate for
urines with a specific gravity of 1.010 to 1.020. Both
methods can be used for serum or urine. The use of
reflectance spectrophotometry has enabled quantitative
measurement of total ketones in a multitest urine dipstick
[4,5].
-Hydroxybutyrate and Acetoacetic acid
Several techniques have been developed that measure
these ketone bodies with high specificity. Acetoacetic acid
and -hydroxybutyric acid can be quantitated by an
 786
Ketones
enzyme assay (Table 1, Method 2) which makes use of the
following reversible reaction:
-HBD, pH 7.0
+
NADH + H+ + AcAc -HB + NAD
pH 8.5-9.5
Where AcAc = acetoacetic acid, -HB = -hydroxybutyric
acid, and -HBD = -hydroxybutyric acid dehydrogenase.
The reaction can be monitored by measuring the
absorbance of NADH at 340 nm [6,7]. At pH 8.5 to 9.5,
the reaction proceeds to the left as written, and the
concentration of -hydroxybutyric acid (-HB) is
quantitated when the increase in absorbance is monitored
at 340 nm. When performed at pH 7.0, the reaction
proceeds to the right, and the amount of acetoacetic acid
(AcAc) present is proportional to the decrease in
absorbance at 340 nm.
A number of assays that employ the above reaction at pH
8.5 to 9.5 have been developed for specifically monitoring
-hydroxybutyric acid for diabetes in the home, clinic, or
acute-care setting [8,9]. The MediSense hand-held device
(Abbott Corporation, Abbott Park, IL, USA) uses capillary
blood (5 L), which is added to a reagent strip, which is
inserted into a reading device. Using the -
hydroxybutyrate dehydrogenase reaction shown above at
alkaline pH, the NAD+ is reduced to NADH, which is
reoxidized to NAD+ by a redox mediator. The current that
is generated by the second reaction is detected by an
electrochemical detector and is directly proportional to the
concentration of -hydroxybutyrate in the sample (Table 1,
Method 3a).
Other such hand-held devices for measurement of -
hydroxybutyric acid include the KetoSite assay (GDS
Technology, Elkhart, IN
[http://www.gdstech.com/products.htm]) and the
Cardiochek ketone assay ( Polymer Technology Systems,
Indianapolis, IN
[http://www.cardiochek.com/docs/pdf/Ket_english.pdf]).
The KetoSite and Cardiochek devices, which use 15 to 25
L of whole blood, employ reagents incorporated into dry-
film and reflectance photometry to measure the reaction in
which the NADH reduces a tetrazolium salt to produce a
colored product (Table 1, Method 3b).
In the United States, the MediSense, KetoSite, and
Cardiochek products are waived tests and are approved for
use in physicians offices and for self-testing in the home.
-HB
+
-HB + NAD NADH + H+ + AcAc
diaphorase
NADH + tetrazolium dye
NAD+ + reduced tetrazolium dye
colored
The GM7 multi-assay analyzer (Analox Instruments
U.S.A., Lunenburg, MA
[http://www.analox.com/frame1.htm]), couples the -
hydroxybutyrate dehydrogenase (3-HBDH) reaction with a
peroxidase (POD) reaction and monitors the consumption
of oxygen.
3HBDH
-HB + NAD+ acetoacetate +NADH + H+
POD
NADH + 02 + H+ NAD+ + H20
This assay is available only on a small analyzer and is not
used for routine clinical situations.
As was discussed earlier, the most frequent clinical
situation requiring the measurement of ketone bodies is
diabetes, both for the diagnosis of acute diabetic
ketoacidosis and for self-monitoring of diabetic therapy.
Because the nitroprusside method for assessment of blood
and urine ketones is used for emergency room needs, for
physicians office screening, or for home use,
overwhelmingly, most tests for ketones, especially blood
ketones (see below), will not be performed in the
traditional laboratory by technologists. The
semiquantitative nitroprusside test in the form of a dipstick
screen is currently the most simple to use in these clinical
situations. Appropriately, this method is overwhelmingly
the most frequently used test method.
In the United States, the nitroprusside test and the hand-
held devices for measuring -hydroxybutyrate are waived
tests, that is, they can be performed by non-technologists
in physicians offices, in clinics, or in patients homes. All
the other test procedures listed in Table 1 are more
appropriate for research situations.
There have been many other methods suggested for the
analysis of ketones, but these are more applicable for
research use, or they are historic in nature and not used at
present. These methods are listed in Appendix A.
Reference and Preferred Method
There are no accepted reference methods for the
measurement of acetoacetic acid, acetone, or -
hydroxybutyric acid.
In healthy persons, the nitroprusside test for acetoacetic
acid accurately reflects total ketones. However, this test
can be misleading in diabetic ketoacidosis, when -
hydroxybutyric acid is the predominant ketone body
formed. Tests for acetoacetic acid may be negative or
weakly positive, thereby underestimating the total load of
ketone bodies and the degree of ketosis. As the diabetic
ketoacidosis is resolved, more acetoacetic acid can be seen
than is found in the initial stages of the crisis as -
hydroxybutyric acid is metabolized to acetoacetate.
However, there is no strong clinical data available that
indicates that measurement of -hydroxybutyric acid has
any significant advantage over the nitroprusside screen for
 787
Ketones
the diagnosis of acute diabetic ketoacidosis. In most cases,
ketoacidosis is detected with the nitroprusside screen, and
the treatment of the acidosis is usually monitored by
measurements of blood pH. In one report [8], the area
under the ROC curves of serum total ketones and capillary
-hydroxybutyric acid were not statistically different,
leading to the conclusion that serum ketone and blood -
hydroxybutyric acid measurement were equally effective
in diagnosing DKA among uncomplicated cases. This
agrees with similar conclusions drawn by an earlier report
[10].
The primary advantage to the nitroprusside screen is its
relative ease of use and reagent stability, and it is the
appropriate test for ketone detection in most clinical
situations. The major drawback to this procedure is that it
cannot detect -hydroxybutyric acid at all and is five- to
tenfold less sensitive for acetone than for acetoacetic acid.
Acetone is rarely positive at serum concentrations less than
5 mmol/L. In addition, although low, undetectable
amounts of ketones are often present in the urine of
healthy individuals, clinically false-positive results can be
associated with the urine of fasting individuals and up to
30% of the urines of pregnant woman [11]. As with any of
the dry-chemical processes, however, the reagent can
deteriorate unless properly protected from air.
The reported sensitivity of the qualitative Ketostix is 50 to
100 mg/L of acetoacetate, whereas the sensitivity for
Acetest is 25 to 50 mg/L. The dipstick methods are linear
up to 1600 mg/L of acetoacetate.
The enzymatic procedures are specific for acetoacetate or
-hydroxybutyrate and have been readily adapted to highly
automated instruments. The reagents for these assays can
be easily prepared by most laboratories, and these methods
are recommended for those laboratories interested in
specifically quantitating the individual serum and urine
ketone bodies. These laboratories would need to comply
with any regulations regarding the use of home brew
reagents (see CLIA 88 in the United States).
The easy-to-use hand-held devices (MediSense, KetoSite,
Analox, and Cardiochek) for measurement of blood -
hydroxybutyrate makes measurement of this analyte more
readily available for most clinical situations. These devices
use small amounts of whole blood (10 to 25 L) and have
acceptable linearity (up to 21 mg/dL and 70 mg/dL for the
KetoSite and Cardiocheck devices, respectively). The
Cardiocheck device reports possible false-positive results
in the presence of ascorbic acid [12].
Specimen
Well-centrifuged urine, serum, or plasma can be used for
the semiquantitative nitroprusside test, and serum or
plasma can be used for the enzymatic assays as well. The
hand-held devices that measure -hydroxybutyric acid can
use whole blood anticoagulated with heparin or EDTA.
Bacterial contamination of urine can hasten the
disappearance of the ketones from urine. In addition,
acetone is a volatile substance and can be lost by
evaporation. Thus urine samples should be well sealed and
refrigerated within 20 to 30 minutes of collection if not
analyzed immediately. Some urine preservative can affect
test results.
Interferences
Nitroprusside Reaction: Total Ketones
Ketostix can give false-positive results in the presence of
large amounts of levodopa [13], and in general, the urinary
ketone assay is reported to give false-positive results in the
presence of drugs containing sulfhydryl groups, such as the
antihypertensive drug captopril, as well as Mesna, N-
acetylcysteine, dimercaprol, and penicillamine [14]. It
should be noted that many of these drugs, such as
captopril, may be used to treat diabetics, and so this assay
must be used with caution in such cases. The presence of
high concentrations of phenylpyruvic acid and
acetaldehyde in urine can also result in false-positive
results with the nitroprusside procedure.
False-negative results have also been reported for acidic
urine containing large amounts of ascorbic acid [15].
The presence of hemolysis may result in discoloration of
the Ketostix test strip and adversely affect interpretation,
whether by manual or automated procedure.
-Hydroxybutyrate
The urinary semiquantitative nitroprusside test is
acceptable for occasional self-monitoring of ketones, but
the measurement of -hydroxybutyric acid will provide
better clinical sensitivity than will urine testing for
detecting ketosis [16]. The advent of simple hand-held
devices applicable for self-testing for the measurement of
blood -hydroxybutyrate will most likely make this assay
the recommended one in the futurefor the detection of
ketosis for home use and possibly for acute ketoacidosis as
well. The assays for the quantitative measurement are
relatively free of interferences. High levels of acetoacetate
have been found to falsely decrease -hydroxybutyrate
concentrations [3,10]. Elevated blood ascorbic acid levels
may cause false positives in one of the hand-held assays
for whole blood -hydroxybutyrate acid concentrations
[12].
Ketones Reference Interval
The reference interval for serum and urinary total ketones
is less than 50 mg/L (approximately < 500 mol/L).
The waived-test instruments report normal serum levels of
-hydroxybutyrate acid to be < 31 mg/L (<0.3 mmol/L),
while a range of < 52 mg/L (<0.5 mmol/L) was reported in
the NACB document. Henry reports a reference interval
for serum acetoacetate of 5 to 30 mg/L (49 to 294 mol/L)
[17], which compares well with results of 3 to 23 mg/L (29
to 225 mol/L) obtained by a laboratory using an
enzymatic procedure [18]. There do not appear to be any
age-related differences in reference intervals for ketone
 788
Ketones
bodies [19]. At these concentrations, serum and urine
samples will be negative by the nitroprusside
semiquantitative screening tests. See p 4, Reference
Intevals section, for acetoacetic acid reference interval and
-hydroxybutyric acid reference interval of the individual
ketone bodies.
Interpretation
Acetoacetic acid, acetone, and -hydroxybutyric acid are
the products of fatty-acid metabolism that accumulate in
large amounts when the livers capacity to metabolize the
final oxidative catabolite of fatty acids, acetyl coenzyme A
(CoA), is overwhelmed. This occurs in states of starvation,
impaired carbohydrate metabolism (absence of insulin,
such as diabetes; or low carbohydrate diet), or acute
ethanol abuse.
In these states, the oxidative capacity of liver cells is
diminished, and the ratio of NAD/NADH is greatly
decreased, resulting in an inability to oxidize or transfer
acetyl CoA or to allow metabolism by the Embden-
Meyerhof pathway. To allow continued metabolism and
recycling of the coenzyme A cofactor, the hepatocytes
begin active ketogenesis by the following series of
reactions:
Thus a series of ketones or ketone bodies (that is, the
ketones plus -hydroxybutyric acid) are formed. The type
and amount of the ketone bodies formed depend on the
oxidative state of the cells. In early, severe diabetic
ketoacidosis or in acute alcoholic ketoacidosis, the cells
are in a more reduced state (that is, decreased
NAD/NADH ratio), and -hydroxybutyric acid is usually
the predominant ketone body that forms. If these states are
associated with a concomitant lactic acidemia, there can be
an even greater tendency toward production of -
hydroxybutyric acid from acetoacetic acid. As the
biochemical status of the diabetic ketoacidotic patient
improves and the NAD/NADH ratio increases, the -
hydroxybutyric acid levels decrease, while the levels of
acetoacetic acid increase. Thus when measurements of
total ketones are made with the nitroprusside method
(urinary dipsticks), the test results may paradoxically
apparently worsen, indicating an increase in ketones even
as the ketoacidosis is resolving.
The ketone bodies appear in urine when the renal threshold
of ~ 25 mg/dL is exceeded.
Both the American Diabetes Association [11] and the
National Association of Clinical Biochemistry (NACB)
[20] recommend that the measurement of blood ketones is
preferred only as an adjunct test for the diagnosis of acute
diabetic ketoacidosis. Blood ketone measurements may
serve as an adjunct test for assessing the severity and
resolution of diabetic ketoacidosis, although measurements
of blood pH are probably more reliable.
It has been recommended that urinary ketone
measurements not be used for the diagnosis of acute
ketoacidosis [13,14].
Generally, the reported proportions of total serum ketone
bodies are 2% acetone, 20% acetoacetic acid, and 78% -
hydroxybutyric acid [17]. The varying pattern of ketones
and ketone bodies produced in diabetics was reported in a
small series of patients (n = 22) with hyperglycemia
(serum glucose ranging from 1870 to 6140 mg/L, mean
3220 mg/L) [18]. None of the patients had an elevated total
CO2 result, and only 6 had a positive (trace, approximately
50 mg/L) result for ketones by the Ketostix test. Only 1
patient had a normal (<2 mmol/L) lactic acid (range 1.92
to 13.7 mmol/L), though all but 2 had normal (<0.15
mmol/L) pyruvic acid levels. -Hydroxybutyric acid levels
were elevated in 5 of the 22 patients (>0.7 mmol/L) and in
4 of the 6 patients with positive Ketostix results. All 6
patients with a positive Ketostix had acetoacetic acid
levels 0.2 mmol/L. These results are summarized in
Table 2.
Ketones Performance Goals
There are no established performance goals for the
quantitative measurement of total ketones or of the
individual ketone bodies.
Coefficients of variation of less than 8% for levels within
the normal range have been reported for the enzymatic
 789
Ketones
procedures for acetoacetate, pyruvate, or - 13
hydroxybutyrate [6,7].
Quantitative total ketone measurement by reflectance
photometry has precision that is acceptable for clinical use.
The reported within- and between-run reproducibility of
this assay was reported as 11.0% to 3.6% and 11.0% to
5.8% for acetoacetate, 8.2% to 9.2% and 10.4% to 16.1%
for acetone, for high- and low-concentration urine pools,
respectively [4]. However, there is no convincing evidence
that the quantitative assay yields more clinically useful
results than the semiquantitative one.
References
1 Online Mendelian Inheritance in Man. Johns
Hopkins University. Available at
<http://www.ncbi.nlm.nih.gov/entrez/dispomim.c
gi?id=248600>
2 Rothera ACH. Note on the sodium nitroprusside
reaction for acetone. J Physiol 1908;37:491-494.
3 Free AH, Free HM. Nature of nitroprusside
reactive material in urine in ketosis. Am J Clin
Pathol 1958;30:7-10.
4 Penders J, Fiers T, Delanghe JR. Quantitative
evaluation of urinalysis test strips. Clin Chem
2002;48:2236-2241.
5 Penders J, Fiers T, Giri M, Wuyts B, Ysewyn L,
Delanghe JR. Quantitative measurement of ketone
bodies in urine using reflectometry. Clin Chem
Lab Med 2005;43:724-9.
6 Li PL, Lee JT, MacGillivray MH, Schaefer PA,
Siegel JH. Direct fixed-time kinetic assays for
beta-hydroxybutyrate and acetoacetate with a
centrifugal analyzer or a computer-backed
spectrophotometer. Clin Chem 1980;26:1713-
1717.
7 Hansen JL, Frier EF. Direct assays of lactate,
pyruvate, beta-hydroxybutyrate and acetoacetate
with a centrifugal analyzer. Clin Chem
1978;24:475-479.
8 Tantiwong P, Puavilai G, Ongphiphadhanakul B,
Bunnag P, Ngarmukos C. Capillary blood beta-
hydroxybutyrate measurement by reagent strip in
diagnosing diabetic ketoacidosis. Clin Lab Sci
2005;18:139.
9 Umpierrez GE, Watts NB, Phillips LS. Clinical
utility of beta-hydroxybutyrate determined by
reflectance meter in the management of diabetic
ketoacidosis. Diabetes Care 1995;18:137-138.
10 Porter WH, Yao HH, Karounos DG. Laboratory
and clinical evaluation of assays for -
hydroxybutyrate. Am J Clin Pathol 1997;107:353-
358.
11 American Diabetes Association. Tests of
glycemia in diabetes: position statement. Diabetes
Care 2002;25:S97-S99.
12 PTS package insert. Available at
<http://www.cardiochek.com/docs/pdf/Ket_englis
h.pdf>
Ketostix package insert. 1983. Ames, Division of
Miles Laboratories, Inc., Elkhart, IN.
14 Csako G. Causes, consequences, and recognition
of false-positive reactions for ketones. Clin Chem
1990;36:1388-1389.
15 Rosenbloom AL, Malone JI. Recognition of
impending ketoacidosis delayed by ketone reagent
strip failure. JAMA 1978;240:2462-2464.
16 Guerci B, Benichou M, Floriot M, Bohme P,
Fougnot S, Franck P, Drouin P. Accuracy of an
electrochemical sensor for measuring capillary
blood ketones by fingerstick samples during
metabolic deterioration after continuous
subcutaneous insulin infusion interruption in type
1 diabetic patients. Diabetes Care 2003;26:1137-
1141.
17 Mayes PA, Robson W. The determination of
ketone bodies. Biochem J 1957;67:11-15.
18 Sacoolidge J, Donohoo R, Gau N. Unpublished
results. Cincinnati, OH: Clinical Chemistry
Laboratory, University of Cincinnati Hospital.
19 Peden VH. Determination of individual serum
ketone bodies, with normal values in infants
and children. J Lab Clin Med 1964;63:332-343.
20 NACB, LMPG. Guidelines and
Recommendations for Laboratory Analysis in the
Diagnosis and Management of Diabetes Mellitus.
Available at
<http://www.nacb.org/lmpg/diabetes/5_diabetes_
keytone.doc>
21 Eriksson CJP. Micromethod of determination of
ketone bodies by dead space gas chromatography.
Anal Biochem 1972;47:235-243.
22 Kimura M, Kobayashi K, Matsuoka A, Hayashi
K, Kimura Y. Head-space gas-chromatographic
determination of 3-hydroxybutyrate in plasma
after enzymic reactions, and the relationship
among the three ketone bodies. Clin Chem
1985;31:596-8.
23 Brega A, Villa P, Quadrini G, Quadri A, Lucarelli
C. High-performance liquid chromatographic
determination of acetone in blood and urine in the
clinical diagnostic laboratory. J Chromatography
1991;553:249-54.
24 Scott-Wilson H. A method for estimating acetone
in animal fluids. J Physiol 1911;42:444-470.
25 Van Slyke DD. Studies of acidosis. VII. The
determination of -hydroxybutyric acid,
acetoacetic acid, and acetone in urine. J Biol
Chem 1917;32:455-493.
26 Procos J. Modification of the spectrophotometric
determination of ketone bodies in blood enabling
the total recovery of -hydroxybutyric acid. Clin
Chem 1961;7:97-106.
27 Tsao MU, Lowrey GH, Graham EJ.
Microdetermination of acetone in biological
fluids. Anal Chem 1959;31:311-314.
28 Nadeau G. The interference of acetone in blood
alcohol determinations: a simple method for the
 790
Ketones

determination of blood acetone (Ravin modified).
Can Med Assoc J 1952;67:158-159.
29 Amlathe S, Gupta VK. Spectrophotometric
determination of acetone using vanillin. Analyst
1990;115:1385-7.
30 Drews PA. Carbohydrate derivatives and
metabolites. In: Henry RJ, Cannon DC,
Winkleman JW, eds. Clinical Chemistry:
Principles and Technics. 2nd ed. Hagerstown,
MD: Harper & Row; 1974:Chapter 26.

Tables
Table 1: Methods for Ketone Analysis
Method 1: Colorimetric; semiquantitative and quantitative
Principle of analysis: Na2Fe(CN)5NO + acetone/acetoacetate purple color
Comments: Most common; frequently used as a stat procedure; sensitivity for acetoacetate five times greater than for
acetone; does not detect -hydroxybutyric acid.
Method 2: Enzymatic; quantitative
Principle of analysis:
__________pH 7.0__________

NADH + H+ + AcAc -hydroxybutyrate dehydrogenase -HB + NAD+

pH 8.5-9.5
Comments: Rare, not useful as a stat procedure; by adjustment of pH and cofactor concentration (that is, NADH or
NAD), reaction used to quantitate AcAc and -HB
Method 3: Enzymatic; quantitative
a. Principle of analysis:
NADH + H+ + AcAc + electrons -Hydroxybutyrate dehydrogenase -HB + NAD+
pH 8.5-9.5
Current generated by re-dox reaction detected by electrochemical detector.
Comments: Possibly useful as an office, stat, or home procedure.

b. Principle of analysis:
NADH + H+ + AcAc + electrons -Hydroxybutyrate dehydrogenase -HB + NAD+
pH 8.5-9.5

NADH + oxidized tetrazolium dye reduced tetrazolium dye + NAD+

Colorless colored

Historical and Research Methods for Acetone (see Appendix A)

Method 4: Gas chromatography; quantitative

Principle of analysis: Acetone is detected by flame ionization detector; AcAc is converted to acetone by heating.
Comments: Rare, not useful as stat test; acetoacetate quantitated by subtraction of nonheated acetone from heated
acetone
Method 5: Acetone; gravimetric or titrimetric, quantitative
Principle of analysis: Acetone is precipitated by mercury salts.
Comments: Historical; measures only acetone
Method 6: Acetone; colorimetric, quantitative
Principle of analysis: see Appendix A below
Comments: Rare; measures only acetone

AcAc, Acetoacetic acid; -HB, -hydroxybutyric acid.

 791
Ketones

Table 2: Pattern of Clinical Results Found in 22 Hyperglycemic Patients [22]*

Glucose: Mean, 3320 mg/L; range, 18706140 mg/L
Ketones, by Ketostix: 6/22 positive (trace)
Lactate: 1/22 positive (>2 mmol/L)
Pyruvate: 2/22 positive (>0.15 mmol/L)
HBA: 5/22 positive (>0.7 mmol/L)
AcAc: 6/22 positive (>0.2 mmol/L)

AcAc, Acetoacetic acid; HBA, -hydroxybutyric acid.

*Individual ketones and ketone bodies measured by enzymatic assays are described elsewhere in the text. Values in
parentheses, Reference intervals.

Figure
Figure 1

Reaction between acetone and the sodium nitroferricyanide (nitroprusside) formulation found in Ketostix (Ames, Division of
Miles Laboratories, Elkhart, IN).
 792
Ketones
Appendix A. Research and Historical Methods for
Measurement of Total Ketones.
Acetone and acetoacetic acid have been quantitated by gas
chromatography (Table 1, Method 4). Acetone is
quantified by a flame ionization detector, and acetoacetic
acid is estimated by measurement of acetone before and
after heating to convert the acetoacetic acid to acetone
[21,22]. The difference represents the amount of
acetoacetic acid present in the sample.
High-performance liquid chromatography [23] has also
been used for quantitation of acetone and acetoacetic acid.
The oldest quantitative methods for the analysis of ketones
employed precipitation of acetone with mercury salts,
followed by gravimetric or titrimetric analysis (Table 1,
Method 5) [24,25]. Colorimetric analyses that have been
used include the reaction of acetone with salicylaldehyde
to form a red color [19, 26] and the formation of
hydrazones by the reaction between the ketones and
dinitrophenylhydrazine [27]. Other colorimetric assays
include the reaction between acetone and vanillin at 50C
to 55C to form divanillalacetone (Table 1, Method 6),
which is red (Amax = 415 nm) under the alkaline
conditions of the reaction [28,29], and the reaction
between nitroferricyanide, ferric chloride, and 2,5-
dichlorobenzene diazonium chloride [30].
The historical methods for urinary ketones based on
precipitation were time consuming and laborious and are
no longer in use. The dinitrophenylhydrazone procedure
requires extraction and is interfered with by ethanol and
acetaldehyde [17]. The salicylate method is not a useful
quantitative test because of the color instability of the
reaction product. The Gerhardt ferric chloride test for
acetoacetate is nonspecific, with false-positive results
occurring with phenols and salicylates, and has poor
sensitivity (250 to 500 mg/L).
The vanillin method is quantitative and fairly specific but
measures only acetone. Its precision is reported to be 15%
for serum acetone levels within the normal range.
793
Lactate Dehydrogenase and Lactate Dehydrogenase Isoenzymes

Lactate Dehydrogenase and Lactate Dehydrogenase Isoenzymes

Mauro Panteghini

Name: Lactate dehydrogenase (LD, L-lactate: NAD+ oxidoreductase)

Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation.
Enzyme number: EC 1.1.1.27
Molecular mass: 134,000 D
Chemical class: Enzyme, protein
Known isoenzymes: LD1, LD2, LD3, LD4, LD5
Biochemical reaction: (See below)
IFCC reference method: [1]

U N F 2 1 -0 2

L a c ta te
CH 3
D ehyd rogen ase CH 3
p H 8 .8 t o 9 . 8 NADH
H C OH + C O +
NAD
C p H 7 . 4 t o 7 .8 C H
O O O O
L -L a c ta te P y ru v a te

Principles of Analysis and Current Usage

i and H (or B), each under separate genetic control. As a
Lactate dehydrogenase (LD) is a hydrogen transfer
enzyme that catalyzes the oxidation of L-lactate (L) to
pyruvate (P) with the mediation of NAD+ as a hydrogen
acceptor. The reaction is reversible, and the reaction
equilibrium is pH dependent, with alkaline pH favoring
the conversion PL and neutral pH favoring the reverse
reaction. Both P and L in excess inhibit enzyme activity,
although the effect of P is greater. The specificity of the
enzyme extends from L-lactate to various related 2-
hydroxyacids and 2-oxo-acids [2]. The catalytic
oxidation of 2-hydroxybutyrate, the next higher
homologue of L, to 2-oxobutyrate is referred to as 2-
hydroxybutyrate dehydrogenase (HBD) activity. EDTA
inhibits the enzyme, perhaps by binding Zn2+; however,
the postulated activator role for zinc ions is not fully
established.
The enzyme has a molecular weight of 134,000 D and is
composed of four peptide chains of two types: M (or A)
i
Lactate Dehydrogenase and Lactate Dehydrogenase
Isoenzymes
Previous and current authors of this method:
First edition: Amadeo J. Pesce, John F. Chapman,
Linda L. Woodard, Lawrence M.
Silverman
Methods book: Amadeo J. Pesce, John F. Chapman,
Linda L. Woodard, Lawrence M.
Silverman
Second edition: Steven C. Kazmierczak
Third edition: Monica Payne, Peter E. Hickman
Fourth edition: Amadeo J. Pesce
Fifth edition: Mauro Panteghini
result, there are five isoenzymes: H4 (designated LD1
because of its faster electrophoretic mobility toward the
anode), H3M1 (LD2), H2M2 (LD3), H1M3 (LD4), and
M4 (LD5). A different, sixth LD isoenzyme, LD-X (also
called LDc), composed of four X (or C) subunits, is
present in postpubertal human testes. A seventh LD,
called LD6, has been identified in the sera of severely ill
patients. The most common method used for separation
of LD isoenzymes is electrophoresis.
LD activity is present in all cells of the body and is
invariably found only in the cytoplasm of the cell.
Enzyme concentrations in various tissues are about 1500
to 5000 times greater than those physiologically found in
serum. Therefore, leakage of the enzyme from even a
small mass of damaged tissue increases the observed
serum activity of LD to a significant extent. Different
tissues show different isoenzyme composition. In the
heart, kidneys (cortex), and erythrocytes, LD1 and LD2
predominate, whereas in liver and skeletal muscle, LD4
and LD5 isoenzymes predominatealthough skeletal
muscle damage may also result in anodic LD patterns.
Isoenzymes of intermediate electrophoretic mobility
account for the LD activity from many sources (e.g.,
spleen, lungs, lymph nodes, leukocytes, and platelets)
[3].
Current routine methods for quantitation of total LD
activity use kinetic spectrophotometry to measure the
interconversion of the coenzyme NAD+ and NADH at
340 nm [3-5]. Older reactions used an indicator dye after
794
Lactate Dehydrogenase and Lactate Dehydrogenase Isoenzymes
a set period of time (end-point) to determine the amount
of reactants consumed or formed [6]. For instance, the
tetrazolium reaction oxidizes NADH using a dye system
to form a colored tetrazolium dye. This method is still
used in some electrophoretic assays for isoenzyme
activity detection.
Reference and Preferred Methods
An LP reference method, optimized for LD1, was
developed by the International Federation of Clinical
Chemistry and Laboratory Medicine (IFCC) [7]. This
method has recently been the basis for developing an
IFCC primary reference procedure for LD at 37C [1].
The most widely used procedures for LD determination
employ the LP reaction, since it is claimed that there
is less dependence on the NAD+ and the L
concentrations and less contamination of NAD+ with
inhibiting products [1,7].
Measurement Conditions [1]
Temperature 37C
Wavelength 340 nm
Buffer, concentration N-methyl-D-glucamine,
325 mmol/L
pH (at temperature) 9.4 (37C)
Volume fraction of sample 0.0435 (1:23)
Concentration of Reagents in the Final Complete
Reaction Mixture
L-(+)-Lactate 10 mmol/L
-NAD+ 10 mmolL
free acid 3.15 mmol/L
lithium salt 6.85 mmol/L
Electrophoretic separation on agarose gels or cellulose
acetate membranes is the procedure most commonly
used to demonstrate LD isoenzymes [8]. After the
isoenzymes have been separated by electrophoresis, a
reaction mixture is layered over the separation medium.
The NADH generated over the LD zones is detected
either by its fluorescence when excited by long-wave
ultraviolet light or by its reduction of a tetrazolium salt
to form a colored formazan.
Specimen
Serum is the preferred specimen for measuring LD
activity. Plasma samples may be contaminated with
platelets, which contain high concentrations of LD [9].
Serum should be separated from the clot as soon as
possible after the specimen has been obtained.
Hemolyzed serum must not be used, because
erythrocytes contain 4000 times more LD activity than
serum. The different isoenzymes vary in their sensitivity
to cold, with LD4 and LD5 being especially labile.
Activity of LD4 and LD5 is lost if the samples are stored
at 20C. Thus serum specimens should be stored at
room temperature, at which no loss of activity occurs for
at least 3 days.
Interferences
Any specimen with visible hemolysis will give falsely
high results because of release of LD from red cells [10-
12].
LD Reference Intervals
Values for LD activity in serum vary considerably,
depending on the direction of the enzyme reaction and
the method used. The reference interval in adult
Caucasian subjects, determined with an assay traceable
to the IFCC reference procedure at 37C, was found to
be 125 to 220 U/L [13]. LD reference limits are higher in
children (180 to 360 U/L) [14].
LD Isoenzymes Reference Intervals
Using an agarose gel technique with fluorometric
quantitation of the generated NADH, the following
reference intervals for isoenzymes were obtained [3].
LD Fraction Activity, U/L* Percent
Total LD 120-250 100
LD1 17-65 14-26
LD2 35-98 29-39
LD3 24-65 20-26
LD4 10-40 8-16
LD5 7-40 6-16
*LP; 37C; pH 9.4
Interpretation
Because of its wide distribution in all tissues, increases
in serum LD activity occur in a variety of clinical
conditions, including myocardial infarction, hepatitis,
hemolysis, and disorders of kidneys, lung, and muscle. A
systematic review of the literature concluded that serum
LD is only relevant in hematology and oncology [15].
Hemolytic anemias significantly increase LD
concentrations in serum. Marked elevations of the LD
activityup to 50 times the upper reference limithave
been observed in the megaloblastic anemias. These
anemias, usually resulting from the deficiency of folate
or vitamin B12, cause the erythrocyte precursor cell to
break down in the bone marrow (ineffective
erythropoiesis), resulting in the release of large
quantities of LD1 and LD2 isoenzymes. These
elevations rapidly return to normal after appropriate
treatment. For monitoring purposes, LD is relevant in
predicting the survival rate (probability of survival) and
duration in Hodgkins disease and non-Hodgkins
lymphoma.
Patients with malignant disease often show increased LD
activity in serum; up to 70% of patients with liver
metastases and 20% to 60% of patients with other
nonhepatic metastases have increased total LD activity.
Notably increased LD is observed in germ cell tumors
(60% of cases), such as teratoma, seminoma of the testis,
and dysgerminoma of the ovary [16]. The percent of
patients with increased LD depended on the stage of the
disease. LD appears to be a useful predictor of outcome
795
Lactate Dehydrogenase and Lactate Dehydrogenase Isoenzymes
in patients with testicular germ cell tumors, melanoma,
and small cell lung cancer.
LD Performance Goals
For a single laboratory, the analytical goals for desirable
performance of LD assays, derived from biological
variation of the enzyme, are as follows [17,18]:
Imprecision (CV): 4.3%
Bias: 4.3%
Total error: 11.4%
At LD activity around the upper reference limit, the
current interlaboratory coefficient of variation (CV)
from the 2007 College of American Pathologists CARB-
B cardiac markers participants survey ranges from
approximately 2% to 6.3% in different peer groups. An
international study assessing the accuracy of LD results
from six diagnostic companies has shown that the results
of none of the companies are within acceptable limits
when compared to the IFCC reference procedure,
implying that current analytical techniques probably
require significant improvement [19].
References
1 Schumann G, Bonora R, Ceriotti F, Clerc-
Renaud P, Ferrero CA, Frard G, et al. IFCC
primary reference procedures for the
measurement of catalytic activity
concentrations of enzymes at 37C. Part 3.
Reference procedure for the measurement of
catalytic concentration of lactate
dehydrogenase. Clin Chem Lab Med
2002;40:643-648
2 McComb RB. The measurement of lactate
dehydrogenase. In: Homburger HA, ed. Clinical
and analytical concepts in enzymology. Skokie,
IL: College of American Pathologists,
1983:157171.
3 Panteghini M, Bais R, van Solinge WW.
Enzymes. In: Burtis CA, Ashwood ER, Bruns
DE, eds. Tietz Textbook of Clinical Chemistry
and Molecular Diagnostics. 4th ed.
Philadelphia, PA: Elsevier Saunders, 2006:597-
643
4 Buhl SN, Jackson KY. Optimal conditions and
comparison of lactate dehydrogenase catalysis
of the lactate-to-pyruvate and pyruvate-to-
lactate reactions in human serum at 25 , 30
and 37 C. Clin Chem 1978; 24: 828-831.
5 Howell BF, McClure S, Schaffer R. Lactate-to-
pyruvate or pyruvate-to-lactate assay for lactate
dehydrogenase: re-examination. Clin Chem
1979;25:269-272
6 Spiegel HE, Symington JA, Hordynsky WE,
Babson AL. Colorimetric determination of
lactate dehydrogenase (L-lactate: NAD
oxidoreductase) activity. In Standard Methods
of Clinical Chemistry, . New York: Academic
Press, Inc.;7:43-46
7 Bais R, Philcox M. Approved recommendation
on IFCC methods for the measurement of
catalytic concentration of enzymes. Part 8.
IFCC method for lactate dehydrogenase (L-
Lactate: NAD+ oxidoreductase, EC 1.1.1.27).
International Federation of Clinical Chemistry
(IFCC). Eur J Clin Chem Clin Biochem 1994;
32: 639655.
8 Moses GC, Ross ML, Henderson AR. Ten
electrophoretic methods compared with a
selected method for quantifying lactate
dehydrogenase isoenzymes in serum. Clin
Chem 1988; 34: 18851890.
9 Bais R, Edwards JB. Plasma lactate
dehydrogenase activity will be increased if
detergent and platelets are present. Clin Chem
1977; 23:1056-1058.
10 Brydon WG, Roberts LG. The effect of
hemolysis on the determination of plasma
constituents. Clin Chim Acta 1972; 41: 435-
438.
11 Frank JJ, Bermes EW, Bickel MS, Watkins BF.
Effect of in vitro hemolysis on chemical values
for serum. Clin Chem 1978; 224: 1966-1970.
12 Blank DW, Kroll MH, Ruddel ME, Elin RJ.
Hemoglobin interference from in vivo
hemolysis. Clin Chem 1985; 31: 1566-1569.
13 Pagani F, Bonora R, Panteghini M. Reference
interval for lactate dehydrogenase catalytic
activity in serum measured according to the
new IFCC recommendation. Clin Chem Lab
Med 2003; 41: 970-971.
14 Freer DE, Statland BE, Johnson M, Felton H.
Reference values for selected enzyme activities
and protein concentrations in serum and plasma
derived from cord-blood specimens. Clin Chem
1979; 25: 565-569.
15 Huijgen HJ, Sanders GTB, Koster RW,
Vreeken J, Bossuyt PMM. The clinical value of
lactate dehydrogenase in serum: a quantitative
review. Eur J Clin Chem Clin Biochem 1997;
35: 569-575.
16 Von Eyben FE. A systematic review of lactate
dehydrogenase isoenzyme 1 and germ cell
tumors. Clin Biochem 2001; 34: 441-54.
17 Ricos C, Garcia-Lario JV, Alvarez V, Caval F,
Domenech M, Hernandez A, et al. Biological
variation database, and quality specifications
for imprecision, bias and total error (desirable
and minimum). The 2004 update.
www.westgard.com/guest26.htm
18 Moses GC, Henderson AR. Biological variance
of total lactate dehydrogenase and its
isoenzymes in human serum. Clin Chem 1984;
30: 1737-1741
19 Jansen R, Schumann G, Baadenhuijsen H,
Franck P, Franzini C, Kruse R, et al. Trueness
verification and traceability assessment of
results from commercial systems for
measurement of six enzyme activities in serum.
An international study in the EC4 framework of
the Calibration 2000 project. Clin Chim Acta.
2006; 368: 160-167.
796
Lactate Dehydrogenase and Lactate Dehydrogenase Isoenzymes

Procedure [1] Assay

Principle
_ compartment held at 37C.
Lactate + NAD+ lactate dehydrogenase pyruvate +
H+ + NADH
The reaction of L and NAD+ is allowed to occur at an
alkaline pH. One follows the reaction by recording the
rate of formation of NADH at 340 nm. The results are
expressed in U/L.
Reagents
Reaction solution [7.30 g (373.8 mmol/L) N-
methyl-D-glucamine, 0.552 g (57.50 mmol/L) lactic
acid, monolithium salt]. Dissolve in about 80 mL
water. Adjust to pH 9.4 (37C) with 2 mol/L
hydrochloric acid. Transfer to a 100 mL volumetric
flask. Fill the water (20C) up to the calibration mark of
the volumetric flask. This solution is stable in a
refrigerator for 1 month.
Start reagent solution [0.240 g (36.23
mmol/L) NAD+, free acid, 0.556 g (78.78 mmol/L)
NAD+, lithium salt, dihydrate]. The NAD+ free acid/
NAD+ lithium salt mixture dissolves rather slowly.
Raising the temperature (up to 40C) speeds up the
dissolving process. Dissolve in about 6 mL water.
Transfer to a 10 mL volumetric flask. Fill water (20C)
up to the calibration mark of the volumetric flask. This
solution is stable in a refrigerator for 1 week.
Note: The use of NAD+ free acid without NAD+ lithium
salt for the preparation of the start reagent solution
reasonably decreases the pH value of the final complete
reaction mixture.
Equipment: A recording spectrophotometer
equipped with a temperature-controlled cuvette
1. Place 2.0 mL of reaction solution into a test
cuvette, and equilibrate in a water bath at 37C for 4 to 5
min.
2. Add 0.1 mL of serum, mix, and incubate for
180 s.
3. Add 0.2 mL of start reagent solution, mix
thoroughly, wait 90 s, and start the automatic recording
of the change in absorbance at 340 nm for additional 180
s.
Calculation
Average the change of absorbance (A) per min
values. If the A values are decreasing or appear
inconsistent (e.g., not linear), repeat the assay with a
diluted specimen.
LD activity (U/L) = A/min ________1________
total vol_
molar absorptivity
sample vol
= A/min _1_ _2.30_ 1000
6.30 0.10
= A/min 3651
Note: The molar absorptivity of NADH is 630 m2/mol
at 339 nm. The method should be linear to 600 U/L.
 797
Lactic Acid

Lactic Acid
Steven C. Kazmierczak
Name: L-Lactic acid, L-lactate
Clinical significance: Refer to Chapter 29, Acid-Base Control and Acid-Base Disorders, in the
5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C3H6O3
Molecular mass: 90.08 D
Merck Index: 5172 to 5174
Chemical class: -Hydroxycarboxylic acid (glucose metabolite)

Structure:
i applications, employ electrochemical detection. The
Principles of Analysis and Current Usage
Measurement of lactic acid, or lactate, in blood is
important because the concentration of this analyte is
related to tissue oxygenation. Lactic acid is considered to
be a reliable indicator of severity, prognosis, and
effectiveness of therapy in patients with sepsis and
multiple organ failure [1,2]. Lactic acid is most commonly
measured by following its oxidation reaction using either
chemical or enzymatic means. Chemical oxidation
methods use either permanganate or manganese dioxide to
degrade lactate to acetaldehyde and CO2 or CO. The
amount of lactate is proportional to the amount of the
products formed. Acetaldehyde can be measured
colorimetrically [3,4,5], by titration [6,7], or by gas
chromatography [8,9]. The CO formed (Table 1, Method
lb) can be determined by titration [10] or gasometric
analysis [11]. The CO2 formed (Table 1, Method lc) can
be quantified gasometrically by a Van Slyke manometer
[12,13].
Enzymatic methods [14,15] employ lactate dehydrogenase
(LD) to oxidize lactic acid to pyruvic acid, with the
formation of NADH (Table 1, Method 2). Hydrazine or
semicarbazide is added to form a complex with the
pyruvate and remove it as a reaction product, thereby
driving the reaction to completion. The reaction is
quantitated by measurement of the formation of NADH by
either absorption spectroscopic or fluorometric methods.
The analysis can be monitored as either an end-point or a
two-point kinetic reaction.
Dedicated lactate analyzers, often used in point-of-care
Methods that use chemical oxidation are accurate and
i
Lactic Acid
Previous and current authors of this method:
First edition: Nancy Gau
Methods edition: Nancy Gau
Second edition: Not updated
Third edition: Steven C. Kazmierczak
Fourth edition: Nancy Gau
Fifth edition: Steven C. Kazmierczak
advantage of using biosensors is that the sample can be
assayed immediately after collection without the need for
sample processing [16]. The principle of measurement is
derived from older methods of lactate oxidation by
ferricyanide, catalyzed by yeast L(+)-lactate
dehydrogenase [17-22] (Table 1, Method 3). The
hexacyanoferrate(II) produced by the reaction is then
oxidized at a platinum electrode against a silversilver
chloride reference electrode. The amount of current
generated at the platinum electrode is a measure of the
concentration of hexacyanoferrate and thus of lactic acid.
A dedicated lactate analyzer that uses an enzyme electrode
sensor concept has been developed (Table 1, Method 4). A
platinum working electrode measures the hydrogen
peroxide formed from the reaction, as follows:
lactate oxidase
Lactate + O2H2O2 + pyruvate
platinum electrode
H2O2 2H+ + O2 + 2e-
The platinum electrode is kept at an electrical potential of
0.70 V with a silversilver chloride reference electrode.
The fabrication of microelectrodes for the measurement of
lactate within single cells has also been described [23]. In
this technique, lactate is measured using an enzyme-linked
assay based upon lactate oxidase and the amperometric
determination of hydrogen peroxide [23].
Reference and Preferred Methods
There is no reference method for lactate.
sensitive but time consuming and laborious. Excessive
oxidation conditions can lead to further reaction of
acetaldehyde and inaccurate results. Thus the reaction
conditions of the methods measuring acetaldehyde must be
carefully controlled. A gas chromatographic method for
measuring the acetaldehyde formed is suitable if the proper
equipment is available. The gas-liquid chromatographic
 798
Lactic Acid

columns, however, are easily contaminated, and care must sodium iodoacetate (final concentration, 0.5 g/L of blood)
be taken to avoid interfering substances such as propylene are stable for up to 2 hours at room temperature [31]. The
glycol and ethanol [9]. iodoacetate does not affect coagulation and thus yields a
suitable serum sample. Heparinized plasma can be used for
The gasometric procedures measuring CO2 produced by lactic acid analysis, but the specimen must be placed on
the oxidation of lactic acid have an advantage over the ice, and the plasma must be separated within an hour of
measurement of acetaldehyde in that excess oxidant, which collection. Plasma samples can also be collected into tubes
can cause further oxidation of the acetaldehyde, does not containing fluoride and oxalate (60 and 12 mmol/L,
affect the formation of CO2. The other gasometric respectively). Lactate is stable for up to 8 hours at room
temperature in tubes collected in this manner [29].
procedure, based on the analysis of CO, is less accurate
and shows a positive bias with both blood and urine
In terms of convenience and accuracy, the recommended
[10,24]. Both gasometric procedures require gasometers,
sample for lactic acid analysis is a serum sample drawn in
which are not routinely available in most laboratories.
a sodium iodoacetate phlebotomy tube. The sample should
be drawn with minimal stasis, preferably without a
Electrochemical devices for lactate analysis are very rapid
tourniquet. If a tourniquet must be used, it should be
(less than 3 min), are automated, and can be used with
released after venepuncture, and the phlebotomist should
whole blood.
then allow several minutes to pass before actually drawing
blood into the phlebotomy tube. Patients should avoid
Enzymatic methods allow direct measurement of lactic
strenuous use of the hand or arm prior to sampling.
acid, are simple to perform, and are adaptable to widely
available automated equipment. For these reasons, the
Interferences
enzymatic procedure employing lactic dehydrogenase is
The presence of hemolysis or high bilirubin concentrations
the method of choice. The Hohorst manual method is an
have been reported to interfere with some methods. High
end-point measurement in the presence of semicarbazide
concentrations of pyruvate and malic acid can interfere
[25]. These reactions generally require a long incubation
with some enzymatic methods [32].
period and are very dependent on the concentration of
various reactants [26].
Lactic Acid Reference Interval
A two-point kinetic method was introduced using an The definition of significant lactic acidosis is defined as a
alkaline pH with hydrazine added to consume the pyruvate blood lactate of 5.0 mmol/L or greater, with an arterial pH
of less than 7.25. In healthy, fasting individuals, less than 2
formed. The method is easily adapted to many automated
analyzers. One limitation of the method is the low upper mmol/L is present in venous blood. Lactate levels in
limit of linearity (5 mmol/L), which requires appropriate arterial blood can be half to two-thirds the level of venous
dilutions of those samples with a moderately elevated blood. These values assume that the patient is at rest;
lactate concentration. The fluorescence method, which higher levels can be seen otherwise. Lactic acid levels
detects NADH by its emission at 450 nm, is an acceptable increase 20% to 50% over baseline fasting values after a
meal [33]. Lactate in capillary blood obtained from
alternative to measurements using absorption
newborns is approximately 50% higher compared with
spectroscopy.
adults [34]. CSF lactate levels usually correlate with levels
seen in blood, although CNS disorders such as bacterial
Specimen
meningitis, cerebrovascular accidents, and intracranial
Rigorous sample processing is necessary for obtaining
hemorrhage can change CNS lactate independently of
accurate lactic acid measurements because blood cells will
those levels seen in blood [35,36]. Cerebrospinal fluid
metabolize glucose to lactic acid, resulting in a positive
lactic acid levels in children 0 to 16 years of age ranged
bias [27,28]. This metabolism is time and temperature
from 1.1 to 2.8 mmol/L [37]. Lactate may also be
dependent. Increases in lactate of 0.4 mmol/L when placed
measured in urine. The normal 24-hour excretion of lactate
on ice and 0.7 mmol/L at room temperature 1 hour
is 5.5 to 22 mmol/day.
following collection have been described [29].
Interpretation
Preservation of lactate concentrations by deproteinization
Lactic acid is a product of the metabolism of pyruvic acid.
of whole blood or serum is neither convenient nor reliable
Healthy individuals produce approximately 1400 mmol of
[30]. Studies on the effect of sodium fluoride and sodium
lactic acid per day, which is produced primarily from
iodoacetate showed an inhibitory effect on glycolysis with
glycolysis and deamination of alanine [38]. Analysis of
either compound and complete inhibition with a mixture of
lactic acid can be used to determine the metabolic basis for
1% sodium fluoride and 1% iodoacetate [28]. Since
an unexplained metabolic acidosis, as determined by a
sodium fluoride can partially inhibit coagulation, the
blood gas analysis. The presence of an elevated anion gap
serum specimens from sodium fluoride phlebotomy tubes
in the absence of an obvious cause of the acidosis, such as
tend to be contaminated with protein clots. One study
renal failure, hyperglycemic ketosis, or sepsis, would
showed that lactic acid levels in samples collected with
prompt a clinician to order a lactic acid analysis [39].
 799
Lactic Acid
An elevated lactic acid level would indicate that the
metabolic acidosis may be a lactic acidosis. Lactic acid is
formed in muscle cells to maintain cellular levels of NAD.
As the oxygen level in muscle cells decreases, the cells
must increasingly depend on anaerobic metabolism by the
glycolytic pathway to generate ATP. To regenerate enough
NAD to allow this pathway to continue metabolizing
glucose, NADH+ is oxidized by reduction of pyruvic acid,
the final product of the Embden-Meyerhof pathway, to
lactic acid by the action of lactate dehydrogenase. Muscle
lactic acid accumulated during periods of tissue anoxia is
released into the general circulation where it can be readily
metabolized by the heart (rich in LD1) and liver.
Serum lactic acid concentration can rise accompanying the
tissue hypoxia associated with vigorous exercise. This can
result in a mild lactic acidosis that disappears as soon as
the exercise ceases. Muscle pain and cramping can
accompany this lactic acidosis. A life-threatening lactic
acidosis can be seen when the tissue hypoxia is caused by
respiratory failure or hypoperfusion states (i.e., shock or
circulatory collapse) [39]. Severe dehydration can also
lead to reduced delivery of oxygen to muscle cells, and
lactic acidosis may often accompany diabetic ketoacidosis
and hyperglycemic type 2 diabetes mellitus. Increased
tissue oxygen consumption, as seen in sepsis and
malignancies, can also result in a lactic acidosis. In all
these cases, the lactic acidosis will remain until the cause
of the reduced delivery of oxygen to tissues is corrected.
This usually requires correction of the respiratory or
cardiac disorder or rehydration of the patient.
The degree of acidemia due to lactate can often help
indicate the severity of the underlying disease. The
survival rate for patients with very elevated blood lactic
acid levels (>10.5 mmol/L) was worse (30%) than for
patients with lower lactate levels (65%) [40]. There is a
report of lactic acidosis (lactate, 18 mmol/L; pH, 7.25)
resulting from the ingestion of large amounts of propylene
glycol [40]. The high lactate level resulted from hepatic
metabolism of the propylene glycol to lactate.
There have been several reports on the relationship
between cerebrospinal fluid (CSF) lactic acid levels and
central nervous system (CNS) infections [41,42]. Increased
CSF lactate is usually associated with decreased CSF
glucose concentrations because of increased anaerobic
glucose utilization. Elevated lactate levels (>4.0 mmol/L)
are associated with CNS infections of bacterial, fungal,
and mycobacterial, but not viral, origin [42]. In known
bacterial meningitis, decreasing CSF lactate levels reflect
successful treatment of the disease. Increased CSF lactic
acid can also result from impaired CNS blood supply
because of space-occupying tumors. Thus elevated CSF
lactate has a high sensitivity for a CNS bacterial infection
but a lower specificity for the disease.
Unless otherwise specified, lactic acidosis refers to
acidosis caused by L-lactic acid. This is the normal isomer
produced in humans. The production of D-lactic acid by
bacteria in the colon can cause D-lactic acidosis. Patients
who have undergone jejunoileal bypass surgery can
present with extremely high plasma concentrations of this
metabolite during acidotic episodes as a result of increased
production by intestinal bacteria due to malabsorption of
carbohydrates [43]. Essentially all of the methods in use in
clinical laboratories utilize L-lactate dehydrogenase, which
does not detect D-lactate. D-Lactate can be measured using
enzymatic assays that utilize D-lactate dehydrogenase.
Lactic Acid Performance Goals
Participant summary reports from 2008 by the College of
American Pathologists indicate that the majority of
laboratories measure lactic acid using an automated
enzymatic assay. Less than 5% report using an electrode-
based system for measurement of lactate.
Acceptable performance criteria for lactic acid requires
that laboratories obtain lactate values that are within 2 SD
or 0.2 mmol/L of the peer-group mean, whichever is
greater. Lactic acid is not currently regulated for
proficiency testing. Interindividual biological variation
measured in 8 healthy individuals over a 5-day period was
determined to be 16.7% [44]. Desirable specifications for
analytical imprecision derived from studies of biological
variation indicate an assay imprecision of no greater than
13.6% [45]. Methods used in clinical laboratories for
measurement of lactate show analytical imprecision that is
well within the limits deemed necessary for medical
decision making. The vast majority of lactate methods
used in clinical laboratories are enzymatic methods
involving the oxidation of lactate to pyruvate. A much
smaller percentage, approximately 5%, utilize methods
involving the oxidation of lactate to pyruvate.
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19. Racine P, Engelhardt R, Higelin JC, Mindt W. An information in CSF. Diag Med 1981;Jan-Feb:23-
instrument for the rapid determination of L-lactate 33.
in biological fluids. Med Instrum 1975;9:11-14. 37. Knight JA, Dudek SM, Haymond RE. Increased
20. Racine P, Klenk HO, Kochsiek K. Rapid lactate cerebrospinal fluid lactate and early diagnosis of
determination with an electrochemical enzymatic bacterial meningitis. Clin Chem 1979;25:809-
sensor: clinical usability and comparative 810.
measurements. J Clin Chem Clin Biochem 38. Marko P, Gabrielli A, Caruso LJ, Mizock BA,
1975;13:533-539. Franklin C. Too much lactate or too little liver? J
21. Soutler WP, Sharp F, Clark DM. Bedside Clin Anesthesia 2004;16:389-395.
estimation of whole-blood lactate. Br J Anaesth 39. Narins RG, Rudnick MR, Bastl CP. Lactic
1978;50:445-450. acidosis and the elevated anion gap (II). Hosp
22. Piquard F, Schaefer A, Haberey P. Bedside Pract 1980;June:91-98.
estimation of plasma lactate. Clin Chem 40. Anderson CT, Westgard JO, Schlingen K,
1980;26:532-533. Birnbaum ML. Contribution of arterial blood
23. Cai X, Klauke N, Glidle A, Cobbold P, Smith lactate measurement to the care of critically ill
GL, Cooper JM. Ultra-low-volume, real-time patients. Am J Clin Pathol 1977;68:63-67.
measurements of lactate from the single heart cell 41. Bland RD, Lister RC, Reis JP. Cerebrospinal
using microsystems technology, Anal Chem fluid lactic acid level and pH in meningitis. Am J
2002;74:908-914. Dis Child 1974;128:151-156.
24. Maver MC. The Schneyer method for the 42. Brook I, Bricknell KS, Overturf GD, Finegold
determination of lactic acid in urine. J Biol Chem SM. Measurement of lactic acid in cerebrospinal
1917;32:71-76. fluid of patients with infections of the central
nervous system. J Infect Dis 1978;137:384-390.
 801
Lactic Acid

43. Bongaerts G, Tolboom J, Naber T, Bakkeren J, 45. Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Severijnen R, Willems H. D-lactic acidemia and Hernndez A, Jimnez CV et al. Current database
aciduria in pediatric and adult patients with short on biologic variation: pros, cons and progress.
bowel syndrome. Clin Chem 1995;41:107-110. Scand J Clin Lab Invest 1999;59:491-500.
44. Panteghini M, Pagani F. Biological variation of
lactate and pyruvate in blood. Clin Chem
1993;39:908.

Table
Table 1: Lactic Acid Methods Summary Table
Method 1: Chemical oxidation with formation of:
Principle of analysis: Permanganate or MnO2 oxidizes lactate to one of several products in one of the following
reactions
Comments: Historical; accurate, precise, and sensitive, but time consuming
Method 1a: Acetaldehyde (CH3CHO); quantitative, end-point
Principle of analysis:
Lactate H2SO4, heat acetaldehyde + H2O + CO
Method 1b: CO (carbon monoxide); quantitative, end-point
Principle of analysis: As in Method 1a
Method 1c: CO2 (carbon dioxide); quantitative, end-point
Principle of analysis: 2 lactate + O2 2 acetaldehyde + 2CO2 + H2O
Method 2: Enzymatic; quantitative, end-point or kinetic
Principle of analysis: Lactate + NAD+ LD pyruvate + NADH + H+
Comments: Automated; accurate and precise; automatable and rapid
Method 3: Electrochemical; quantitative, end-point
Principle of analysis:

Lactate + 2Fe(CN)63 pyruvate + 2H+ + 2Fe(CN)64
2Fe(CN)64 Pt. electrode 2FE(CN)63 + 2e
Comments: Automated; accurate, precise, and sensitive, rapid analysis, but equipment not readily available
Method 4: Enzyme electrode sensor; quantitative, end-point
Principle of analysis:
Lactate + O2 LO H2O2 + pyruvate
H2O2 2H+ + O2 + 2e
Current measured is proportional to hydrogen peroxide concentration
Comments: Semiautomated
K, Kinetic analysis mode; EP, end point analysis mode; LD, lactate dehydrogenase; LO, lactate oxidase.

Procedure: Enzymatic Analysis for Lactic Acid
Editors Note: While the following assay was developed
for a specific analyzer, it can easily be adapted to any
automated spectrophotometer.
Principle
This method is based on the enzymatic oxidation of lactate
to pyruvate by lactate dehydrogenase in the presence of
NAD. The reaction is as follows:
LD, pH 9.8
L-Lactate + NAD + pyruvate + NADH + H+
At this pH, equilibrium favors oxidation of lactate to
pyruvate. If hydrazine is added, it reacts with pyruvate and
removes the pyruvate as it is formed, further shifting the
equilibrium to the right. The rate of increase in absorption
of the NADH formed, measured at 340 nm, is related to
the concentration of lactic acid in the sample. Instructions
are given for an ABA-100 and a centrifugal analyzer.
However, this procedure is easily adapted to any random
access analyzer.
Reagents
1. TrisEDTAhydrazine buffer (499, 11.9, and
226 mmol/L, respectively). Dissolve 60.5 g of
hydroxymethylaminomethane (Tris) and 4 g of disodium
ethylenediaminetetraacetate in 800 mL of distilled water.
Add 11 mL of hydrazine hydrate. Adjust the pH to 9.8, and
dilute to 1 L with distilled water. Stable 6 months at 4C.
2. Beta-NAD (Product no. 7004, Sigma Chemical
 802
Lactic Acid

Co., St. Louis, MO). Preweigh 65.4 mg (0.1 mmol) beta- 5. Set 0 absorbance on position 1 with zero knob.
NAD into test tubes. Cover, and store at 4C. Stable for at 6. Push calibrate button in, and set to 0.500 with
least 1 month. Dissolve preweighed NAD into 3 mL of scale knob.
distilled water before preparing the working reagent. 7. Return carousel to starting position, and push run
3. Lactate dehydrogenase (Boehringer button.
Mannheim Catalog no. 127230). An ammonium sulfate 8. When carousel begins to move, spin it back to the
suspension of purified rabbit muscle LD, approximately starting position. It will now print first absorbance
550 U/mg. readings.
4. Working reagent. Mix 27 mL of buffer, 3 mL of 9. After second print (5 min later), perform
dissolved NAD, and 40 L of LD solution. Mix. Stable 24 calculations.
h at 4C.
5. Stock lactate standard (20 mmol/L). Dissolve Calculation
192 mg of lithium L(+)-lactate (available from the 1. Calculate change of absorbance (A) for each
National Bureau of Standards) in 100 mL of distilled cuvette. Subtract first print from second print.
water. Stable 6 months at 4C. 2. Use the following formula:
6. Working standards
2 mmol/L. 1 mL of 20 mmol/L standard, diluted Absorbance UNK
volumetrically to 10 mL with distilled water. Conc STD Conc UNK (nmole / L)
5 mmol/L. 2.5 mL of 20 mmol/L standard, diluted AbsorbanceSTD
volumetrically to 10 mL with distilled water.
Both standards are stable for 2 months at 4C. 3. For unknowns (UNK) less than 5 mmol/L, use the
Assay (Reaction Conditions for Lactate Analysis on ABA- 2 mmol/L standard (STD) for the calculation. For
100 Table ) unknowns greater than 5 mmol/L, use the diluted sample
Equipment: ABA-100 (Abbott Diagnostics, for calculation, again either the 2 or 5 mmol/L standard
Chicago, IL) (click here).
1. Test parameters: Notes
Temperature 37C 1. The between-run coefficient of variation for the
Mode End-point ABA-100 enzymatic method is 8.9% at a lactate
Normal/fast reaction rate Normal concentration of 4 mmol/L.
Direction Up 2. This method has also been adapted to a
Time 5 min centrifugal analyzer (CentrifiChem 400, Baker
Revolutions 3 Instruments, Allentown, PA). Use the following
Filter 340/380 parameters on this instrument:
Syringe plate 1:51 Pipettor Analyzer
Decimal 0.000 10 L of sample 30C
Calibration factor 0.500 50 L of diluent 340 nm
Set after initial dispersing. 350 L of reagent t0 at 3 sec
Zero setting 0
(place reagent in 0 cuvette) t at 4 min
2. Follow normal operational procedures according
Automatic mode
to Abbott ABA-100 instrument manual.
Terminal Concentration
3. Analyze both the 2 and 5 mmol/L lactate
Follow normal pipetting and operational guidelines for the
standards in each run. Analyze patient samples, undiluted
CentrifiChem as outlined in the CentrifiChem procedure
and diluted fivefold with distilled water.
manual. Set print to 2 or 5 mmol/L standard, according to
4. After the last sample is aspirated and dispensed,
the approximate concentration of the unknown.
push stop button. Go to test mode.
803
Lead
Lead
Gus Koerbin
Name: Lead
Clinical significance: Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: Pb
Molecular weight: 207.2 D
Merck Index: 5227
Chemical class: Metal
i urine sample [7,10]. After sample preparation, the
Principles of Analysis and Current Usage
Lead, a periodic-system Group IVB metal, occurs in
three oxidation states, 0, +2, and +4. The +2 oxidation
state is the most widely occurring form of the inorganic
salts of lead, whereas Pb(IV) is the most important state
in organolead compounds. A complete discussion on
lead regarding sources of occupational exposure has
been published [1].
The method historically used to analyze lead in both
biological and environmental samples is the dithizone
compleximetric method (Table 3, Method 1) [2-4]. The
sample, 10 mL of blood or 25 mL of urine, tissues, or
particulates, is ashed by acid oxidation to remove
organic material. After ashing, the sample is redissolved
in acidified water. The pH is adjusted to 9-10, cyanide is
added to eliminate reaction with other metals, and then
the sample is reacted with diphenylthiocarbazane to form
the red leaddithizonate complex. The complex is
extracted into chloroform, and the absorbance of the
complex is measured spectrophotometrically at 510 nm
(Figure 1).
Many methods of sample preparation, concentration, and
spectrophotometric techniques for atomic absorption
spectroscopy (AAS) have been reported [5-11].
However, all have the common features of (1) a
digestion or treatment of the sample to allow separation
of the lead from the sample matrix, (2) aspiration of the
ionic lead into a flame or heating of the ionic lead
solution by electrical means, where it is reduced to the
atomic state, and (3) spectrophotometric quantitation by
measurement of the light absorbed by the lead at its
characteristic resonance frequency of 283.3 nm [5-8,10].
A sample-ashing procedure using acid oxidation similar
to that used for the dithizone method [2-4] (Table 3,
Method 2a) typically requires a 10-mL blood or 50-mL
i
Lead
Previous and current authors of this method:
First edition: M. Wilson Tabor
Methods edition: M. Wilson Tabor
Second edition: M. Wilson Tabor
Third edition: M. Wilson Tabor
Fourth edition: M. Wilson Tabor, Tania Carreon-
Valencia
Fifth edition: Gus Koerbin
solution of ionic lead is aspirated into an air-acetylene
flame, where it is reduced to the atomic state.
Quantitation is accomplished by measurement of the
light absorbed by the lead at 283.3 nm.
Although numerous variations in this AAS procedure
have been published, two are noteworthy. The first
features co-precipitation of the lead with bismuth
hydroxide after sample digestion [8]. After
centrifugation and supernatant fluid removal, the
precipitate is dissolved in acid for aspiration into the
flame. The second method features chelation of the lead
with pyrrolidine dithiocarbamate after sample digestion
[7,10]. This lead chelate is extracted into methyl isobutyl
ketone (MIBK), and then the MIBK extract is aspirated
into the flame.
In addition to these variations, a blood micromethod for
flame AAS is also widely used [12,13]. This procedure,
the Delves microscale technique [9], does not use the
acid oxidation sample-ashing procedure. In the Delves
cup method, 10 L of blood, obtained by a finger stick,
is added to a special nickel cup that contains hydrogen
peroxide to partially oxidize the sample. After the
sample preparation procedure, the cup is inserted into the
AAS flame for the lead determination (Table 3, Method
2b).
Other major variations in the classical flame AAS
procedures now widely used are the flameless or
electrothermal AAS procedures that are otherwise
referred to as the graphite furnace [6] and carbon rod
[11] techniques (Table 3, Method 2c). Some
electrothermal procedures [6] feature the sample-ashing
by acid oxidation, as described for the classical AAS
methods, whereas in others a microsample, 5 L, is
introduced directly into the instrument [11]. The
electrothermal AAS procedures feature a graphite tube
or carbon rod held on the optical axis of the AAS unit.
The tube is protected from the atmosphere by flowing
nitrogen or argon. After sample introduction, the
graphite tube or carbon rod is heated by electrical
resistance successively to a drying temperature of
100C, followed by a charring temperature of 400C,
and finally to an atomization (reduction) temperature of
2000C to 2500C. During atomization, a high
concentration of Pb0 is formed in the light path, but the
804
Lead

atoms quickly escape by diffusion. Lead absorbs light at
both 280.2 and 283.3 nm, and the absorbance is
proportional to the lead concentration in the sample.
The third general category of lead analytical methods is
electrochemical. These anodic stripping voltammetry
(ASV) instrumental methods have become widely
accepted methods for lead analysis [14-17] (Table 3,
Method 3). In this methodology, ionic lead in blood or
urine samples is first reduced to elemental lead by a
negative potential applied to a working mercury
electrode. The reduced lead is deposited on the mercury
electrode.
Pb+2 + 2 e- Pb0 (deposited on mercury electrode)
After a preselected time, the working electrode potential
is moved toward more positive values, thereby causing
the reoxidation of the lead from the mercury electrode.
From this oxidation the resulting anodic current, which
is proportional to the concentration of lead, is measured.
Pb0 (from mercury electrode) Pb2+ + 2 e-
The ASV technique has become a reliable and
reproducible method for microliter-sized blood samples
because of the design of an efficient mercurygraphite
composite electrode. This electrode has been
incorporated into equipment for the semiautomated
determination of lead in blood, urine, water, foods, and
reagents. Sample preparation for ASV is facilitated by
the use of the Metexchange reagent, a chromate-based
digestion solution containing an antifoaming surfactant.
The sample preparation is simplified to the addition of
sample to the Metexchange reagent. The instruments are
designed to measure the current generated during a
specific window of voltage during which lead is
stripped off the mercury-graphite electrode. More
accurate values may be obtainable if the current change
during analysis is plotted on a strip recorder. Usually the
peak area of the lead peak is used for calculations, but
peak height can be used as well. Two ASV methods are
recommended by The National Institute for
Occupational Safety and health (NIOSH), one requiring
100 L of blood and the other requiring 1.0 mL of urine
[14-16]. Blood ASV methods are widely applicable to
routine monitoring of biological samples during
treatment for lead intoxication and for studies of lead
exposure in children and in workers who are
occupationally exposed, such as in the smelting and
battery industries.
There are several nonroutine methods for the analysis of
lead in a variety of sample matrices [12,13]. These
methods include electron microprobe, x-ray
fluorescence, neutron activation analysis, and mass
spectrometry. All require the use of expensive facilities
and a high degree of expertise for the analyst. In neutron
activation analysis for lead, the sample is irradiated with
fast neutrons, energy greater than 1 MeV, converting the
stable 206Pb isotope to the metastable radioisotope
207Pb. Quantitation is accomplished by measurement of
the gamma rays emitted from the radionuclide 207Pb
isomeric transition to the stable 206Pb isotope. The
theory and practical applications of this technique have
been described in numerous reviews [17].
Reference and Preferred Methods
There is no reference method for the determination of
blood lead. There is a definitive method for lead
determination in both whole blood and urine, employing
isotope dilution inductively coupled plasma mass
spectrometry (ICP/MS) [18] (Table 3, Method 4). This
method has been employed by the Centers for Disease
Control and Prevention (CDC) for establishing lead
concentrations in their samples for the licensure and
proficiency testing program [19].
The choice of methodology for the
determination of lead in a particular laboratory is
dependent on the following several factors: the
availability of equipment, the number of samples to be
analyzed per day, the purpose for the analysis, and
experience of those undertaking the analysis.
The atomic absorption method has been
published as a selected method of the American
Association for Clinical Chemistry [8] and has shown
accuracy, in terms of relative recovery, of 98.8% 1.0%
for lead in blood, 99% 4.5% for lead in urine, and 97%
3.6% for lead in hair.
The dithizone procedure is subject to certain
interferences, but the method has the advantage of being
thoroughly tested and evaluated. Disadvantages of the
method include use of large quantities of scrupulously
cleaned glassware, large quantities of reagents, and the
procedure is highly susceptible to contamination from
outside sources and is tedious and time consuming.
Atomic absorption spectroscopy is recommended as a
general procedure. The utility of this method is its wide
applicability. AAS methods using an electrothermal
furnace (flameless AAS) have the advantage of this
greater sensitivity of one to two orders of magnitude
over flame AAS procedures [6,11]. Disadvantages of the
electrothermal AAS procedure are that sample matrix
effects are more prominent than in flame atomic
absorption, analysis is time consuming, and the
equipment is expensive compared to flame AAS. The
flame AAS procedure utilizing the MIBK extraction
technique offers several advantages [7], such as speed of
analysis, no requirement for a high level of technical
skill, and no known interferences. The main
disadvantage is the requirement for larger sample sizes
than those in some of the other AAS [9] and ASV [4,16]
procedures. The principal advantage of the bismuth
hydroxide precipitation method [8] is its speed, making
it practical for routine monitoring of biological samples
during treatment for lead intoxication.
The Delves cup method [9] has the advantage of small
sample size, speed, and the requirement for only a
minimum of technical competence. Disadvantages
include interferences from background absorptions and
matrix effects and variations in lot-to-lot cup quality.
805
Lead
ASV methods have high sensitivity and are less
expensive per analysis than other methods in terms of
equipment cost and time required for analysis. The
principal disadvantage is that thallium interferes because
the currentvoltage peak for this element is not resolved
from lead when the two metals are at comparable
concentrations. The ASV methods are recommended for
laboratories having a need to do lead analyses on a
routine basis, particularly for doing monitoring studies.
The AAS methods are recommended for laboratories
having a need to do not only lead but also other metals
on a routine basis, particularly in doing analyses on
samples in addition to blood and urine.
Specimen
Whole blood is the sample of choice, but urine can be
used for determining lead in humans. Blood must be
collected in lead-free heparinized vacutainers. EDTA
may also be used as an anticoagulant. Caution should be
exercised during sample handling to prevent any external
contamination. No differences in blood lead
concentrations have been observed between properly
collected venous and capillary specimens. Heparinized,
refrigerated blood samples are stable for 2 weeks, but
EDTA blood samples are stable for several months if
frozen at 20C. The sample is suitable for analysis by
all four methods listed in Table 3. If EDTA is used as an
anticoagulant, 1.4 mg of calcium chloride per milliliter
of blood must be added to enhance lead recovery from
the samples [20].
For the extraction/AAS procedure utilizing ammonium
pyrrolidine dithiocarbamate as a chelating agent [7,10],
the calcium chloride should be added to the sample after
the addition of the chelating agent [21].
Urine samples must be collected in lead-free borosilicate
or polyethylene bottles. At least 50 mL should be
collected and the specific gravity measured. The sample
must be preserved by the addition of 500 mg of thymol
per liter of urine. The urine is stable for 1 week if
refrigerated. The sample is suitable for analysis by all
three methods.
Interferences
Some lots of heparin used as an anticoagulant for blood
lead measurements have been reported to contain lead
[22]. Lead AAS measurement by direct aspiration of
urine has been shown to give falsely elevated results.
This interference is caused by nonspecific absorption of
urinary constituents such as calcium, creatinine, and urea
and may falsely increase the result by up to 100 g/L.
By subtracting the absorbance reading at 220 nm (non-
lead peak) from the lead peak at 217 nm, correction for
this interference is achieved. This correction is
applicable to both direct aspiration of urine and
determinations where there is co-precipitation by
bismuth [23]. The dithizone procedure is subject to
interference from tin(II), bismuth, and thallium. The
ASV methods are subject to interference from thallium,
because the currentvoltage peak for this element is not
resolved from that for lead when the two metals are at
comparable concentrations.
Lead Reference Intervals
There are two classes of persons who must be considered
in regard to lead exposure: those exposed occupationally
and those exposed environmentally. The Occupational
Safety and Health Administration (OSHA) has two
standards for lead: 29 CFR 1926.62 covers construction
industry activities, and 29 CFR 1910.1025 covers
general industry such as foundries and the manufacture
of lead-containing products [24,25]. These laws require
the employer to keep lead exposures at or below 50
g/m3 of air. This level is called the Permissible
Exposure Limit (PEL), based on an 8-hour time-
weighted average (TWA).The World Health
Organization (WHO) has stated that blood lead levels >
30 g/dL indicate significant exposure. The maximum
allowable blood lead concentration in workers has been
set at 60 g/dL (2.9 mol/L), a level at which health
effects are observed. The typical range for healthy non-
exposed adults is 10 to 20 g/dL (0.48 to 0.96 mol/L),
with values for children being lower [1,22,26]. Blood
and urine lead concentrations have been reported to be
about 10% to 20% higher in males than females [26].
Interpretation
The pathological effects of lead have been recognized
for centuries. Many physiological systems, including
renal, nervous, reproductive, endocrine, immune, and
hemopoietic, are affected. These effects have been the
subject of numerous reviews and research publications.
For example, Damstra [27] has presented an overview of
the toxicological properties of lead, and Mahaffey [28]
has presented an overview of sources of human exposure
and bioavailability.
Exposure to lead, either acute or chronic,
presents a variety of signs, symptoms, and chemical
evidence. The exposed person may be asymptomatic or
symptomatic. A number of signs and symptoms of
exposure have been observed. For exposed adults, Lane
et al. [29] listed tiredness, lack of energy, constipation,
slight abdominal discomfort, anorexia, altered sleep,
irritability, anemia, pallor, and less frequently diarrhea
and nausea. More severe signs and symptoms include
colic, reduction of muscle power, muscle tenderness,
paresthesia, and symptoms or signs of neuropathy or
encephalopathy. A short list, in descending order of
frequency of presentation, has been published for
exposed adults [30]. These include abdominal pain,
constipation, vomiting, non-abdominal pain, seizures,
paresthesia, psychological symptoms, and diarrhea. The
symptoms most frequently seen in children are
irritability, vomiting, abdominal pain, ataxia, anorexia,
behavioral changes, speech disturbances, seizures,
intercurrent fever, and dehydration [31,32].
A number of reviews of the clinical
presentation of lead exposure have been published
[13,33]. Other biochemical parameters indicative of lead
806
Lead

exposure include delta-aminolevulinic acid, delta- Blood lead concentration 20-24 g/dL (0.97-1.16
aminolevulinic acid dehydratase, and porphyrins. mol/L). Minimal data exists on chelation therapy for
Toxic Levels of Lead in Blood children in this range, and children probably should not
There is no safe blood lead concentration. Virtually any be chelated except in the context of approved clinical
amount of lead in blood is now considered to have some trials.
degree of toxicity. Table 1 below summarizes the effects
of lead on children as blood lead levels increase. Note Blood lead concentration 25-44 g/dL (1.21-2.12
that low levels of lead have very subtle effects; only at mol/L). In this blood lead range, treatments vary from
high levels are signs and symptoms obvious. clinic to clinic. Some practitioners will treat children at
this level with one or more of the drugs listed in the table
Table 1: Lowest Blood Levels at Which Clinical above, on an outpatient basis.
Effects are Observed in Children
Blood lead Blood lead concentration 45-69 g/dL (2.17-3.33
concentration (g/dL): Effect:
mol/L). Children with levels greater than 45 g/dL
All Lead deposited into bones
should be referred for immediate appropriate chelation
Less than 10 Production of red blood cells
slows down therapy.
1520 Abnormally high amount of
EP in the blood When chelation therapy ceases, some rebound may
Below 25 Subtle behavioral and occur. The action of the chelators has caused
learning changes mobilization of lead from tissue stores, and this process
5060 Loss of nerve signals from may continue after chelation therapy ceases
brain to muscle
80100 Brain damage (lead Lead Performance Goals
encephalopathy) Survey data from the 2007 College of American
Pathologists Participant Summary Report shows
Available Treatments for Lead Poisoning imprecision values (% coefficient of variation) for
The most effective drugs to treat lead poisoning are measurement of lead at a mean concentration of 21.2
chelating agents that attach to the lead in a form which g/dL range from 7.4% for anode stripping voltammetry
can leave the body through the urine. As lead is removed to 10.2% for methods employing ICP/MS.
from the blood, lead moves out of tissues into the blood
to be in turn bound and removed. All drugs, including Acceptable Clinical Laboratory Improvement
chelators, have potential side effects and are used with Amendments of 1988 (CLIA-88) performance criteria
caution. Several different drugs of this type are used in for measurement of lead in blood require that
the treatment of lead poisoning and are described below. laboratories be accurate to within 4 g/dL or 10% of
the peer group mean. A problem associated with
Table 2: Chelating Agents Used in Treating Children obtaining accuracy and precision in all methods of
With Lead Poisoning analysis is a lack of adherence to method protocols [12].
Product name: Calcium disodium EDTA, or versenate
In terms of accuracy, an overestimation of lead is
Generic name: Calcium disodium edetate, or
probably attributable to lead contamination of glassware,
edetate calcium
Chemical name: Calcium disodium reagents, or equipment and inadequate blank correction.
ethylenediaminetetra-acetic acid, [[N,N'-1,2- An underestimation is probably attributable to
ethanediyl-bis[N-(carboxymethyl)- incomplete analytical recovery in extraction or
glycinato](4)-N,N',O,O',ON,ON']-, disodium, concentration steps and nonlinearity of calibration
hydrate, (OC-6-21)-Calciate(2-) curves.
Abbr: CaNa2 EDTA
References
Product name: BAL in oil 1 U.S. Department of Health & Human Services.
Generic name: Dimercaprol Criteria for a recommended standard:
Chemical name: 2,3-dimercapto-1-propanal
occupational exposure to inorganic lead,
Abbr.: BAL
revised criteria. NIOSH, DHEW Publication
Product name: Cuprimine
Generic name: D-Penicillamine No. 78-158. Washington, DC: 1978; U.S.
Chemical name: 3-Mercapto-D-valine Government Printing Office.
Abbr.: D-penicillamine 2 Lead in blood and urine, Method no. P&CAM
Product name: Chemet 102. In: Taylor DG, manual coordinator.
Generic name: Succimer NIOSH Manual of Analytical Methods. 2nd ed.
Chemical name: Meso-2,3- Washington, DC: U.S. Government Printing
Dimercaptosuccinic acid Office; 1977:1.102-1 to 9.6.
Abbr: DMSA 3 American Society for Testing and Materials.
Standard test method for lead in the atmosphere
807
Lead
by colorimetric dithizone procedure: gaseous
fuels: coke: atmospheric analyses. Method D-
3112-77. Philadelphia: American Society for
Testing and Materials; 1982;26:628-37.
4 Rice EW, Fletcher DC, Stumpf A. Lead in
blood and urine. Stand Methods Clin Chem.
1965;5:121-9.
5 General procedure for metals, Method no.
P&CAM 173. In: Taylor DG, manual
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Methods. 2nd ed. Washington, DC: U.S.
Government Printing Office; 1977:1.173-1 to
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6 Lead in air or blood, Method no. P&CAM 214.
In: Taylor DG, manual coordinator. NIOSH
Manual of Analytical Methods. 2nd ed.
Washington, DC: US. Government Printing
Office; 1977:1.214-1 to 6.
7 Lead in blood and urine, Method no. P&CAM
262. In: Taylor DG, manual coordinator.
NIOSH Manual of Analytical Methods. 2nd ed.
Washington, DC: U.S. Government Printing
Office; 1977:1.262-1 to 4.
8 Kopito L, Schwachman H. Measurement of
lead in blood, urine and scalp hair by atomic
absorption spectrometry. Stand Methods Clin
Chem. 1972;7:151-62.
9 Marcus M, Hollander M, Lucas RE, Pfeiffer
NC. Micro-scale blood lead determinations in
screening: evaluation of factors affecting
results. Clin Chem. 1975;21:533-556
10 Yeager DW, Cholak J, Henderson EW.
Determination of lead in biological and related
material by atomic absorption
spectrophotometry. Environ Sci Technol.
1971;5:1020-1022.
11 Subramanian KS, Meranger JC. A rapid
electrothermal atomic absorption
spectrophotometric method for cadmium and
lead in human whole blood. Clin Chem.
1981;27:1866-71
12 Boone J, Hearn J, Lewis S. Comparison of
interlaboratory results for blood lead with
results from a definitive method. Clin Chem.
1979;25:389-393
13 Christian GD. The biochemistry and analysis of
lead. Adv Clin Chem. 1976;18:289-326
14 Searle B, Chan W, Davidow B. Determination
of lead in blood and urine by anodic stripping
voltammetry. Clin Chem. 1973;19:76-80
15 Lead in blood, Method no. P&CAM 195. In:
Taylor DG, manual coordinator. NIOSH
Manual of Analytical Methods. 2nd ed.
Washington, DC: U.S. Government Printing
Office; 1977:1.195-1 to 7.
16 Lead in urine, method no. PP&CAM 200. In:
Taylor DG, manual coordinator. NIOSH
Manual of Analytical Methods. 2nd ed.
Washington, DC: U.S. Government Printing
Office; 1977:1.200-1 to 8.
17 Filby RH, Shah KR. Activation analysis and
applications to environmental research. Toxicol
Environ Chem Rev. 1984;2:1-44
18 Barnes IL, Murphy TJ, Gramlich JW, Shields
WR. Lead separation by anodic deposition and
isotope ratio mass spectrometry of microgram
and smaller samples. Anal Chem.
1973;45:1881-4
19 Licensure and Proficiency Testing Division,
Bureau of Laboratories, Centers for Disease
Control and Prevention. Blood lead proficiency
testing. Atlanta: Public Health Service, U.S.
Department of Health and Human Services.
20 Centers for Disease Control and Prevention.
Blood lead analysis 1981. Document no.
34Q205158208. Atlanta: U.S. Department of
Health and Human Services; May 1982.
21 Zinterhofer LJM, Jatlow PI, Fappiano A.
Atomic absorption determination of lead in
blood and urine in the presence of EDTA. J Lab
Clin Med. 1971;78:664-74
22 Crick J, Flegal AR. Contaminant lead in blood-
collection tubes for trace-element studies. Clin
Chem. 1992;38:600-1
23 Segal RJ. Nonspecificity of urinary lead
measurements by atomic absorption
spectroscopy. Clin Chem. 1969;15:1124
24 U.S. Department of Labor, Occupational Safety
and Health Administration. Regulations
(Standards - 29 CFR) Lead. 1926.62. 2006
(online). Available at:
http://www.osha.gov/pls/oshaweb/owadisp.show_docum
ent?p_table=STANDARDS&p_id=10641 Accessed
2008 Mar 6.
25 U.S. Department of Labor, Occupational Safety
and Health Administration. Regulations
(Standards - 29 CFR) Lead. 1910.1025. 2006
(online). Available at
http://www.osha.gov/pls/oshaweb/owadisp.sho
w_document?p_table=STANDARDS&p_id=10
030 Accessed 2008 Mar 6.
26 National Research Council, National Academy
of Sciences. Airborne lead in perspective:
report of the Committee on Biological Effects
of Atmospheric Pollutants of the National
Research Council, National Academy of
Sciences. Washington DC: National Academy
of Sciences, Printing and Publishing Offices;
1972.
27 Damstra T. Toxicological properties of lead.
Environ Health Perspect. 1977;19:297-307.
28 Mahaffey KR. Quantities of lead producing
health effects in humans: sources and
bioavailability. Environ Health Perspect.
1977;19:285-295
29 Lane RE. Diagnosis of inorganic lead
poisoning: a statement. BMJ. 1968;4:501.
808
Lead

30 Dagg JH, Goldberg A, Smith JA, Lochhead A.
The relationship of lead poisoning to acute
intermittent porphyria. Q J Med. 1965;34:163-
75.
31 Centers for Disease Control and Prevention.
Increased lead absorption and lead poisoning in
young children. J Pediatr. 1975;87:824.
32 Sachs HK, Blanksma LA, Murray EF,
OConnell MJ. Ambulatory treatment of lead
poisoning: report of 1155 cases. Pediatrics.
1970;46: 389-96.
33 Granick JL, Sassa S, Kappas A. Some
biochemical and clinical aspects of lead
intoxication. Adv Clin Chem. 1978;20:287-339.

Tables
Table 3: Methods of Lead Analysis
Method 1: Spectrophotometric dithizone
Type of analysis: Colorimetric, quantitative
Principle: After ashing, Pb2+ + dithizone form a complex absorbing at 510 nm
Usage: Blood, urine, tissues, environmental
Comments: Widely used, very hard to do with good precision
Method 2a: Atomic absorption spectrometry (AAS)Classical AAS
Type of analysis: Spectrophotometric, quantitative
Principle: Ashing of lead sample, ionized lead is reduced in flame; absorbs light at 283.3 nm
Usage: Blood, urine, tissues, environmental
Comments: Straightforward; applicable to variety of macrosamples and sizes
Method 2b: Atomic absorption spectrometry (AAS)Delves microscale technique
Type of analysis: Spectrophotometric, quantitative
Principle: Same as in (a), except sample is oxidized in microcup by H2O2
Usage: Blood, urine
Comments: As above and additionally applicable to microsamples
Method 2c: Atomic absorption spectrometry (AAS)Graphite furnace or carbon rod (FAAS)
Type of analysis: Spectrophotometric, quantitative
Principle: Same as in (a), except sample is dried, ashed, and vaporized on a graphite rod
Usage: Blood, urine
Comments: As for 2b, widely used
Method 3: Anodic stripping voltammetry
Type of analysis: Electrochemical, quantitative
Principle: Ionic lead plates as Hg amalgam; at 430 mV Pb reoxidized with increased amperage proportional to
lead concentration
Usage: Blood, urine
Comments: Simple to do, most reliable over wide concentration ranges, widely used
Method 4: Isotope-dilution inductively coupled plasma mass spectrometry
Type of analysis: Mass spectrometry, quantitative
Principle: Extraction by anion exchange chromatography with isotopic analysis
Usage: Blood, urine
Comments: NIST definitive method
809
Lead

Table 4: Comparison of Assay Conditions for Lead Analysis

Assay 1: Dithizone
Temperature: Ambient
pH: >11
Final concentration of reagent components: Dithizone: 6.25 mol/L
Sample volume (blood): 10 mL
Linearity: 0 to 5 g/mL
Precision: 6%
Time of reaction: Hours
Major interferences: Tin, bismuth, thallium
Assay 2: Atomic absorption (Moero)
Temperature: 2000+
pH:
Final concentration of reagent components: Ammonium pyrrolidine dithiocarbamate: 0.12 mol/L
Sample volume (blood): 10 mL
Linearity: 01000 ng/mL
Precision: 5%
Time of reaction: Minutes
Major interferences:
Assay 3: Anodic stripping
Temperature: Ambient
pH: <1.0
Final concentration of reagent components: Perchloric acid: 0.35%
Sample volume (blood): 100 L
Linearity: 6 to 1000 ng
Precision: 5% at 600 ng/mL
Time of reaction: Minutes
Major interferences: Thallium

Figure
Lead: Figure 1

Spectra of dithizonelead complex; method described in text. Dashes, dithizone; dots with dashes, 5 g of extracted lead
added; solid line, 25 g of extracted lead.
810
Lead
Procedure: Preparation of Reagents, Standards,
Glassware
Cleaning of Glassware
Reagents
1. Hot chromic acid. Dissolve 25 g (0.25 mol) of
reagent-grade chromic trioxide (Caution: suspected
carcinogen!) in 2.5 L of reagent-grade sulfuric acid. Stir
with a Teflon-coated stirring bar. Heat to 50C before
use. Stable for 6 months at room temperature. When the
solution changes color from red to green, it should be
discarded. Dispose according to appropriate
environmental guidelines.
2. Nitric acid soaking solution. Dilute
concentrated nitric acid (reagent grade) with an equal
volume of glass- or double-distilled water. Stable for 1
year at room temperature.
Method
All glassware must be rendered lead free before
use.
1. Wash in detergent in tap water, and follow with
tap- and distilled-water rinses.
2. Soak in hot chromic acid, and follow with tap-
and distilled-water rinses.
3. Soak in the nitric acid soaking solution for 30
minutes, and follow with tap-, distilled-, then double-
distilled-water rinses.
Notes. The entire procedure must be followed
when glassware is being initially prepared for use in the
lead analysis. For each successive use of equipment that
has previously been subjected to the entire cleaning
procedure, it is necessary to use only the nitric acid
cleaning procedure of step 3. However, glassware must
be committed to lead analysis to avoid contamination
from other sources. Also, polyethylene laboratory ware,
used for storage of standards and so on, can be
effectively cleaned when it is subjected to the above
procedure.
Ashing of the Sample
Reagent
1. Redistilled nitric acid (Ultrex grade).
Method
1. Add 10 mL of a sample of heparinized blood to
a 125 mL borosilicate glass beaker, followed by
7 mL of redistilled nitric acid. For urine, use a
25 mL sample.
2. Evaporate the sample at 130C, just to the point
of dryness, and cover the beaker with a lead-
free watch glass.
3. After cooling, add successive portions of the
nitric acid, that is, 2.0, 1.5, 1.0, and 0.5 mL, by
sliding the watch glass only sufficiently to
facilitate each new addition of acid.
4. After each addition and as soon as the residue
becomes light colored, heat the sample to
400C just long enough to blacken the sample.
5. Cool the sample, add an additional portion of
the acid, and repeat the heating procedure.
6. Finally, the residue will remain pale yellow or
light brown after it has been heated for 5 to 10
minutes at the 400C temperature.
Standards
Stock aqueous lead solution
1. To prepare a 1 mg/mL lead standard stock
solution, dry lead nitrate (reagent grade) at 100C for 2
hours, cool, dissolve 1.5980 g in nitrite acid (Ultrex
grade) diluted 1:10 with water, and then dilute to 1 L
with the 1:10 acid.
2. From this 1 mg/mL solution, make working
standards of 1, 2, 5, and 10 g of lead per
milliliter of solution by appropriate dilutions
with 1:10 nitric acid.
3. The 1 mg/mL stock solution is stable for at least
6 months, whereas the working standard
solutions should be made fresh on a weekly
basis. Commercial standards sold are usually
1% in HNO3 and are stable only for 6 to 8
months at room temperature. Our experience
shows that standards made up in 10% HNO3 are
stable for several years stored at room
temperature.
Blood Standard
1. Obtain heparinized expired blood from the local
blood bank to be used as a working laboratory
blood standard stock. The sample is preserved
by refrigeration.
2. Each day that an analysis is to be performed,
spike 6 10-mL aliquots to 0, 1, 2, 5, 10, and 20
g lead per milliliter, respectively, using the 1
mg/mL aqueous stock solution.
3. Run these samples along with the aqueous
working standards and the blood specimens
being analyzed.
It should be noted that accurate records must be
maintained as to the analytical results obtained from
analysis of this blood standard. This continuous
laboratory record will allow for the long-term
monitoring of method performance. Further, as one
supply of expired blood is being depleted, a new supply
should be obtained and run so that an overlap of the
blood standards is maintained.
Urine Standard
1. Prepare a standard by obtaining urine from a
nonexposed person.
2. Preserve the sample by the addition of Ultrex
nitric acid to a final concentration of 10% (v/v)
and store at 4 C.
3. On the day the analysis is to be performed,
spike 6 25-mL aliquots to 0, 1, 2, 5, 10, and 20
g of lead per milliliter, respectively, using the
1 mg/mL aqueous stock solution.
811
Lead
4. Run these samples along with the aqueous
working standards and the urine specimens
being analyzed. The same procedure described
for the recording of the laboratory blood
standard and the initiation of the use of a new
urine stock should be applied to the laboratory
urine standard.
Procedure: Spectrophotometric Method [8]
Principle
+ ammonium hydroxide. Dilute to 1 L. Stable for 1 year at
Ashed sample is solubilized in a KCN-NH4 solution,
and the lead is extracted into a dithizoneCHCl3
(chloroform) solution. The leaddithizonate complex is
back extracted into nitric acid, which is then made
alkaline with KCN-NH4+ and reextracted into dithizone
CHCl3 reagent. The complex is quantitated
spectrophotometrically at 510 nm.
Reagents
1. Nitric acid. Double distilled, Ultrex grade.
Stable for 1 year at room temperature.
2. Sodium diethyldithiocarbamate (0.177
mol/L) 4%. Dissolve 4 g of sodium
diethyldithiocarbamate in 100 mL of distilled water.
Stable for 1 month at ambient temperature.
3. Ammonium hydroxide, concentrated 15.1
mol/L. High-purity. Specific gravity, 0.92.
4. Hydrochloric acid (6 mol/L). Dilute redistilled
concentrated HCl, Ultrex grade, with an equal volume of
water. Stable for several years at room temperature.
5. Dithizone extraction solution (390 mol/L).
Dissolve 100 mg of dithizone in 1 L of chloroform (ACS
reagent grade or better). Stable for 1 month at room
temperature.
6. Sodium citrate (0.548 mol/L). Dissolve 125 g
of sodium citrate eicosadecahydrate in 400 mL of
distilled water. Adjust to pH 9 to 10 with ammonium
hydroxide and bring to 500 mL volume. Extract for 50
min with a 10 mL portion of the dithizone extraction
solution. Trace amounts of lead in the citrate solution
will turn the green dithizoneCHCl3 solution red.
Discard chloroform phase, and repeat extraction until the
chloroform extract remains green. Extract the citrate
solution repeatedly with chloroform to remove dissolved
dithizone until the extract is colorless. Remove the last
traces of chloroform by pipetting. Stable for 6 months at
4C. Check for pH change and microbial growth
monthly.
7. m-Cresol purple indicator. Dissolve 1 g in
100 mL of distilled water. Stable for 1 year at room
temperature.
8. Hydroxylamine solution (9.3 mol/L). Dissolve
20 g of hydroxylamine in 65 mL of distilled water.
Stable for 2 years at room temperature.
9. Working hydroxylamine solution (6.06
mol/L). To 65 mL of hydroxylamine solution add a few
drops of m-cresol purple indicator. Then add ammonium
hydroxide until the indicator turns yellow; add 5 to 10
mL of 4% solution of sodium diethyldithiocarbamate to
combine metal impurities; mix; then extract repeatedly
with chloroform until the excess carbamate has been
removed; add redistilled 6 M HCI until the indicator
turns pink; and then adjust the volume to 100 mL with
double distilled water. Stable for 6 months at room
temperature.
10. 10% potassium cyanide solution (1.53
mol/L). Dissolve 10 g in 100 mL of distilled water.
Extract as for sodium citrate. Bring to 500 mL volume.
Stable for 1 year at room temperature.
11. Potassium cyanideammonia solution (0.306
mol/L KCN, 2.25 mol/L NH4OH). Mix 200 mL of
10% KCN solution and 150 mL of concentrated
room temperature.
12. Dithizone reagent (6.25 mol/L). Dissolve 16
mg of dithizone in 1 L of chloroform. Prepare fresh
daily.
Assay
Equipment: Spectrophotometer with a band pass < 10
nm, capable of reading at 510 nm; pH meter.
1. The ash from each standard and sample is
warmed with 2.0 mL of redistilled,
concentrated nitric acid to dissolve the residue,
and then 25 mL of distilled water is added. The
solution is heated at 50C to 60C until
clarified.
2. Add 1.0 mL of hydroxylamine solution,
followed by 4 mL of sodium citrate solution.
Using a pH meter, adjust the titrated sample
solution to pH 9 to 10 with ammonium
hydroxide.
3. After a quantitative transfer to a 125 mL
separatory funnel containing 5 mL of potassium
cyanide solution, add 5 mL of the chloroform
solution of the dithizone reagent. The sample is
extracted by being shaken for 2 min; then the
layers are allowed to separate.
4. The chloroform layer is drawn off into a second
separatory funnel containing 30 mL of dilute (1
part acid to 99 parts double distilled water)
nitric acid. Successive 5 mL portions of the
chloroform solution are added to the first
funnel, and the extraction process is repeated
until the extraction dithizone reagent remains
green.
5. Combine the chloroform extracts in the second
funnel, extract with the nitric acid, and allow
the layers to separate.
6. Discard the chloroform (bottom) layer, and then
add 6.0 mL of the ammoniacyanide mixture to
the aqueous layer (nitric acid), and add exactly
15.0 mL of the standard dithizonechloroform
reagent solution. Shake 2 minutes, and allow
the layers to separate.
7. Carefully decant the chloroform solution into a
spectrophotometer tube and read at 510 nm.
Calculation
812
Lead
Plot A for each blood or urine standard (vertical axis)
510
versus micrograms of lead per standard (horizontal axis).
Determine the slope of the curves, dividing the A by
510
gPb: (A /gPb).
510
(A Sample) (A Blank)
510 510
Net amountof Pb in sample
A / g Pb
510
Net amount of Pb = g of Pb/mL
Sample volume
For blood:
gPb/mL 1000 = gPb/L of blood
For urine:
0.024/ (specific gravity of urine 1.000) gPb/mL
1000 = gPb/L urine
(Note: corrected to specific gravity of 1.024)
Procedure: Atomic Absorption Spectroscopy
Principle
The blood or urine sample is ashed and extracted into
methyl isobutyl ketone, and the organic layer is passed
through the flame of the atomic absorption instrument
where the Pb++ is reduced to Pb0. The amount of light
from a hollow cathode lamp absorbed by the lead atoms
is proportional to the concentration of lead in the sample.
Reagents
1. Nitric acid. Concentrated, redistilled, Ultrex
grade. Stable for 1 year at room temperature.
2. Phenol red indicator. Dissolve 0.1 g of
phenol red in 100 mL of distilled water. Stable for 1 year
at room temperature.
3. Ammonium citrate buffer, pH 8.5 (4.16
mol/L citrate, 12.12 mol/L ammonia). Add 400 g of
citric acid (2.08 mol) to 100 mL of distilled water.
Dissolve by slowly adding 400 mL of concentrated
ammonium hydroxide (6.06 mol). Allow to cool, and
adjust to pH 8.5. Stable for 6 months at 4C. Check for
pH change and microbial growth monthly.
4. Dithizone extraction solution (390 mol/L).
Dissolve 100 mg of dithizone in 1 L of chloroform.
Stable for 2 months at room temperature.
5. 10% potassium cyanide solution (1.53
mol/L). Dissolve 10 g in 100 mL of distilled water.
Extract repeatedly with successive 10 mL aliquots of
dithizone extraction solution until the chloroform
remains green. Extract repeatedly with chloroform until
the extract is colorless. Remove the last traces of
chloroform by evaporation. Stable for several years at
room temperature.
6. Ammonium pyrrolidine dithiocarbamate
(0.12 mol/L). Dissolve 2 g of ammonium pyrrolidine
dithiocarbamate in 100 mL of distilled water. Stable at
6C for 6 months.
7. Methyl isobutyl ketone. Shake 500 mL of
methyl isobutyl ketone (ACS reagent grade or better)
with water. Separate organic phase with separatory
funnel. Prepare fresh before use.
Assay
Equipment: Any recent-model flame atomic absorption
spectrophotometer, preferably one equipped with an
automatic sampler.
1. Sample preparation. Ash a 10 mL sample of
heparinized blood or a 50 mL sample of urine.
(See Ashing of the sample).
2. When all of the organic matter has been
destroyed, add 1.0 mL of concentrated
redistilled nitric acid and 5.0 mL of distilled
water to the beaker and slowly apply heat at
50C to 60C. When the residue has dissolved,
transfer the solution to a clean 50-mL
volumetric flask.
3. Add two drops of phenol red indicator and then
5.0 mL of ammonium citrate buffer, pH 8.5.
Add concentrated ammonium hydroxide drop
by drop until the solution turns red, and then
add 1.0 mL of 10% potassium cyanide solution.
Add 1 mL of 2% ammonium pyrrolidine
dithiocarbamate, and mix the entire solution
well.
4. Add 4.0 mL of water-saturated methyl isobutyl
ketone, and extract the sample by shaking for
30 sec. Add distilled water until the organic
layer is brought up into the neck of the flask.
5. Set the atomic absorption spectrophotometer
(check manufacturers instructions) to measure
absorption at 283.3 nm, and start the air-
acetylene flame. Reduce the acetylene flow so
that an oxidizing flame is obtained while the
water-saturated methyl isobutyl ketone layer is
being aspirated.
6. Zero the instrument against a reagent blank.
Use at least two standards to calibrate the
instrument if using the calibrate mode.
7. Aspirate each sample and record data.
8. If instrument is used in the absorbance mode,
record absorbance once a steady-state signal is
achieved.
Calculations
If the instrument is set in concentration mode, results are
obtained in concentration units. If the absorption mode is
used, calculate concentration by direct proportions with
one of the standards or directly from a calibration curve.
Calibration curve. Pipet 0.5 mL of each working
aqueous standard into individual 50 mL volumetric
flasks. Process each diluted solution according to the
procedure, from step 2 (first evaporation). Construct a
calibration curve of absorbance versus micrograms of
lead.
Blood: gPb/L blood = g from calibration curve (10
mL sample) 100
Urine:g of Pb/L = g from calibration curve(50 mL
813
Lead
sample) 1000 0.024(SG of urine -1)
Procedure: Anodic Stripping Voltammetry [14]
Principle
Sample is ashed by nitric, perchloric, sulfuric acid
mixture. The resultant solution is quantitated by
measurement of the current at 430 mV after reduction
and plating of lead.
Reagents
1. Acids. Double-distilled, concentrated nitric
acid (Ultrex grade); double-distilled, concentrated
sulfuric acid (Ultrex grade); and double-distilled 70%
perchloric acid.
2. Acid ashing solution. Combine 32.7 mL of
double-distilled concentrated nitric acid with 65.3 mL of
double-distilled 70% perchloric acid and 2.0 mL of
double-distilled concentrated sulfuric acid. Stable for 6
months at room temperature.
3. 0.35% perchloric acid (0.35 g/L). Add 1 mL
of double-distilled, concentrated (70%) perchloric acid
to 199 mL of water. Stable for 1 year at room
temperature.
Assay
Equipment: Model 3010 atomic stripping voltameter
(Environmental Science Associates, or equivalent);
Ultrasonics mixer model W-140-E sonifier (Heat
Systems, Ultrasonics Inc., or equivalent).
1. Mix the sample by rinsing the ultrasonic mixer
tip thoroughly in distilled deionized water, and
then place the tip into a blood sample within 2
cm of the bottom of the Vacutainer tube, and
mix at the maximum permissible power for 20
to 30 sec. If a sample in a Vacutainer tube sits
for more than 10 min after it has been mixed,
remix it before taking an aliquot for analysis.
2. Pipet 100 L of the mixed blood or standard
into an analysis cell. Rinse the pipet once with
distilled, deionized water, and add the rinse to
the analysis cell. To prepare the pipet for reuse,
rinse the pipet four times with distilled
deionized water, and discard the rinse water.
Since some batches of the micropipets may
have an appreciable contamination with lead, it
is best to use the same tip throughout a series of
samples and to rinse it between each sample.
Duplicate 100 L blood samples should be
taken for analysis.
3. Pipet 300 L of acid ashing solution into the
analysis cell containing the sample. Place the
cell in the digestion rack on a hot plate at 220C
to 240C. The sample will initially turn yellow
but will clear in about 10 min. A reflux line will
be established about halfway up the cell. At the
end of 30 minutes, there will be approximately
100 to 200 L of acid left in the bottom of the
cell, with most of the nitric acid having
evaporated. The digestion should be continued
for an additional 30 to 60 min until the acid is
almost completely evaporated. Remove the cell
from the hot plate, cool it, and add 4.5 mL of
0.35% perchloric acid.
4. For urine samples, reconstitute urine ash with
4.5 mL of 0.35% perchloric acid, and continue
with the assay.
5. Following the manufacturers instructions for
the instrument, analyze the sample solution
under the following conditions (voltages given
with respect to a Ag/AgCl reference electrode):
plating potential, 780 mV; plating time, 10 to
30 min (being sure that plating time for samples
and standards are exactly the same); sweep rate
of +60 mV per second; current range of 0.2 to
1.0 mA, full scale. The stirring rate for the
sample solution during plating should not be
lower than 170 rpm/min, since at lower rates,
significant variations in the amount of lead
plated on the electrode may occur. As the
sample is stripped, a cadmium peak may occur
first at 600 mV, followed by the lead peak at
430 mV and a copper peak at 50 mV.
Calculations
One prepares a standard curve for each sample type,
urine or blood, by plotting lead peak height in
microamperes (A) (measured when one draws a
baseline to connect the flat portions of the tracing before
and after the lead peak at 430 mV and then draws a
perpendicular line from the apex of the peak to the
baseline; the length of this perpendicular is the peak
height) versus the nanograms of lead in the standard
sample. A response factor is calculated from the standard
curve, that is, ngPb/A. The amount of lead in a sample
is then calculated as follows:
ngPb = Peak height for sample (in A) Response
factor (in ng/A)
For urine:
gPb/L = Amt. (in ng) of Pb in urine (Volume [in mL]
of urine) 1000
Corrected gPb/L urine = gPb/L 0.024 (Specific
gravity of sample) 1.000
For blood:
gPb/L blood = Amt. (in ng) of Pb in blood (Volume [in
mL] of sample) 1000
Note: Using the ESA model 3010 stripping voltameter
without a strip recorder, some workers have observed a
negative interference caused by high levels of copper in
pregnant women. Measuring the lead peak with a
recorder prevented this interference.
814
Lipase

Lipase
Ming Jin

Name: Lipase, triacylglycerol acylhydrolase (LPS)

Clinical significance: Refer to Chapter 34, The Pancreas: Function and Chemical
Pathology, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular weight: Approximately 48 kDa
Enzyme commission number: EC 3.1.1.3
Chemical class: Enzyme
Isoforms: At least two; most likely posttranslational variants

Principles of Analysis and Current Usage

i point measurements (Table 1, Method 1b) and developed
The measurement of serum lipase activity has long been
recognized as useful for diagnosing pancreatitis.
Unfortunately, early methods for lipase were plagued by
technical difficulties, making accurate measurement of
the enzyme problematic and its clinical utility unreliable.
However, advances in our understanding of lipase action
have greatly improved the reliability of lipase
measurements and subsequently have improved the
clinical utility of lipase for diagnosing pancreatitis.
Lipases are enzymes that hydrolyze glycerol esters of
long-chain fatty acids. They act on the glycerol bonds
(ester linkage) to liberate two molecules of fatty acid and
one molecule of -monoglyceride (Figure 1).
Essential to the understanding of lipase methodology is
the fact that the enzyme acts only at an ester-water
interface. Thus lipase-assay substrates must be
established as emulsions. The reaction rate will increase
with the dispersion of the emulsion (surface area).
Failure to achieve a suitable ester-water interface will
permit the action of nonlipase enzymes (carboxylic ester
hydrolase, aryl-ester hydrolase, and lipoprotein lipase).
The use of short-chain fatty acid triglyceride substrates,
which are water soluble, will also permit this false lipase
reaction to occur.
Titrimetric Method
Titration methods were the first practical assays. The
classical technique of Cherry and Crandall [1] was based
on the incubation of serum for 24 hours at 37C, with a
50% emulsion of olive oil and 5% gum acacia in a pH
7.0 phosphate buffer. The liberated fatty acid was then
quantified by titration against 0.05 M NaOH with a
phenolphthalein indicator being used (Table 1, Method
1a).
Alternative detection methods to replace titration
methods have been developed. For example, Tietz and
Fiereck [2] did considerable work using pH meter end-
i
Lipase
Previous and current authors of this method:
First edition: Michael D.D. McNeely
Methods edition: Michael D.D. McNeely
Second edition: Michael D.D. McNeely
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Ming Jin
a method using pH stat apparatus [3]. A more recently
developed variation of the pH technique involves
measurement of the rate of change of pH as the fatty
acids are liberated [4]. This method requires a very
sensitive pH measurement deviceone that can measure
pH changes of less than 0.02 for values in the normal
range. This procedure employs olive oil as the substrate
but does not use bile acids or colipase to activate lipase.
The reaction is very rapid, with most analyses being
completed in 3 minutes.
Colorimetric Method
The method of Massion and Seligson [5] quantitates the
liberated fatty acids after 1 h of incubation by extracting
them and allowing them to change the color of an
appropriately buffered methyl red indicator (Table 1,
Method 2).
Immunological Techniques
A number of immunochemical assays have been
reported for measurement of lipase. These include both
quantitative enzyme immunoassay procedures [6] and a
semiquantitative latex-agglutination procedure (Table 1,
Method 3a). Antibodies to lipase are bound to latex
particles, and these particles are mixed with the sample.
If lipase is present, agglutinated clumps of particles
appear on the reaction slide [7].
A noncompetitive immunoactivation procedure has been
described [8]. This assay uses Fab-antibody fragments
against pancreatic lipase that are covalently coupled to
horseradish peroxidase. In the presence of lipase, the
peroxidase catalyzes a color-producing reaction, the rate
of which is proportional to the lipase concentration in the
sample (Table 1, Method 3b).
Coupled Enzyme Spectrophotometric Method
Several coupled enzymatic assays have been developed
for the measurement of serum lipase. One enzymatic
assay utilizes a micellar-solubilized long-chain fatty
acid, 1,2-diglyceride (Table 1, Method 4a) [9]. Lipase
cleaves the substrate to form 2-monoglyceride and fatty
acids. The 2-monoglyceride is subsequently hydrolyzed
to glycerol by a specific 2-monoglyceride lipase
(MGLP). In the final reaction step, the chromogen
system (4-aminophenazone/N-ethyl-N-[2-hydroxy-3-
sulfopropyl-m-toluidine] [TOOS]) is oxidized to a violet
dye, with peak absorption at 550 nm. The reaction
mixture also contains colipase and deoxycholate.
815
Lipase
A different enzymatic approach has been adapted to the
dry-film technology (Table 1, Method 4b) [10]. This
method employs 1-oleyl-2,3-diacetoyl glycerol adsorbed
onto TiO2 particles as the substrate. The TiO2 particles
act to provide the surface needed for lipase activity. In
addition, an emulsifying agent and porcine colipase are
present to increase lipase activity. Lipase will act on the
asymmetric triglyceride to release the oleyl fatty acid
and the water-soluble [11,12] diacetoyl glycerol. The
latter molecule diffuses into another film layer of the
reaction slide, where an esterase clears both acetate
groups to release glycerol, which can then be quantitated
by an enzymatic method. When one employs a lag
period before measuring the glycerol formed,
endogenous glycerol is consumed and will not interfere
in the assay.
A new synthetic substrate, 1,2-O-dilauryl-rac-glycero-3-
glutaric acid-[4-methylo-resorufin]-ester) has been used
for lipase measurement [13]. Lipase hydrolyzes the
substrate in an alkaline medium to an unstable
dicarbonic acid ester that spontaneously hydrolyzes to
glutaric acid and methylresorufin. Methylresorufin is
measured at 570 nm (Table 1, Method 4c).
These coupled enzymatic assays based on the
measurement of free glycerol can be adversely affected
if the patient sample contains increased concentrations of
free glycerol. Certain drugs and medications have been
found to contain high concentrations of this compound.
However, if a lag period is employed before the
measurement of formed glycerol, endogenous glycerol is
consumed and does not interfere with the assay.
Turbidimetric Method
A completely different analytical approach that utilized
clearing of an emulsion was introduced by Vogel and
Zieve [14] and has been modified successfully by others
[15]. In this method, the turbidity of the oil-water
emulsion, monitored at 340 or 400 nm, is reduced as
lipase hydrolyzes the triglyceride molecules. The
technique requires a highly stable, reproducible substrate
with an appropriate initial absorbance. The sample is
added to the substrate, and kinetic measurements are
made turbidimetrically or nephelometrically (Table 1,
Method 5).
Most assays in use today employ colipase to enhance
lipase measurements. Colipase increases the reaction rate
of lipase reactions by allowing the enzyme better access
to the substrate [16]. Although variable amounts of
colipase have been reported to be released into
peripheral blood during acute pancreatitis [17], colipase
is added to ensure sufficient activation of the pancreatic
isoenzyme [18]. The suggested optimal reaction mixture
for lipase includes triolein or olive oil substrate (100
mL/L), colipase (6 mg/L), CaCl2 (8.5 mmol/L), and a
bile salt, sodium glycocholate (35 mmol/L) [19].
Reference and Preferred Methods
There is no reference method for lipase assay. The
titrimetric method using pH measurement techniques has
been proposed as a useful reference measurement
procedure in the evaluation of new lipase methods [13],
but the technique of this method is too complex for
routine use.
Methods using substrates other than long-chain fatty acid
triglycerides must be viewed with great suspicion. The
use of purified olive oil substrate, extraction of long-
chain fatty acid triglycerides, and quantitation against
pure standards is recommended as a standard technique.
The modified method of Massion and Seligson [5]
(detailed in the following paragraphs) has these
characteristics carried out with microsamples in less than
2 hours.
The turbidimetric methods, by detecting emulsion
clearing, are fast and can be automated [19], but the
stability and reproducibility of the substrate is extremely
critical. An important deficiency of these methods is that
because of instrument limitations, the substrate
concentrations are far below the Km (Michaelis-Menten
constant) of the reaction. This makes the standardization
of these procedures more difficult and the reproducibility
poorer than the methods utilizing triglyceride or
diglyceride substrates. Modifications such as increasing
the concentrations of colipase and bile salts and
continuous reaction-rate monitoring have led to
increased assay performance [19]. The other deficiency
is the limited range of linearity.
The optimized titrimetric method uses pH measurement
techniques [2,4] by titrating liberated fatty acid with a
pH stat instrument during the reaction. This procedure
appears to be reasonably precise and rapid. Good
correlation has been shown between the pH
measurement and turbidimetric methods. This method is
suggested to be a reference method.
Colorimetric methods that employ either chromogenic
long-chain fatty acid substrates or colorimetric
determination of glycerol are being used with increased
frequency [9,10,21]. They have the advantage of
minimum sample volume requirements (<25 L), good
precision, wide dynamic range, and ease of automation.
Coupled enzyme spectrophotometric methods with
different substrate are widely used in clinical
laboratories and are run on automated instruments.
It is important that all new methods for lipase
measurement be compared to a technique employing an
olive oil substrate with a long-chain fatty acid
measurement of known clinical utility.
In order to standardize methods, reference materials
have been prepared from human pancreatic juice and
recombinant technology. These have been standardized
using a titrimetric procedure [22].
Specimen
Serum and plasma samples are suitable for use, since
both give equivalent results [23]. Specimens are stable
for at least 1 week at room temperature, up to 3 weeks at
4C, and for several years at 20C [24]. Heating of
816
Lipase
specimens has been found to destroy enzyme activity
[25]. Repeated freezing and thawing should be avoided.
Interferences
The different methodologies used for lipase
measurements have their own associated interferences.
Sample hemolysis does not generally interfere with any
of the described methods. Rheumatoid factor has been
shown to interfere with the turbidimetric procedures
because of aggregation of sample components [26].
Approximately 3% to 5% of specimens analyzed by
emulsion-clearing methods will display an anomalous
absorbance increase due to this phenomenon. By
avoidance of freezing or by using polyethylene glycol
(PEG) to precipitate protein (mostly IgM), the frequency
of these problems can be reduced [27].
Coupled enzymatic assays based on the determination of
glycerol can be adversely affected if the patient sample
contains abnormally increased concentrations of glycerol
that may be found in certain drugs and medications [28].
In addition to these analytical interferences, a variant
form of lipase consisting of lipase bound to IgG has been
described [28]. This macrolipase was found to result in
greatly increased lipase activity; the clinical significance
of this variant, if any, remains to be determined.
Lipase Reference Interval
Reference intervals for lipase activity in healthy
individuals are largely method dependent. The upper
reference limit for turbidimetric procedures is
approximately 200 U/L, and the upper reference limit for
the colorimetric assays is approximately 60 U/L. The
upper reference limit for the coupled enzyme methods
varies, depending upon the choice of substrate and the
measurement conditions. Lipase activities in serum
remain constant until about age 60, when values may
rise by as much as 20% [29]. There are no differences
between males and females.
Interpretation
Serum lipase measurement is used to diagnose acute
pancreatitis. Many recent studies have shown lipase to
have greater diagnostic utility in the evaluation of
pancreatitis when compared to less specific amylase [30-
33]. Reported sensitivities and specificities of lipase for
diagnosing pancreatitis range from 60% to 100% and
81% to 97%, respectively [33-35]. After the onset of
acute pancreatitis, serum lipase increases within 4 to 8
hours, reaches a peak at approximately 24 hours and
may take 8 to14 days to return to normal levels.
Lipase is present in at least two molecular forms,
designated L1 and L2, that are probably posttranslational
variants. L2 is absent in normal persons but present in
high activity in patients with pancreatitis. The L2
isoform has been found to have better diagnostic
efficiency than total lipase; however, further studies are
needed to evaluate the clinical utility of this more
specific marker.
Various drugs have been implicated in causing
pancreatitis. A strong association between therapy with
valproic acid and pancreatitis has been described [36].
Therapy with 2,3-dideoxyinosine (ddI), a promising
therapeutic agent for human immunodeficiency virus
infections, has been implicated in causing pancreatitis in
approximately 10% of patients taking the drug [37]. In
addition, infection with human immunodeficiency virus
by itself is associated with acute pancreatitis. It appears
that the virus has a direct damaging effect on the
exocrine pancreas.
Renal insufficiency in patients without pancreatitis may
result in increases of lipase of up to six times the upper
reference interval limit. Values greater than 10 times the
upper reference interval limit are considered to be
pathognomonic for pancreatitis [30]. Obstruction of the
pancreatic duct may also increase serum lipase activity.
Lipase Performance Goals
Lipase is not regulated under Clinical Laboratory
Improvement Amendments (CLIA). The criteria for
acceptable performance for lipase assay by College of
American Pathologists (CAP) Survey standards is within
30% of the mean value of laboratory peer groups. Data
on lipase assay with different methodology from the
CAP Survey Participant Summary Report in C-A 2007
indicate CVs generally below 10% at the mean of lipase
activities around 100 U/L.
The variety of methods used to measure lipase activities
results in a wide range of activities obtained from
identical specimens. Up to 20-fold differences may be
seen in identical survey materials analyzed with different
instrument/reagent combinations.
References
1. Cherry IS, Crandall IA Jr. The specificity of
pancreatic lipase: its appearance in the blood
after pancreatic injury. Am J Physiol 1932; 100:
266-273.
2 Tietz NW, Fiereck EA. Measurement of lipase
in serum. In: Cooper GR, editor. Standard
methods of clinical chemistry. New York:
Academic Press; 1972. vol. 7, p. 19-31.
3 Tietz NW, Repique EV. Proposed standard
method for measuring lipase activity in serum
by a continuous sampling technique. Clin Chem
1973; 19: 1268-1275.
4 Cerolotti F, Bonini PA, Murone M, Barenghi L,
Luzzana M, Mosca A et al. Measurement of
lipase activity by a differential technique. Clin
Chem 1985; 31: 257-260.
5 Massion CG, Seligson D. Serum lipase: a rapid
photometric method. Am J Clin Pathol 1967;
48: 307-313.
6 Rood FW, Coleman PF. Solid-phase enzyme
immunoassay for the quantitation of pancreatic
lipase in human serum. Clin Chem 1986; 32:
1129-1130.
7 Moller-Peterson J, Klaerke M, Dati F, Toth T.
Immunochemical qualitative latex agglutination
test for pancreatic lipase in serum evaluated for
817
Lipase
use in diagnosis of acute pancreatitis. Clin
Chem 1985; 31: 1207-1210.
8 Van Ingen HE, Sanders GTB. Clinical
evaluation of pancreatic lipase mass
concentration assay. Clin Chem 1992; 38: 2310- 24
2313.
9 Fassati P, Ponti M, Paris P, Berti G, Tarenghi
G. Kinetic colorimetric assay of lipase in serum. 25
Clin Chem 1992; 38: 211-215.
10 Mauck JC, Weaver MS, Stanton C.
Development of a Kodak Ektachem clinical
chemistry oxide for serum lipase. Clin Chem 26
1984; 30: 1058.
11 MacDonald RP, LeFave RO. Serum lipase
determination with an olive oil substrate using a
three-hour incubation period. Clin Chem 1962;
8: 509-519. 27
12 Roe JH, Byler RE. Serum lipase determination
using a one-hour period of hydrolysis Anal
Biochem 1963; 6: 451-460.
13 Panteghini M, Bais R, van Solinge WW. 28
Enzymes. In: Burtis CA, Ashwood RA, Bruns
DE, editors. Tietz textbook of clinical chemistry
and molecular diagnostics. 4th ed. St. Louis
MO: Elsevier Saunders; 2006. pp 597-643. 29
14 Vogel WC, Zieve L. A rapid and sensitive
turbidimetric method for serum lipase based
upon differences between the lipases of normal 30
and pancreatitis serum. Clin Chem 1963; 9:
168-181.
15 Shipe JR, Savory J. The simultaneous kinetic
measurement of amylase and lipase using an 31
automatic sampling fluoronephelometer. Clin
Chem 1973; 19: 645.
16 Borgstrm B, Erlanson-Albertsson C, Weilock
T. Pancreatic colipase: chemistry and
physiology. J Lipid Res 1979; 20: 805-816. 32
17 Junge W, Leybold K. Detection of colipase in
serum and urine of pancreatitis patients. Clin
Chim Acta 1982; 123: 293-302.
18 Arzoglou PL, Lessinger JM, Frard G. Plasma
lipase properties as related to pancreatic 33
condition. Clin Chem 1986; 32: 50-52.
19 Tietz NW, Astles JR, Shuey DF. Lipase activity
measured in serum by a continuous-monitoring
pH-stat technique - an update. Clin Chem 1989;
35: 1688-1693. 34
20 Feld RD, Witte DL, Barrett DA. Kinetic
determination of serum lipase activity with the
Abbott ABA-100. Clin Chem 1976; 22: 607-
610.
21 Robbrecht JH, DeBuyzere ML, Delanghe JR. 35
Wake UV colorimetric pancreatic lipase assay
with 1,2-dilinoleoylglycerol as substrate
evaluated. Clin Chem 1989; 35: 1540-1541.
22 Lessinger J-M, Parashou S, Arzoglou P, Ramos
P, Chapus C, Dufaux J et al. Determination of 36
lipase catalytic activity in two reference
materials: BCR 693 and BCR 694 by titrimetry
at constant pH. Clin Chem Lab Med
2004;42:62-66 37
23 Doumas BT, Hause LL, Simuncak DM,
Breitenfeld D. Differences between values for
plasma and serum in tests performed in the
Ektachem 700 XR analyzer, and evaluation of
"plasma separator tubes (PST)". Clin Chem
1989; 35: 151-153.
Tietz NW, Shuey DF. Lipase in serum-the
elusive enzyme: an overview. Clin Chem 1993;
38: 1167-1185.
Hussain S, Dasgupta A. Effect of heat treatment
on sera for deactivation of AIDS virus on
commonly monitored serum analytes. Clin
Chem 1991; 37: 932.
Kannisto J, Lalla M, Lukkari E.
Characterization and elimination of a factor in
serum that interferes with turbidimetry and
nephelometry of lipase. Clin Chem 1983; 29:
96-99.
Bilodeau L, Grotte DA, Preese LM. Glycerol
interference in serum lipase assay falsely
indicates pancreas injury. Gastroenterology
1992; 103: 1066-1067.
Stein W, Bohner J, Bahlinger M. Macrolipase-a
new member of the family of immunoglobulin-
linked enzymes. J Clin Chem Clin Biochem
1987; 25: 837-843.
Mohiuddin J, Katrak A, Junglee D, Green MF,
Dandona P. Serum pancreatic enzymes in the
elderly. Ann Clin Biochem 1984; 21: 101-104.
Lott JA, Lu CJ. Lipase isoforms and amylase
isoenzymes: assays and application in the
diagnosis of acute pancreatitis. Clin Chem
1991; 37: 361-368.
Werner M, Steinberg WM, Pauley C. Strategic
use of individual and combined enzyme
indicators for acute pancreatitis analyzed by
receiver-generator characteristics. Clin Chem
1989; 35: 967-971.
Van Lente F, Kazmierczak SC.
Immunologically-derived pancreatic amylase,
pancreatic lipase, and total amylase compared
as predictors of pancreatic inflammation. Clin
Chem 1989; 35: 1542.
Kazmierczak SC, Van Lente F, Hodges ED.
Diagnostic and prognostic utility of
phospholipase A activity in patients with acute
pancreatitis: comparison with amylase and
lipase. Clin Chem 1991; 37: 356-360.
Van Lente F, Kazmierczak SC.
Immunologically-derived pancreatic amylase,
pancreatic lipase, and total amylase compared
as predictors of pancreatic inflammation. Clin
Chem 1989; 35: 1542.
Panteghini M, Pagani F. Diagnostic value in
measuring pancreatic lipase and the P3 isoform
of the pancreatic amylase isoenzyme in serum
of hospitalized hyperamylasemic patients. Clin
Chem 1989; 35: 417-421.
Lott JA, Bond LA, Bobo RC, McClung HJ,
Murray RD. Valproic acid-associated
pancreatitis: report of three cases and a brief
review. Clin Chem 1990; 36: 395-397.
Lerch MM, Alder G. Acute pancreatitis. Curr
Opin Gastroenterol 1992; 8:817-823.
818
Lipase

Table
Table 1: Methods of Lipase Analysis
Method 1: Titrimetric Method
Principle of analysis:
Reaction A: Triglyceride lipasemonoglyceride + 2 fatty acids
a. Reaction A with fatty acid + NaOH titration to neutrality using phenolphthalein indicator
b. Reaction A with titration of released acids using a pH meter to assess end point
Comments:
a. Used; Cherry-Crandall method
b. Rarely used; requires a pH meter accurate to 0.01 units
Method 2: Colorimetric Method
Principle of analysis: Reaction A with released fatty acids changing the color of a pH indicator, methyl red
Comments: Rarely used
Method 3: Immunological Techniques
a. Latex agglutination
b. Immunoactivation
Principle of analysis:
a. Antibody-coated latex beads aggregate if lipase is present in sufficient quantity
b. Competitive binding assay using radiolabeled lipase; anti-lipase Fab' fragments linked to horseradish
peroxidase (HRP); HRP activity increased by presence of lipase
Comments:
a. Rarely used; semiquantitative
b. Rarely used
Method 4: Coupled Enzyme Spectrophotometric Method
Principle of analysis:
a. 1,2-Diglyceride + H2O lipase 2-monoglyceride + fatty acid
2-Monoglyceride + H2O 2-monoglyceride lipase glycerol + fatty acid

Glycerol + ATP glycerol kinase glycerol 3-phosphate + ADP

Glycerol 3-phosphate + O2 glycerol phosphate oxidase dihydroxyacetone phosphate + H2O2

2H2O2 + 4-aminophenazone + TOOS peroxidase quinone monoimine dye + 4 H2O

b. 1-Oleoyl-2,3-diacetoyl glycerol on TiO2 particles lipaseoleic acid + 2,3-diacetoyl glycerol

2,3-Diacetoyl glycerol diacetinase glycerol + acetate

Glycerol is then quantitated by enzymatic reaction

c. 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester
lipase 1,2-o-dilauryl-rac-glycerol + glutaric acid-(6-methylresorufin)-ester
OH glutaric acid + methylresorufin (absorbs at 570 nm)
Comments:
Commonly used
Method 5: Turbidimetric Method
Principle of analysis: Turbidimetric or nephelometric monitoring of decrease in size of emulsion of substrate
after action of lipase
Comments: Widely used; very dependent on availability of stable, reproducible substrate
819
Lipase
Figure
Figure 1: Hydrolysis of triglyceride by lipase
Procedure: Turbidimetric Method for Serum Lipase
(Modified) [30]
Principle
The clearing of an emulsion of olive oil is measured
turbidimetrically at 340 nm.
Reagents
1. Tris-deoxycholate buffer
Dissolve 3.0 g (0.025 mol) of
tris(hydroxymethyl)aminomethane and 6.0 g (0.014 mol)
of sodium deoxycholate in distilled water, and dilute to a
final volume in 1 L. Adjust to pH 8.8 with concentrated
HCl. Stable for 6 months at 4C to 8C.
2. Olive oil (reagent grade, Fisher Scientific Co.)
Separate the olive oil from the fatty acids by passing it
through alumina (80 to 200 mesh, catalog number A-
540, Fisher Scientific Co.) contained in a column or
separatory funnel plugged with glass wool. One volume
of oil is purified with an equal volume of alumina.
Check the fatty acid content of the oil by pipetting a
0.050 mL aliquot into 3 mL of methyl red reagent,
shaking, and centrifuging. If the absorbance (A502 nm)
of the methyl red layer is more than 0.020 in a 10 mm
cuvette, the oil must be passed through more alumina.
The oil is stable for at least 1 week at 4C to 8C.
3. Olive oil emulsion
Dissolve 1.0 g of purified olive oil in 100 mL of absolute
ethanol. Slowly add 4 mL of this solution, while stirring,
to 100 mL of Tris-deoxycholate buffer. The absorbance
of this reagent at 340 nm should be adjusted by the
addition of more Tris-deoxycholate buffer until it is
within the measurable range of the photometer.
4. Standard
A lyophilized serum with known lipase activity is used.
Measurement Procedure
Equipment: A 10 nm band-pass spectrophotometer
with temperature-controlled cuvette.
1. To 1 mL of emulsion, add 50 L of sample,
standard, or control. Mix immediately.
2. Read the absorbance of each tube at 340 nm at
1 minute (A1) and again at 5 minutes (A5).
Calculation
LPS unknown = (A1 A5) unknown x LPS standard
(A1 A5) standard
820
Lipoprotein (a)
Lipoprotein (a)
Gregory A. Hobbs i
Name: Lipoprotein (a); Lp(a)
Clinical significance: Associated with coronary heart disease, Refer to Chapter 37, Coronary artery
disease, in the 5th edition if Clinical Chemistry: Theory, Analysis, Correlation.
Chemical class: Lipoprotein
Principles of Analysis and Current Usage
Lipoprotein (a) (Lp[a]) was first discovered in 1963 [1].
It is a distinct type of lipoprotein whose physiological
function is not well understood. Its protein component
consists of one molecule of apoB-100 attached to one
molecule of apo(a). The latter protein is found only in
Lp(a). It is apo(a) that distinguishes the particleLp(a)
otherwise closely resembles low density lipoprotein
(LDL). Apo(a) in turn closely resembles plasminogen,
sharing extensive amino-acid sequence homology [2].
Differences in the size of apo(a) within and between
individuals, as well as variations in its carbohydrate
content and oxidation state [3], lead to significant
variability in the size of Lp(a).
In density gradients, Lp(a) bands with LDL, being of
similar density, and may be detected by lipoprotein
electrophoresis. The most widely used assay for Lp(a) is
an ELISA assay.
Though numerous studies point to Lp(a) as an
independent predictor of coronary heart disease [4-8],
current usage is limited because of the lack of
standardization of an Lp(a) assay. Standardization has
been challenging, owing chiefly to the heterogeneity in
particle size and composition.
Reference and Preferred Methods
There is no reference method listed by the International
Federation for Clinical Chemistry. The most specific
method is to assay amino-acid concentration of purified
Lp(a) [9,10]. Slight racial and sex differences have been
reported [11]. Immunometric analysis includes ELISA
[12-15], DELPHIA, immunonephelometry, and
turbidimetry [16]. Commercially available assays
include an immunoturbidimetric test [17-19], which can
be run on automated chemistry analyzers.
Specimen
Serum or plasma is generally acceptable as a specimen.
Some workers [9] have claimed the preparation of a
primary standard stable at 70C for more than 1 year. It
has been shown that controls are stable at 70C for 5
years, but not plasma samples [20]. Others have found
that Lp(a) is relatively stable at room temperature up to
24 hours after venipuncture but less so when processing
i
Lipoprotein (a)
New method:
Fifth edition: Gregory A. Hobbs
was delayed for 36 hours [21]. It is probably wise to
assay for Lp(a) shortly after venipuncture or within 36
hours after storage at 4C.
Interferences
It is claimed [18] that hemoglobin (<500 mg/dL),
conjugated bilirubin (<30 mg/dL), unconjugated
bilirubin (<30 mg/dL), plasminogen (<200 mg/dL),
apoB (<200 mg/dL), and triglycerides (<1500 mg/dL) do
not interfere with assays for Lp(a). No apo(a) size
dependency was observed. Wako [19] found no
significant interference from ascorbic acid (<50 mg/dL),
triglyceride (Intralipid < 2%), and rheumatoid factor
(<510 U/mL). However, they did find some interference
from hemoglobin and bilirubin.
Reference Interval
Owing to the lack of standardization, each laboratory
must determine its own reference interval. This should
be done with a suitably large population and should be
done in consultation with cardiologists who will be using
the results. A widely used reference interval is < 30
mg/dL, but the above-noted racial differences may affect
clinical interpretation. The reference interval is method
specific, and if changing methods, a new interval must
be established.
The principle confounding variable among reference
materials is the size of the apo(a) moiety [10]. Methods
insensitive to this variable are more easily calibrated. An
international reference standard has been developed [22].
Although a value has been assigned, there is
considerable variation in this value among assays. A
value transfer procedure has been described [1]
Interpretation
Some authors [23] recommend that Lp(a) only be
measured in certain high-risk groups and then only by
specialized lipid laboratories. An elevated Lp(a) is
associated with increased risk for coronary heart disease
[4-8] and angina pectoris [24].
Lp(a) Performance Goals
Recovery and correlation experiments are complicated
by the lack of standardization. In general, one should be
able to meet or exceed the claims of the assay
manufacturer.
821
Lipoprotein (a)
Assays vary with respect to the upper limit of linearity,
from 80 to 150 mg/dL [17-19]. Evaluation of linearity
has been addressed elsewhere [22,25]; data should at a
minimum be graphed and appear to be linear on visual
inspection. All calibrators should fall within the linear
portion of the curve.
A coefficient of variation (CV) of less than 5% should
be achievable, but for some kits, imprecision will be
greater at low concentrations (usually using the low
control).
The limit of detection should be less than 5 mg/dL,
though some manufacturers claim 1 mg/dL [19].
References
1 Berg K. A new serum type system in man: the
Lp system. Acta Pathol Microbiol Scand
1963;59:362-382
2 McLean JW, Tomlinson JE, Kuang WJ, Eaton
DL, Chen EY, Fless GM et al. cDNA sequence
of human apolipoprotein(a) is homologous to
plasminogen. Nature 1987;330:132-137.
3 Naruszewicze M, Giroux L-M, Davignon J.
Oxidative modification of Lp(a) causes changes
in the structure and biological properties of
apo(a). Chem Phys Lipids 1994;67/68:167-174.
4 Dahlen GH, Guyton JR, Mohammad A, Farmer
JA, Kautz JA, Gotto AM. Association of levels
of lipoprotein Lp(a), plasma lipids, and other
lipoproteins with coronary artery disease
documented by angiography. Circulation
1986;74:758-765.
5 Armstrong VW, Cremer P, Eberle E, Manke E,
Schulze F, Wieland H et al. The association
between serum Lp(a) concentrations and
angiographically assessed coronary
atherosclerosis. Atherosclerosis 1986;62:249-
257.
6 Hearn JA, DeMaio SJ Jr, Roubin GS,
Hammarstrom M, Sgoutas D. Predictive value
of lipoprotein(a) and other serum lipoproteins
in the angiographic diagnosis of coronary artery
disease. Am J Cardiol 1990;66:1176-1180.
7 Rosengren A, Wilhelmsen L, Eriksson E,
Risberg B, Wedel H. Lipoprotein (a) and
coronary heart disease: a prospective case-
control study in a general population sample of
middle-aged men. Brit Med J 1990;301:1248-
1251.
8 Kamstrup PR, Benn M, Tybjaerg-Hansen A,
Nordestgaard BG. Extreme lipoprotein(a) levels
and risk of myocardial infarction in the general
population. Circulation 2008;117:176-184.
9 Kostner GM, Ibovnik A, Holzer H, Grillhofer
H. Preparation of a stable, fresh frozen primary
lipoprotein(a) (Lp[a]) standard. J Lipid Res
1999;40:2255-2263.
10 Marcovina SM, Albers JJ, Scanu AM, Kennedy
H, Giacully F, Berg K et al. Use of a reference
material proposed by the International
Federation of Clinical Chemistry and
Laboratory Medicine to evaluate analytical
methods for the determination of plasma
lipoprotein(a). Clinical Chemistry
2000;46:1956-1967.
11 Marcovina SM, Albers JJ, Wijsman E, Zhang
ZH, Chapman NH, Kennedy H. Differences in
Lp(a) concentration and apo(a) polymorphs
between black and white Americans. J Lipid
Res 1996;37:2569- 2585.
12 Labeur C, Michlels G, Bury J, Usher DC,
Rosseneu M. Lipoprotein(a) quantified by an
enzyme-linked immunosorbent assay with
monoclonal antibodies. Clin Chem
1989;35:1380-1384.
13 Abe A, Maeda S, Makino K, Seishima M,
Shiimokawa K, Noma A, Kawade M. Enzyme-
linked immunosorbent assay of lipoprotein(a) in
serum and cord blood. Clin Chim Acta
1988;30:31-40.
14 Yeo KH, Walmsley TA, Owen MC, George
PM. A competitive ELISA for lipoprotein(a).
Clin Chim Acta 1992;205:213-222.
15 Morikawa W, Iki R, Terano T, Funatsu A,
Sugiuchi H, Uji Y, Okabe H. Measurement of
Lp(a) with a two-step monoclonal competitive
sandwich ELISA method. Clin Biochem
1995;28:269-275.
16 Tiran A, Tiran B, Hojas S, Kostner GM,
Wilders-Truschnig MM. Immunoquantification
of lipoprotein(a): comparison of nephelometry
and electroimmunodiffusion. J Clin Lab Anal
1993;7:256-262.
17 Lp(a), Package Insert. Rev. 2007-09. Kamiya
Biomedical Company. Seattle, WA.
18 Lp(a), Package Insert. Rev. 2005-12. Point
Scientific Inc. Canton, MI.
19 Autokit Lp(a), Package Insert. Rev.
1/9903IBD00K. WAKO Chemicals. Richmond,
VA.
20 Simo JM, Camps J, Vilella E, Gomez F, Paul
A, Joven J. Instability of lipoprotein(a) in
plasma stored at 70C: effects of
concentration, apolipoprotein(a) genotype, and
donor cardiovascular disease. Clin Chem
2001;47:1673-1678.
21 Pai JK, Curlan GC, Cannuscio CC, Rafai N,
Ridker PM. Stability of novel plasma markers
associated with cardiovascular disease:
processing within 36 hours of specimen
collection. Clin Chem 2002;48:1781-1784.
22 Passey RB, Bee DE, Caffo A, Erikson JM.
Evaluation of the Linearity of Quantitative
Analytical Methods: Proposed Guideline.
NCCLS Document EP6-P. Villanova, PA:
National Committee for Clinical Laboratory
Standards; 1986.
23 Marcovina SM, Levine DM, Lippe G.
Lipoprotein(a). In: Rifai N, Warnick GR, eds.
Structure, Measurement, and Clinical
Significance in Laboratory Measurement of
Lipids, Lipoproteins, and Apolipoproteins.
Washington DC: AACC Press; 1994.
24 Rifai N, Ma J, Sacks FM, Ridker PM,
Hernandez WJL, Stampfer MJ, Marcovina SM.
822
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Study. Clin Chem 2004;50:1364-1371.
 823
Lithium
Lithium
Danni L. Meany and William Clarkei
Name: Lithium
Clinical significance: Treatment of acute mania and long-term prophylaxis of recurrent mania
and depressive episodes Refer to Chapter 47, Nervous System, in the 5th edition
of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: Li
Molecular weight: 6.94 D
Merck Index: 5343
Chemical class: Metal
Structure: Li
Reference method: FAAS
Principles of Analysis and Current Usage
Lithium has been used for treatment of acute mania and
long-term prophylaxis of recurrent mania and depressive
episodes [1, 2]. Because it has a clearly defined therapeutic
range, monitoring of serum lithium concentrations has
been well documented to improve its usage for therapeutic
management of depression and for mood stabilization.
Current methodologies for lithium analysis include flame
atomic absorption spectroscopy (FAAS), flame atomic
emission spectroscopy (FAES), ion-selective electrodes
(ISEs), and colorimetric assays. Although FAAS and
FAES are still used in a few clinical laboratories (less than
3% of the participants of the College of American
Pathologists [CAP] survey in 2007), they now have been
replaced largely by ISEs and colorimetric methods, so they
are only briefly described here. Readers who desire more
information on FAAS and FAES can refer to the previous
version of this chapter.
Flame Atomic Emission Spectroscopy (FAES)
Determination of lithium concentrations using FAES is
based on characteristic emission of lithium atoms at 670.8
nm when given sufficient energy by hot flame. This
characteristic wavelength is selected to provide sufficient
sensitivity and specificity for lithium. Under controlled
conditions, the emission intensity at 670.8 nm is directly
proportional to the number of lithium atoms and therefore
lithium concentration in a sample. This serves as the basis
for lithium quantification using FAES [3].
Flame Atomic Absorption Spectroscopy (FAAS)
FAAS uses the same characteristic wavelength as FAES
for lithium analysis. However, there is an important
difference between these two techniques. In FAES, lithium
atoms in the flame are excited to a high energy level so
they emit light, and it is the emitted light that is measured.
In FAAS, lithium atoms in the flame are kept at a low
energy level so that they are capable of absorbing radiation
at a very narrow bandwidth corresponding to lithiums
own characteristic wavelength. In FAAS, a hollow lithium
cathode lamp is used to produce the characteristic emission
i
at 670.8 nm. When the emitted light from the lamp enters
the flame, some of it is absorbed by the ground-state
lithium atoms in the flame, which results in a decrease of
light intensity. FAAS is approximately 100 times more
sensitive than FAES, because in FAAS most of the lithium
atoms are in the ground state, whereas in FAES only a
small fraction of lithium atoms contribute to emission [3].
Ion-Selective Electrodes (ISEs)
ISE technology for lithium was established in the late
1980s [4,5]. Before 1987, lithium was measured by FAES
in about 90% of clinical laboratories and by FAAS in the
rest. Because lithium analysis using ISEs is simple, rapid,
and only requires a small serum sample without requiring
prior manipulations, it gained in popularity very quickly.
This was evidenced by the CAP participant summary
report that the number of participants using ISEs for
lithium analysis had increased from less than 20 in 1987 to
540, or 25% of the participants, in 1989. Currently, 25% of
the 2007 CAP participants use ISEs for lithium analysis.
Similar to sodium and potassium analysis by ISEs, an
electrical potential that is proportional to lithium
concentration in the sample is generated between a lithium
ISE and a reference electrode, and measured by a
potentiometer. However, the introduction of lithium ISEs
was much later than sodium and potassium ISEs, because
early lithium ISEs responded excessively to many common
ions found in serum, such as Na+, K+, Mg++, Ca++, H+ and
NH4+ [6,7]. Interference from Na+ was particularly
troublesome because the molar ratio of lithium to sodium
i
Lithium
Previous and current authors of this method:
First edition: Robert L. Murray
Methods edition: Robert L. Murray
Second edition: Robert L. Murray
Third edition: Steven C. Kazmierczak,
Fourth edition: Steven C. Kazmierczak
Fifth edition: Danni L. Meany, William Clarke
 824
Lithium
ions in serum is about 1:1500 [4]. One approach to
mitigate the positive bias from Na+ was to use algorithms
in microprocessor circuits to correct for it [4,5]. However,
a more definitive way to achieve a high sensitivity for
lithium was to change the composition of the ion-selective
membrane and add selectivity-enhancing additives in the
ionophores [8,9]. For example, addition of
trioctylphosphine oxide to the ionophore dodecylmethyl-
14-crown-4, improves both sensitivity and selectivity of
lithium ISEs [9]. The drawback of improving lithium ISEs
with additives may be its increased sensitivity to protons.
To prevent errors caused by loss of carbon dioxide, serum
samples for lithium analysis by ISEs should be handled
anaerobically [4,7].
Colorimetric Methods
Although colorimetric methods are the most recently
introduced approach for lithium analysis, they are the most
widely used methods in clinical laboratories. Their use is
promoted by the availability of automated multiple-
channel analyzers in rapid-response and core laboratories,
potential for workstation consolidation, and labor savings
if lithium analysis can be included on common, existing
analytic instrumentation. According to the 2007 CAP
participant summary report, over 75% of the participants
use one of the following colorimetric methods described
here: crown ether method, substituted porphyrin method,
and crown formazan method.
In the crown ether method, lithium binds to a crown ether
dye, which causes a colorimetric shift of peak absorbance
from 400 nm to 600 nm. The shift of the peak absorbance
is then used quantitatively to correlate with lithium
concentrations [10,11]. Overall, this method was capable
of quantitating serum lithium with adequate precision and
correlated well with ISE-based methods.
In the substituted porphyrin method, lithium chelates with
a substituted porphyrin compound and decreases its
absorbance of visible light at alkaline pH. This method
determines the lithium concentration by calculating the
difference of absorption of an unknown sample at 505 and
480 nm against that of a calibrator [12]. A comparison of
this method with an ISE-based method showed excellent
agreement.
Crown formazan dyes have been reported previously for
colorimetric lithium analysis. The method discussed here
is based on the use of the crown formazan dye, 7-nitro-2,
12-dicarboxyl-16, 17-dihydro-5H, 15H-dibenzo (b, i) (1,
11, 4, 5, 7, 8) dioxatetraazacyclotetradecine (shown in
Figure 1), which reacts rapidly with lithium in an alkaline
mixture of water and dimethyl sulfoxide to form a
monovalent binary complex with increased absorbance at
540 nm [11,13,14]. This mixture was found not to cause
precipitation of serum proteins [13]. Similarly to the
substituted porphyrin method, it determines lithium
concentration by the difference of absorption at 540 and
700nm. Correlation between this method and the FAAS
method was acceptable.
Reference and Preferred Methods
The reference method for lithium analysis is based on
FAAS [15], although both FAES and FAAS methods are
equally precise at clinically significant concentrations [16].
The preferred method for serum lithium analysis has
changed from flame photometry (FAAS and FAES) to
ISEs to the current interest in colorimetric methods
because of the reasons described earlier in this chapter.
The performance of all these methods meets acceptable
performance criteria for lithium by the Clinical Laboratory
Improvement Amendments of 1988 (CLIA-88), so the
choice of methodology can be based on available
instrumentation, degree of automation available, and
logistic considerations.
Specimen
Because lithium reaches its steady-state concentration in
serum approximately 10 to 12 hours after the last dose in a
patient on chronic dosing, the standard draw time for
lithium therapeutic monitoring is at least 10 to 12 hours
after the evening dose for patients on a twice-daily dose
regime [1]. Serum and plasma are commonly used
specimens, although specimen requirement may be method
dependent. If plasma is used, sodium heparin Vacutainer is
recommended, and lithium heparin Vacutainer should be
avoided. Serum and plasma should be separated from
cellular blood components if storage of more than 4 hours
is anticipated. Once separated, lithium is stable for 8 hours
at room temperature, for 2 days at 4C, and for months if
frozen. Serum separator tubes are unacceptable because
lithium concentration may be reduced when sample is
stored in a separator tube for a prolonged period of time
(>2 to 6 hrs). Hemolyzed specimens should be rejected for
analysis.
Interferences
Analytical interferences are method dependent and should
be determined on an individual basis. In general, FAAS
and FAES methods are less prone to interferences than
ISEs and colorimetric methods. FAES results are
unreliable for urine analysis only when extreme
concentrations of potassium and hemoglobin are present in
urine. ISEs and colorimetric methods are prone to
interferences from both endogenous and exogenous
sources. As stated previously, the endogenous interfering
ions for ISEs mainly include Na+ and H+. In the crown
ether method, a slight sodium-dependent positive bias in
lithium determination was evident at 157 mmol/L, but only
clinically significant at physiologically extreme
concentrations (>188 mmol/L). Very high concentration of
hemoglobin (>325 mg/dL) in grossly hemolyzed
specimens were also found to cause a clinical significant
positive bias [10]. However, potassium, bilirubin, and
triglycerides did not cause detectable interference with
lithium analysis. In the substituted porphyrin method,
hemolysis, icterus, lipemia, and high and low sodium
levels did not significantly interfere. In the crown
 825
Lithium
formazan method, the crown formazan dye reacts with no
other serum cations except for sodium because of its weak
affinity. To minimize this problem, excess sodium ions
were added in reagents so that specimen-to-specimen
difference in sodium concentrations would have minimal
effects [13]. Interferences from other sources were
minimal.
The exogenous interfering compounds for ISE and
colorimetric methods are the frequently prescribed drugs
such as carbamazepine, n-acetylprocainamide,
procainamide, quinidine, lidocaine, and valproic acid [1].
However, these interferences are concentration dependent
and typically do not introduce clinically significant errors.
Moreover, one ISE method has been shown to be
positively biased by when the blood sample was collected
in a red-top plastic Vacutainer Plus Tube (Becton
Dickinson) containing a silica clot activator and silicone
surfactant [17]. Some ISEs introduce a positive bias in the
presence of high calcium concentrations (>8.9 mmol/L).
Lithium Reference Intervals
Following the initiation of lithium therapy, dosage
adjustments should be based on serum concentrations
determined on a biweekly or weekly basis. Once steady-
state concentrations and symptom remission are achieved,
the lithium concentration should be monitored every 1 to 3
months [18]. Suggested lithium therapeutic concentrations
are illustrated in Table 2. Serum lithium concentrations
between 1.5 and 2.5 mmol/L are considered mildly toxic,
and between 2.5 and 3.5 mmol/L moderately toxic [2].
Urine lithium estimation is of extremely limited use except
to verify a patients compliance or monitor changes in
lithium renal-excretion patterns.
Lithium Reference Intervals
Prophylactic control of mania: 0.6-1.2 mmol/L [19]
Treatment of acute mania: 0.8-1.5 mmol/L [20]
Long-term control of manic-depressive illness:
0.4 to 0.8 mmol/L [21]
Mild toxicity: 1.5-2.5 mmol/L [2]
Moderate toxicity: 2.5-3.5 mmol/L [2]
Interpretation
Lithium has been effectively used for treatment of acute
mania and bipolar disorders for 3 decades, although its
exact mechanism of action is unknown [1,2]. The effects
of lithium on depolarization-provoked and calcium-
dependent release of dopamine and norepinephrine from
presynaptic nerve endings in the central nerve system,
neuronal secondary messenger pathways, and distribution
of Na+, Ca2+, and Mg2+ across neuronal membranes all
have been suggested to contribute to its therapeutic effects
[22-24].
Lithium is available in both immediate and slow-release
formulations. After ingestion, lithium ions are almost
completely absorbed in the gastrointestinal tract. Peak
concentrations occur within 2 to 4 hours after
administration of immediate-release formulations. Slow-
release formulations are associated with later and lower
peak concentrations. Lithium is neither metabolized nor
bound to proteins. Approximately 95% of lithium is
excreted by the kidneys through two phases: the first phase
clears up to two thirds of an acute dose within 6 to 12
hours, followed by a second-phase elimination over the
next 12 hours [24]. Lithium is also able to cross the
placenta and is excreted in breast milk. It should be used
with caution in pregnant and breastfeeding women [25].
Lithium has a low therapeutic index. Toxicity of lithium is
proportional to concentrations just above the therapeutic
range, although some patients may display signs of toxicity
at the upper end of it. Lithium intoxication most
commonly occurs in the settings of hyponatremia, fluid-
volume depletion, and use of diuretics or angiotensin-
converting enzyme inhibitors when renal excretion of
lithium is reduced [20]. The clinical manifestations of mild
lithium intoxication include irregular coarse tremors,
muscle weakness, nausea, vomiting, and diarrhea. Severe
lithium toxicity can lead to seizures, stupor, and coma.
Moreover, the complications of chronic lithium use
include renal insufficiency, nephrogenic diabetes insipidus,
leukocytosis, goiter, and hypothyroidism [2,24].
Treatments for lithium intoxication include fluid repletion
to restore sodium and water balance in hyponatremia, oral
charcoal administration to decrease intestinal absorption
[26], and hemodialysis for severe lithium toxicity [27,28].
Lithium Performance Goals
Acceptable performance criteria for lithium by CLIA-88
are target value 0.3 mmol/L or 20%, whichever is
greater. Nevertheless, more stringent performance goals,
such as less than 3% and 5% analytical imprecision, have
been reported elsewhere [1,29]. Currently, according to the
2007 CAP survey, the imprecision for all the lithium
measurements range from 3% to 10% for specimens with
an average lithium concentration of 1.48 mmol/L.
References
1 Linder MW, Keck PE Jr. Standards of laboratory
practice: antidepressant drug monitoring, National
Academy of Clinical Biochemistry. Clin Chem.
1998;44:1073-1084.
2 Orsulak PJ. Agents for the treatment of bipolar
disorder. In: Shaw LM, editor. The Clinical
Toxicology Laboratory: Contemporary Practice
of Poisoning Evaluation. Washington, DC:
American Association for Clinical Chemistry;
2001:237-245.
3 Kricka LJ. Optical techniques. In: Burtis CA,
Ashwood ER, Bruns DE, editors. Tietz Textbook
of Clinical Chemistry and Molecular Diagnostics.
St Louis: Mosby; 2006:61-91.
4 Okorodudu AO, Burnett RW, McComb RB,
Bowers GN Jr. Evaluation of three first-
 826
Lithium
generation ion-selective electrode analyzers for
lithium: systematic errors, frequency of random
interferences, and recommendations based on
comparison with flame atomic emission
spectrometry. Clin Chem. 1990;36:104-110.
5 Bertholf RL, Savory MG, Winborne KH,
Hundley JC, Plummer GM, Savory J. Lithium
determined in serum with an ion-selective
electrode. Clin Chem. 1988;34:1500-1502.
6 Oesch U, Ammann D, Simon W. Ion-selective
membrane electrodes for clinical use. Clin Chem.
1986;32:1448-1459.
7 Kitazawa S, Kimura K, Yano H, Shono T.
Lithium-selective polymeric membrane electrodes
based on dodecylmethyl-14-crown-4. Analyst.
1985;110:295-299.
8 Kimura K, Oishi H, Miura T, Shono T. Lithium
ion selective electrodes based on crown ethers for
serum lithium assay. Anal Chem. 1987;59:2331-
2334.
9 Bastard G. Bastard replies. Phys Rev Lett.
1988;60:2561.
10 Gorham JD, Walton KG, McClellan AC, Scott
MG. Evaluation of a new colorimetric assay for
serum lithium. Ther Drug Monit. 1994;16:277-
280.
11 Christenson RH, Mandichak JJ, Duh SH,
Augustyn JM, Thompson JC. Clinical
performance characteristics of a new photometric
lithium assay: a multicenter study. Clin Chim
Acta. 2003;327:157-164.
12 Lyon AW, Whitley C, Eintracht SL. Analytic
evaluation and application of a novel
spectrophotometric serum lithium method to a
rapid response laboratory. Ther Drug Monit.
2004;26:98-101.
13 Thompson JC. Development of an automated
photometric assay for serum lithium and use of
binding equilibrium expressions to optimize
results. Clin Chim Acta. 2003;327:149-156.
14 Gruson D, Lallali A, Furlan V, Taburet AM,
Legrand A, Conti M. Evaluation of a new lithium
colorimetric assay performed on the dade behring
dimension X-pand system. Clin Chem Lab Med.
2004;42:1066-1068.
15 Pybus J, Bowers GN Jr. Serum lithium
determination by atomic absorption spectroscopy.
In: McDonald RP, editor. Standard Methods of
Clinical Chemistry. New York: Academic Press;
1970:189-192.
16 Levy AL, Katz EM. A comparison of serum
lithium determinations using flame photometry
and atomic absorption spectrophotometry. Clin
Chem. 1969;15:787.
17 Sampson M, Ruddel M, Albright S, Elin RJ.
Positive interference in lithium determinations
Table 1: Lithium Methods Summary
from clot activator in collection container. Clin
Chem. 1997;43:675-679.
18 NIMH/NIH Consensus Development Panel.
Consensus Development Conference Statement.
Mood disorders: pharmacologic prevention of
recurrences. Am J Psychiatry. 1985;142:469-476.
19 Roberts WL, McMillin GA, Burtis CA, Bruns
DE. Reference information for the clinical
laboratory. In: Burtis CA, Ashwood ER, Bruns
DE, editors: Tietz Textbook of Clinical Chemistry
and Molecular Diagnostics, Philadelphia:
Saunders; 2006:2251-2318.
20 Okusa MD, Crystal LJ. Clinical manifestations
and management of acute lithium intoxication.
Am J Med. 1994;97:383-389.
21 Boton R, Gaviria M, Batlle DC. Prevalence,
pathogenesis, and treatment of renal dysfunction
associated with chronic lithium therapy. Am J
Kidney Dis. 1987;10:329-345.
22 Baldessarini RJ, Vogt M. Release of 3H-
dopamine and analogous monoamines from rat
striatal tissue. Cell Mol Neurobiol. 1988;8:205-
216.
23 Chen G, Huang LD, Zeng WZ, H KM. Mood
stabilizers regulate cytoprotective and mRNA-
binding proteins in the brain: long-term effects on
cell survival and transcript stability. Int J
Neuropsychopharmacol. 2001;4:47-64.
24 Baldessarini RJ, Tarazi FI. Drugs and the
treatment of psychiatric disorders: psychosis and
mania. In: Hardman JG, Limbird LE, editors.
Goodman & Gilmans The Pharmacological Basis
of Therapeutics. New York: McGraw-Hill;
2001:485-520.
25 Schou M. Lithium treatment during pregnancy,
delivery, and lactation: an update. J Clin
Psychiatry. 1990;51:410-413.
26 Linakis JG, Lacouture PG, Eisenberg MS, Maher
TJ, Lewander WJ, Driscoll JL et al.
Administration of activated charcoal or sodium
polystyrene sulfonate (Kayexalate) as gastric
decontamination for lithium intoxication: an
animal model. Pharmacol Toxicol. 1989;65:387-
389.
27 Jacobsen D, Aasen G, Frederichsen P, Eisenga B.
Lithium intoxication: pharmacokinetics during
and after terminated hemodialysis in acute
intoxications. J Toxicol Clin Toxicol. 1987;25:81-
94.
28 Hauger RL, OConnor KA, Yudofsky S, Meltzer
HL. Lithium toxicity: when is hemodialysis
necessary? Acta Psychiatr Scand. 1990;81:515-
517.
29. Fraser CG. Desirable standards of performance
for therapeutic drug monitoring. Clin Chem.
1987;33:387-389.
 827
Lithium

Method 1: Flame atomic absorption spectrophotometry; quantitative

Principle of analysis: Absorption of light at 670.8 nm by Li0
Comments: Serum, plasma, urine, red blood cells

Method 2: Flame atomic emission spectrophotometry; quantitative

Principle of analysis: Emission of light at 670.8 nm by Li0
Comments: Serum, plasma

Method 3: Ion-selective electrodes; quantitative

Principle of analysis: Measurement of voltage difference generated by Li+ in contact with lithium ionophore
Comments: Serum, plasma, whole blood

Method 4: Colorimetric; quantitative

Principle of analysis:
Lithium + crown ether (400 nm) dye complex (600 nm)
Comments: Serum, plasma

Method 5: Colorimetric; quantitative

Principle of analysis: Lithium bound to substituted porphyrin compound, resulting in decrease of absorbance of
visible light at alkaline pH
Comments: Serum, plasma; this is currently the most popular method in diagnostic laboratories.

Method 6: Colorimetric; quantitative

Principle of analysis: Lithium bound to crown formazan dye, 7-nitro-2, 12-dicarboxyl-16, 17-dihydro-5H, 15H-
dibenzo [b, i] [1, 11, 4, 5, 7, 8] dioxatetraazacylotetradecine in an alkaline mixture of water and dimethyl sulfoxide to
form a monovalent binary complex with increased absorbance at 540 nm
Comments: Serum, plasma

Figure

Figure 1. Structure of crown formazan dye under conditions of the assay and with lithium bound within
the pocket of the 14-membered ring [13].
828
Luteinizing Hormone

Luteinizing Hormone
Gregory Hobbs

Name: Luteinizing hormone, LH

Clinical significance: Refer to Chapter 44, Pregnancy, and Chapter 50, The Gonads, in the 5th
Edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular weight: 30,000 D
Half-life: 30 minutes
Chemical class: Protein

i anti- LHcoated microparticles, forming an antigen-

Principles of Analysis and Current Usage
Luteinizing hormone (LH), a glycoprotein with a antibody complex. This mixture is transferred to a
molecular weight of approximately 30,000 daltons, is reaction cell, where the microparticles bind irreversibly
produced by the anterior pituitary. Like follicle- to a glass fiber matrix. After washing to remove
stimulating hormone (FSH), LH is also present as a unbound materials, an anti- LH antibody conjugated to
heterodimer with two different subunits, and The alkaline phosphatase is added, which binds to the
subunit (molecular mass 14000 D) is common to all antigen-antibody complex. After again washing the
gonadotropins and also to thyroid-stimulating hormone matrix cell to remove unbound materials, the substrate,
(TSH). The sub-unit (118 amino acids) is specific for methylumbelliferyl phosphate, is added and the
LH because of its amino-acid sequence and carbohydrate fluorescent product, methylumbelliferone, is measured.
content [1]. Thus immunoassays are specific for the
Roche Diagnostics produces an assay based on
subunit. Early immunoassays used radioisotopes.
electrochemiluminescence detection technology (ECL)
Although radioimmunoassays are available, current
[6,7]. This is a sandwich-based immunoassay that
immunoassays use enzyme-linked immunosorbent assay
involves two incubations. The first involves the sample,
(ELISA) [2], proprietary enzyme immunoassay (EIA)
a biotinylated monoclonal antibody specific for LH, and
methods, or chemiluminescent methods.
a second LH-specific monoclonal antibody labeled with
a ruthenium complex. An antibody-antigen-antibody
The Vitros immunodiagnostic method [3] uses an
sandwich forms during this incubation. In the second
immunometric assay technique. The LH in the sample is
incubation, streptavidin-coated microparticles are added,
captured by a biotinylated antibody on one binding site,
and the sandwich complex becomes bound to the
while a second antibody labeled with horseradish
microparticle by way of the biotin-streptavidin
peroxidase binds to a different site. The antibody
interaction. The reaction mixture is aspirated into a
complex is captured by streptavidin bound to plastic
measuring cell where the microparticles are captured
wells. The peroxidase bound to the solid phase is used to
magnetically on the surface of an electrode. Unbound
catalyze the oxidation of a luminol derivative, with the
substances are removed by a wash step. ECL works by
production of light.
the removal of the magnet and supply of voltage to the
electrode. The ruthenium in the complexes and
The Siemens (formerly DPC) procedure is a two-site
tripropylamine in the residual buffer are oxidized. The
sandwich chemiluminometric immunoassay [4]. LH in
two compounds react to produce an excited state of
the patient serum binds to an acridinium ester (AE)-
ruthenium. The spontaneous decay results in emission of
labeled mouse monoclonal antibody (Lite Reagent). A
a photon of light at 620 nm.
second antibody specific for LH attached to magnetic
particles captures the complex. The acridinium ester
Unfortunately, these assays are still not standardized [8],
remaining on the solid phase is reacted chemically to
and results are not comparable across methods.
release light. A direct relationship exists between the
Reference ranges also vary with method.
amount of LH in the patient sample and the amount of
relative light units (RLUs) detected by the Advia
Reference and Preferred Methods
Centaur system.
There is no established reference method.
Abbott Diagnostics produces an LH method based on its
It is preferred that any selected method be standardized
microparticle enzyme immunoassay (MEIA) technology
to a World Health Organization (WHO) reference
[5] for its AxSYM platform. In this method, LH binds to
material. The current practice is to use the second WHO
International Standard LH 80/552.
i
Luteinizing Hormone (LH)
Previous and current authors of this method: Sensitive LH methods are available from various
First edition: Not done manufacturers. As an example, we describe below the
Second edition: Not done assay available on the ACS 180 system, which is also
Third edition: Not done performed on the Bayer Centaur. Both systems are now
Fourth edition: Amadeo J. Pesce offered by Siemens Medical Solutions.
Fifth edition: Gregory Hobbs
829
Luteinizing Hormone

Specimen in moderate amounts, does not interfere with most

The choice of serum or plasma is dependent in part on
the manufacturer of the test system. For example, for the
ACS 180 system, serum is the preferred specimen, while
for the Vitros or Abbott systems, serum, EDTA, or
heparinized plasma are all acceptable. However, some
systems show significantly increased results when
Interferences
Heterophile antibodies such as human anti-mouse
antibody can interfere in many immunoassays. The
presence of hemolysis, lipemia, or icterus, when present
immunoassay methods. Abbott notes that the sample
should be free of fibrin, which may cause erroneous
results [9]; for this reason, complete clot formation
should take place.
Reference Interval
Selected reference interval values for the LH
concentration in serum using the WHO International
Standard 80/552 in milli-international units/mL (mIU/L)
are:

Bioserve Abbott Roche

Patient Population ELISA Ortho Siemens AxSYM Elecsys
Females
Follicular phase 20 2.6-12 0.8-25.8 1-18 2.4-12.6
Midcycle peak 40-200 27-93 25-57.3 24-105 14-95.6
Luteal phase 20 0.8-15 0.8-27.1 0.4-20 1.0-11.4
Postmenopausal 20-100 13-85 39.7-103.5 15-62 7.7-58.5
Males 3-12 1.6-9.7 1.3-12.9 2-12 1.7-8.6

gel-separator tubes compared with specimens collected 3 Ortho Clinical Diagnostics. VITROS
in plain evacuated tubes. The sample should be Immunodiagnostic Products LH Reagent Pack.
refrigerated if not analyzed immediately. Samples may Instructions for Use. Publication Number
be refrigerated for up to 5 days (Abbott recommends 24 J03784. Rochester, NY: Ortho Diagnostics;
hours). Samples may be stored at 20C but not frozen 2002.
and thawed repeatedly. 4 Bayer HealthCare LLC. LH ADVIA Centaur
Assay Manual. 111736, Rev. J, 2005.
Interferences 5 Abbott Laboratories Diagnostic Division. LH in
Heterophile antibodies such as human anti-mouse Abbott AxSYM system, 34-2540/R8, Abbott
antibody can interfere in many immunoassays. The Park, IL 60064, 2003.
presence of hemolysis, lipemia, or icterus, when present 6 Roche Diagnostics. GmbH, D-68298. LH,
in moderate amounts, does not interfere with most Luteinizing Hormone, 11818252001v13.
immunoassay methods. Abbott notes that the sample Mannheim, Germany, 2005-06.
should be free of fibrin, which may cause erroneous 7 Roche Diagnostics website.
results [9]; for this reason, complete clot formation <http://us.labsystems.roche.com/products/ecl/ec
should take place. ltech.shtml>
8 Sturgeon, CM Ellis, AR. Standardization of
Interpretation FSH, LH, and hCG: current position and future
LH is made in the anterior lobe of the pituitary gland. prospects. Mol Cell Endo 2007;260-262:301-
LH production and release is under the control of the 309.
hypothalamus through gonadotropin-releasing hormone 9 Abbott Laboratories Diagnostics Division.
(GnRH). In women, LH and FSH are responsible for the Abbott AxSYM Sytem: LH. Abbott Park, IL:
physiological changes of the ovaries during the Abbott Diagnostics; 2003.
menstrual cycle. Under the influence of FSH, LH, and 10 Kicklighter EJ Norman RJ. The gonads. In:
estrogens, the follicles mature during the follicular Kaplan LA, Pesce AJ, eds. Clinical Chemistry:
phase. Theory, Analysis, and Correlation. St Louis:
In men, while FSH acts directly on the Sertoli cells in Mosby; 1989:650-663.
the spermatogenic tubule, LH influences spermato- 11 Liwnicz BH, Liwnicz RG. The
genesis indirectly by increasing testosterone synthesis in hypothalamopituitary system. In: Kaplan LA,
the adjacent Leydig cells. Pesce AJ, eds. Clinical Chemistry: Theory,
Analysis, Correlation. 3rd ed. St Louis: Mosby;
LH and the other pituitary gonadotropin, FSH, play a 1989:613-619.
critical role in maintaining the normal function of the 12 Scott MG, Ladenson JH, Green ED, Gast MJ.
male and female reproductive systems. Hormonal evaluation of female infertility and
reproductive disorders. Clin Chem
References 1989;35:620-629.
1 Niewoehner CB. Endocrine Pathophysiology. 13 Butt WR, Blunt SM. The role of the laboratory
Madison, CT: Fence Creek; 1998. in the investigation of infertility. Ann Clin
2 BioServ Diagnostics (Rostock, Germany) Biochem 1988;25:601-609.
website. <http://www.bioserv-diagnostics.com>
830
Luteinizing Hormone
Procedure: Siemens ACS:180
Editors note: the following procedure is used only
as an example of an automated immunoassay for
LH, and is not intended to promote this assay or
any other assay.
PUBLISHERS NOTE: this material, derived from the
Siemens package insert, cannot replace the
information provided by the manufacturer for the
analysis of this analyte on their instrument. Please
refer all questions to the manufacturer.
Luteinizing Hormone (LH) in Serum on the
Siemens Automated Chemiluminescence System
(ACS)-180
Principle
The Siemens ACS LH assay is a two-site
chemiluminometric (sandwich) immunoassay which
uses constant amounts of two antibodies that have
specificity for the intact LH molecule. The first antibody
or Lite Reagent is a polyclonal sheep anti-LH antibody
labeled with acridinium ester (AE). The second
antibody, or Solid Phase, is a monoclonal mouse anti-LH
antibody covalently coupled to paramagnetic particles. A
direct relationship exists between the LH in the sample
and the RLUs detected by the ACS:180 systems.
This assay is intended for the quantitative determination
of luteinizing hormone (LH) in serum using the Siemens
Automated Chemiluminescence System (ACS). Results
from this assay can be combined with other results to
assess the integrity of the pituitary-gonadal axis.
Specimen Collection and Handling
Serum is the recommended sample type for this assay.
Cap and refrigerate specimens if testing is not done
immediately, or freeze specimens if testing is not done
within 2 days. Freeze specimens only once, and mix
thoroughly after thawing. No further patient preparation
is required.
Assay Reagents
For In-Vitro Diagnostic Use.
Caution:
1. Discard opened assay reagents that are at room
temperature (20C to 30C) for a total of 40 hours. Do
not use these reagents to calibrate the ACS:180 systems
or assay samples.
2. Do not use kit components beyond expiration
date.
3. Do not mix different lot numbers of reagents.
Contents
Catalog Number Contents
672270 Six vials of LH Lite Reagent
Six vials of LH Solid Phase
One Master Curve card
or
672271 One vial of LH Lite Reagent
50
One vial of LH Solid Phase
One Master Curve card
Assay Reagents
Assay Reagents
Reagent Volume/Ingredients Storage/Stability
LH Lite 5 mL/vial; polyclonal sheep anti-LH 2C8C; until
Reagent antibody (~1.344 mg/vial) labeled on the date vial
acridinium ester in buffer, labeled with label or
sodium azide, and microbicides cumulative 40
hours at room
temperature
LH Solid 20 mL/vial; monoclonal mouse anti- 2C8C; until
Phase LH antibody (~1.066 mg/vial) expiration date
covalently coupled to pasramagnetic on the vial label
a articles in buffer, sodium azide, and or cumulative
microbiocides 40 hours at room
s temperature
CAUTION: Sodium azide can react with copper and lead
plumbing to form explosive metal azides. On disposal,
flush with a large volume of water to prevent the buildup
of azides.
BIOHAZARD: These reagents are intended for use with
human serum. Components from biological sources were
used in the manufacture of this product. It is
recommended that appropriate biosafety precautions be
followed to prevent the transmission of infectious
agents.
Preparing the Assay Reagents
1. For best results, thoroughly mix the Solid Phase by
inverting the vial before each use. Visually inspect
the bottom of the vial to ensure that all the particles
are dispersed and suspended. If foaming occurs,
rim the top of the vial with applicator sticks to
remove the foam.
2. Place a septum cap on the Lite Reagent and on the
Solid Phase vials.
3. Prewarming or bringing the reagents to room
temperature before use is not required.
Master Curve Calibration
The ACS LH assay requires a Master Curve calibration
when you use a new lot number of Lite Reagent and
Solid Phase. For each new lot of Lite Reagent and Solid
Phase, use the Master Curve card to enter the Master
Curve information at the System
Management/Definitions/Master Curve screen. The
Siemens LH assay is standardized against the World
Health Organization (WHO) 2nd IRP 80/552 Reference
Material.
Two-Point Calibration Interval
The ACS LH assay requires a two-point calibration
every 28 days. Also, perform a two-point calibration
when Number
you change lot numbers of Lite Reagent and Solid
of Tests
Phase300
or when your controls are repeatedly out of range.
Quality Control
For detailed information about entering quality-control
(QC) information, refer to Section 7, Calibration and
Quality Controls, in your ACS:180 Plus and ACS:180
Reference Manual. Enter quality-control information at
the System Management/Definitions/Controls screen.
831
Luteinizing Hormone
To monitor system performance and chart trends,
analyze three levels of QC materials for LH at least once
a day, usually at the beginning of the working day. Each
separate tray should have at least one level of QC pool to
detect between-tray drifts. This policy is in accordance
with CLIA 88 requirements and Ciba-Cornings
recommendations that a minimum of three levels of
controls be run at least one time during a 24-hour period
and when a two-point calibration is performed.
Please refer to the laboratorys QC manual for the
procedure for the reconstitution of these pools and the
interpretation of the QC results. All QC data must be
entered into the LIS.
Assay Procedure
For detailed information about operating the ACS:180
systems, refer to Section 6, Operating the ACS:180, in
your ACS:180 Plus and ACS:180 Reference Manual.
NOTE:
If automatic tray and cup assignment is on, use the
printed worklist to help you load calibrators, controls,
and patient samples into the correct tray and cup
positions.
If automatic tray and cup assignment is off, and
your controls and patient samples are all barcoded, you
can load samples in any position.
1. Schedule the requested tests or profiles for each
calibrator, control, or patient sample.
2. Prepare and load ACS Calibrator B, if required.
a. Reconstitute the low and high calibrators
according to the preparation instructions in the
ACS Calibrator B product insert.
b. Dispense the low and high calibrator into a
sample cup labeled with the appropriate
barcode label.
c. Load the sample cups in any position on the
sample tray.
Ensure that the low calibrator precedes the
high calibrator on the sample tray.
3. Prepare and load the controls.
a. Prepare the controls according to the
instructions in the control product insert.
b. Dispense the controls into a labeled sample
cup.
c. Load the sample cup in the appropriate
position on the sample tray.
4. Prepare the primary tubes or sample cups, and load
them on the sample tray.
This assay requires 100 mL of sample for a single
determination. This volume does not include the
unusable volume in the sample cup or the
additional volume required when performing
duplicates or other tests on the same sample.
5. If automatic dilution is required, dispense Multi-
Diluent 1 into a sample cup labeled with the
appropriate barcode label, and load the sample cup
on the sample tray.
If manual dilution is required, refer to Procedural
Notes.
6. Load the Lite Reagent and Solid Phase in adjacent
positions on the reagent tray.
7. Press START. The ACS:180 system:
a. dispenses 100 mL of sample into a cuvette
b. dispenses 100 mL of Lite Reagent and
incubates it for 5 min at 37C
c. dispenses 400 mL of Solid Phase and
incubates it for 2.5 min at 37C
d. separates, aspirates, and washes the cuvette
with reagent water
e. dispenses 300 mL each of Reagent 1 and
Reagent 2 to initiate the
chemiluminescent reaction
f. prints results according to the print option you
selected, which is described in Section 5,
Defining System Parameters, in your ACS:180
Plus and ACS:180 Reference Manual.
Calculating Results
For detailed information about how the ACS:180
calculates results, refer to Section 2, Understanding
the ACS:180, in your ACS:180 Plus and ACS:180
Reference Manual.
LH reference IntervalsSiemens
As with all diagnostic tests, each laboratory should
establish its own reference intervals for the diagnostic
evaluation of patient results.
To determine the Siemens ACS LH reference intervals,
the data in the following table were generated.
832
Luteinizing Hormone

Expected Values
Mean Observed Range
Category N (mIU/mL) (mIU/mL)
Females
Normally menstruating
Follicular phase 63 5.0 0.825.8
Midcycle peak 8 43.0 25.057.3
Luteal phase 73 4.2 0.8-27.1
Pregnant 35 <0.1 <0.11.4
Postmenopausal
No hormone therapy 82 65.0 39.7103.5
Hormone therapy 12 25.5 12.047.8
Contraceptives 15 6.2 1.011.9
Ovarian failure 15 73.8 23.4123.0
Turners syndrome 5 70.5 64.375.7
Polycystic ovary disease 61 52.9 16.6114.0
Males
13-20 years 25 8.2 4.69.4
20-70 years 100 4.1 1.312.9
>70 years 25 30.6 11.356.4
Testicular failure 16 61.2 45.487.2
Klinefelters syndrome 8 52.5 44.267.7
Children 45 2.8 1.05.9

Procedural Notes endogenous LH values, whereas administration of

1. Dispose of hazardous and biologically
contaminated materials according to your
institutions practices. Discard all materials in a
safe and acceptable manner and in compliance with
all federal, state, and local requirements.
2. Samples greater than 200 ng/mL must be diluted
and then repeated to obtain accurate results. Use
the ACS Multi-Diluent 1 to dilute samples.
3. Patient samples can be diluted automatically by the
ACS:180 system or manually. For automatic
dilutions, ensure that you load the ACS Multi-
Diluent 1 and that you have defined values for
Dilution Setpoint and Dilution Ratio in System
Management.
4. For detailed information about setting system
parameters, refer to Section 5, Defining System
Parameters, and Section 6, Operating the
ACS:180, in your ACS:180 Plus and ACS:180
Reference Manual.
5. Manually dilute LH patient samples when patient
results exceed those values obtained by automatic
dilution or when your laboratory protocol requires
manual dilution, not automatic dilution.
6. Use ACS Multi-Diluent 1 to manually dilute
patient samples, and then load the diluted sample
onto the sample tray, replacing the neat sample.
7. Ensure that you mathematically correct the results
for the dilution.
8. To program automatic dilutions at the System
Management/Definitions/Tests screen, Siemens
recommends you select a 1:2 or 1:5 dilution ratio
for optimal performance of the ACS LH assay.
Limitations of Procedure
Administration of gonadotropin-releasing hormone
(GnRH), clomiphene citrate, and human menopausal
gonadotropin (hMG) to females can result in elevated
estrogens or testosterone can result in decreased
endogenous LH values. Administration of hMG to males
can result in elevated endogenous LH values, whereas
administration of testosterone decreases endogenous LH
values.
Patient samples with high LH levels can cause a
paradoxical decrease in the RLUs (high-dose hook
effect). In this assay, patient samples with LH levels as
high as 1500 mIU/mL will assay greater than 200
mIU/mL.
It is well known that heterophilic antibodies in human
serum can react with reagent immunoglobulins,
interfering with in-vitro immunoassays. Patients
routinely exposed to animals or to animal serum
products can be prone to this interference, and
anomalous high values can be observed. Additional
information may be required for diagnosis.
Specimens have an insignificant
that are effect on the ssay up to
hemolyzed 500 mg/dL of hemoglobin
lipemic 3000 mg/dL of triglycerides
icteric 20 mg/dL of bilirubin
Performance Characteristics
Specificity
The cross-reactivity of the ACS LH assay with TSH, -
LH, human chorionic gonadotropin (hCG), -hCG, FSH,
prolactin, and human growth hormone (hGH) was
determined by adding these hormones to samples
containing LH. The level of LH in the samples was then
determined.
833
Luteinizing Hormone

Performance Characteristics
LH Value Without LH Value With
Cross-Reactant Cross-Reactant (mIU/mL) Cross-Reactant (mIU/mL)
TSH, 1000 mIU/mL 4.8 5.0
10.2 11.6
71.5 70.7
-LH, 5 ng/mL 4.3 4.1
9.2 9.7
90.7 91.1
hCG, 200,000 mIU/mL 3.2 3.8
9.4 10.6
99.9 92.9
-hCG, 200,000 mIU/mL 2.9 2.9
10.5 10.5
69.3 65.6
FSH, 200 mIU/mL 2.7 2.6
9.4 9.4
77.2 79.0
Prolactin, 400 ng/mL 3.2 3.2
9.1 9.5
59.4 67.4
hGH, 100 ng/mL 3.1 3.4
9.2 10.7
57.8 60.2
Sensitivity LH is secreted by the anterior pituitary in response to
GnRH secreted by the hypothalamus [11]. In males, LH
Siemens reports that this assay has a minimum is also called interstitial cellstimulating hormone
detectable concentration of 0.09 mIU/mL. (ICSH) [10]. In both males and females, LH secretion is
regulated by a balance of positive and negative feedback
Reportable Range mechanisms involving the hypothalamic-pituitary axis,
The Siemens LH assay measures LH concentrations up the reproductive organs, and the pituitary and sex steroid
to 200 mIU/mL. hormones [10,12,13]. LH and the other pituitary
gonadotropin, FSH, play a critical role in maintaining
Interpretation the normal function of the male and female reproductive
LH is a glycoprotein hormone having two subunits. The systems. Table 1 shows the target tissues and actions of
subunit is similar to those of FSH, hCG, and TSH. The LH in females and males [10-13].
subunit is different from those of the other gycoprotein
hormones and confers its biochemical specificity [10].

Table 1. LH Target Tissues and Actions

Category Target Tissues Actions
Females Theca cells of the ovarian Stimulates production of androgens
follicles that FSH converts to estradiol during
the follicular phase
Graafian follicle Acts synergistically with FSH to cause
ovulation during midcycle peak
Corpus luteum Stimulates formation of the corpus luteum
after ovulation
Stimulates progesterone secretion during
the luteal phase
Males Leydig cells in the interstitial Stimulates testosterone secretion
tissue of the testes

Abnormal LH levels with corresponding increased or
decreased levels of FSH, estrogens, progesterone, and
testosterone are associated with a number of pathological
conditions [11]. Increased LH levels are associated with
menopause, primary ovarian hypofunction, and
polycystic ovary disease in females and primary
hypogonadism in males. Decreased LH levels are
associated with primary ovarian hyperfunction in
females and primary hypergonadism in males.
834
Lysozyme
Lysozyme
Arnold L. Schultz
Name: Lysozyme, muramidase, N-acetylmuramide glycanohydrolase,
N-acetylmuramyl hydrolase, globulin G1
Clinical interpretation:
Enzyme number: EC 3.2.1.17
Molecular mass: 14,400 D
Merck Index: 5457
Chemical class: Protein
Principles of Analysis and Current Usage
Lysozyme catalyzes the hydrolysis of the N-
acetylmuramic acid (14) N-acetylglucosamine
linkages in the polysaccharide component of the cell
wall of Micrococcus lysodeikticus. The original method
for the determination of lysozyme activity measured the
time required for the sample to lyse part or all of a full-
grown culture plate of Micrococcus lysodeikticus
(method 1, Lysozyme Methods Summary Table).[1]
The two most commonly used procedures for the assay
of lysozyme are based upon the lytic action of the
enzyme disrupting the bacterial cell wall resulting in the
dispersion of the cell contents and the clearing of the
turbid bacterial suspension (method 2, Lysozyme
Methods Summary Table). The photometric method for
the determination of lysozyme activity is based upon the
decrease in turbidity with time when the sample is added
to a suspension of Micrococcus lysodeikticus.[2]
Turbidity may be monitored at 540, 640, or 645 nm. In
another variation of this technique, the cell lysis reaction
is monitored by nephelometry using white light.[3]
The agarose plate or lysoplate technique uses a
suspension of the bacteria in solidified agar contained in
a Petri dish.[4]
Sample wells, which are cut into the agar, are filled, and
the plate is allowed to stand at room temperature for 12
to 18 h. During this time period a cleared zone of lysis
develops in the initially translucent agarose gel (method
3, Lysozyme Methods Summary Table). The diameters
of the cleared zones are related to the log of the
concentration of lysozyme in the unknown by means of a
series of standards.
Several other procedures for the determination of
lysozyme activity in body fluids have been investigated.
Lysozyme activity has been measured with a
viscosimetric assay, employing glycol chitin as
substrate[5] (method 4, Lysozyme Methods Summary
Table). Lysozyme action on the glycol chitin substrate
results in decreased viscosity of the substrate solution,
and the change in viscosity can be related to the
lysozyme concentration. In addition to hydrolysis,
lysozyme catalyzes extensive transglycosylation. This
reaction has been used to relate the rate of release of
methylglycosamide from 14C-labeled chitobiose to
lysozyme concentration[6] (method 5, Lysozyme
Methods Summary Table). An immunoassay based on
C:\5th Edition Print Revised\Lysozyme_2009-06-24.doc
the specific inactivation of human lysozymeT4-
bacteriophage conjugate has been used to detect
enzymatically inactive forms of lysozyme in addition to
the biologically active forms[7] (method 6, Lysozyme
Methods Summary Table). Immunochemical
quantitation of lysozyme has been performed by
immunoelectrophoresis; the lysozyme in serum or urine
is electrophoresed into antibody-containing agarose gel,
and the immunoprecipitates are stained with Coomassie
Brilliant Blue R-250[8] (method 7, Lysozyme Methods
Summary Table). Two radioimmunoassays (method 10,
Lysozyme Methods Summary Table) for lysozyme have
been described.[9,10]
A method using living Micrococcus lysodeikticus cells
loaded with trimethylphenylammonium ions (TMPA+)
as a marker has been reported.[11] This method utilized
a TMPA+ selective electrode to measure TMPA+
released by lysis (method 8, Lysozyme Methods
Summary Table). The initial rate of potential change is a
function of the rate of lysis and is directly proportional
to the lysozyme activity. A chromophoric substrate has
been evaluated as an alternative to the bacterial cell wall
methods.[12] The substrate used was 3,4-dinitrophenyl
tetra-N-acetyl--chitotetraoside (method 9, Lysozyme
Methods Summary Table). Upon incubation with serum
samples in a sodium citrate buffer, the substrate is
converted to 3,4-dinitrophenol. The increase in
absorbance at 410 nm is measured with respect to time.
Reference and Preferred Methods
Most of the methods described for the determination of
lysozyme activity in body fluids have significant
limitations including substrates that are not available
commercially, lack of sensitivity, necessity for pure
human standards that are not readily available, need for
specialized instrumentation, and laborious procedures.
The two most widely accepted techniques for the
determination of lysozyme activity are the turbidimetric
method[13] and the lysoplate diffusion method.[14] Both
of these methods are strongly affected by several factors
that must be controlled to give reproducible results. The
lysoplate method is influenced by the pH and ionic
strength of the gel medium. The turbidimetric method is
affected by the technique used to prepare the bacterial
cell suspension. Both methods are affected by the pH of
the reaction mixture, by the nature and concentrations of
835
Lysozyme
the salts and ions present, and by the reaction
temperature.[15]
Although both the turbidimetric and lysoplate methods
are relatively easy to perform and the results of the two
methods correlate well,[16] the lysoplate method may
offer an important clinical advantage.[14] Several
investigators using the lysoplate procedure have reported
significantly elevated lysozyme activity in sera from
patients with active Crohns disease.[17-19] However,
when the turbidimetric method is used, the serum
lysozyme activity does not readily differentiate Crohns
disease from other types of bowel inflammation.[20-23]
Specimen
Lysozyme activity has been measured in serum, urine,
stool, cerebrospinal fluid, gastric juice, bile, saliva,
mothers milk, amniotic fluid, sperm, tears, transudates,
pus[24] and pleural fluid.[25]
EDTA plasma or serum may be used. Heparin interferes
with lysozyme activity through formation of heparin
lysozyme complexes. Serum should be separated from
the clot within 2 h of collection. Delayed separation of
the serum can result in spuriously high levels of
lysozyme, presumably because of the breakdown of
leukocytes.[4] Serum lysozyme is stable for at least 1
month at C if not repeatedly frozen and thawed.[25]
At pH 4.5 to 6.3, urinary lysozyme is stable for 4 days at
room temperature. The higher the pH of the urine, the
more rapidly the enzyme activity decreases. Lysozyme
in urine is stable for 8 days at 4 C and for 18 days at
20 C.[25] It has been reported that serum and urine
samples that have been dried or pretreated on filter paper
are stable for 13 weeks at room temperature.[26]
Tears are collected by having the patient stare at a strong
light from a slitlamp. When tears have collected in the
lower cul-de-sac, they are drawn into a micropipet by
capillary action, with care being taken not to damage the
conjunctiva.[27] Because of their very high lysozyme
activity, tears are diluted 1:20 with the phosphate buffer
before they are assayed.[24]
Interferences
There is a slight increase in lysozyme activity in icteric,
lipemic, and hemolyzed serum when lysolyme is
measured by the lysoplate method.
Lysozyme reference Intervals[24]
1. Serum, 4 to 15.6 g/mL (0.28 to 1.1 mol/L)
2. Urine, 0 to 1.4 g/mL (0 to 0.097 mol/L)
3. Tears, 1151 to 1383 g/mL (80 to 96 mol/L)
Lysozyme activity is independent of age or sex.
Interpretation
In healthy persons serum and urine levels of lysozyme
are usually insignificant. Serum and urine levels are
higher in the Chdiak-Higashi syndrome and in some
leukemias. In the leukemias they are low in M2
(myeloblastic leukemia with maturation), M3
(hypergranular progranulocytic leukemia), and M6
(erythroleukemia); intermediate in M4 (myelomonocytic
leukemia); and high in M5 (monocytic histiocytic
leukemia).[28] Lysozyme, which is placed in
circulation, is rapidly cleared by the kidney. Thus, in
those diseases in which large amounts of lysozyme are
released, both serum and urine measurements may be
made.
Lysozyme Performance Goals
The between-run precision of the chromophoric method
varies from 8% for high concentrations of lysozyme to
5% for values within the reference interval. The
sensitivity of the lysoplate method is 0.1 g/mL.
The sensitivity of the turbidometric method is at least 0.5
g/mL, and its reproducibility is 18% at 0.5 g/mL and
4.4 at 25 g/mL.[29]
References
1 Fleming A. On a remarkable bacteriolytic
element found in tissues and secretions. Proc
Roy Soc 1922;93:306-317.
2 Boasson EH. On the bacteriolysis by lysozyme,
J Immunol 1938;34:281-93.
3 Borgen J, Romslo I. Lysozyme determined in
serum and urine by a simple nephelometric
method. Clin Chem 1977;23:1599-1601.
4 Osserma, EF, Lawlor DP. Serum and urinary
lysozyme (muramidase) in monocytic and
monomyelocytic leukemia. J Exp Med
1966;124:921-51.
5 Lundbla G, Hultin E. Human serum lysozyme
(muramidase). I. Viscosimetric determination
with glycol chitin and purification by selective
adsorption. Scand J Clin Lab Invest
1966;18:201-8.
6 Dahlquist FW, Borders CL, Jacobson G,
Raftery MA. The stereospecificity of human,
hen, and papaya lysozymes. Biochemistry
1969;8:694-700.
7 Maron E, Bonavida B. A sensitive
immunoassay for human lysozyme in biological
fluids. Biochim Biophys Acta 1971;229:273-5.
8 Johansson BG, Malmquist J. Quantitative
immunochemical determination of lysozyme
(muramidase) in serum and urine. Scand. J Clin
Lab Invest 1971;27:255-61.
9 Peeters TL, Depraeter YR. Vantrappen GR.
Radioimmunoassay for urinary lysozyme in
human serum from leukemic patients Clin
Chem 1978;24:2155-7.
10 Thomas MJ, Russo A, Craswell P, Ward M,
Steinhardt I. Radioimmunoassay for serum and
urinary lysozyme. Clin Chem 1981;27:1223-6.
11 DOrazio P, Meyerhoff ME, Rechnitz GA.
Membrane electrode measurement of lysozyme
using living bacterial cells. Anal Chem
1978;50:1531-4.
12 Turner GA, Ghneim H, Freeman J. Evaluation
of 3,4-dinitrophenyltetra-N-acetyl--
chitotetraoside as a substrate for the
836
Lysozyme
measurement of lysozyme in normal and
pathological sera. Ann Clin Biochem
1979;16:51-3.
13 Litwack G. Photometric determination of
lysozyme activity. Proc Soc Exp Biol Med
1955;89:401-3.
14 Peeters TL, Vantrappen GR. Factors
influencing lysozyme determinations by the
lysoplate method. Clin Chim Acta 1977;74:217-
55.
15 Smolelis AN, Hartsell SE. Factors affecting the
lytic activity of lysozyme. J Bacteriol
1952;63:665-74.
16 Zucker S, Hanes DJ, Vogler WR, Eanes RZ.
Plasma muramidase: a study of methods and
clinical applications. J Clin Lab Med
1970;75:83-92.
17 Schussheim A, Josephson AS, Greenwald RA.
Serum lysozyme in inflammatory bowel
disease. Gastroenterology 1976;70:632-4.
18 Falchuk KR, Perrotto JL, Isselbacher KJ. Serum
lysozyme in Crohns disease, a useful index of
disease activity. Gastroenterology 1975;69:893-
6.
19 Falchuk KR, Perroto JL, Isselbacher KJ. Serum
lysozyme in Crohns disease and ulcerative
colitis. N Engl J Med 1975;292:395-7.
20 Peeters TL, Vantrappen G, Geboes K. Serum
lysozyme levels in Crohns disease and
ulcerative colitis. Gut 1976;17:300-5.
21 Pruzanski W, Marcon N. Lysozyme in Crohns
disease. N Engl J Med 1975;293:611-2.
22 Peeters TL, Geboes K, Vantrappen GR. Serum
lysozyme levels in Crohns disease (cont.). N
Engl J Med 1975;292:1349-50.
23 Pounder RE, Avella JR, McCallum H,
Misiewicz JJ. Serum lysozyme in inflammatory
bowel disease. Lancet 1975;2:228-9.
24 Hankiewicz J, Swierczek E. Lysozyme in
human body fluids. Clin Chim Acta
1974;57:205-9.
25 Klockars M, Pettersson T, Riska H, Hellstrm
PE, Norhagen A. Pleural fluid lysozyme in
human disease Arch Intern Med 1979;139:73-7.
26 Maeda K, Ito K, Yamaguchi N. A simple
lysoplate method of lysozyme determination
with samples dried on filter paper. Clin Chim
Acta 1980;100:175-81.
27 Sen DK, Sarin GS, Mani K, Saha K.
Immunoglobulins in tears of normal Indian
people. Br J Ophthalmol 1976;60:302-4.
28 Miale JB. Laboratory medicine hematology. 6th
ed, St. Louis (MO): CV Mosby Co; 1982. p
722-3.
29 Daniels JC, Fukushima M, Larson DL, Abston
S, Ritzmann SE. Studies on muramidase
(lysozyme). I. Serum and urine muramidase
activity in burned children. Tex Rep Biol Med
1971;29:13-39.
837

Lysozyme

Tables

Methods of Lysozyme Analysis

Method 1: Micrococcus lysodeikticus; agar plate, qualitative, semiquantitative
Principle of analysis: Inhibition of growth; time of lysis of cells in plate proportional to concentration
Comments: Many body fluids; historical
Method 2: Micrococcus lysodeikticus; turbidimetric, photometric
Principle of analysis: Lysozyme dissolves suspended cells. Rate at which cell suspension clears is proportional
to concentration
Comments: Serum, urine, tears; linear to only 15 g/mL
Method 3: Micrococcus lysodeikticus; agarose diffusion
Principle of analysis: Cells immobilized in agarose. Diameter of zone cleared of bacterial suspension varies with
lysozyme concentration
Comments: Serum, urine, tears, many other body fluids; recommended procedure
Method 4: Glycol chitin; viscosimetric
Principle of analysis: Enzyme hydrolyses polysaccharide. Decrease in viscosity proportional to enzyme
concentration
Comments: Serum; not used in clinical studies
Method 5: Transglycosylation; liquid scintillation radioassay
Principle of analysis: 14C-labeled chitobiose forms methylglycosamide in presence of enzyme
Comments: Experimental; laborious; substrate not readily available; not used in clinical studies
Method 6: Bacteriophage conjugate; immunoassay
Principle of analysis: Inactivation of lysozymebacteriophage conjugate with antilysozyme; competition by
sample lysozyme
Comments: Serum, urine, tears, saliva; sensitive, laborious; substrate not readily available; not used in clinical
studies
Method 7: Electrophoresis; immunochemical
Principle of analysis: Electrophoresis of antigen into antibody-containing agarose gel to form precipitate
Comments: Serum, urine; laborious, lacks sensitivity
Method 8: Micrococcus lysodeikticus; electrochemical
Principle of analysis: Release of trimethylammonium ions from cells; measured with a selective electrode
Comments: Experimental; laborious; substrate and instrumentation not readily available; good sensitivity and
precision
Method 9: 3,4-Dinitrophenyl tetra-N-acetyl--chitotetraoside; photometric
Principle of analysis: Chromophoric substrate read at 410 nm
Comments: Serum; lacks sensitivity; substrate not readily available
Method 10: Radioimmunoassay
Principle of analysis: Competitive assay using 125I-Labeled lysozyme
Comments: Serum, urine; reagents not readily available; good sensitivity and precision
Method 11: Nephelometry; immunochemical
Principle of analysis: Light scattering by antigenantibody complex
Comments: Serum, urine; reagents not readily available; good sensitivity and precision
838
Lysozyme
Figures
Lysozyme: Figure 1
Standard curve for agarose gel cell lysis assay. Cleared diameter versus lysozyme concentration. Notice that the x axis is a
three-cycle, logarithmic scale.
Procedure: Measurement of Lysozyme Activity by
Agarose Gel Cell Lysis Assay (Lysozyme Table:
Reaction conditions)
Principle
Lysozyme diffuses through an agarose gel containing a
suspension of Micrococcus lysodeikticus. A cleared zone
ring of lysis develops in the initially translucent agarose
gel.
Reagents
1. Sodium phosphate, dibasic, 0.067 mol/L.
Dissolve 9.47 g of anhydrous dibasic sodium phosphate
in 1 L of deionized water. Stable for 6 months at 2 to 6
C.
2. Potassium phosphate, monobasic, 0.067
mol/L. Dissolve 9.07 g of monobasic potassium
phosphate in 1 L of deionized water. Stable for 6 months
at 2 to 6 C.
3. Phosphate buffer, 0.067 mol/L, pH 6.3. Mix
224 mL of 0.067 mol/L dibasic sodium phosphate and
776 mL of 0.067 mol/L monobasic potassium phosphate.
Check the pH with a pH meter. Stable for 2 months at 2
to 6 C.
4. Agarose, 10 g/L. Dissolve 10 g of agarose in 1
L of 0.067 mol/L phosphate buffer, pH 6.3, kept at a
temperature of 60 to 70 C, with stirring. Stable for 1
month stored in a glass-stoppered bottle at 2 to 6 C.
5. Buffered substrate. Prepare a uniform
suspension of Micrococcus lysodeikticus by mixing 500
mg of dried Micrococcus lysodeikticus (Sigma Chemical
Co., St. Louis, MO) in 5 mL of phosphate buffer on a
vortex mixer. Add the uniform suspension of the
organism to 1 L of molten (60 to 70 C) 10 g/L agarose.
Discard after use.
6. Substrate plates. Pour freshly prepared buffer
substrate into a Petri dish, 85 mm in diameter, to a depth
of 4 mm. After the agarose solidifies, cut 16 sample
wells 2 mm in diameter in four rows of four, 15 mm
apart. The plates are stable for 2 weeks stored inverted at
2 to 6 C in plastic bags.
7. Stock lysozyme solution, 400 g/mL (27.8
mol/L). Dissolve 20 mg of grade I, 3crystallized,
chicken egg white lysozyme, 40,000 units/mg (Sigma
Chemical Co.) in 50 mL of 8.5 g/L sodium chloride in
siliconized glassware. Stable at 2 to 6 C for 6 days.
8. Working lysozyme standards, 3, 15, 30, 60,
and 120 g/mL. Prepare a 120 g/mL standard by
diluting 3.0 mL of the stock lysozyme solution to 10.0
mL with 8.5 g/L sodium chloride. The remainder of the
working standards are prepared by serially diluting the
120 g/mL standard with 8.5 g/L sodium chloride.
Prepare fresh daily.
Assay
Equipment: Enlargerviewer to measure zones of
clearance.
1. Allow the necessary number of lysoplates to
come to room temperature in their plastic bags.
2. Allow the samples to come to room
temperature.
3. Fill the sample wells with a volume of 25 L in
as short a time interval as possible. Each filled
plate should contain the 5 working lysozyme
standards as well as the samples to be assayed.
Apply serum, plasma, and urine undiluted;
839

Lysozyme

dilute tears 1:20 with 0.067 mol/L phosphate Notes

buffer, pH 6.3. 1. A lysoplate test kit is commercially available
4. Carefully cover the plates tightly, and return from Kallestad Laboratories, Inc. (Austin, TX).
them to their plastic bags. 2. The standardization of the lysozyme activity
5. Incubate the plates at room temperature on a determination is a matter of concern. An unexplained
level surface for 12 to 18 h. The time of finding of a specific activity ratio of human
development of the lysoplates is not critical. lysozyme/hen white lysozyme activity between 8:1 and
12:1 for the lysoplate method has been observed[4] For
Calculation turbidimetric methods a ratio of 3:1 to 4:1 has been
1. At the end of the incubation period, measure the reported.[8] Methods for the isolation and purification
cleared-zone ring diameters that have of human lysozyme have been developed.[4,8]
developed to the nearest 0.1 mm with an However, the techniques used are beyond the scope of
enlarger-viewer (available from Kallestad many routine clinical chemistry laboratories. The
Laboratories, Inc., Austin, TX). All standard commercially available lysoplate test kit does contain
and sample ring diameters should be read in the reference materials prepared from pooled human urine.
same order and at the same time intervals that
were required to fill the sample wells.
2. For each lysoplate, plot the concentration of
each working standard on the logarithmic axis
against the corresponding cleared-zone ring
diameters on the linear axis of three-cycle
semilogarithmic graph paper. A typical
standard curve is presented in Lysozyme:
Figure 1.
3. For each sample, calculate the lysozyme
activity from the samples cleared ring diameter
and its intersection with the corresponding
value in micrograms per milliliter from the
reference curve.
4. For samples whose values are higher than 120
g/mL, dilutions in 8.5 g/L sodium chloride
must be made and the test repeated. The
resulting concentration is then multiplied by the
dilution factor. The dilution factor for the initial
determination of tears is 20.
 840
Magnesium
Magnesium
Steven C. Kazmierczak
Name: Magnesium
Clinical significance: Refer to Chapter 33, Bone Disease, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Atomic formula: Mg
Atomic mass: 24.31 D
Merck Index: 5469
Chemical class: Alkaline earth element
Forms: Free Mg2+, protein bound or in inorganic complexes
Principles of Analysis and Current Usage i
Magnesium, like calcium, has offered a challenge to
analysts because of the ease of contamination and
problems with binding to serum proteins. Serum
magnesium accounts for only 0.3% of the bodys
magnesium. Approximately 30% to 35% of the total
magnesium ion concentration in serum is protein bound;
the remainder is free, or ionized [1].
The original methods used for magnesium analysis were
precipitation techniques [2-4]. In these, the magnesium
was precipitated from solution in the form of salts such as
MgNH4PO4. The amount of precipitate, measured
gravimetrically or by analysis of phosphorus in the
precipitate, was directly related to the magnesium
concentration. These manually performed precipitation
methods are no longer in use.
Compleximetric techniques using EDTA [5] and other
chelators [1,6] have been developed but are rarely used.
Sensitive fluorometric assays employing calcein [1], o,o-
dihydroxyazobenzene [7], and 8-hydroxyquinoline are also
available [1].
In the latter method (Table 1, Method 1), a chelated
complex forms, has an excitation maximum at 420 nm, and
fluoresces at 530 nm. Many fluorescence procedures
employ chelating agents such as EGTA to inhibit
2+
interference from Ca .
Magnesium can be measured by flame photometry, since
its band emission peaks at 370 or 383 nm, but this is not a
iMagnesium
Previous and current authors of this method:
First edition: E. Christis Farrell
Methods edition: E. Christis Farrell
Second edition: E. Christis Farrell
Third edition: Steven C. Kazmierczak
Fourth edition: Steven C. Kazmierczak
Fifth edition: Steven C. Kazmierczak
widely used procedure [1]. Spectrophotometric methods
for measurement of magnesium in biological fluids
following deproteinization have been developed. Several
of these methods have been automated and are in
widespread use today.
Gindler and Heth [9] introduced the use of calmagite (1-[1-
hydroxy-4-methyl-2-phenylazo]-2-naphthol-4-sulfonic
acid), a metallochromic dye, for direct determination of
magnesium without deproteinization (Table 1, Method 2).
The alkaline, blue-colored working reagent forms a pink
magnesium-calmagite complex, and the intensity of the
color produced is measured at 532 nm. EGTA is used to
2+
prevent Ca interference, and KCN is used to inhibit
heavy-metal complexes. Polyvinylpyrrolidone and its 9-
ethylene oxide adduct (Bion PVP, Bion Ne-9) have been
employed to prevent serum proteins from shifting the
absorbance maxima of the Mg-calmagite complex [10].
The reaction is quite rapid and completed in 60 seconds.
This method has been automated.
Methylthymol blue, which has been used for calcium
analysis [1], has also been employed for the analysis of
magnesium. Again, specific calcium chelators are used to
increase the specificity of the analysis. The color is
quantitated bichromatically at 510 and 600 nm (Table 1,
Method 3).
Another direct spectrophotometric method is based on the
reaction between magnesium and the dye Titan yellow
(Clayton Yellow, Thiazol yellow, 2,2-[(diazoamino)-di-p-
phenylene]bis[6-methyl-7-benzothiazolesulfonic acid]
disodium salt). Under the alkaline conditions of the
reaction, the colloid Mg(OH)2 forms [1,11].
The colloidal particles adsorb the reactive fraction of the
Titan yellow dye to form a red complex (lake) measured at
540 nm (Table 1, Method 4). To prevent precipitation of
the dye-colloid complex, stabilizers such as polyvinyl
alcohol have been added to the reaction mixture. The
stabilizers also increase the intensity of the color [1]. This
method is rarely used.
 841
Magnesium
Another method for analysis of magnesium in serum and
urine utilizes the chelating agent chlorophosphonazo III
(CPZ) (Table 1, Method 5) [12]. The reaction is initiated
by the introduction of sample to a dye reagent containing
CPZ and EGTA. The CPZ selectively complexes the
magnesium present in the sample while EGTA chelates
calcium. This first step results in an absorbance decrease at
550 nm and an absorbance increase at 675 nm. In the
second phase of the assay, reagent containing EDTA is
added, which removes magnesium from the dye complex
with a resultant absorbance change. Less than 5%
interference has been reported from samples containing up
to 200 mg/L calcium [12].
The majority of magnesium determinations performed in
clinical laboratories measure the concentration of total
magnesium present. However, the most important fraction
is the free, or ionized, magnesium, because this is the
physiologically active fraction. Ionized magnesium is in
equilibrium with protein bound and organic or inorganic
complexed forms. The development of synthetic neutral
carriers for determination of ionized magnesium have
enabled measurement of this form of magnesium. The
selectivity of these ionophores allows for the measurement
of ionized magnesium even in the presence of
pathophysiological concentrations of calcium. Intracellular
determination of magnesium interference by calcium with
the ionophore is generally ignored, since the ratio of
magnesium to calcium within the cell is approximately
1000:1 [13].
Enzyme assays for magnesium have been described (Table
1, Method 8) [14,15]. These assays are based on the
principle that an Mg-ATP complex must be present to
enable the phosphorylating enzyme to transfer the high-
energy phosphate from the ATP to another molecule. The
reaction rate is followed by monitoring the rate of NADPH
formation at 340 nm. In this assay, it is presumed that the
Mg2+ binds to the ATP in solution and remains bound to
the ADP reaction product.
Reference and Preferred Methods
Measurement of magnesium with use of flame atomic
absorption spectrometry (Table 1, Method 6) has been
recognized as the traditional reference method for
magnesium [16], but ion chromatography has been
considered as a candidate reference method [17].
Magnesium has a strong spectral emission or absorption
line at 285.2 nm, which can be readily isolated and used to
measure magnesium concentration specifically in
biological fluids. The definitive method of magnesium
27
analysis is neutron activation with Mg (Table 1, Method
7).
When comparing methods or comparing the results of
proficiency studies, one must be aware of the use of four
different systems of units for the reporting of magnesium
concentrations:
0.5 mmol/L = 1.0 mEq/L = 1.22 mg/dL = 12.2 mg/L
Methods that relied on manual precipitation techniques
were relatively imprecise and difficult to automate. The
fluorescent techniques, though sensitive, suffer from
problems of both background fluorescence and
fluorescence quenching. Quenching effects in urine
samples render fluorescence methods unsuitable for
analysis in urine.
The direct spectrophotometric analyses, adapted to
automated techniques, are fairly accurate and precise. The
Titan yellow dye method is adversely affected by the
impurity of the dye. Only a portion of the dye is reactive
with magnesium [1,18], and the degree of purity can affect
the sensitivity and accuracy of the method. Titan yellow
methods appear to show a 10% to 15% positive bias when
compared to precipitation methods [19]. The precision (%
coefficient of variation [CV]) of these assays for serum
magnesium values in the normal range is 8% to 14%.
The calmagite method has been adapted to many
automated analyzers. The reagents for the calmagite
reaction are more stable separately and should therefore be
added in two separate steps, or the reagent should be
mixed immediately before use. It may be advantageous to
take an absorbance reading of the dye plus serum before
adding the alkaline cyanide reagent to develop the color.
Subtracting the blank reading from the final reaction color
gives a change of absorbance proportional to magnesium
concentrations up to 40 mg/L. In addition, it has been
reported that the calmagite will precipitate if left in contact
with plastic lab ware for more than 15 to 20 minutes [20].
On the basis of precision and accuracy, atomic absorption
is the preferred method.
Specimen
Serum must be separated from the clot as soon as possible,
or the level of magnesium will increase because of its
elution from red blood cells [1]. Plasma is also an
acceptable specimen. Serum or plasma separated from red
cells or stored in gel-barrier tubes are stable for 7 days at
room temperature or 4C and indefinitely at 20C [21].
Binding of magnesium to proteins and ligands in serum
and plasma is pH dependent. For measurement of ionized
magnesium concentrations, some suggest simultaneous
measurement of pH to allow adjustment of magnesium
concentration to a pH of 7.40 [22]. Urine should be
acidified to pH 1 with concentrated HCl. If a precipitate
forms, shake, mix, acidify, and warm to 60C to redissolve
it [1].
 842
Magnesium
Interferences
2+
Erythrocytes contain about three times the Mg found in
2+
plasma, and the Mg is free within the interior of the cell,
not bound to the membrane, as is calcium [1]. Thus in
general, hemolyzed samples are unacceptable for analysis.
The presence of calcium gluconate in serum because of
intravenous administration can cause results of the Titan
yellow method to be 35% low [1]. Silicon, found in
vacutainer tubes, does not interfere with measurements.
Blood anticoagulated with citrate, oxalate, or EDTA is
unacceptable because these compounds can chelate
magnesium ions. Specimens with increased bilirubin or
those that are lipemic have been found not to interfere.
Magnesium Reference Interval
Urinary excretion of magnesium is diet dependent, but
normally equals about one third of daily intake [1]. Serum
and cerebrospinal fluid magnesium concentrations are
shown below. Pregnancy typically results in a slow,
progressive decrease in serum magnesium to about 10%
below nonpregnant values [23]. Aging has not been found
to have any appreciable effect on serum magnesium
concentrations. There is no appreciable variation in the
patients magnesium levels throughout the day if exercise
is avoided, and there is no difference between fasting and
nonfasting magnesium levels. Newborns have essentially
the same serum concentrations of magnesium as adults.
Magnesium Reference Intervals in different
biological fluids
Sample/Method Mg2+
Serum and cerebrospinal fluid
Titan yellow [1]
for serum 18.2329.3 mg/L
(1.52.4 mEq/L)
Average value [1]
for CSF 24.432.0 mg/L
(no reported sex difference) (2.02.7 mEq/L)
Atomic absorption 15.825.5 mg/L
(1.32.6 mEq/L)
Urine 7.3 12.2 mg/24 hrs
(6.0 10.0 mEq/24 hrs)
(3.0 5.0 mmol/24
h )
Interpretation
Magnesium is a cofactor for many intracellular enzymes,
including all those that use ATP as a substrate. It is present
in all tissues and bone and is about equally distributed
between soft tissue and bone.
A major cause of hypermagnesemia is inappropriate
dosage of therapeutic agents such as magnesium sulfate.
Hypomagnesemia is caused by either decreased intake or
loss of magnesium. Often hypomagnesemia is associated
with hypocalcemia. Signs of magnesium deficiency
include increased neuromuscular and cardiac excitability.
Signs of magnesium excess include antagonism of nerve-
impulse transmission, with consequent muscle weakness.
Some of the main causes of hypo- and hypermagnesemia
are shown in Table 2.
Ionized magnesium represents approximately 70% of total
magnesium concentration. Although the proportion may
vary from subject to subject, it has been found to be
remarkably consistent in sequential samples from a
particular individual [24]. Ionized magnesium
concentrations may show significant changes from normal,
despite no changes in total serum magnesium
concentrations.
Magnesium Performance Goals
A review of results of quality-control surveys and linear
regression analyses of several adaptations of the calmagite
reaction reveal a rather small, negative proportional bias (r
= 0.96) for the calmagite method compared to atomic
absorption [25]. The methylthymol blue color reaction
usually shows a closer correlation with atomic absorption.
The precision, %CV, of the methylthymol blue (MTB)
method for serum magnesium concentrations within the
interval is less than 5%. The coefficient of variation for the
calmagite methods for serum magnesium concentrations
within normal reference limits is less than 8%. Proposed
limits for measurement of magnesium suggest a limit for
analytical error of 2.4% or less [17]. Clearly, improvement
in the precision of magnesium methods is indicated.
The acceptable Clinical Laboratory Improvement
Amendments of 1988 (CLIA 88) performance criteria for
magnesium require that laboratories obtain magnesium
values that are within 25% of peer-group laboratories.
Precision goals based on biovariability data indicate a
maximum standard deviation of 0.02 mg/dL for serum
magnesium [26]. Intra-individual variation obtained in
healthy adults over a 4-week period found a %CV of 3.4
[27]. The vast majority of magnesium methods used in the
clinical laboratory involve use of calmagite, methylthymol
blue, or Xylidyl Blue. Chlorophosphonazo III and
arsenazol dye are used less frequently. A very small
percent of labs (<1%) utilize an enzymatic method for
magnesium.
References
1 Henry RJ, Cannon DC, Winkelman JW. Clinical
Chemistry Principles and Techniques. 2nd ed.
New York: Harper & Row; 1974
2 Kramer B, Tisdall FF. A single technique for the
determination of calcium and magnesium in small
amounts of serum. J Biol Chem. 1921;47:475-
481.
3 Briggs AP. A colorimetric method for the
 843
Magnesium
determination of small amounts of magnesium. J
Biol Chem. 1922;52:349-355.
4 Denis W. The determination of magnesium in
blood, plasma and serum. J Biol Chem.
1922;52:411-415.
5 Schwartzenbach G, Bidermann W, Banserter F.
Komplex-one VI: neue einfache Titriermethoden
zur Bestimmung der Wasserhrte. Helv Chim
Acta. 1946;29:811-818.
6 Thiers RE. Magnesium (fluorometric). In: Meites
S, ed. Standard Methods in Clinical Chemistry.
Vol 5. New York: Academic Press; 1965
7 Brien M, Marshall RT. An automated
fluorometric method for the determination of
magnesium in serum and urine using o,o-
dihydroxyazobenzene: studies on normal and
uremic subjects. J Lab Clin Med. 1966;68:701-
712.
8 Schachter D. The fluorometric estimation of
magnesium in serum and urine. J Lab Clin Med.
1959;54:763-768.
9 Gindler EM, Heth DA. Colorimetric
determination with bound Calmagite of
magnesium in human blood serum (abstract). Clin
Chem. 1971;17:663.
10 Pierce Magnesium Stat Kit, Pierce Chemical Co.,
Rockford, IL; insert revised Feb. 1979.
11 Basinski DH. Magnesium (Titan yellow). In:
Meites S, ed. Standard Methods in Clinical
Chemistry. Vol 5. New York: Academic Press;
1965:137.
12 Dixon DJ, Denton J, Kaufman RA. New
magnesium method for the Cobas Chemistry
Systems using chlorophosphonazo III. Clin Chem.
1990;36:1068
13 Elin RJ. Laboratory tests for the assessment of
magnesium status in humans. Magnes Trace
Elem. 1992;10:172-181.
14 Tabata M, Kido T, Totani M, Marachi T. Direct
spectrophotometry of magnesium in serum after
reaction with hexokinase and glucose-6-
phosphate dehydrogenase. Clin Chem.
1985;31:703-705.
15 Wimmer MC, Artiss JD, Zak B. A kinetic
colorimetric procedure for quantifying
magnesium in serum. Clin Chem. 1986;32:629-
632.
16 Kulpmann WR, Ruschke D, Buttner J, Paschen K.
A candidate reference method for the
determination of magnesium in serum. Clin Chem
Clin Biochem. 1989;27:33-39.
17 Thienpont LM, Van Nuwenborg JE, Stockl D. Ion
chromatography as reference method for serum
cations. J Chromatogr. 1997;789:557-568.
18 King HGC, Pruden G. Component of commercial
Titan yellow most reactive towards magnesium:
its isolation and use in determining magnesium in
silicate minerals. Analyst. 1967;92:83.
19 Simonsen DG, Westover LM, Wertman M. The
determination of serum magnesium by the
molybdivanadate method for phosphate. J Biol
Chem. 1947;169:39-47.
20 Khayam-Bashi K, Liu TZ, Walter V.
Measurement of serum magnesium with a
centrifugal analyzer. Clin Chem. 1977;23:289.
21 Wilding P, Zilva JA, Wilde CE. Transport of
specimens for clinical chemistry analysis. Ann
Clin Biochem. 1977;14:301-306.
22 Rayana MC, Burnett RW, Covington AK,
DOrazio P, Fogh-Andersen N, Jacobs E et al.
Guidelines for sampling, measuring and reporting
ionized magnesium in undiluted serum, plasma or
blood: Internation Federation of Clinical
Chemistry and Laboratory Medicine (IFCC):
IFCC Scientific Division, Committee on Point of
Care Testing. Clin Chem Lab Med. 2005;43:564-
569.
23 Lind T. Clinical chemistry of pregnancy. Adv Clin
Chem. 1980;21:1-24.
24 Altura BT, Altura BM. Role of magnesium in
pathophysiological processes and the clinical
utility of magnesium ion selective electrodes.
Scand J Clin Lab Invest. 1996;56(suppl 224):211-
221.
25 Cohen SA, Daza JE. Calmagite method for
determination of serum magnesium modified
[letter]. Clin Chem. 1977;23:289.
26 Fraser CG, Peterson PH, Ricos C, Haeckel R.
Quality specifications. In: Haeckel R, ed.
Evaluation Methods in Laboratory Medicine. R.
New York: VCH Publishers; 1993:87-99.
27 Gonzlez-Revaldera J, Garca-Bermejo S,
Menchn-Herreros A, Fernandez-Rodriguez E.
Biological variations of Zn, Cu, and Mg in serum
of healthy subjects. Clin Chem. 1990;36:2140-
2141.
 844
Magnesium

Table 1: Methods for Magnesium Analysis

Method 1: Titration of fluorescent complex
Principle of analysis:
a. Mg2+ + calcein Mg-calcein complex (fluorescent; ex, 420 nm;
em, 530 nm)
b. Mg2+ + o,o-dihydroxyazobenzene fluorescent complex
Comments:
a. Stat or small laboratories, difficulty with fluorescence background noise, as well as quenching
b. Rarely used
Method 2: Calmagite
Principle of analysis:
Comments: Widely used in labs without Du Pont aca or atomic absorption; adapted to many automated analyzers;
slight negative bias; positive shift caused by lipemia
Method 3: Methylthymol blue
Principle of analysis: Mg2+ + methylthymol blue complex at 510 and 600 nm
Comments: Du Pont aca, most commonly reported method; good correlation with atomic absorption
Method 4: Titan yellow
_
Principle of analysis: Mg2+ + 2NaOH + Titan yellow PVP Mg(OH) Titan yellow (red lake; colloidal ppt)
2
Comments: Infrequently used; only one eighth sensitivity of calmagite
Method 5: Chlorophosphonazo III
_
Principle of analysis: Mg2+ + CPZ Mg-CPZ complex EDTAMg-EDTA + CPZ
Comments: Frequently used; minimal interference from Ca2+
Method 6: Atomic absorption
Principle of analysis:

Comments: Used in a number of routine laboratories; excellent accuracy, reference method

Method 7: Neutron activation isotope dilution
Principle of analysis: Uses magnesium isotope 27Mg
Comments: Definitive method
Method 8: Enzyme colorimetric
Principle of analysis: Enzyme transfer of phosphate from MgATP to substrate is dependent on Mg2+ concentration
in serum; reaction monitored by coupling to indicator system such as NADPH or peroxidase
Comments: Newly developed; potential as new automated method

Table 2: Magnesium Changes in Disease

High magnesium Low magnesium
Uremia Mg2+-deficiency tetanus
Acute and chronic renal failure Loss of gastrointestinal secretions
Chronic glomerulonephritis Malabsorption syndrome
Addisons disease Diuretic therapy
Intensive antacid therapy Diabetic acidosis
Oxalate poisoning Treatment of diabetic coma
Lupus erythematosus Parenteral fluids (prolonged administration)
Multiple myeloma Hyperparathyroidism
Rickets
 845
Magnesium

Procedure: Magnesium by Atomic Absorption temperature. Stable 3 months.

Spectrophotometry 4. Stock magnesium standard, 1000 mg/L (1
Adapted from Diagnotes, Lufkin Medical Laboratories, mg/mL, 82.2 mEq/L). Commercially available;
Minneapolis, MN, 55440. alternatively, dissolve 100 mg of magnesium metal
(commercially available in powder or ribbon form) in 2
Principle mL of 1 mol/L HCl in a 100 mL volumetric flask. After
The magnesium determinations by atomic absorption metal has completely dissolved, dilute to 100 mL with
spectroscopy are based on the fact that atoms of an distilled, deionized water. Store in a polyethylene bottle at
element in the ground, or unexcited, state absorb light of 4C. Stable for 12 months.
the same wavelength as that emitted by the element in the 5. Working standards
excited state. Each element has its own characteristic
absorption or resonance lines, and no two elements are Mg Stock Mg
known to have an identical resonance line. mg/L (mEq/L) (mL)
10 (0.82) 1
To avoid interference by phosphate ions, magnesium in 20 (1.64) 2
serum or urine is determined after sufficient dilution with 30 (2.47) 3
lanthanum oxide solution. 40 (3.28) 4
50 (4.11) 5
Reagents
Use special atomic-absorption glassware and water that are Assay
free of calcium and magnesium contamination.
1. Stock lanthanum, 50 g/L (314 mmol/L) in 4 Equipment: Absorption spectrophotometer with a calcium
mol/L HCl. Weigh 58.64 g of lanthanum oxide (La2O3), magnesium cathode lamp. Follow manufacturers
and wet with about 50 mL of deionized water. Add 250 instructions for proper analytical procedure and for
mL of concentrated HCl slowly and cautiously under hood. establishing a proper airacetylene fuel ratio.
Mix to dissolve powder. Dilute to 1 L with deionized A pipetter dilutor is used to prepare 1:50 dilutions
water. Store in dark at room temperature. Stable 6 months. of samples with the appropriate lanthanum solution.
2. Working lanthanum diluent for serum, 1 g/L Note
(6.28 mmol/L). Dilute 20 mL of stock lanthanum to 1 L The dilution (1:50) of serum or urine prevents protein
with deionized water. Store in dark bottle at room interference.
temperature. Stable 3 months.
3. Working lanthanum diluent for urine, 5 g/L
(31.4 mmol/L). Dilute 100 mL of stock lanthanum to 1 L
with deionized water. Store in dark bottle at room
846
Maternal-Fetal Screening
Maternal-Fetal Screening
Peter OLeary and Barry Lewis
Clinical significance: Refer to Chapter 44, Pregnancy, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
i 35 years and considered to be of advanced maternal age.
Principles of Analysis and Current Use
Screening Overview
Screening is the systematic application of a test to
identify asymptomatic subjects at sufficient risk of a
disorder who are likely to benefit from further diagnostic
investigation. Common screening practices include
looking for hypertension by measurement of blood
pressure, atherosclerosis by measuring plasma
cholesterol concentrations, and cervical cancer by the
Papanicolaou screen. The necessary parameters for a
good screening program [1] are:
(a) The condition is common and well defined
(b) Simple tests detect with high specificity and
sensitivity, so there are few false positives and
few false negatives
(c) The screening tests are cost effective
(d) Diagnostic confirmatory tests are available
(e) Ethically acceptable options for intervention or
treatment
Screening for fetal genetic disorders such as
aneuploidies and neural tube defects (NTD) fits within
the screening criteria. We inherit half of our
chromosomes from each parent when a sperm fertilizes
an ovum. The normal human chromosomal complement
is 46, or 23 pairs (22 pairs of autosomes and 2 sex
chromosomes), with each parent contributing one
chromosome per pair. The fertilized cell divides to form
an embryo and eventually the fetus. Cells with 46
chromosomes are called euploid. If the ovum or sperm
is missing a chromosome (45) or has an extra
chromosome (47), this is referred to as aneuploidy. The
most common aneuploidies are trisomy of chromosome
21 (Down syndrome), trisomy 18, and trisomy 13, 45XO
(Turners syndrome, affecting 1 in 2500 girls) and
47XXY (Klinefelters syndrome, affecting about 1 in
600 to 800 boys). Most fetuses with any of these
chromosomal abnormalities are usually lost early in
pregnancy.
The risk of an adverse pregnancy outcome, such as a
chromosomal anomaly, increases with maternal age. In
the 1980s, just 5% of pregnant women were older than
i
Maternal-Fetal Screening
Previous and current authors of this method:
First edition: Paul T. Russell
Methods edition: Not updated
Second edition: Paul T. Russell
Third edition: Steven C. Kazmierczak
Fourth edition: Jerzy Stanek, Lawrence A. Kaplan
Fifth edition: Peter OLeary, Barry Lewis
However in recent times, many women are choosing to
delay pregnancy, and the proportion of pregnant women
older than 35 now approaches 20%.
One in every 700 pregnancies has an NTD such as spina
bifida or anencephaly, which occur when the fetal brain
or spine fail to develop normally during the first 4 weeks
of pregnancy. Spina bifida, which constitutes 50% of
NTDs, is caused by an incomplete covering of the neural
tube, leaving an open gap (open NTD) or skin-covered
split (closed NTD) on the spinal cord. While a third of
babies with spina bifida live beyond 10 years, 20% die
in the first year, and the remainder are stillborn or die
soon after birth. Related defects include anencephaly
(small or absent neural tissue in the brain) and
encephalocele (a malformation of the brain and skull
allowing protrusion of brain tissue). Practically all
babies with anencephaly or encephalocele are stillborn
or die soon after birth. The majority of NTDs can be
prevented if the mother takes folic acid supplements (0.5
mg daily for 1 month before conception and up to 3
months after conception) or consumes a diet rich in
folate. In 2007, 54 countries had documented national
regulations for mandatory wheat flour, and this has been
associated with a reduction in the prevalence of children
with spina bifida and other NTDs [2]. About 95% of all
NTDs occur in families with no history of these defects.
The birth prevalence [3], including number of live
births, stillbirths, and terminations of pregnancy per
10,000 of selected fetal genetic disorders is as follows:
Spina bifida 3.39 (Atlanta, USA, 1999-2003)
Trisomy 21 17.44
Trisomy 18 4.67
Trisomy 13 1.88
Analyte Description
In the past decade, several biochemical and ultrasound
markers have been developed as part of a prenatal
screening algorithm for fetal chromosomal and structural
abnormalities. The American College of Obstetricians
and Gynecologists has stated that first-trimester
screening using measurements of both nuchal
translucency by ultrasound and biochemical markers
provide an effective screening test for Trisomy 21 in the
general population [4]. Nuchal translucency (NT) refers
to the size of a fluid accumulation at the back of the fetal
neck. An increased measurement of NT is strongly
associated with an increased risk of Trisomy 21.
Currently the recommended maternal serum markers for
Trisomy 21 [5] in the first trimester include pregnancy-
associated placental protein-A (PAPP-A) and free -
human chorionic gonadotropin (free -hCG). Second
trimester -fetoprotein (AFP), total hCG or free -hCG,
847
Maternal-Fetal Screening
and unconjugated estriol (uE3) constitute the so-called
Triple screen. The inclusion of inhibin-A [6] in a four-
test panel is called the Quadruple test. Screening for
NTDs by measuring maternal serum AFP can be offered
in the second trimester either as a stand-alone test or as
part of a Triple or Quadruple screen for fetal aneuploidy.
AFP ( globulin) is the major blood protein of early
fetal life, later to be replaced by albumin. AFP is
produced in the yolk sac, gastrointestinal tract, and liver
and is excreted into amniotic fluid by the immature fetal
kidneys. AFP passively enters into maternal circulation,
increasing the maternal serum AFP level. The AFP
concentration peaks in fetal blood between 12 and 14
weeks and in maternal serum between 28 and 32 weeks
of gestation. An abnormal increase in maternal serum
AFP is the basis of the serum screening test for NTDs.
Leakage into the amniotic fluid is negligible in cases of
closed NTDs, and the maternal serum AFP is not
increased.
The hormone hCG is a glycoprotein composed of two
non-covalently linked polypeptides: the alpha (-hCG)
and beta (-hCG) subunits. The individual subunits lack
biological activity but become active when linked to
form the intact complex. The subunit of hCG contains
92 amino acids and is identical to the subunit of the
other glycoproteins, luteinizing hormone (LH), follicle
stimulating hormone (FSH), and thyroid-stimulating
hormone (TSH). The subunit is composed of 145
amino acids, which distinguishes it from the other
glycoproteins. In addition to intact hCG, free -hCG
subunits, free subunits, and -core subunits are present
in the circulation. Since most immunoassays detect both
intact and free -hCG, they are designated total hCG
assays. During pregnancy, hCG is produced by the
trophoblast layer of the placenta. Total hCG and free -
hCG concentrations increase in maternal blood to reach
a peak at 8 to 10 weeks and then decline to 10% to 15%
of the peak concentrations and plateau from 20 weeks
until term [7, 8]. The designation -hCG commonly
represents the total and free subunit of hCG. Assays
which measure either total or free -hCG have been
used, although free -hCG assays appear to provide
slightly improved predictive value in first- and second-
trimester screening programs [9,10].
Estriol is produced by a unique biosynthetic pathway
during pregnancy, demonstrating the interdependency of
fetus, placenta, and mother. Dehydroepiandrosterone
sulfate (DHAS), the major steroid produced by the fetal
adrenal gland, undergoes 16-hydroxylation (16OH-
DHAS) in the fetal liver and adrenal glands. When this
steroid reaches the placenta, the sulfate side chain is
cleaved by placental sulfatase, and 16OH-DHA is
aromatized to form estriol, most of which is conjugated
in the maternal liver for excretion through the urine. The
concentration of plasma estriol rises 1000-fold during
pregnancy [11]. The protein-bound, uE3, with a half-life
of only 20 min, provides the most specific reflection of
the output of the fetoplacental unit at the time of
phlebotomy [12].
Inhibin is a glycoprotein consisting of an subunit
linked to either a A subunit (inhibin A) or to a B
subunit (inhibin B). Serum inhibin A concentrations
begin to increase early in pregnancy, with the feto-
placental unit being the major source. Very high inhibin
A concentrations are found at the end of pregnancy.
Several studies have established that measurements of
inhibin A in maternal serum are useful for the evaluation
of fetuses with Trisomy 21 [13].
PAPP-A measured during the first trimester has been
advocated as one of the best biochemical markers in
early screening to test for Trisomy 21. PAPP-A is one of
several glycoprotein proteases produced by placental
trophoblast cells that interacts with insulin-like growth
factorbinding protein (IGFBPs), specifically IGFBP-4.
Once IGF-I and IGF-II are released from their IGFBPs,
they promote fetal growth and development through
metabolic and differentiation pathways [14]. This
provides a biological rationale for PAPP-A influencing
feto-placental growth and development, and particularly
for an association between low PAPP-A and poor
pregnancy outcome. In uncomplicated pregnancies,
serum PAPP-A concentrations increase with gestational
age until term. Low levels of maternal serum PAPP-A
are associated with fetal Trisomy 21.
Other serum analytes that are currently under
investigation include ADAM12 [15] and
hyperglycosylated hCG (hCG-H) [16]. Human ADAM
12 (a disintegrin and metalloproteinase) is a placentally-
derived glycoprotein that appears to be involved in
growth and differentiation. Similar to PAPP-A, ADAM
12 acts on insulin-like growth factorbinding proteins
(IGFBP-3 and IGFBP-5) to regulate the bioavailability
and action of IGF-1 and II. Early in the first trimester (8
to 10 weeks), maternal serum ADAM12 is decreased in
Trisomy 21 pregnancies, relative to unaffected
pregnancies, and then ADAM 12 increases relative to
unaffected pregnancies by 16 weeks of gestation.
Population modeling showed that a combination of
ADAM 12 and PAPP-A measured at 8 to 9 weeks,
combined with NT and free -hCG measured at 12
weeks, could achieve a detection rate of 97% at a 5%
false-positive rate.
An essential component of successful embryo
implantation is hCG-H; it has been used as an early
marker of early abnormal pregnancy. In Trisomy 21, a
defect in cytotrophoblast differentiation into
syncytiotrophoblast cells results in an accumulation of
cytotrophoblasts, the hCG-H producing cells. Variable
results have been reported when using hCG-H to predict
Trisomy 21 in the first and second trimesters of
pregnancy. Assays with increased specificity for the
hyperglycosylated isoform of hCG-H, with low sialic
acid content, may achieve Trisomy 21 detection rates of
80% in both the first and second trimester [16].
Brief Description of Pathology
848
Maternal-Fetal Screening
Most NTDs are malformations, although some of them
are disruptions (e.g., in amniotic band syndrome). They
may be closed (e.g., spina bifida occulta) or open (e.g.,
complete rachischisis or open encephalocoele). If not
lethal (all cephalad NTDs are lethal), they cause
irreversible disability. The maternal serum AFP test
detects approximately 80% of NTDs. Increased maternal
serum AFP can occur for other reasons, such as multiple
pregnancy, omphalocele, gastroschisis, placenta accreta,
and may also be associated with pregnancy-induced
hypertension, miscarriage, preterm delivery, intrauterine
growth restriction, intrauterine fetal death,
oligohydramnios, and abruptio placentae. To increase
specificity for NTDs, maternal serum AFP is often
adjusted for maternal weight, race, multiple gestation,
insulin-dependent diabetes mellitus, and prior family
history of NTDs.
Most Trisomy 21 cases are formed spontaneously and
are noninheritable. They result from meiotic
nondisjunction. A minority occurs in offspring of
carriers of balanced translocations. The rate of the
nondysjunction forming trisomy 21 increases with
maternal age. Trisomy 21 is associated with fetal
developmental defects, especially cardiac and
neurological abnormalities.
The Screen
Screening Overview
The maternal serum analysis serves only to screen for
diseasethat is, to identify those pregnancies at
increased risk of the more common fetal anomalies such
as Trisomy 21 and NTDs, which might benefit from
diagnostic testing through chorionic villous sampling or
amniocentesis. Invasive diagnostic tests require
amniocytes or trophoblast tissue to undertake karyotype
or other physicochemical measurements, and these
procedures are associated with a small risk (0.5 to 1%)
of spontaneous fetal loss.
To facilitate informed decision making about the options
available, women and their partners should be provided
with clear written and verbal information from a suitably
qualified health professional. The information provided
should be available before pregnancy occurs or as early
as possible in pregnancy before women are faced with
making decisions. Counseling is a necessary prerequisite
to ensure women have a thorough understanding of the
screening test and its accuracy. Women should be
informed of:
The practical aspects of screening, including the
conditions that are being screened
The type of tests, the timing of tests, and the
approximate costs involved
The possibility that ultrasound scans may be
diagnostic of anomalies other than those for which
the screening programs are designed
The probabilistic nature of results, including
residual risk and the offer of a follow-up diagnostic
test if an increased risk (positive) result is
obtained
Laboratories have a responsibility for monitoring
screening-program performance and should maintain
data on analyte medians, detection rate, screen-positive
rate, maternal age distribution of the screened
population, uptake of screening and prenatal diagnosis
tests, and pregnancy outcome data. The level of posttest
counseling support needed may vary with the type of
result and the resources of the referring practitioner to
deal with the issues surrounding an abnormal result.
Women receiving an increased-risk test result, in
particular, require adequate posttest counseling.
Maternal screening programs for fetal anomalies
evolved from the standard practice of offering diagnostic
amniocentesis or chorionic villous sampling (CVS) and
karyotyping to most women over the age of 35 years,
based on the known association between advanced
maternal age and increased risk of delivering a baby
with Trisomy 21. However, in 1984, this diagnostic
intervention detected only 30% of Downs-affected
pregnancies, because 70% of cases were born to the 95%
of women younger than 35 years of age. Using first-
trimester markers (NT, PAPP-A, and free -hCG) or
second-trimester maternal serum markers in algorithms
that account for gestational age and maternal age, a risk
for NTD or Trisomy 21 can be calculated for the fetus.
Several commercial software programs are available for
generating risk estimates. An additional level of
complexity results when several screening markers are
combined together (for example, maternal age, NT, and
serum marker concentrations), each with a different unit
of measurement and contribution to the ultimate risk.
Maternal age becomes increasingly influential over a
womens reproductive age. A practical way to combine
several markers concurrently is to convert each
measurement into a risk estimate and then combine the
risks into an individual risk for NTD or Trisomy 21. The
program will usually be adjusted so that approximately
5% of women screened will be identified as increased
risk and offered further diagnostic tests.
First-trimester screens are performed between 10 and 13
weeks of gestation. PAPP-A provides a higher
discrimination between affected and unaffected
pregnancies at around 10 to 11 weeks of gestation,
whereas NT is usually more straightforward when
performed around 12 to 13 weeks of gestation. The
second-trimester screens should be performed between
14 and 22 weeks of gestation. However, because of the
possible need for repeat sampling and amniocentesis,
times earlier than 22 weeks are preferred. Most
laboratories recommend that second trimester screening
be performed at 15 to 18 weeks.
To enable comparison of results between laboratories,
reference intervals for normal pregnancies are calculated
around the medians for each gestational week for each
analyte. The observed results are then divided by the
appropriate median and reported as the multiple of the
median, or MoM. The statistical use of medians, as
opposed to mean values, allows for simple manipulation
of non-Gaussiandistributed data. The median values are
849
Maternal-Fetal Screening
calculated for each week of gestation. For a laboratory to
have statistically reliable data, at least 100 screening
results for each gestational week is recommended. The
most difficult item to obtain is an accurate gestational
age; therefore, a high-level ultrasound is recommended
when screening results appear to be positive or
borderline.
The accuracy of screening results is improved when the
gestational age is calculated by ultrasonography or by
certain last menstrual period. A common cause of false-
positive and false-negative results is misdating of the
pregnancy.
First-Trimester Screening (913 Weeks)
First-trimester screening for Trisomy 21, based on
maternal age, PAPP-A and free -hCG, and
measurement of nuchal translucency has been shown to
detect around 85% of affected pregnancies with a false-
positive rate of approximately 5% [17, 18].
Single Screen for NTDs (1420 Weeks)
Measurement of only one analyte, maternal AFP, is used
as a screen to detect open NTDs, but other fetal
pathologies can give raised AFP levels. The screening
cutoff for open NTDs is defined as 2.5 MoM for AFP,
with elevated values requiring further investigation by
ultrasound or other diagnostic tests.
Triple Screen for Chromosomal Defects (1420 Weeks)
This screen measures maternal serum AFP, total or free
-hCG, and uE3, and correcting for the maternal age
(most important risk factor), weight, and race, an overall
estimate of risk for Trisomy 21 can be calculated. Low
AFP and uE3 MoMs and high hCG MoMs are
associated with an increased risk for Trisomy. A Triple
screen can achieve a detection rate of 69% at a 5% false-
positive rate. In addition to Trisomy 21 detection, other
chromosomal abnormalities may be detected. Maternal
serum uE3 is decreased both in fetal trisomy 18 and in
Trisomy 21. However, maternal serum hCG is low in
trisomy 18.
Quadruple Screen for Chromosomal Defects (1420
Weeks)
The Quadruple screen is performed exactly like the
Triple screen but with the addition of inhibin A. The
multiple-marker combination with the highest screening
performance currently available is AFP, uE3, hCG, and
inhibin A, together with maternal age (so-called quad
marker screen). With this combination, a detection rate
of 80% at a 5% false-positive rate can be achieved. Like
hCG, inhibin A concentrations tend to be increased in
Trisomy 21 pregnancies. Addition of inhibin A as the
fourth biochemical marker in screening has been
suggested to reduce the false-positive screening rate to
2% to 3% while increasing the detection rate.
Another screening approach that has been proposed is
termed integrated screening [19], which uses first- and
second-trimester markers to adjust a womans age-
related risk of having a child with Trisomy 21. A
variation on this strategy is the serum integrated screen,
which combines first-trimester serum PAPP-A and
second-trimester Triple or Quadruple screen into a risk-
calculation algorithm. An alternative strategy is the
contingent sequential screen, which uses the results of
first-trimester screening (nuchal translucency, PAPP-A,
and free -hCG) to categorize pregnancies into high,
moderate, and low risk. Those women at high risk
(greater than 1:30) are offered diagnostic chorionic
villus sampling, and those at lowest risk (risk less than
1:1500) are reassured and offered no further testing.
Those women at moderate risk (between 1:30 and
1:1500) are offered second-trimester screening (AFP,
free -hCG or total hCG, uE3, and inhibin-A), with the
final risk calculated on all markers. The relative
performance of the different strategies, each with a
screen-positive rate of 5%, is shown in Table 2.
Estimates from the two most recent such studies,
SURUSS, conducted primarily in the United Kingdom,
and FASTER [17, 18], conducted in the United States,
are remarkably similar (Table 2).
Reference and Preferred Methods
A number of analytical techniques for measurement of
AFP, hCG, uE3, and inhibin A have been described. The
American College of Medical Genetics has published
technical standards and guidelines for prenatal screening
for Trisomy 21 [20] and open NTDs [21] covering pre-
analytical, analytical, and results-reporting phases of the
screening program. When changing analytical methods,
median values must be reestablished using the new
method.
The U.S. Food and Drug Administration (FDA) has not
licensed any kits for Trisomy 21 screening. While many
kits have Class II approval for other clinical
applications, the manufacturers cannot make claims
about screening for Trisomy 21 [20]. On the other hand,
the FDA licenses AFP kits as an aid in the diagnosis of
open NTDs. They are classified as Class III devices and
approved for the measurement of AFP in maternal serum
and amniotic fluid samples during the second trimester.
Both immunometric and radioisotope assay formats are
available to measure AFP in the range 25 to 150 IU/mL
[21].
Conventionally, AFP assays are calibrated in either mass
(ng/mL) or International Units (IU/mL). Most hCG
assays are calibrated as IU/mL against the 1st
International Reference Preparation (equivalent to the
3rd International Standard). Inhibin A assays are usually
reported as pg/mL and free -hCG are calibrated either
as IU/mL or ng/mL.
Responses to Abnormal Screening Results
There are many reasons for increased maternal serum
AFP results (false-positive values) that are not
associated with NTDs or chromosomal abnormalities.
The most important are gestational age and fetomaternal
transfusion.
In the case where the maternal serum AFP is slightly
elevated, the first step is to confirm the gestational age,
850
Maternal-Fetal Screening
by ultrasound if possible, and then look for fetal
structural anomalies such as an NTD.
If the maternal serum AFP is increased, an
amniocentesis can be considered. Clear AF with no
blood contamination may be sent to the laboratory for
AFP measurement and cell culture (for chromosomal
analysis). If the amniotic fluid AFP concentration is
increased, some laboratories will undertake an assay for
acetylcholinesterase, although this measurement has
been largely superseded by modern ultrasound
techniques capable of detecting open NTDs. If the
cholinesterase test is positive, an open NTD is most
likely to be present, and ultrasonography and genetic
counseling are recommended.
A positive maternal serum screen for fetal chromosomal
abnormalities is an indicator for referral to genetic
counseling. Even if the estimated risk is considered low,
there is still a chance that the pregnancy may not yield a
normal outcome. The false-negative rate for Trisomy 21
will be around 1:2000 to 1:3000, and the risk of an
adverse outcome with an increased screening risk, even
when Trisomy 21 is excluded by fetal chromosome
analysis, is still around 1:15. Potential adverse outcomes
include spontaneous pregnancy loss, poor fetal growth,
preterm birth, and neonatal death.
Specimen
Serum is the specimen of choice for the initial maternal
screen. Tests for AFP, hCG, uE3, and inhibin A can be
readily performed on serum. Amniotic fluid may be
required for positive AFP screen confirmation. AFP and,
if necessary, acetylcholinesterase assays may be
performed on the amniotic fluid. The fluid must be free
from fetal blood contamination. For samples suspicious
for blood contamination, a fetal hemoglobin assay may
be performed to determine if this has occurred. Part of
the amniotic sample may be used for karyotyping, and
aseptic technique is essential for this type of specimen.
Interferences
The usual interferences for each of the analytes as
described in their respective chapters apply to the
determination of this possibility for each analyte.
However, the purpose of these tests is to obtain a risk
estimate of the potential for a genetic defect. Because of
the constantly changing concentrations of these analytes
during pregnancy, the major error in the risk calculations
is the estimate of gestational age [30]. Another possible
error is amniotic fluid contamination with fetal blood. In
this case, a fetal hemoglobin measurement may
determine if this has occurred.
Interpretation
Screening for NTDs
There are differences in the upper limits of AFP MoM
values between whites and African Americans, and
between patients with and without insulin-dependent
diabetes mellitus. Most commonly, increased maternal
serum AFP is not due to an NTD. Maternal serum AFP
concentrations may also be observed in:
Fetomaternal transfusion
Placental hemorrhage
Intrauterine fetal demise
Fetuses < 1500 grams
Fetal hydrops
Hemangiomas
Pregnancy complications (including intrauterine
growth retardation, hypertension,
oligohydramnios, placenta previa)
Fetal malformations such as gastroschisis,
gastrointestinal atresia, congenital nephrosis
Tumors: hepatocellular carcinoma and yolk-sac
tumor
Nonneoplastic conditions: ataxia-telangiectasia
syndrome, acute viral hepatitis, chronic active
hepatitis, cirrhosis of the liver.
Because of the variety of other conditions that can lead
to increased maternal serum AFP, further tests may be
required to exclude other causes of a positive screen for
NTDs [22]. These include testing for cocaine abuse that
increases risk for abruption, viral titers, and lupus
anticoagulant measurement to identify autoimmune
disease that might lead to placental thrombosis.
Maternal serum AFP results are adjusted for:
Maternal weight: obese women have spuriously lower
median values due to a dilution effect [23]. However,
note that recent studies demonstrate that these women
have a two- to threefold increased risk of fetal NTD
[24].
Race: African Americans have higher median
values than whites.
Twin gestation: Higher maternal serum median
values are observed. However, amniotic fluid
AFP values are not affected. An adjustment is
made to the maternal serum AFP by dividing
the value by 1.5.
Insulin-dependent diabetes mellitus: This
population has a 20% lower median value of
AFP. Therefore, the patient-specific MoM
value for a woman with insulin-dependant
diabetes mellitus should be divided by 0.8.
Efficacy of Screening
Using a maternal serum cutoff of 2.5 MoM, the
detection rate for anencephaly is expected to be 95% or
greater, and 65% to 80% for open spina bifida [25]. In
twin pregnancy, the detection rate is approximately 50%
at the 5% false-positive rate (even after correcting for
multiple gestation). In general about 1% to 2% of all
women tested undergo amniocentesis. For every 15
women with unexplained increased serum AFP, only
one will have a fetus with some form of NTD.
Confirmatory Laboratory Testing
Amniotic fluid AFP and acetylcholinesterase are
confirmatory laboratory tests. A qualitative test for
acetylcholinesterase may be performed using gel
electrophoresis [26]. However, the polyacrylamide gel
electrophoresis method is rarely performed because of
its complexity and has been replaced by
ultrasonography, which is now the definitive test for
851
Maternal-Fetal Screening
open NTDs.
Screening for Chromosomal Defects
Historically, maternal age of 35 years or older at the
time of delivery has been used to identify women at the
highest risk of having a child with Trisomy 21, and these
women have been offered genetic counseling and
diagnostic amniocentesis or chorionic villous sampling.
However, the introduction of maternal serum screening
and nuchal translucency measurements have
significantly improved the detection rate and decreased
the screen-positive rate, with a decreased risk of
spontaneous fetal loss. The American College of
Obstetricians and Gynecologists has recommended that
all women should be offered aneuploidy screening
before 20 weeks of gestation, regardless of maternal age
[4]. The following patterns occur in cases of
chromosomal defects.
The risk for a woman having a baby with a
chromosomal defect is calculated from serum values,
gestational age, and maternal age. The risk can be
expressed at the estimated time of deliver or at the date
the sample is drawn. Approximately 25% of fetuses with
chromosome defects will spontaneously abort between
the date of screening and term [27]. Because the risks
will be different, the report must clearly state whether
the risk is at term or at the date of screening. Corrections
of analyte values for weight, race, diabetes, and multiple
gestation are made in the same manner as the NTD risk
assessment. For hCG, the effect of weight is many times
that of race [23]. For twin gestation, the hCG MoM
value is divided by 2.0. For uE3, the MoM values are
corrected for weight, and a factor of 1.7 is used to
correct for twin gestation. The results are expressed as a
statistical risk for abnormality, which is based on an age-
associated risk. This value is multiplied by a likelihood
ratio derived from the AFP, hCG and uE3
measurements. It is clear then, that the use of multiple
analytes increases detection rates of aneuploidies [28].
Dual positivity at NTDs/Trisomy 21 screening may
anticipate numerous, frequently severe, pregnancy
complications [29].
Performance Goals
The overall performance of a screening program is
determined by the serum analyte assay characteristics,
the risk cutoff level, whether dating was done by
ultrasound or dates, and the distribution of maternal ages
in the population being screened. The CLIA guidelines
[31] provide a framework for assessing satisfactory
performance that incorporate internal and accredited
external quality-control monitoring procedures to
demonstrate satisfactory analytical performance.
Performance goals for detection rates (DR) and false-
positive rates (FPR) by screening programs that
incorporate maternal age and gestational age estimated
by dates or ultrasound in the risk calculation are shown
in Table 5.
It is sometimes impractical to determine the detection
rate by monitoring the outcome of all pregnancies, but
laboratories can use the screen-positive rate as a
reflection of the false-positive rate. The screen-positive
rate is sensitive to precision and accuracy of the assay,
assay drift, and inappropriate median values for the
unaffected population. According to the CLIA
guidelines, the screen-positive rate, established from 300
to 500 specimens, should fall within limits obtained by
large, experienced screening programs [17, 18]. The
screen-positive rate in the current data set can be
compared with the rate observed in data collected over
the past 6 months. Any exceptions should be
investigated for shifts in median values, or assay drifts.
Calculation of the median MoM for each analyte should
be undertaken at least monthly to determine whether any
significant deviation from 1.00 has occurred. The
median of the MoM values used in the risk calculation
may be adjusted for gestational age, maternal weight,
diabetes, smoking status, and ethnicity and should lie
within 5% of the target value of 1that is, the median
adjusted MoM value should lie between 0.95 and 1.05.
References
1. Wilson JMG, Jungner, G. Principios y metodos del
examen colectivo para identificar enfermeda.
Boletin de la Oficina Sanitaria Panamericana
1968;65:281-393.
2. Morbidity and Mortality Weekly Report.
2008;57(01):8-10. Available at
<http://www.cdc.gov/mmwr/preview/mmwrhtml/m
m5701a4.htm> Accessed June 2008.
3. International Clearinghouse for birth defects
surveillance and research MACDPAR.
2005;Chapter 5, p276. Available at
<http://www.icbdsr.org/filebank/documents/Report2
005.pdf> Accessed June 2008.
4. ACOG. Practice bulletin: clinical practice
guidelines for obstetrician-gynecologists: No. 77.
Obstet Gynecol 2007;109:217-227.
5. Wald N, Kennard A, Hackshaw A, McGuire A.
Antenatal screening for Downs syndrome. Health
Technol Assess UK 1998;2:1-112.
6. Spencer K, Wallace EM, Ritoe S. Second-trimester
dimeric inhibin-A in Downs syndrome screening.
Prenat Diag 1996;16:1101-1110.
7. Bateman BG. Vaginal sonography findings and
hCG dynamics of early intrauterine and tubal
pregnancies. Obstet Gynecol 1990;75:421-427.
8. Braunstein GD, Rasor J, Adler D, Wade ME. Serum
human chorionic gonadotropin levels throughout
normal pregnancy. Am J Obstet Gynecol
1976;126:678-681.
9. Palomaki G. A summary analysis of Down
syndrome markers in the late first trimester. Adv
Clin Chem 2007;43:177-210.
10. Sancken U, Bahner D. Comparison of triple-risk
assessment of fetal trisomy 21, including total
human choriogonadotropin (hCG) or its free beta-
subunit (free beta hCG). Fetal Diagn Ther
2003;18:122-127.
11. Klopper A. Criteria for the selection of steroid
assays in the assessment of fetoplacental function.
852
Maternal-Fetal Screening
In: Klopper A, ed. Plasma Hormone Assays in the
Evaluation of Fetal Well-Being. New York:
Churchill Division of Longmens Press; 1976:20-35.
12. Distler W, Gabbe SG, Freeman RK, Mestman JH,
Goebelsmann U. Estriol in pregnancy. V.
Unconjugated and total plasma estriol in the
management of pregnant diabetic patients. Am J
Obstet Gynecol 1978;130:424-431.
13. Canick JA, MacRae AR. Second-trimester serum
markers. Seminar Perinatol 2005;29:203-208.
14. Lawrence JB, Oxvig C, Overgaad MT, Sottrup-
Jensen L, Gleich GJ, Hays LG, Yates JR, Conover
CA. The insulin-like growth factor (IGF)-dependent
IGF binding protein-4 protease secreted by human
fibroblasts is pregnancy associated plasma protein-
A. Proc Natl Acad Sci USA 1999;96:3149-3153.
15. Spencer K. Aneuploidy screening in the first
trimester. Am J Med Genet 2007;145:18 -32.
16. Cole LA. Hyperglycosylated hCG. Placenta
2007;28:977-986.
17. Wald NJ, Rodeck C, Hackshaw AK, Walters J,
Chitty L, Mackinson AM. First and second trimester
antenatal screening for Downs syndrome: the
results of the Serum, Urine and Ultrasound
Screening Study (SURUSS). J Med Screen
2003;10:56-104.
18. Malone FD, Canick JA, Ball RH, Nyberg DA,
Comstock CH, Bukowski R, Berkowitz RL, Gross
SJ, Dugoff L, Craigo SD, Timor-Tritsch IE, Carr
SR, Wolfe HM, Dukes K, Bianchi DW, Rudnicka
AR, Hackshaw AK, Lambert-Messerlian G, Wald
NJ, DAlton ME. First- and Second-Trimester
Evaluation of Risk (FASTER) Research
Consortium. First-trimester or second-trimester
screening, or both, for Downs syndrome. N Engl J
Med 2005;353:2001-2011.
19. Wald NJ, Watt HC, Hackshaw AK. Integrated
screening for Downs syndrome based on tests
performed during the first and second trimesters. N
Engl J Med 1999;341:461-467.
20. Palomaki GEBL, McDowell GA. Technical
standards and guidelines: prenatal screening for
Down syndrome. Genet Med 2005;7:344-354.
21. Bradley LA, Palomaki GE, McDowell GA.
Technical standards and guidelines: prenatal
screening for open neural tube defects. Genet Med
2005;7:355-369.
22. Elias S, Simpson, JL. Maternal Serum Screening for
Fetal Genetic Disorders. New York: Churchill
Livingstone; 1992.
Table 1: Screening Tests, Analytes and Conditions in Maternal-Fetal Screening Programs.
Weeks Analyte
First-trimester 9-13 PAPP-A
maternal Free -hCG or total hCG
serum
markers
First-trimester 10-13 Nuchal translucency (NT) Determine gestational age by crown-rump Multiple
ultrasound
measurements
Second trimester 14-20 AFP
23. Reynolds TM, Vrankren G, Van Neuten J. Weight
correction of MoM values: which method? J Clin
Pathol 2006;59:753-758.
24. Werber MM, Louik C, Shapiro S, Mitchell AA. Pre-
pregnant weight in relation to risk of neural tube
defects. JAMA 1996;275:1089-1092.
25. Knight GJ, Palomaki GE. Epidemiologic monitoring
of prenatal screening for neural tube defects and
Down syndrome. Clin Lab Med 2003;23:531-551.
26. Goldfine C, Miller WA, Haddow JE. Amniotic fluid
gel cholinesterase density ratios in fetal open
defects of the neural tube and ventral wall. BJOG
1983;90:238-240.
27. Morris JK, Mutton DE, Alberman E. Revised
estimates of the maternal-age-specific live birth
prevalence of Downs syndrome. J Med Screen
2002;9:2-6.
28. Palomaki GE, Knight, GJ, McCarthy JE, Haddow
JE, Donhowe JM. Maternal serum screening for
Down syndrome in the United States: a 1995
survey. Am J Obstet Gynecol 1997;176:1046-1051.
29. Zanini R, Tarantini M, Cerri V, Jacobello C, Bellotti
D, Lancetti S, Scalchi S, Groli C, Bianchi UA.
Dual positivity for neural tube defects and Down
syndrome at maternal serum screening: gestational
outcome. Fetal Diagn Ther 1998;13:106-110.
30. Benn PA, Borgida A, Horne D, Briganti S, Collins
R, Rodis JF. Down syndrome and neural tube defect
screening: the value of using gestational age by
ultrasonography. Am J Obstet Gynecol
1997;176:1056-1061.
31. National Committee on Clinical Laboratory
Standards. Maternal serum screening: approved
standard. NCCLS Document I/LA25-A. Wayne,
PA: NCCLS; 2004.
32. Haddow JE, Palomaki GE, Knight GJ, Foster DL,
Neveux NL. Second-trimester screening for Downs
syndrome using maternal serum dimeric Inhibin A.
J Med Screen 1998;5:115-119.
33. Summers AM, Langlois S, Wyatt P, Wilson RD.
Prenatal screening for fetal aneuploidy. J Obstet
Gynaecol Can 2007;29:146-161.
Measurement Conditions
Detected
Correct for maternal weight, adjust for Trisomy 21
gestational age. Trisomy 18
Trisomy 13
Anencephaly
length or biparietal diameter. pregnancy
Some structural
defects
Correct for maternal weight, adjust for NTDs
853
Maternal-Fetal Screening

Free -hCG or total hCG gestational age. Trisomy 21

uE3 Trisomy 18
Inhibin A Trisomy 13

Table 2: Summary Data for First- and Second-Trimester Trisomy 21 Screens from the Prospective SURUSS
[17] and FASTER [18] trials.
Analytes Detection Rate (%) Optimal Time
for 5% (Gestation Weeks)
False-Positive Rate
First trimester NT only 64-70 12-13
Combined NT, PAPP- 82-87 11-13
A, free -hCG
Second trimester Triple screen (AFP, 69 15-17
hCG, uE3)
Quadruple screen 81 15-17
(AFP, hCG, uE3,
inhibin-A)
First and second Serum integrated 85-88 Serum PAPP-A at 11 weeks and
trimester (PAPP-A + Quadruple screen at 15-17 weeks
Quadruple screen)
Integrated (NT, PAPP- 94-96 All results withheld until the second
A + Quadruple trimester
screen)
Contingenta (NT, 88-94 First trimester screen for all with
PAPP-A + Quadruple screen only offered to
Quadruple screen) those with a moderate risk
Sequential 95 Step-wise testing, where 1.5% of
women screened in first trimester
identified at high risk are offered
invasive diagnostic testing; the
remaining women are offered second-
trimester Quadruple screening.
a
NT, PAPP-A and free -hCG are used to stratify a high-risk group (risk greater than 1:30) who are offered invasive
diagnostic testing, whereas women at low risk (risk less than 1:1500) are not offered further testing. Those at
intermediate risk are offered second-trimester Quadruple or Triple analyte screening
854
Maternal-Fetal Screening

Table 3: Screening Program Framework for Fetal Anomalies, Including Trisomy 21 and Neural
Tube Defects.
Steps Protocols
Preanalytical Appropriate written patient information about:
Disorders detected by screening
Difference between screening and diagnostic tests
Program performance (detection and false-positive rates)
Risk and benefits of screening
Costs
Consent process for physician to discuss with patient
The next steps if the test is positive
Specimen Laboratory requirements for specimen collection, transport:
Patient demographics
Gestational age
Maternal weight, race, and age
Relevant clinical history of insulin-dependent diabetes, multiple pregnancy,
past history of NTDs or Trisomy 21 fetuses
AFP, hCG, Inhibin A and uE3 are stable in sera stored for several days at
4C to 8C and at 20C for several years; uE3 is not stable in whole
blood, and samples should be centrifuged and separated within 8 hours of
collection; free -hCG is unstable in specimens exposed to high daytime
temperatures or sunlight.
Analysis Result turnaround time
Clear arrangement for transmitting results to physician
Risk cutoffs explicitly defined in terms of detection rate and false-positive
rate
Quality Control Commercial control materials have the advantage of long-term stability for
monitoring assays over at least a year, and there is reduced risk of transmission
of bloodborne infectious agents. Standard within- and between-run assay
performance should be monitored.
Other factors that Gestational age dating method: calculation based on the first day of the last
modify the risk menstrual period, or (preferably) ultrasound measurement of the fetal
estimate crown-rump length (CRL) or biparietal diameter (BPD) are used.
Computer-based risk algorithms are available commercially or developed
in-house but must be validated carefully before introduced into routine
service.
Repeat testing is discouraged as part of screening unless the sample timing
or validity is suspected to be incorrect.
Risk algorithm Patient specific risks are generated from the analytical MoM values
adjusted for maternal weight, race, relevant past medical history. The risk
is calculated from the multiplying the likelihood ratios for each parameter
falling into an affected or unaffected Gaussian distribution and the a-priori
risk for Trisomy 21 based on the maternal age.
Results The patient-specific risk can be reported as risk at term or risk at the
time of screening, noting that approximately 25% of Trisomy 21 fetuses
will spontaneously abort between early second trimester and term.
855
Maternal-Fetal Screening

Table 4. Common Maternal Serum Marker Patterns With Fetal Trisomies

Trisomy 21 Trisomy 18
Serum and 45, X and 45, X
marker with hydrops Trisomy 13 without hydrops
AFP low low low
hCG high normal low
uE3 low low low

Table 5: Current Screening Options and Screening Performance Goals.

DR and FPR
Second trimester risk cutoff 1:300 at term [32] Dates Ultrasound
AFP, uE3, hCG 68/5.6 72/5.6
AFP, uE3, hCG, Inhibin-A 73/4.3 76/4.4
First trimester [33]
NT, PAPP-A, free-hCG 83/5.0
DR, Detection rate; FPR, False Positive Rate

Figure 1: Ultrasound image of a fetus showing

the nuchal translucency (NT) measurement
856
Metanephrines, Urine
METANEPHRINES (URINE)
Brett McWhinney
Name: Metanephrines: normetanephrine (NM), 3-o-methyl-norepinephrine;
metanephrine (M), 3-o-methylepinephrine
Clinical significance: Refer to Chapter 51, Adrenal Hormones and Hypertension, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation
Molecular formula: NM, C9H13NO3; M, C10H15NO3
Molecular mass: NM, 183.20 D; M, 197.23 D
Merck Index: NM, 6549; M, 5777
Chemical class: o-Methylated catechol derivative
i
Principles of Analysis and Current Usage
The metanephrines (metanephrine and normetanephrine)
are present in very low amounts in normal urine, and as
a result, a number of sensitive measuring techniques
have been developed.
Metanephrines are found in urine both in the free and
conjugated form, primarily as sulfates. The excretion
rate of conjugated urinary normetanephrine and
metanephrine is about ten times that of the free
metanephrines [1,2]. The conjugates are hydrolyzed by
being boiled in acid before sample extraction and
included in the total metanephrines measured.
The most widely used method for the measurement of
total metanephrines is that developed by Pisano in 1960
[1]. It involves the separation of total metanephrines
from other urinary components by cation-exchange
chromatography, followed by oxidation with periodate to
yield vanillin, which is measured photometrically at 360
nm (Table 1, Method 1). This method does not
distinguish between metanephrine (M) and
normetanephrine (NM). The same process using a
liquid-liquid extraction rather than ion-exchange
chromatography can be used to measure urinary 4-
hydroxy-3-methoxymandelic acid (HMMA), also known
as vanillylmandelic acid (VMA). The extent of
adsorption and elution of metanephrines from cation-
exchange columns will vary. Gitlow [2] sought to
overcome this problem by employing two cation-
exchange columns for one urine specimen and pooling
the eluates.
The Pisano method was modified by Gupta et al. [3] who
replaced cation-exchange chromatography with
differential solvent extraction. An internal standard was
incorporated to correct for procedural losses.
Interference from some related compounds was
negligible.
i is extracted and converted to its volatile silyl derivative
Urine Metanephrines
Previous and current authors of this method:
First edition: Not done
Methods edition: Charles Sohr, Lawrence A. Kaplan
Second edition: Not updated
Third edition: Not updated
Fourth edition: Charles Sohr
Fifth edition: Brett McWhinney
Other methods have been developed in an attempt to
increase the analytical sensitivity and specificity of the
Pisano-based method. These include fluorometric [4-10],
gas chromatographic [11-14], and high-performance
liquid chromatographic (HPLC) methods [15-20]. The
fluorescence methods (Table 1, Method 2) are both
based on the oxidation and tautomerization of M and
NM to their fluorescent trihydroxyindole derivatives. It
is assumed [9,10] that the oxidation products are the
same as those derived from the oxidation of epinephrine
and norepinephrine in the trihydroxyindole method
adrenolutin and noradrenolutin. The fluorescence of the
trihydroxyindole products is measured at 510 to 520 nm
for NM and 520 to 531 nm for M, after excitation at 405
to 410 nm for NM and 415 to 422 nm for M. Many of
these methods employ a two-column extraction
procedure (Table 1, Method 2a). In this procedure, the
total hydrolyzed metanephrine fraction is extracted on a
strong cation exchanger, and then M and NM are
fractionated on another ion-exchange column. The
separated fractions are oxidized by iodine or periodate to
their trihydroxy derivatives. Some procedures [8]
separate the conjugated total metanephrine fraction from
the free fraction and then hydrolyze before separating M
and NM in each fraction. This allows fractionation and
quantitation of conjugated and free metanephrines.
Several methods employ only a single cation-exchange
chromatography step (Table 1, Method 2b) to purify
total metanephrines partially. The amounts of M and NM
in the eluate are estimated by oxidation of the eluate at
different pH values [7,9]. At pH 5 to 7, both NM and M
will be oxidized, whereas at a lower pH (pH 1.5), only
M will be oxidized.
The gas-chromatography (GC) techniques (Table 1,
Method 3) typically require either column
chromatography [11] or a liquid-liquid extraction [12] to
extract other catecholamines differentially from the
metanephrines. The metanephrines can then be
converted to vanillin by periodate oxidation. The vanillin
by N,o-bis(trimethylsilyl) acetamide [12]. The
derivatized compounds are separated on 3% OV-1
columns, and the vanillin derivative is detected by flame
ionization. Alternatively, the trifluoroacetyl derivatives
of NM and M can be prepared, separated on 3% QF-
1:2% XF-1105 columns, and quantitated using an
857
Metanephrines, Urine
electron-capture detector [11]. A mass spectrometer has
also been used as a detector [13,14].
HPLC separation (Table 1, Method 4) and quantitation
of total metanephrines (conjugated and free) is usually
performed after acid hydrolysis to convert all the
metanephrines into the free form. Following this, the
metanephrines are partially purified by loading the
hydrolyzed urine on a cation-exchange column and, after
washing to remove catecholamines, eluting with
ammonium hydroxide. This eluate is then transferred to
an anion-exchange column for further cleanup. The
metanephrines are then eluted with acetate buffer and
can be analyzed directly by HPLC. The mobile phase
usually consists of a phosphate buffer, ion-pairing
reagent octane sulfonic acid, and a small amount of 7.5%
acetonitrile. The most commonly used detector has been
an electrochemical detector [17-20], but fluorescence
detection has also been employed [15,16].
Standardization can be achieved by direct, external
standardization or by internal standardization. Internal
standards employed include 3-methoxy-4-
hydroxybenzylamine [20] and 3-hydroxy-4-
methoxyphenethylamine [19].
Immunoassays have been developed for measurement of
metanephrines (Table 1, Method 5). However, because
of their small molecular size, and because the
metanephrines lack sufficient immunogenic properties,
the development of specific antibodies has been difficult
[21,22]. One technique used to counteract this limitation
is to use the N-acetylated derivatives of metanephrines.
By reaction with Bolton-Hunter reagent [3-(p-
hydroxyphenyl)propionic acid N-hydroxysuccinimide
ester], both metanephrines are readily converted to their
N-acylhemisuccinates. These acetylated derivatives have
excellent immunogenic properties and allow for the
production of specific antibodies.
Reference and Preferred Methods
There is no accepted reference method for the
measurement of urinary metanephrines.
The colorimetric assay lacks specificity, giving rise to
both false-positive and false-negative results [2,23]. In
addition, the method is very tedious, requiring excellent
control over the pH of the hydrolysis step and over the
conditions for oxidizing M and NM to vanillin. The
method also lacks sensitivity for metanephrine levels
found in a healthy population (<500 g/L). However, the
method is technically fairly simple and can easily be
adopted by most laboratories. It may be useful in a
population-screening situation.
The fluorometric methods are quite sensitive (0.25 to
0.35 g) but require an extensive number of
chromatographic steps to ensure specific and accurate
results. Excellent care must be taken with glassware to
ensure minimum background fluorescence from
detergents and other chemicals.
The gas chromatographic (GC) methods have the
capability for accurate analysis but require several
purification steps and derivatization. The GC-mass
spectrometers are highly specific and sensitive but
require equipment that is not available in most
laboratories.
The HPLC techniques are becoming more popular
because of their ability to separate M and NM quickly
(less than 15 and up to 30 min) with almost no
interference from any drug or natural biochemical. The
sensitivity of the HPLC methods with electrochemical
detection is in the nanogram range with between-run
coefficients of variation reported to be less than 10%.
Because the HPLC analysis provides highly specific
measurements of both M and NM, and HPLC equipment
is rapidly becoming routine equipment in many clinical
chemistry laboratories, it is the recommended method
for the analysis of urinary metanephrines.
Specimen
The recommended specimen is a complete 24-hour urine
collection saved in a container containing 25 mL of
concentrated HCl. Boric acid is not an acceptable
preservative. The specimen should be refrigerated during
collection. Properly collected urine (pH < 3) can be
stored at 4C to 8C for weeks and frozen at 20C for
many months.
Interferences
Interferences with the colorimetric procedure can result
from radiopaque x-ray contrast dye, the measurement of
other phenoxy acids, and certain drugs, such as
propranolol and the triamterene diuretics. The
fluorometric assay is subject to interference from -
methyldopa.
Metanephrines Reference Interval
Using the spectrophotometric assay, less than 1 mg of
total metanephrines are excreted in 24 hours.
The ranges listed below indicate that significant
differences may be obtained in laboratories, even when
using similar methods. There are no reported sex or race
differences for the urinary excretion of metanephrines.
Interpretation
The three catecholamines of physiological importance in
the human are epinephrine (adrenaline), norepinephrine
(noradrenaline), and dopamine (3-hydroxytyramine).
The important metabolites are 4-hydroxy-3-
methoxymandelic acid (HMMA or VMA), homovanillic
acid (HVA), metanephrine, and normetanephrine
858
Metanephrines, Urine

Total Urinary Metanephrine Excretion

Age (years)
01 (HPLC) <0.87
Metanephrine <1.84
Normetanephrine
12 (HPLC) <1.04
(g/mg creatinine) <1.38creatinine)
(g/mg
24 (HPLC) <0.44 <0.81
47 (HPLC) <0.26 <0.67
7Adult (HPLC) <0.17 <0.41
g/24 hours g/24 hours
Adult (HPLC) [23,24] <395 <930
Adult (HPLC) [25] <300 <355
Adult (GC) [25] <260 <340
Adult (GC-MS) [26] <255 <620
Adult (ELISA) [22] <275 <320
concentration of 153 pmol/L measured by HPLC. For
normetanephrines at 671 pmol/L measured in plasma by
Interpretation HPLC, intra- and interassay coefficients of 4.7% and
The three catecholamines of physiological importance in 12%, respectively, have been reported [28]. The
the human are epinephrine (adrenaline), norepinephrine intraindividual variation of metanephrine and
(noradrenaline), and dopamine (3-hydroxytyramine). normetanephrine in urine of patients with
The important metabolites are 4-hydroxy-3- pheochromocytoma was found to be 15% (range 1% to
methoxymandelic acid (HMMA or VMA), homovanillic 34%) and 17% (range 0% to 35%) [29].
acid (HVA), metanephrine, and normetanephrine.
References
Testing for the catecholamines and their metabolites is 1 Pisano JJ. A simple analysis for
important in the diagnosis of the cause and extent of normetanephrine and metanephrine in urine.
certain neurological and endocrinological disorders such Clin Chim Acta 1960;5:406-414.
as orthostatic hypotension and pheochromocytoma. 2 Gitlow SE, Mendlowitz M, Bertani LM. The
biochemical technique for detecting and
Pheochromocytomas exhibit excessive storage and establishing the presence of
release of catecholamines. The majority of pheochromocytoma. Am J Cardiol
pheochromocytomas secrete norepinephrine, though 1970;26:270.
secretion of large amounts of epinephrine and dopamine 3 Gupta RN, Price D, Kearne, PM. Modified
is not uncommon. In some cases, epinephrine alone is Pisano method for estimating urinary
secreted; in these cases, the tumor is almost always metanephrines. Clin Chem 1973;19:611.
localized in the adrenal medulla. 4 Weil-Malherbe H, Smith ERB. The estimation
of metanephrine, normetanephrine, and 3,4-
The recommended screening tests for dihydroxymandelic acid in urine. Pharmacol
pheochromocytoma are urinary HMMA (VMA) and Rev 1966;18:331-341.
fractionated metanephrines. The positive predictive 5 Kahane Z, Vestergaard P. Fluorometric assay
value of elevated urinary metanephrines for for metanephrine and normetanephrine in urine.
pheochromocytoma is approximately 95%. When J Lab Clin Med 1967;70:333-342.
combined with an elevated VMA excretion rate, the 6 Kahane Z, Vestergaard P. Column
positive predictive value is approximately 100% (click chromatographic separation and fluorometric
here for VMA chapter). Because of paroxysmal estimation of metanephrine and
secretion of catecholamines and their metabolites from normetanephrine in urine. Clin Chim Acta
tumors, borderline results may be obtained. The analysis 1960;25:453-458.
should be repeated on a new specimen, and perhaps 7 Anton AH, Sayer DF. Distribution of
fractionated catecholamine analysis should also be metanephrine and normetanephrine in various
performed. animals and their analysis in biologic material. J
Pharmacol Exp Ther 1966;153:15-29.
Metanephrines Performance Goals 8 Taniguchi K, Kakimoto Y, Armstrong M.
Long-term precisions (% coefficient of variation [CV]) Quantitative determination of metanephrine and
for metanephrines and normetanephrines measured in normetanephrine in urine. J Lab Clin Med
urine by HPLC were found to be 5.4% at a concentration 1964;64:469-484.
of 1.01 mol/L and 3.8% at a concentration of 4.35 9 Bigelow LB, Weil-Malherbe H. A simplified
mol/L hour, respectively [27]. For metanephrines method for the differential estimation of
measured in plasma, intra- and interassay CVs of 7.0% metanephrine and normetanephrine in urine.
and 12%, respectively, have been reported at a Anal Biochem 1968;26:92-103.
859
Metanephrines, Urine

10 Dalmaz Y, Peyrin L. Specific ion-exchange
chromatography and fluorometric assay for
urinary 3-o-methyldopamine. J Chromatogr
1976;116:379-394.
11 Bertani LM, Dziedzic SW, Clarke, DD, Gitlow
SE. A gas chromatographic method for the
separation and quantitation of normetanephrine
and metanephrine in human urine. Clin Chim
Acta 1970;30:227-233.
12 Calseyde JF, van de Scholtis RJ, Schmidt NA,
Leyten CJ. Gas chromatography in the
estimation of urinary metanephrines and VMA.
Clin Chim Acta 1971;32:361-366.
13 Muskiet FA, Thomasson CG, Gerding AM,
Fremouw-Ottevangers DC, Nagel GT, Wolthers
BG. Determination of catecholamine and their
3-o-methylated metabolites in urine by mass
fragmentography with use of deuterated internal
standards. Clin Chem 1979;25:453-460.
14 Cenfell C, Binder SR, Khayam-Bashi H.
Quantitation of urinary normetanephrine and
metanephrine by reversed-phase extraction and
mass-fragmentography analysis. Clin Chem
1982;28:25-28.
15 Mell LO. Reverse-phase high pressure liquid
chromatography of urinary catecholamines and
related acidic metabolites. In: Hank GL, ed.
Biological/Biomedical Applications of Liquid
Chromatography. Chromatography Science
Series 10. New York: Marcel Dekker;
1979:619-631.
16 Freeman VS, Kiser EJ. Urinary total
metanephrines by high-pressure liquid
chromatography with fluorometric detection
[abstract]. Clin Chem 1984;30:1045.
17 Shoup RE, Kissinger PT. Determination of
urinary normetanephrine, metanephrine, and 3-
methoxytyramine by liquid chromatography,
with amperometric detection. Clin Chem
1977;23:1268-1274.
18 Bertani-Dziedzic LM, Krstulovic AM, Dziedzic
SW, Gitlow SE, Cerqueira S. Analysis of
urinary metanephrines by overset-phase high-
performance liquid chromatography and
electrochemical detection. Clin Chim Acta
1981;110:1-8.
19 Binder SR, Sivorinovsky G. HPLC
measurement of urinary metanephrines using
ion-moderated partition
chromatography[abstract]. Clin Chem
1984;30:1045.
20 Orsulak PJ, Kizuka P, Grab E, Schildkrant JJ.
Determination of urinary normetanephrine and
metanephrine by radial-compression liquid
chromatography and electrochemical detection.
Clin Chem 1983;29:305-309.
21 Wassell J, Reed P, Kane J, Weinkove C.
Freedom from drug interference in new
immunoassays for urinary catecholamines and
metanephrines. Clin Chem 1999;45:2216-2223.
22 Wolthers BG, Kema IP, Volmer M, Wesemann
R, Westermann J, Manz B. Evaluation of
urinary metanephrine and normetanephrine
enzyme immunoassay (ELISA) kits by
comparison with isotope dilution mass
spectrophotometry. Clin Chem 1997;43:114-
120.
23 Amery A, Conway J. A critical review of
diagnostic tests for pheochromocytoma. Am
Heart J 1966;73:129-133.
24 Graham PE, Smythe GA, Edwards GA, Lazarus
L. Laboratory diagnosis of pheochromocytoma:
which analytes should we measure? Ann Clin
Biochem 1993;30:129-134.
25 Rosano TG, Swift TA, Hayes LW. Advances in
catecholamine and metabolite measurements for
diagnosis of pheochromocytoma. Clin Chem
1991;37:1854-1867.
26 Kema IP, Meiborg G, Nagel GT, Stob GJ,
Muskiet FA. Isotope dilution ammonia-
chemical ionization mass fragmentographic
analysis of urinary 3-o-methylated
catecholamine metabolites. Rapid sample
cleanup by derivatisation and extraction of
lyophilised samples. J Chromatogr. B. Biomed
Appl 1993;671:181-189.
27 Atef H, Prosser C, LeGatt D, Froment S,
Cembrowski GS. Medically allowable
imprecision in measuring urinary
metanephrines. Clin Biochem 2001;34:159-160.
28 Roden M, Raffesberg W, Raber W, Bernroider
E, Niederle B, Waldhusl W, Gasic S.
Quantification of unconjugated metanephrines
in human plasma without interference by
acetaminophen. Clin Chem 2001;47:1061-1067.
29 Gerlo EAM, Stevens C. Urinary and plasma
catecholamines and urine catecholamine
metabolites in pheochromocytoma: diagnostic
value in 19 cases. Clin Chem 1994;40:250-256.

Table 1: Methods of Analysis of Urinary Metanephrines

Method 1: Spectrophotometry
Principle of analysis: Metanephrines, separated by cation-exchange chromatography, are oxidized to vanillin,
which is measured at 360 nm. Measures total metanephrines.
Comments: Common; poor specificity and sensitivity; very laborious
Method 2: Fluorometry
Principle of analysis: Oxidation of metanephrines partially purified by cation-exchange chromatography to
trihydroxyindole derivatives; quantitation of fluorescence at 510 to 531 nm after excitation at 405 to 422 nm
Comments: Frequent; very good sensitivity and, with column separation, good specificity
Method 2a: Fluorometrytwo-column
Principle of analysis: Total metanephrines separated by cation-exchange chromatography are fractionated into
860
Metanephrines, Urine

normetanephrine (NM) and metanephrine (M) before the oxidation reaction and fluorometric analysis.
Comments: Direct quantitation of NM and M; has also been used to estimate conjugated and free fractions
Method 2b: Fluorometrydifferential pH
Principle of analysis: Total metanephrines separated by cation-exchange chromatography are oxidized to vanillin
at pH 5 to 7 and pH 1.5. At high pH both NM and M are oxidized; at low pH only M is oxidized.
Comments: Same as for 2a
Method 3: Gas chromatography
Principle of analysis: Partially purified metanephrines are oxidized to vanillin, which is derivatized; derivatives
separated by gas chromatography and vanillin peak are detected by flame ionization. Measures total
metanephrines.
Comments: Rare; good sensitivity and specificity; fairly labor intensive
Method 4: High-performance liquid chromatography
Principle of analysis: Metanephrines are converted to free form by acid hydrolysis and partially purified by
cation-exchange/anion-exchange chromatography. Separation into NM and M fractions by reversed-phase HPLC,
with detection by fluorescence or electrochemical detectors.
Comments: Common; excellent sensitivity and very good specificity; reasonably rapid; preferred method
Method 5: Immunoassay (RIA or ELISA)
Principle of analysis: Antibodies produced against N-acetylated derivatives of metanephrine or normetanephrine
by coupling to bovine serum albumin as hapten
Comments: Requires overnight incubation
2. 70 mM Ammonium Pentaborate Buffer
Procedure: Fractionated Urinary Metanephrines by Ammonium Pentaborate. Store at room
High-Performance Liquid Chromatography (HPLC) temperature.
Ethylenediaminetetra acetic acid di-sodium
salt
Principle a) Dissolve 40 g ammonium
Reverse-phase isocratic high-performance liquid pentaborate and 1.0 g EDTA in 900 mL of
chromatography (HPLC) coupled with electrochemical
water.
detection (ECD) is utilized to determine the
b) Make up to 1000 mL.
concentration of urinary metadrenalines. Metadrenalines c) Store at room temperature.
are also referred to as metanephrines. Sample
purification is a two-step procedure: cation exchange and
3. 2 M Ammonium Hydroxide (Make fresh
anion-exchange extraction. Urinary metanephrines are weekly.)
all converted to the free, unconjugated form by acid Ammonia Solution
hydrolysis. The samples are then passed through Highly Corrosive. Use gloves when
disposable columns containing cation-exchange resin, handling in fume cupboard.
and the resin-bound metanephrines are then eluted onto a) Add 67.5 mL of ammonia solution to
anion-exchange columns. The columns are washed, and 400 mL water.
the metanephrines eluted with acetate buffer. A small b) Make up to 500 mL.
volume of this eluate is injected onto the isocratic HPLC c) Store at room temperature.
system. An Atlantis C18 reverse-phase column separates
the individual metanephrine peaks and the internal 4. 1 M Hydrochloric Acid.
standard peak (IS). Peak resolution is improved by the Concentrated HCl (10 M)
addition of an ion-pairing reagent, 1-octane sulfonic Highly Corrosive. Use gloves when
acid, to the mobile phase. The metanephrines (METS) handling.
are quantitated via ECD by being oxidized to their a) Add 100 mL concentrated HCl to
corresponding anthraquinones (i.e., the oxidation of the 800 mL of water.
phenolic group) at a fixed voltage potential, the resulting b) Make up to 1000 mL.
flow of electrons being measured as current. The electric c) Store at room temperature.
current produced is proportional to the molar
concentration of the METS over a wide linear range. 5. 1 M Acetic Acid.
Concentrated CH3COOH (17 M)
Reagents Highly Corrosive. Use gloves when
Note: All water used is deionized (18 megohm handling.
resistivity) a) Add 15 mL concentrated acetic acid
to 200 mL of water.
1. 0.05 M HCl b) Make up to 250 mL.
HCl (Assay 31.5-33.0%) (10M) MW 36.5 c) Store at room temperature.
Highly Corrosive. Use gloves when
handling. 6. 0.2 M Ammonium Acetate Buffer pH
a) Add 5 mL concentrated HCl to 950 6.0 (Urine Metanephrines Elution Buffer)
mL water. Make up to 1000 mL.
Ammonium Acetate. Store at 4C.
b) Store at room temperature.
861
Metanephrines, Urine

a) Dissolve 15.4 g of ammonium Stock Internal Standard 500 M: MW 18906.

acetate in 950 mL of water. a) Weigh out 0.10 g of 4-hydroxy-3-
b) Adjust the pH to 6.00 with 1 M methoxybenzylamine.
acetic acid. b) Make up to 100 mL of 0.05 M HCl.
c) Make up to 1000 mL with water. c) Store frozen for up to 6 months.
d) Store at 4C. Working Internal Standard 2.5 M:
a) Dilute stock internal standard 1:200
7. Mobile Phase with 0.05 M HCl.
(A) Stock Mobile Phase Solution b) Store at 4C.
Sodium dihydrogen orthophosphate
dehydrate (AR Grade). MW 156.01. 2. Aqueous Metanephrine Standards:
Final concentration 0.8 M. Stock Standards (500 M solutions):
Ethylenediaminetetra acetic acid di-sodium Metanephrine MW 233.7; Sigma #M-8625
salt. Final concentration 134 M. i) Dissolve 0.1168 g in 1000 mL 0.05 M
1-Octanesulphonic acid sodium salt. MW HCl.
216.3. Final concentration 21 mM. ii) Store at 20C.
All reagents stored at room temperature.
Normetanephrine MW 219.7; Sigma #N-7127
a) Dissolve in 1500 mL of water: 248 g i) Dissolve 0.1098 g in 1000 mL 0.05 M
sodium dihydrogen orthophosphate (OSA) HCl.
b) 0.10 g EDTA 9.0 g OSA4C. ii) Store at 20C.
Expires in 6 months.
3-Methoxytyramine MW 203.7, Sigma #M4251
i) Dissolve 0.1019 g in 1000 mL 0.05 M
CALIBRATION STANDARDS
HCL.
LOW MEDIUM HIGH
ii) Store at 20C.
Vol. Conc. Vol. Conc. Vol. Conc.
(L) (M) (L) (M) (L) (M) 3. Calibration Standards:
Normetanephrine 400 0.8 2000 4.0 4000 8.0 a) Prepare three calibration
Metanephrine 250 0.5 1250 2.5 2500 5.0 standards from the Stock Standards as per the
3- 400 0.8 2000 4.0 4000 8.0 table below, making up each standard to 250 mL
Methoxytyramine with 0.05 M HCl.
b) Store 1.0-mL aliquots at -20C.
c) Make up to 2 liters with water.Store at
(B) Working Mobile Phase Quality Control
Biorad Lyphochek 1 and Lyphochek 2 are the
Sodium dihydrogen orthophosphate (AR controls used. Store at 4C.
Grade). Final concentration 0.08 M.
Acetonitrile HPLC grade. Final Prepare fresh weekly in 10 mL of 0.05 M HCl.
concentration 8.5% Place on a roller mixer for 10 min prior to use.
EDTA. Final concentration 13 M Store at 4C while in use. Discard after 7 days.
OSA. Final concentration 2.1 mM
Equipment
a) Add 200 mL of mobile-phase stock
solution and 170 mL of acetonitrile to 1.
a 2-L volumetric flask. a) pH Meter
Ross Combination pH electrode, 0-14 pH, Model
b) Add 1500 mL of water. 815600

c) Adjust pH to between 3.95 and 4.05 b) BioRad Cation-exchange resin

with phosphoric acid. columns; BioRad catalog number 1956017 (100
columns/kit).
d) Make up to 2 liters with water. Store at 4C.

e) Filter and de-gas under vacuum c) BioRad Anion-exchange resin columns;

through a 0.45-m filter. BioRad catalog number 1956018 (100 columns/kit).
Store at 4C.
f) Store at 4C. d) Waters 24-port extraction manifold attached
to a vacuum pump.
2. HPLC SYSTEM:
Standard Solutions
1. Internal Standard 4-Hydroxy-3- a) Pump
Methoxybenzylamine (HMBA):
862
Metanephrines, Urine

Waters Model 2690 solvent delivery system. 8. Wash each column with 2 10 mL
Flow rate 1.5 mL/min. deionized water. Discard eluate.

b) Autosampler 9. Place numbered anion-exchange

Waters Model 2690 integrated autosampler. columns in plastic rack under the
appropriate cation-exchange column.
c) Column
Waters stainless steel Atlantis C18 10. Pipette 8.0 mL of 2 M ammonium
Cartridge Column 150 mm 4.6 mm hydroxide into each upper cation-
with 5 m packing. Part No. 186001344. exchange column, and allow to drain
completely through both columns. Apply
d) Data Manager a short vacuum to remove all the 2 M
Waters Empower Build No. 2154 ammonium hydroxide from the cation-
Chromatography Manager Project Metanephrines, Urine exchange column. Remove and save the
cation-exchange columns for washing.
e) Detector:
ESA Coulochem II 5200A Coulometric Detector 11. Attach the anion columns to the 24-port
(ECD) with a Model 5021 Conditioning Cell and a manifold in the positions previously
Model 5011 High Sensitivity Cell. occupied by its corresponding cation
Method 1Settings as shown in table below: column. Rinse the anion-exchange
N.B. These values must be determined for each columns with 2 10 mL deionized water.
cell and will vary depending on the age of cells AECD SETTINGS
and mobile phase. pGuard Cell
pCell potential +500 mV
l
PROCEDURE Channel 1
y
Cell potential +350 mV
Sample Extraction Ion Exchange Full scale range 500 nA
a
1. Aliquot 0.5 mL Standards Mets and 3 Filter 5 sec
MT Standards (3 levels) (stored in 2.25- sSignal output voltage 1 V
mL aliquots), QC (2 levels), and hBaseline offset 0%
patients urine into numbered 16 150 oChannel 2
mm glass tubes. rCell potential 250 mV
tFull scale range 500 nA
2. Add 100 L of internal standard (10 M Filter 5 sec
HMBA) and 0.25 mL of 1.0 M vSignal output voltage 1 V
hydrochloric acid to each tube, and cap aBaseline offset 0%
firmly. c
uum to remove all the water from the
3. Place the tubes in heater block set at anion column.
120C under a fume hood for 45 min.
12. Place appropriately numbered 5 LP tubes
4. Remove the tubes, and allow to cool. into the plastic rack, and add 400 L of 1
M acetic acid. Place the rack and tubes
5. Bio-Rad METS cation columns are under the corresponding columns. Add 5
removed from the fridge. The caps and mL of 0.2 M ammonium acetate buffer
tails are removed and the columns (metanephrine elution buffer) to each
placed on the 24-port vacuum extraction column, and allow metanephrines to
device to drain. elute. If a column does not drain, apply a
short vacuum to start the flow.
6. Transfer the samples to 25 mL LP, add
6.0 mL 70 mM ammonium pentaborate 13. Transfer the contents of each tube to
buffer, and mix. Adjust the pH to 6.5 numbered glass autosampler vials, and
0.10 using NaOH or HCl (5 M &1 M). inject 60 L.

7. Pour each sample onto separate labeled 5.3 System Settings Summary
cation columns which have been placed
onto the 24-port extraction manifold. Injection volume: 60 L
Allow to drain. If a column does not Flow rate: 1.5 mL/min
drain, apply a short vacuum to start the Run time: 25 mins
flow. Discard eluate. ECD Method Number: 1
Recycle mobile phase: yes
863
Metanephrines, Urine

CALCULATIONS Where: PH = peak height of chromatographic

peak
Empower measures the peak heights, calculates
UNK = analyte of interest in unknown
the ratios of analytes to the IS, and constructs a
STD = analyte of interest in standard
standard curve of this ratio versus concentration
IS = internal standard
in mol/L, mol/day and Creat ratios. The
concentrations of the unknowns are then
Multiply the results obtained by the 24-hour urine
determined from this standard curve, using
volume to obtain the output as mol/24 hours.
their calculated peak height ratios. Check the
internal standard peak heights. Variations of
10% to 20 % are to be expected, but low Notes
recoveries of the internal standard suggest some 1. No interference has been seen in these HPLC
problem with the sample clean-up. systems, and no dietary restrictions are
necessary for persons to be tested.
For manually calculating the concentration of 2. Optimal working electrode potential can vary with
an analyte, use the following formula: time and should be checked at varying
intervals.
PH UNK PH ISSTD 3. Limit of Detection = 0.05 mol/L
Analyte concentration (mol/L)
PH STD PH ISUNK
864
Metanephrines, Urine

IS

400.00

300.00
Normetadrenaline 3 Methoxy Tyramine

200.00
Metadrenaline

mV100.00

0.00

-100.00

-200.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
Minutes

Figure 1: Ion-pair high-performance liquid chromatographic (HPLC) analysis by method described in text; electrochemical
detector (ECD) response versus elution time. A, Normetanephrine (6.15 minutes), metanephrine (8.55 minutes), internal
standard (12.5 minutes) and 3 methoxy tyramine (21 minutes).
865
Methotrexate

Methotrexate
Michael A. Pesce
Name: Methotrexate
Clinical significance: Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C20H22N8O5
Molecular mass: 454.46 D
Merck Index: 5861
Structure:

Methotrexate

NH2 CH3 CO2H

N CH2N CONHCH(CH2)2CO2H
N

N N
Chemical class: Pteridine derivative, a structural analog of folic acid
i
Principles of Analysis and Current Usage
The fluorometric assay (Table 2, Method 1) involves
Methotrexate is given in both high and low doses as a protein precipitation with trichloroacetic acid and
cancer chemotherapeutic agent [1]. High-dose therapy is extraction into n-butanol. After re-extraction into an
often followed by leucovorin rescue (folinic acid, 5- aqueous phase, methotrexate is oxidized to 2,4-
formyl tetrahydrofolate). Monitoring is required to diaminopteridine-6-carboxylic acid by the reaction
establish the necessity and duration of the leucovorin scheme shown below.
rescue and to avoid renal damage [2-5]. In this latter
circumstance, concentrations of the order of 108 M
must be measured to establish that the drug is present at
a low and safe level. Methotrexate has a chemical
structure containing a pteridine ring with para-
aminobenzoic acid and glutamate moieties. Methotrexate
is metabolized to 7-hydroxy methotrexate, 2,4-diamino-
N10-methylpteroic acid (DAMPA) and methotrexate
polyglutamates. The pharmacological and physical
properties of methotrexate are summarized in Table 1,
and the ultraviolet absorbance spectrum is shown in
Figure 1.

Methotrexate may be measured in serum by

spectrofluorometric, spectrophotometric, high-
performance liquid chromatographic, and
immunochemical techniques.

i
Methotrexate
Previous and current authors of this method:
First edition: Not done
Methods edition: Michael A. Pesce
Second edition: Not updated
Third edition: Not updated
Fourth edition: Michael A. Pesce
Fifth edition: Michael A. Pesce
866
Methotrexate
Fluorescence is measured at an emission wavelength of
450 nm after excitation at 370 nm [6].
The spectrophotometric assay is based on the binding of
methotrexate to the enzyme dihydrofolate reductase
(DHFR), which results in inhibition of the catalytic
activity of DHFR (Table 2, Method 2) [7,8]. The affinity
of methotrexate for DHFR is much greater than the
natural substrate, dihydrofolic acid, for the binding sites
on the enzyme. Methotrexate concentration is
determined by its ability to inhibit the reaction shown at
at the bottom of this page.
The change in absorbance of NADPH is measured at a
wavelength of 340 nm. The decrease in DHFR activity
attributable to inhibition by methotrexate is directly
related to the concentration of methotrexate in the
sample. The methotrexate concentration is calculated by
use of a calibration curve made with aqueous standards.
The general procedure for high-performance liquid
chromatography (HPLC) techniques involves protein
precipitation of the serum sample, followed by injection
of an aliquot of the supernatant onto a reversed-phase
C18 column (Table 2, Method 3). Methotrexate and its
metabolites, 7-hydroxy methotrexate and DAMPA, are
detected by absorption of ultraviolet radiation at a
wavelength of 308 nm [9]. Protein precipitation can be
eliminated when the sample is passed through a
pretreatment column [10,11]. HPLC procedures have
also been developed that either oxidize or reduce
methotrexate and use fluorescence detection for
quantitation of methotrexate [12,13]. Methotrexate is
oxidized by potassium permanganate (see above) to
yield 2,4-diamino pteridine-6-carboxylic acid, which is
detected at an excitation wavelength of 275 nm and an
emission wavelength of 410 nm [12]. Methotrexate can
also be detected after reduction to 2,4-diamino-6-
methylpteridine by the reaction shown at the top of the
following page.
Fluorescence of the 2,4-diamino-6-methylpteridine is
detected at an emission wavelength of 463 nm after
excitation at 367 nm [13].
HPLC can also be used to measure methotrexate
polyglutamates in erythrocytes. Two procedures are
used. The first approach measures methotrexate
polyglutamates as a function of the total methotrexate
concentration. Methotrexate polyglutamates are
converted to methotrexate by the enzyme gamma-
glutamyl hydrolase, followed by precipitation with
perchloric acid and injection of the extract into a C18
column. The second approach measures the individual
methotrexate polyglutamates by precipitation of the
packed cells with perchloric acid and injection of the
supernatant into the C18 column. In both procedures,
methotrexate is measured by photo-oxidation with
ultraviolet irradiation in the presence of hydrogen
peroxide, followed by fluorometric detection of the
reaction products [14]. Capillary zone electrophoresis
867
Methotrexate
has also been used to detect methotrexate, 7-
hydroxymethotrexate, DAMPA, and methotrexate
polyglutamates in blood [15].
The immunochemical assays are classified as
heterogeneous or homogeneous procedures. The
heterogeneous methods can be divided into enzyme
immunoassays and radiolabeled procedures.
In the heterogeneous enzyme immunoassays (Table 2,
Method 4), methotrexate is labeled with an enzyme, such
as luciferase [16] or beta-galactosidase [17], and
competes with methotrexate in the sample for binding
sites on the antibody. One can separate the bound and
free labeled drug by using a second antibody, and
determine the activity of the enzyme-bound fraction
either by a bioluminescent reaction using luciferin and
ATP as the substrate or spectrophotometrically using o-
nitrophenyl--D-galactopyranoside as the substrate and
measuring the production of o-nitrophenol at a
wavelength of 420 nm.
An enzyme immunoassay was developed in which
methotrexate bound to polystyrene spheres competes
with methotrexate in the sample for an antibody labeled
with the enzyme peroxidase [18]. The amount of
peroxidase bound to the polystyrene spheres is
determined by measurement of the oxidation product of
o-phenylenediamine at a wavelength of 492 nm and is
related to the concentration of methotrexate in the
sample.
The radiolabeled procedures are divided into competitive
protein binding (Table 2, Method 5) and
radioimmunoassays. The competitive protein-binding
procedure is based on a competition between 3H-labeled
[19] or 125I-labeled [20] methotrexate and methotrexate
in the sample for binding sites on the enzyme DHFR.
The unbound drug is separated by absorption to
charcoal, and after centrifugation to pellet the charcoal,
the supernatant containing the radiolabeled drug bound
to DHFR is counted in a gamma-ray scintillation
counter. The amount of radioactivity in the supernatant
is related to the concentration of methotrexate in the
sample.
The radioimmunoassay methods are based on a
competition between 3H- or 125I-labeled methotrexate
and methotrexate in the sample for binding sites on an
antibody (Table 2, Method 6). The radiolabeled
methotrexate bound to the antibody is separated from the
unbound radiolabeled drug by precipitation of the
complex by the addition of a second antibody [21].
The homogeneous immunoassay procedures include an
enzyme-multiplied immunoassay technique (EMIT) and
a fluorescence polarization immunoassay (FPIA).
With the EMIT assay, methotrexate in the sample
competes with methotrexate covalently linked to an
enzyme (glucose-6-phosphate dehydrogenase) for
binding sites on an antibody (Table 2, Method 7). When
the methotrexate-enzyme complex is bound to the
antibody, the enzyme activity is inhibited. The activity
of the unbound enzyme-drug complex is determined by
measurement of the amount of NADH formed by the
conversion of glucose-6-phosphate to 6-
phosphogluconate, with the concurrent reduction of
NAD+ to NADH. The change in absorbance is measured
at a wavelength of 340 nm and is proportionally related
to the amount of methotrexate present in the sample
[22,23].
The FPIA assay involves competition between
fluorescein-labeled methotrexate and drug in the sample
for binding sites on an antibody (Table 2, Method 8).
The fluoresceinmethotrexate antibody complex is a
large molecule and rotates slowly. When plane-polarized
light strikes this molecule, the emitted fluorescent light
retains its polarization. In contrast, the unbound labeled
methotrexate rotates rapidly, and the fluorescent light is
depolarized. The polarization is related to the amount of
fluorescein-bound complex and is inversely proportional
to the concentration of methotrexate present in the
sample [24].
Reference and Preferred Methods
There is no formal reference method for methotrexate,
but HPLC is the de facto reference method for
measurement because it separates methotrexate from its
metabolites and can be used for both drug monitoring
and pharmacokinetic studies. When HPLC is used to
measure methotrexate levels in serum with ultraviolet or
fluorescent detection, analytical sensitivity can be 10
nmol/L [11]. The within-run coefficient of variation
(CV) at a methotrexate concentration of 5 mol/L, was
9.7% [9]. HPLC can also be used to measure
methotrexate polyglutamate concentrations in
erythrocytes. The inter-day CV of the method ranged
between 5.4% and 9.1% at a methotrexate polyglutamate
concentration of 25 nmol/L. The limit of quantification
for each of the methotrexate polyglutamates was 5
nmol/L [14]. HPLC methods are not commonly used to
measure methotrexate concentrations because they are
time consuming and require expertise in the
interpretation of the chromatograms and in
troubleshooting of the HPLC system.
A 2007 College of American Pathologists Participant
Summary Report showed that the FPIA procedure was
used by 96% of laboratories, with EMIT making up the
remaining methods. FPIA is the preferred method for
routine monitoring of methotrexate.
The FPIA method is a rapid and automated procedure
that can detect methotrexate at levels of 50 nmol/L. The
FPIA method uses 50 or 80 L of sample, and the
methotrexate results are obtained in about 18 minutes.
The linearity of the method is up to 1 mol/L. A
significant advantage of this FPIA procedure is that the
Abbott TDx system automatically dilutes samples.
Methotrexate concentrations can be measured in
undiluted samples and at dilutions of 10-, 100-, and
1000-fold. The software program automatically
calculates the methotrexate concentration that is in the
range of the calibration curve in the diluted sample. The
868
Methotrexate
inter-imprecision CV of the method (n = 9) for
methotrexate at levels of 0.08 and 0.79 mol/L were
9.1% and 3.0%, respectively [24].
The EMIT procedure is used by 4% of the laboratories to
measure methotrexate. The EMIT method is a rapid and
automated procedure that can be adapted to most clinical
chemistry systems. The inter-day imprecision CV of the
method was 7.9% at a methotrexate concentration of 0.8
mol/L [22]. The lower limit of detection with the EMIT
assay is 0.3 mol/L, and the linearity of the method is up
to 2 mol/L.
The following methods are not commonly used for
routine measurement of methotrexate. The
spectrofluorometric method is a nonspecific assay that is
subject to interferences by folate and methotrexate
metabolites because their chemical structures are similar
to that of methotrexate. In addition, drugs not chemically
related to pteridines (e.g., penicillin and ampicillin) will
also give a false-positive result [7]. The
spectrofluorometric method has a lower limit of
detectability of 1000 nmol/L, is a long, tedious, manual
assay and is not used for monitoring of methotrexate.
The spectrophotometric, enzyme-inhibition assay is rapid
and has been automated using discrete analyzers [22,25-
27]. Choosing the right source of the enzyme DHFR is
essential for obtaining accurate results. When DHFR is
obtained from bacterial sources, such as Lactobacillus
casei, the drug trimethoprim binds to the enzyme, giving
false-positive results [28]. This does not occur when
DHFR is obtained from bovine liver. The cross-reactivity
of this assay with methotrexate metabolites is 100% with
methotrexate diglutamate and triglutamate, 10% with
DAMPA, and 1% with 7-hydroxymethotrexate [29]. There
is no cross-reactivity with folate metabolites. The method
is not subject to interferences from bilirubin at
concentrations of 100 mg/L and hemoglobin at
concentrations of 1000 mg/L [22]. The assay is sensitive
to methotrexate at concentrations of 20 nmol/L. The
within-run CVs at methotrexate concentrations of 0.65 and
1.90 mol/L were 6.12% and 4.74%, respectively [22].
The spectrophotometric enzyme inhibition method
requires preparation of the reagents each time that
methotrexate is measured, which limits its use in the
clinical laboratory.
With the heterogeneous enzyme immunoassay
procedures, methotrexate can be detected at nanomolar
concentrations. There is no cross-reactivity with 7-
hydroxymethotrexate or folate metabolites, but there is
40% cross-reactivity with DAMPA [17]. These
procedures are time consuming, require extensive
reagent synthesis by the laboratory, and are not useful
for routine monitoring of methotrexate.
For the competitive protein-binding radiolabeled assay,
the enzyme DHFR must be obtained from bovine liver
because of the binding of trimethoprim to DHFR
obtained from Lactobacillus casei [30]. The lower limit
of detectability is 10 nmol/L, and there is no cross-
reactivity with folate metabolites. The cross-reactivity
with 7-hydroxymethotrexate was 4% and with DAMPA
it was 20% [31].
The radioimmunoassay procedures are the most sensitive
assays available and can measure less than nanomole
quantities of methotrexate. There is no cross-reactivity
from folate metabolites and trimethoprim. There is 5%
cross-reactivity with 7-hydroxymethotrexate and 100%
cross-reactivity with DAMPA and methotrexate
diglutamate and triglutamate [29]. The competitive
protein-binding and radioimmunoassays are time-
consuming manual procedures and are not widely used
to measure methotrexate.
Specimen
Serum, cerebrospinal fluid, plasma, or erythrocytes can
be used for measurement of methotrexate. One report
suggests that in patients with very low methotrexate
concentrations, the drug may be detectable in capillary
plasma but not in venous plasma [32]. Methotrexate is
stable for 5 days in serum samples stored at 23C, 4C,
or 20C [22] and for 6 days in plasma stored at 4C or
at room temperature [33]. Methotrexate is stable in blood
for 3 days at room temperature or for 6 days if blood is
stored at 4C [33]. Methotrexate polyglutamates are
stable for 48 hours in blood that is in contact with a cold
pack [14].
Interferences
With the EMIT assay, there is 100% cross-reactivity
with DAMPA. The amount of cross-reactivity obtained
with 7-hydroxymethotrexate depends on when the
sample is drawn after high dose methotrexate infusion.
Samples drawn at 42 and 66 hours overestimate
methotrexate concentrations by 5% and 31%,
respectively. There is greater cross-reactivity in the 66-
hour specimens because of the higher concentration of 7-
hydroxymethotrexate [34]. There is no cross-reactivity
from leucovorin, 5-methyltetrahydrofolate, 5-
fluorouracil, and 6-mercaptopurine [22]. The EMIT
assay shows no interference from bilirubin at
concentrations of 100 mg/L, but there is a negative
interference from hemoglobin at concentrations greater
than 1000 mg/L [22].
With the FPIA assay, there is 0.6% cross-reactivity with
7-hydroxy methotrexate and 44% cross-reactivity with
DAMPA [24]. There is no cross-reactivity from
leucovorin, trimethoprim, 5-methyltetrahydrofolate, 5-
fluorouracil, or 6-mercaptopurine. There is no
interference with the FPIA assay from bilirubin at
concentrations of 200 mg/L, hemoglobin at
concentrations of 8000 mg/L, or triglycerides at
concentrations of 6000 mg/L [24].
Methotrexate Therapeutic Interval
Serum concentrations after high-dose methotrexate
infusion are typically monitored at 24, 48, and 72 hours
following therapy to help gauge the potential for
toxicity. Concentrations over a 72-hour period following
a single-bolus infusion may differ by two orders of
magnitude. Serum concentrations indicative of potential
toxicity are as follows:
869
Methotrexate
Hours post dose Methotrexate concentration, mol/L
24 >5
48 >0.5
72 >0.1
Interpretation
High-dose MTX therapy is used in non-Hodgkins
lymphoma, osteogenic sarcoma, breast carcinoma, acute
lymphocytic leukemia, and epidermoid cancer of the
head and neck. Methotrexate is used in low doses to treat
rheumatoid arthritis and other chronic inflammatory
diseases.
Methotrexate acts as an antitumor agent by
competitively inhibiting the intracellular enzyme
dihydrofolate reductase (DHFR), which prevents the
conversion of dihydrofolate to tetrahydrofolate. As a
result, it blocks the production of reduced folates and
inhibits DNA, RNA, and purine synthesis. Leucovorin is
given to rescue cells from the toxic effects of
methotrexate because it replenishes the reduced folate
pool by a pathway independent of the enzyme DHFR.
Methotrexate is metabolized intracellularly to
polyglutamate derivatives by the enzyme
folypolyglutamate synthase. Polyglutamate derivatives
of methotrexate cannot be transported extracellularly
unless they are hydrolyzed to the monoglutamate by the
enzyme gammaglutamyl hydrolase. Up to 7
polyglutamate forms of methotrexate can be measured in
erythrocytes. Within the cell, these methotrexate
polyglutamates are potent inhibitors of the enzyme
DHFR. They also inhibit the enzyme thymidylate
synthase, resulting in a decrease in purine and DNA
synthesis. These polyglutamate derivatives remain in the
cell in the absence of extracellular methotrexate and
increase the possibility of cytotoxicity. Low-dose
methotrexate therapy is routinely used to treat
rheumatoid arthritis. The antiinflammatory properties of
methotrexate are probably due to the binding of
methotrexate polyglutamates to 5-aminoimidazole-4-
carboxamide ribonucleotide (A1CAR) transformylase,
which leads to accumulation of adenosine in the blood.
Adenosine activates extracellular adenosine receptors
which inhibit the reproduction of tumor necrosis factor
alpha, IL6, and IL8 and increase the secretion of the
antiinflammatory cytokine IL10 [35]. Routine
monitoring of erythrocyte methotrexate polyglutamate
levels has been proposed in rheumatoid arthritis patients
because of the correlation between methotrexate
polyglutamate concentrations and clinical efficacy.
The major route of elimination of methotrexate and its
metabolites is by the kidneys. Metabolites have been
detected in both the plasma and urine of patients
receiving high-dose therapy. The major metabolite, 7-
hydroxymethotrexate, is formed in the liver by the
enzyme aldehyde oxidase. It is an ineffective inhibitor of
DHFR and has a half-life two to three times longer than
that of methotrexate.
The slow clearance of 7-hydroxymethotrexate results in
plasma concentrations exceeding those of methotrexate
at 24 and 48 hours after therapy. Therefore, falsely
elevated methotrexate values will be obtained with
analytical methods that exhibit substantial cross-
reactivity with 7-hydroxymethotrexate. The minor
metabolite DAMPA is formed in the intestine by
bacterial cleavage of glutamate. It is a weak inhibitor of
DHFR and is usually not detected in plasma.
The metabolism of methotrexate to 7-
hydroxymethotrexate and DAMPA represents
detoxification pathways, because they are not effective
inhibitors of the enzyme DHFR. Carboxypeptidase G2 is
an enzyme that rapidly hydrolyzes methotrexate to the
inactive metabolite DAMPA and can be used to rescue
patients who exhibit delayed clearance of methotrexate
because of renal dysfunction. After treatment with
carboxypeptidase G2, very high DAMPA concentrations
are obtained. Since DAMPA significantly cross-reacts
with the FPIA and EMIT assays, another method,
preferably HPLC, should be used to monitor
methotrexate levels in patients receiving
carboxypeptidase G2 therapy [36].
Methotrexate is sparingly soluble in water at pH < 7.0,
whereas 7-hydroxymethotrexate is about fourfold less
soluble than methotrexate. Precipitation of methotrexate
and 7-hydroxymethotrexate in the renal tubules at acid
pH causes renal dysfunction and delayed excretion of
methotrexate, which increases the risk of toxicity.
Therefore, in patients receiving high-dose therapy, urine
must be alkalinized so that drug toxicity is minimized.
Methotrexate toxicity is related to the level and duration of
exposure to the drug. To identify patients at risk for toxicity,
one should measure methotrexate at various times after
administration of the drug. Several studies have shown that
there is a high risk of toxicity if the drug concentration is
greater than 5 mol/L or 10 mol/L at 24 hours [1,37] or
greater than 0.5 mol/L [2], 1 mol/L [3], or 0.9 mol/L [4]
at 48 hours after high-dose infusion. Therefore, monitoring
methotrexate levels identifies patients likely to develop
toxicity and allows the clinician to determine (1) the length of
time between doses and (2) the amount of leucovorin that
must be administered. It has been suggested that drug levels
be monitored and leucovorin doses adjusted until the
methotrexate concentrations are less than 50 nmol/L [5].
Pharmacogenetics can play a role in understanding the
large interpatient variability in therapeutic responses to
methotrexate. Methotrexate pharmacogenetics has
focused on the enzymes involved in folate metabolism
and the transport of methotrexate into and out of cells.
For example, the enzyme methylene tetrahydrofolate
reductase (MTHFR) catalyzes the conversion of 5-10
methylene tetrahydrofolate to 5-methyl tetrahydrofolate.
A MTHFR 677CT polymorphism, which is an alanine to
valine substitution, results in a variant MTHFR enzyme
with decreased enzyme activity. As a result of this
polymorphism, patients treated with methotrexate have
an increased risk of hepatotoxicity [38].
870
Methotrexate
Pharmacogenetics can also be used to predict drug
resistance to methotrexate.
Cellular resistance to methotrexate can be caused by a
polymorphism in the DHFR gene, which significantly
increases the enzyme activity of DHFR; by a variant
folypolyglutamate synthase enzyme, which decreases the
polyglutamylation of methotrexate; or by a variant
gamma glutamyl hydrolase enzyme, which increases the
hydrolysis of the methotrexate polyglutamates to the
monoglutamate. In the future, pharmacogenetic pre-
screening of methotrexate treated patients may be able to
prevent drug toxicity and customize treatment to
optimize clinical efficacy.
Methotrexate Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report shows
imprecision values (% coefficient of variation) for
methotrexate measurements performed by FPIA to be
less than 8% in samples, with methotrexate
concentrations of approximately 3 and 29 mol/L. The
CVs with the EMIT procedure are usually higher than
those obtained with FPIA. Acceptable performance
criteria (CLIA-88) for measurement of methotrexate
require that laboratories be accurate to within 3SD or
10% of the peer group mean, whichever is greater.
The FPIA assay meets these goals and is the method of
choice for measuring methotrexate.
References
1 Isacoff WH, Townsend, CM, Eilber FR, Forster
T, Morton DL, Block JB. High methotrexate
therapy of solid tumors: observations relating to
clinical toxicity. Med Pediatr Oncol 1976; 2:
319-325.
2 Isacoff WH, Morrison PF, Aroesty J, Willis,
KL, Block JB, Lincoln TL. Pharmacokinetics
of high-dose methotrexate with citrovorum
factor rescue. Cancer Treat Rep 1977; 61: 1665-
1674.
3 Nirenberg A, Mosende C, Mehta BM, Gisolfi
AL, Rosen G. High-dose methotrexate with
citrovorum factor rescue: predictive value of
serum methotrexate concentrations and
corrective measures to avert toxicity. Cancer
Treat Rep 1977; 61: 779-783.
4 Stoller RC, Hande KR, Jacobs SA, Rosenberg
SA, Chabner BA. Use of plasma
pharmacokinetics to predict and prevent
methotrexate toxicity. N Engl J Med 1977; 297:
630-634.
5 Jolivet J, Cowan KH, Curt GA, Clendeninn NJ,
Chabner BA. The pharmacology and clinical
use of methotrexate. N Engl J Med 1983; 309:
1094-1104.
6 Kinkade JM, Vogler WR, Dayton PG. Plasma
levels of methotrexate in cancer patients as
studied by an improved
spectrophotofluorometric method. Biochem
Med 1974; 10: 337-350.
7 Overdijk B, Van Der Kroef WMJ, Visser AAM,
Hooghwinkel GJM. The determination of
methotrexate in serum and urine. Clin Chim
Acta 1975; 59: 177-182.
8 Falk LC, Clark DR, Kalman SM, Long TF.
Enzymatic assay for methotrexate in serum and
cerebrospinal fluid. Clin Chem 1976; 22: 785-
788.
9 Cairnes DA, Evans WE. High-performance
liquid chromatographic assay of methotrexate,
7-hydroxymethotrexate, 4-deoxy-4-amino-N10-
methylpteroic acid and sulfamethoxazole in
serum, urine and cerebrospinal fluid. J
Chromatogr 1982; 231: 103-110.
10 Tong WP, Wisnicki JL, Horton J, Ludlum DB.
A direct analysis of methotrexate,
dichloromethotrexate and their 7-hydroxy
metabolites in plasma by high pressure liquid
chromatography. Clin Chim Acta 1980; 107:
67-72.
11 Breithaupt H, Kuenzlen E, Goebel G. Rapid
high-pressure liquid chromatographic
determination of methotrexate and its
metabolites 7-hydroxymethotrexate and 2,4-
diamino-N10-methyl-pteroic acid in biological
fluids. Anal Biochem 1982; 121: 103-113.
12 Nelson JA, Harris BA, Decker WJ, Farquhar D.
Analysis of methotrexate in human plasma by
high-pressure liquid chromatography with
fluorescence detection. Cancer Res 1977; 37:
3970-3973.
13 Deen WM, Levy PF, Wei J, Partridge RD.
Assay for methotrexate in nanomolar
concentrations with simultaneous direction of
citrovorum factor and vincristine. Anal
Biochem 1981; 114: 355-361.
14 Dervieux T, Orentas Lien D, Marcelletti J,
Pischel K, Smith K, Walsh M, Richerson R.
HPLC determination of erythrocyte
methotrexate polyglutamates after low-dose
methotrexate therapy in patients with
rheumatoid arthritis. Clin Chem 2003; 49:
1632-1641.
15 Kuo CY, Wu HL, Kou H, Chiou SS, Wu DC,
Wu SM. Simultaneous determination of
methotrexate and its eight metabolites in human
whole blood by capillary zone electrophoresis. J
Chromatogr 2003; A1014: 93-101.
16 Wannlund J, Azari J, Levine L, DeLuca M. A
bioluminescent immunoassay for methotrexate
at the subicomole level. Biochem Biophys Res
Commun 1980; 96: 440-445.
17 Al-Bassam MN, OSullivan MJ, Bridges JW,
Marks V. Improved double-antibody enzyme
immunoassay for methotrexate. Clin Chem
1979; 25: 1448-1452.
18 Ferrua B, Milano G, Ly B, Guennec JY,
Masseyeff R. An enzyme immunoassay design
using labelled antibodies for the determination
of haptens: application to methotrexate assay. J
Immunol Methods 1983; 60: 257-268.
19 Arons E, Rothenberg SP, Costa M, Fischer C,
Igbal MP. A direct ligand-binding radioassay
871
Methotrexate
for the measurement of methotrexate in tissues
and biological fluids. Cancer Res 1975; 35:
2033-2038.
20 Methotrexate radioassay kit for the
determination of methotrexate in serum,
plasma, uring and cerebrospinal fluid. The
Enzyme Center Inc, Malden, MA 02148.
21 Paxton JW, Ropwell FJ. A rapid, sensitive and
specific radioimmunoassay for methotrexate.
Clin Chim Acta 1977; 80: 563-572.
22 Pesce MA, Bodourian SH. Enzyme
immunoassay and enzyme inhibition assay of
methotrexate with use of the centrifugal
analyser. Clin Chem 1981; 27: 380-384.
23 Finley PR, Williams RJ, Griffith F, Lichti DA.
Adaptation of the enzyme-multiplies
immunoassay for methotrexate to the
centrifugal analyser. Clin Chem 1980; 26: 341-
343.
24 Pesce MA, Bodourian SH. Evaluation of a
fluorescence polarization immunoassay
procedure for quantitation of methotrexate.
Ther Drug Monit 1986; 8: 115-121.
25 Brown LF, Johnson GF, Witte DL, Feld RD.
Enzymatic inhibition assay for methotrexate
with a discrete analyser, the ABA-100. Clin
Chem 1980; 26: 335-338.
26 Finley PR, Williams RJ. Methotrexate assay by
enzymatic inhibition, with use of the centrifugal
analyser. Clin Chem 1977; 23: 2139-2141.
27 Imbert AM, Pignon T, Lena N. Enzymatic
assay for methotrexate with a centrifugal
analyzer (Cobas-Bio). Clin Chem 1983; 29:
1317-1318.
28 Bock JL, Pierce R. Trimethoprim interference
in methotrexate assays. Clin Chem 1980; 26:
1510-1511.
29 Howell SK, Wang YM., Hosoya R, Sutow
WW. Plasma methotrexate as determined by
liquid chromatography, enzyme-inhibition
assay, and radioimmunoassay after high-dose
infusion. Clin Chem 1980; 26: 734-737.
30 Hande K, Gober J, Fletcher R. Trimethoprim
interferes with serum methotrexate assay by the
competitive protein binding technique. Clin
Chem 1980; 26: 1617-1619.
31 Buice RG, Evans WE, Karas J, Nicholas CA,
Sidhu P, Straughn AB et al. Evaluation of
enzyme immunoassay, radioassay, and
radioimmunoassay of serum methotrexate, as
compared with liquid chromatography. Clin
Chem 1980; 26: 1902-1904.
32 Bomelburg TH, Ritter J, Schellong G. Analysis
of methotrexate concentrations in serum:
comparison of capillary and venous blood
samples. Klin Padiatr 1987; 199: 230-232.
33 Limelette N, Ferry M, Branger S, Thuillier A,
Fernandez, C. In-vitro stability study of
methotrexate in blood and plasma samples for
routine monitoring. Ther Drug Monit 2003; 25:
81-87.
34 Fotoohi K, Skarby T, Soderhall S, Peterson C,
Albertioni. Interference of 7-
hydroxymethotrexate with the determination of
methotrexate in plasma samples from children
with acute lymphoblastic leukemia employing
routine clinical assays. J Chromatogr 2005; B
817: 139-144.
35 Grim J, Chladek,J, Martinkova J.
Pharmacokinetics and pharmacodynamics of
methotrexate in non-neoplastic diseases. Clin
Pharmacokinet 2003; 42: 139-151.
36 Buchen S, Ngampolo D, Melton RG, Hasan C,
Zoubek A, Henze G, Bode U, et al.
Carboxypeptidase G2 rescue in patients with
methotrexate intoxication and renal failure. Br J
Cancer 2005; 92: 480-487.
37. Corn WR, Taylor RH, Pratt CB. Methotrexate:
Therapeutic use and serum concentration
monitoring. In Taylor WJ and Finn AL (eds):
Individualizing Drug Therapy: Practical
Applications of Drug Monitoring. New York,
Gross, Townsend, Frank, Inc. 1981;1:149-173.
38. Stamp L, Roberts R, Kennedy M, Barclay M,
ODonnel J, Chapman P. The use of low-dose
methotrexate in rheumatoid arthritisare we
entering a new era of therapeutic drug
monitoring and pharmacogenomics? Biomed
Pharmacother 2006; 60: 678-687.
872
Methotrexate

Table 1: Pharmacological and Physical Properties of Methotrexate

Structural formula:

Molecular weight: 454.46 D

Melting point: 185C to 204C
Solubility in water: At pH 5.0, 0.44 mg/mL; at pH 7.0, 8.90 mg/mL
pKas: 4.8, 5.5
Dose: 50 to 250 mg/kg of body weight or > 1 g/m2 for high-dose therapy
Protein binding: 30% to 70%
Volume of distribution: 0.87 L/kg of body weight
Major route of elimination: Renal
Half-life:
Biphasic t, 2.06 h; t, 10.4 h
Triphasic t, 0.75 h; t, 3.5 h; t, 10.4 h
Metabolites:
Serum Major: 7-hydroxymethotrexate
Minor: 2,4-diamino-N10-methyl pteroic acid (DAMPA)
Erythrocytes Methotrexate polyglutamates, n=2-7
873
Methotrexate

Table 2: Methods of Methotrexate Analysis

Method 1: Fluorometric; spectrofluorometric
Principle of analysis: Protein precipitation; extraction and oxidation with potassium permanganate
Comments: Not used; manual nonspecific assay; subject to interferences by folate and methotrexate metabolites
Method 2: Enzymatic inhibition; spectrophotometric
Principle of analysis: Inhibition of enzyme DHFR by methotrexate and measurement of residual enzyme activity
by addition of dihydrofolate and NADPH
Comments: Not commonly used; automated; sensitive to 20 nmol/L; cross-reactivity is 100% with methotrexate
diglutamate and methotrexate triglutamate and 10% with DAMPA; there is no interference from folate derivatives
or from 7-hydroxy methotrexate.
Method 3: High-performance liquid chromatography; HPLC.
Principle of analysis: Isolation by reversed-phase column chromatography
Comments: Used for pharmacokinetic studies; sensitive to 50 nmol/L; separates and detects methotrexate, 7-
hydroxymethotrexate, DAMPA, and intracellular methotrexate polyglutamates
Method 4: Enzyme immunoassay; spectrophotometric or bioluminescent
Principle of analysis: Binding of an enzyme to methotrexate; competition between enzyme-bound drug and free
drug for antibody; measures activity of antibody-bound enzyme
Comments: Not commonly used; sensitivity at nmol/L level; reagents must be prepared by laboratory; no
interference from folate derivatives or 7-hydroxymethotrexate; DAMPA cross-reacts
Method 5: Competitive protein binding; radiolabeled assay
Principle of analysis: Binding of 3H or 125I to methotrexate; competition between radiolabeled drug and free
drug for antibody; measures amount of radiolabeled drug bound to antibody
Comments: Not commonly used; sensitive to 10 nmol/L; see comments for enzyme inhibition assay
Method 6: Radioimmunoassay; radiolabeled assay
Principle of analysis: Binding of 3H or 125I to methotrexate; competition between radiolabeled drug and free
drug for antibody; measures amount of radiolabeled drug bound to antibody
Comments: Not commonly used; sensitive to 5 nmol/L; no interference from folate metabolites or 7-
hydroxymethotrexate; DAMPA, methotrexate diglutamate, and methotrexate triglutamate cross-react
Method 7: Enzyme-multiplied immunoassay; spectrophotometric
Principle of analysis: Binding of glucose-6-phosphate dehydrogenase to methotrexate; competition between
enzyme-bound drug and free drug for antibody; measures activity of enzymedrug complex not bound to antibody
Comments: An infrequently used automated procedure for monitoring methotrexate; sensitive to 300 nmol/L; no
interference from bilirubin at concentrations of 100 mg/L or folate metabolites; hemoglobin at concentrations of
greater than 1000 mg/L decreases methotrexate values; DAMPA and 7-hydroxy methotrexate cross-react
Method 8: Fluorescence immunoassay; fluorescence polarization
Principle of analysis: Binding of fluorescein to methotrexate; competition between fluorescein-bound drug and
free drug for antibody; measures polarization of bound antibody complex
Comments: Most commonly used method and automated procedure; sensitive to 50 nmol/L; no interference from
folate metabolites or 7-hydroxy methotrexate; DAMPA cross-reacts

DAMPA, 2,4-Diamino-N10-methylpteroic acid; DHFR, dihydrofolate reductase; NADPH, reduced nicotinamide adenine
dinucleotide phosphate.
874
Methotrexate

Figure
Figure 1: UV absorbance spectrum of methotrexate.

Absorbance spectrum of methotrexate in 0.1 M KOH (pH 13). At this pH, the molar absorptivities of
methotrexate are as follows: 370nm = 7100; 302nm = 22,000; and 257nm = 23,000 mol L1 cm1. (From
Seeger DR et al: J Am Chem Soc 71:1753-1759, 1949.)
875
Methylmalonic Acid
Methylmalonic Acid
Kevin Carpenter & Kathryn Green
Name: Methylmalonic acid (MMA)
Clinical interpretation: Refer to Chapter 52, Diseases of Genetic Origin, in the 5th edition of
Clinical Chemistry: Theory, Analysis, Correlation.
Molecular formula: C4H6O4
Molecular mass: 118.09 D
Chemical class: Dicarboxylic acid
i comparison of the developed spot with an appropriate
Principles of Analysis and Current Usage
Methylmalonic acid (as its CoA ester) is an intermediate
in the propionate degradation pathway (Figure 1).
Deficient activity of the enzyme responsible for the
conversion of methylmalonyl CoA to succinyl CoA
(methylmalonyl CoA mutase) results in the organic
aciduria known as methylmalonic aciduria, with a
classical presentation of neonatal onset metabolic
acidosis, hyperammonemia, and poor outcome if
untreated. The enzyme requires adenosylcobalamin as a
cofactor, so inborn errors in the cellular uptake and
processing of cobalamin will also result in increased
methylmalonic acid (MMA) in plasma and urine. In both
methylmalonyl CoA mutase and cobalamin defects,
concentrations of MMA in plasma and urine are
significantly elevated. However, in recent times a mild
elevation in MMA has been shown to be a sensitive
marker for dietary cobalamin deficiency, and in this
application, a sensitive specific assay for MMA in
plasma is required.
The earliest methods for the quantitation of urinary
methylmalonic acid (MMA) were based on its reaction
with diazotized p-nitroaniline. The emerald green color
that developed after alkalinization with sodium
hydroxide was measured at 620 nm [1] (Table 1, Method
1). Various modifications have been published to
improve specificity, but these procedures are of historic
interest only.
Separation of methylmalonic acid from interfering
compounds was achieved by a variety of
chromatographic techniques. Diethyl ether extractions of
acidified urine were concentrated by evaporation and
then applied to thin-layer chromatographic plates coated
with silica gel. An alternative method utilized an
aluminum-backed, cellulose thin-layer plate, developed
by ascending chromatography in n-butanolacetic acid
water [2]. MMA could be semiquantitated by
i
Methylmalonic Acid
Previous and current authors of this method:
First edition: Not done
Methods edition: Zulfikarali H. Verjee
Second edition: Not updated
Third edition: Not updated
Fourth edition: Theodore Dashman, Zulfikarali H.
Verjee
Fifth edition: Kevin Carpenter, Kathryn Green
standard (Table 1, Method 2). A simple and rapid gas
chromatographic method for the quantitative
determination of urinary methylmalonic acid, applicable
on a routine clinical basis, has been described [3]. The
method is based on the isolation of a mono- to oligo-
carboxylic acid fraction from urine by a series of ether
extractions and the subsequent gas chromatography
(GC) analysis of these acids as their trimethylsilyl
(TMS) esters and ethers.
Since many organic acidurias present with a similar
clinical picture, it is now commonplace to perform urine
organic-acid analysis by gas chromatography or gas
chromatography/mass spectrometry (GC/MS) in
symptomatic patients (Table 1, Method 3). These
methods may be qualitative, but if MMA is found to be
increased, it may be easily quantitated, especially when
using GC/MS, since a stable isotope internal standard is
commercially available. Goodman and Markey [4]
described a standard organic-acid method that employs
ethyl acetate and ether extractions and TMS
derivatization. Although solvent extraction GC or
GC/MS methods are usually performed in urine, they
can be adapted to the much lower levels seen in plasma,
especially where GC/MS is used in the selected ion-
monitoring mode in conjunction with stable isotope
internal standards [5]. Solid-phase extraction has also
proved to be a suitable sample preparation technique for
plasma [6].
The other major technique in use is liquid
chromatography (LC) coupled to tandem mass
spectrometry (MS/MS) (Table 1, Method 5). A method
for the determination of MMA as the di-n-butyl ester
derivative in plasma and urine using LC-MS/MS has
been described [7]. Further refinements of this procedure
[8], based on the extraction of dicarboxylic acids from
samples with methyl-tert-butyl ether and derivatized
with butanolic HCL to form dibutyl esters have been
found to be specific for MMA without interference from
succinic acid. With all dibutyl ester methods, care must
be taken to ensure the volatile product is not lost when
removing the excess derivatizing agent [9].
Reference and Preferred Methods
No reference method is available. Although the GC/MS
method is widely used, LC-MS/MS is becoming more
commonplace in the clinical laboratory and has the
advantage of increased throughput over GC methods.
876
Methylmalonic Acid
The thin-layer chromatography methods are qualitative
or semiquantitative, cumbersome, and time consuming.
Compounds other than MMA also couple with the
stabilized diazotate of dianisidine (fast blue B).
However, the solvent system used in TLC gives clear
and discrete separation of methylmalonic acid from these
compounds.
GC/MS is a sensitive and specific method, allowing
accurate quantitation of the low levels seen in normal
plasma. GC with other detector systems may be used,
but selection of a suitable internal standard not present in
normal body fluids may be troublesome.
LC-MS/MS using isotope-labeled internal standards is
the method of choice if the instrumentation is available.
It offers excellent sensitivity, with low sample volumes
required, and can be used for both diagnosis of inborn
errors of metabolism and micronutrient studies.
Specimen
Traditionally, a random urine sample has been the
specimen of choice for detection of inborn errors of
metabolism. Classical methylmalonic aciduria is likely
to be detected as part of an organic-acid profile, and
excretion is markedly elevated. Milder increases are
more likely to be associated with cobalamin defects or
deficiency, and although theoretically the higher values
in urine should make it suitable for monitoring, many
studies have used plasma or serum. In all sample types,
MMA is stable for many months at 20C.
Interferences
The major interference in MMA estimation comes from
the naturally occurring, structurally related isomer,
succinic acid, which is a product of MMA degradation
and usually present at concentrations greater than that of
MMA [8]. Interference from succinic acid is not usually
an issue in GC/MS-based methods, since the two
isomers are adequately separated chromatographically.
Tandem MS methods have relied upon using different
transitions for MMA and succinate, although complete
freedom from interference requires a short
chromatographic run, as well as careful selection of
transitions.
Methylmalonic Acid Reference Interval
Urinary excretion of MMA in newborns can be up to 83
mg/g creatinine (79.4 mol/mmol creatinine) (97.5
percentile, n = 57) [10]. Excretion falls with increasing
age, so that above 10 years, the upper limit of normal is
22.4 mg/g (21.6 mol/mmol creatinine). Upper limit of
normal excretion in adults is 3.8 mg/g (3.7 mol/mmol
creatinine) [5]. Similarly, plasma or serum MMA
concentrations are higher in infants younger than 6
months of age (0.12 to 1.57 mol/L) than in older
children and adults (0.06 to 0.34 mol/L) [9]. There is
generally a good consensus between studies with an
upper limit of normal between 0.26 and 0.35 mol/L.
Aging in males does not affect MMA concentrations in
serum, but females older than 60 years had slightly
higher values than younger women [11].
Interpretation
Like many of the organic acidurias, methylmalonic
aciduria has a spectrum of clinical presentation and
disease severity, depending on the residual enzyme
activity. In the most severe neonatal form, patients
generally present acutely with a common set of clinical
and laboratory results. The signs and symptoms include
lethargy, failure to thrive, recurrent vomiting,
dehydration, respiratory distress, and muscular
hypotonia. If untreated, coma and death ensue. The most
common laboratory findings at onset of clinical illness
are metabolic acidosis, ketosis, hyperammonemia,
hypoglycemia or hyperglycemia, leukopenia,
thrombocytopenia, and anemia.
Methylmalonic acid is a catabolic product of the amino
acids valine, isoleucine, threonine, and methionine. Its
excretion is greatly increased after a protein load in
patients who cannot metabolize MMA further. The
therapy for such patients, therefore, is the administration
of a protein-restricted diet. Carnitine and oral antibiotic
therapy to reduce propionate derived from gut flora are
also used.
Gross elevations of methylmalonic acid are seen in the
urine of patients with congenital methylmalonic
acidemia (50 to 1000 times normal). The defective
enzyme is mitochondrial L-methylmalonyl CoA mutase,
which catalyzes the conversion of L-methylmalonyl CoA
to succinyl CoA. Isolated defects of adenosyl cobalamin
production lead to methylmalonic aciduria biochemically
indistinguishable from mutase defects. Methylmalonic
aciduria is accompanied by homocystinuria in early
processing defects in vitamin B12 (cobalamin)
metabolism. Absence of both the coenzymes
methylcobalamin (MeCbl) and adenosylcobalamin
(AdoCbl) leads to a combined deficiency of the activity
of methionine synthetase and methylmalonyl CoA
mutase, which leads to accumulation and excretion of
homocysteine and methylmalonic acid.
The milder end of the spectrum of inborn errors affecting
methylmalonyl CoA mutase may have a benign course,
but patients are at risk of acute presentation during
intercurrent illness and catabolic stress.
Severe vitamin B12 deficiency can lead to marked
elevations of MMA in urine and plasma, although
typically not to the levels seen in classical mutase
defects. Mild elevations of MMA in plasma or serum are
considered by many to be a better marker for subclinical
cobalamin deficiency than direct estimates of B12 levels.
Other causes of mild elevations in plasma or serum
levels are impaired renal function [12] and thyroid
disease [13].
Methylmalonic Acid Performance Goals
To be able to detect vitamin B12 deficiency, methods
should be able to accurately measure concentrations of
methylmalonic acid of approximately 1 mol/L in urine
and 0.1 mol/L in serum. The GC method has good
precision (coefficient of variation [CV] = 7.8% for
MMA concentration of 0.2 mol/L in plasma) [6]. A
877
Methylmalonic Acid
study of performance across 13 laboratories found
improvements were needed to reduce the analytical
imprecision of most laboratories, and attention must be
focused on calibration issues [14].
References
1 Giorgio AJ, Plaut GWE. A method for the
colorimetric determination of urinary
methylmalonic acid in pernicious anaemia. J
Lab Clin Med. 1965;66:667-676.
2 Bhatt HR, Green A, Linnell JC. A sensitive
micromethod for the routine estimation of
methylmalonic acid in body fluids and tissues
using thin layer chromatography. Clin Chim
Acta. 1982;118:311-321.
3 Gibbs BF, Itiaba K, Mamer OA, Crawhall JC,
Cooper BA. A rapid method for the analysis of
urinary methylmalonic acid. Clin Chim Acta.
1972;38:447-453.
4 Goodman S, Markey S. Diagnosis of organic
acidurias by GC-MS. Lab Res Methods Biol
Med. 1981;6:105-114.
5 Marcell PD, Stabler SP, Podell ER, Allen RH.
Quantitation of methylmalonic acid and other
dicarboxylic-acids in normal serum and urine
using capillary gas-chromatography mass-
spectrometry. Anal Biochem. 1985;150:58-66.
6 Kushnir MM, Komaromy-Hiller G.
Optimization and performance of a rapid gas
chromatography-mass spectrometry analysis for
methylmalonic acid. J Chromatogr B Biomed
Sci Appl. 2000;741(2):231-241.
7 Magera MJ, Helgeson JK, Matern D, Rinaldo P.
Methylmalonic acid measurement in plasma
and urine by stable-isotope dilution and
electrospray tandem mass spectrophotometry.
Clin Chem. 2000;46:1804-1810.
8 Kushnir MM, Komaromy-Hiller G, Shushan B,
Urry FM, Roberts WM. Analysis of
dicarboxylic acids by tandem mass
spectrometry. High-throughput quantitative
measurement of methylmalonic acid in serum,
plasma, and urine. Clin Chem. 2001;47:1993-
2001.
9 Green AK. Unpublished.
10 Guneral F, Bachmann C. Age-related reference
values for urinary organic acids in a healthy
Turkish pediatric population. Clin Chem.
1994;40:862-868.
11 Rasmussen K, Moller J, Lyngbak M, Pedersen
AM, Dybkjaer L. Age- and gender-specific
reference intervals for total homocysteine and
methylmalonic acid in plasma before and after
vitamin supplementation. Clin Chem.
1996;42:630-636.
12 Lindgren A. Elevated serum methylmalonic
acid: How much comes from cobalamin
deficiency, and how much comes from the
kidneys? Scand J Clin Lab Invest. 2002;62:15-
19.
13 Chong YY, Gupta MK, Jacobsen DW, Green R.
Serum homocysteine and methylmalonic acid
are not reliable indicators of cobalamin or folate
deficiency in patients with abnormal thyroid
function. Blood. 1993;82:94.
14. Pfeiffer CM, Smith J, Miller D, Gunter EW.
Comparison of serum and plasma
methylmalonic acid measurements in 13
laboratories: an international study. Clin Chem.
1999;45:2236-2242.
878
Methylmalonic Acid

Table 1: MMA Methods Summary

Method 1: Colorimetric urine assay

Principle: Reaction of MMA with diazotized p-nitroaniline to generate emerald green color.
Usage: Of historical interest only.

Method 2: Thin-layer chromatography

Principle: Thin-layer chromatographic separation followed by localization with diazo reagent.
Usage: Largely superseded by GC or LC/MS methods.

Method 3: Gas chromatography/mass spectrometry of organic acids

Principle: Organic acids are extracted from urine using organic solvent (ethyl acetate/diethyl ether), converted to
TMS esters, and separated on capillary GC. Positive peak identification by mass selective detector.
Usage: Widely used as most common method for detecting all organic acidurias.
Comments: Method can be adapted to low levels seen in plasma by using stable isotope dilution quantitation and
selected ion monitoring.

Method 4: Liquid chromatography/tandem mass spectrometry

Principle: Methyl malonate is extracted from urine or plasma by solid-phase or solvent extraction, converted to
the dibutyl ester by reaction with butanolic HCl, and analyzed by tandem mass spectrometry, using stable isotope
internal standard. Interference from succinate is avoided by a short chromatographic separation.
Usage: Method of choice if instrumentation is available.
Comments: Excellent sensitivity and high throughput make this assay suitable for micronutrient studies.

Procedure: Gas Chromatography/Mass 9. Calibration standards are prepared

Spectrometry
Principle
Based on methods published by Rasmussen
[11] and Kushnir [6], a rapid and sensitive GC/MS assay
for methylmalonic acid determination in serum and
plasma is described. The assay utilizes an anion-
exchange, solid-phase extraction, trimethylsilyl
derivatization, and stable isotope internal standard
quantitation. The method is sensitive enough to allow
accurate quantitation of levels seen in normal
individuals.
Reagents
1. Methanol, A.R.
2. Acetonitrile, A.R.
3. Methyl tert-butyl ether (MTBE),
A.R.
4. Formic acid, A.R.
5. MTBSTFA (N,(tertiary
butyldimethylsylyl)-N-
methyltrifluoro-acetamide).
6. Methylmalonic acid stock standard
(MMA), 10 mmol/L in methanol.
Place 118.1 mg of MMA in a 100-mL
volumetric flask, add methanol to
dissolve, and bring to volume with
methanol. Store at 20C.
7. d3 Methylmalonic acid (Cambridge
Isotopes DLM-387) stock standard
(d3MMA), 1 mmol/L in methanol.
Place12.1 mg of d3MMA in a 100-mL
volumetric flask, add methanol to
dissolve, and bring to volume with
methanol. Store at 20C.
8. MMA and d3MMMA working
standards at 10 and 15 mol/L,
respectively, prepared
acetonitrile.
at 0.2, 0.5, 1.0 and 2.0 mol/L in
dialyzed human plasma.
Assay
Equipment: A suitable GC-MS system capable
of operation in electron-impact ionization and selective
ion-monitoring mode is required. The gas
chromatograph is fitted with a 5% phenylmethyl-silicone
capillary column, 25 m 0.20 mm, 0.33-m film
thickness. Solid-phase extraction is achieved using
strong anion-exchange columns (e.g., Bond Elut, SAX)
1. In a screw-capped glass tube, 100 L of the
internal standard d3MMA and 1 mL of
acetonitrile is added to each calibrator, control,
and test sample.
2. The tubes are vortexed for 20 sec and
centrifuged for 5 min at 2000 g.
3. The contents of the tubes are transferred onto
the SPE columns, previously conditioned with 3
mL methanol and 5 mL distilled water.
4. The columns are washed by sequential addition
of 10 mL deionized water, 5 mL of methanol, 2
mL of MTBE, and dried after each wash with
vacuum for 3 min.
5. The analytes are eluted with 5 mL of elution
solvent consisting of 3% formic acid in MTBE.
6. The eluent is evaporated to dryness under a
stream of nitrogen at 35C.
7. The residue is reconstituted with 25 L of
MTBSTFA and 25 L acetonitrile and the tubes
incubated at 55 5C for 5 min.
8. GC/MS parameters:
The mass selective detector is used in
the electron-impact ionization mode
in at 70 eV.
Helium is utilized as a carrier gas.
879
Methylmalonic Acid

Injection port temperature 270C. Calculation

Initial column temperature 100C,
ramped at 18C/min to 160C,
ramped at 50C/min to 300C, with a
final hold of 2.5 min.
Interface temperature 300C.
Split injection, ratio of 1:20, and
injection volume 1 L.
The ions monitored for the derivatives
are: m/z 289 for MMA and m/z 292
for d3MMA
Results are calculated by calculation of the ratio
of MMA to d3MMA signal peak areas in unknowns read
against a standard curve.
Notes:
1. MTBSTFA gives superior signal for the M-57
ion compared to the corresponding M-15 ion
for TMS derivatives, but either may be used.
2. The method adequately separates succinate
from MMA, allowing accurate quantitation.

Figure

Isoleucine
Valine
Methionine

Adenosylcobalamin
Propionyl CoA Methylmalonyl CoA Succinyl CoA

Cholesterol Methylmalonic acid

Odd Chain FA

Figure 1. Simplified pathway of propionate precursors and degradation. Conversion of methylmalonyl CoA to succinyl
CoA requires adenosylcobalamin as a cofactor.
 880

Mycophenolic Acid

Mycophenolic Acid
Michal J Figurski, Magdalena Korecka, Leslie M Shawi

Name: Mycophenolic acid, MPA

Systematic (IUPAC) name: (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-
dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid
Clinical significance: Immunosuppressant for prevention of acute rejection of allograft in
transplant patients Refer to Chapter 54, Laboratory Evaluation of the
Transplant Recipient, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation
Molecular formula: C17H20O6
Molecular weight: 320.34 D
CAS number: 24280-93-1
Structure:

Principles of Analysis and Current Usage
Mycophenolic acid is a fungal antibiotic, initially isolated
from a strain of Penicillium brevicompactum by the Syntex
company (now in the Roche group) in the late 19th
century. It is widely used to prevent allograft rejection in
solid organ transplant patients. Currently, it is approved for
prevention of acute rejection in renal, heart, and hepatic
transplants and is used in various combinations with other
immunosuppressants. There are ongoing clinical trials
evaluating MPA in other administration protocols, as well
as for other organs, such as pancreas, intestine, lungs, and
also bone marrow [1].
In addition, mycophenolic acid is under investigation as an
immunomodulator in treatment of autoimmune diseases
such as lupus nephritis (Phase III study as of June 2007),
pemphigus vulgaris (Phase III study as of June 2007),
myasthenia gravis (failed Phase III study) [2], immune
thrombocytopenic purpura, autoimmune hepatitis and IgA
nephropathy [3-5].
According to the International Proficiency Testing Scheme
for MPA, provided by Analytical Services International
(ASI, London, UK), 45% of participating laboratories
analyze mycophenolic acid concentrations using EMIT,
33% using HLPC/UV, and 20% using mass spectrometry;
2% of participants report use of other methods [6].
According to the 2007 College of American Pathologists
(CAP) survey [7], participating laboratories analyze MPA i
concentrations either with HPLC/UV (60%) or mass
spectrometry (40%). The CAP survey shows no use of the
EMIT assay for MPA, since it is not approved by the U.S.
Food and Drug Administration (FDA) for use in the United
States.
Historically, the first method for determination of
mycophenolic acid, published in 1968, was based upon
separation of various antibiotics by paper chromatography
and then isolation of MPA using thin-layer
chromatography [8]. Several other methods for analysis of
MPA were developed in the following years, including
turbidimetric analysis, thin-layer chromatography, and gas
chromatography [9-11]. However, all these methods
involved a complex sample preparation step. In a report
from a Syntex study published in 1990, a simple
HPLC/UV method for analysis of mycophenolic acid was
first mentioned, although no details were given [12]. This
method was described later in 1994 [13]. High sensitivity
for MPA in this method was achieved using liquid-liquid
extraction from plasma and detection with ultraviolet light
of 215 and 304 nm wavelength.
i
Mycophenolic acid
New method
Fifth edition: Michal J Figurski, Magdalena Korecka,
Leslie M Shaw
 881
Mycophenolic Acid
Later on, many other HPLC methods were developed for
analysis of mycophenolic acideither alone, with its
metabolites, or as a part of whole panels of
immunosuppressive drugs. The methods utilizing
ultraviolet detection are mainly applied to analysis of total
mycophenolic acid and/or its glucuronide metabolite
(MPAG) concentration. For analysis of free MPA or the
total concentration of other metabolites, such as
mycophenolic acid acyl-glucuronide (AcMPAG), mass
spectrometric detection is more suitable. Mass
spectrometric detection is applied exclusively in the
systems where multiple drugs are analyzed simultaneously.
Starting from the year 2001, immunoassay methods for
analysis of MPA were introduced and gradually gained
popularity because of their simplicity, low initial
investment cost and lack of sample pretreatment step.
Chromatographic Methods
Sample Preparation and Chromatography
Sample preparation, the first step in chromatographic
analysis of MPA, is the removal of proteins and other large
molecules that could compromise the performance of the
chromatographic column. Several of the most commonly
used methods are described below.
A simple and cost-effective method is protein precipitation
(PP), in which the sample is mixed with a precipitating
agent, centrifuged, and the supernatant is recovered. The
precipitating agent can be methanol [13], acetonitrile [14],
or sodium tungstate [15]. This process additionally frees
mycophenolic acid from albumin binding and allows for
analysis of the total concentration of MPA. However, this
method does not remove any small-molecule impurities,
which may interfere with the chromatographic analyses of
MPA.
Other methods of protein removal include solid-phase
extraction (SPE) [16-19] or liquid-liquid phase extraction
(LLE) [13, 20]. These methods allow for substantial
sample clean-up and increase in concentration, which often
results in increased sensitivity and a broader calibration
range for HPLC/UV methods. However, a substantial
drawback of these methods is their complexity and
required workload. In the case of SPE, an additional
drawback is the high cost of disposable cartridges.
Plasma can also be injected directly onto HPLC without
any sample pretreatment [21]. A system of two columns
and a switching valve is employed for sample clean-up,
with mobile phase composed of acetonitrile and phosphate
buffer (1:1 v/v).
Ultrafiltration (UF) is a necessary step in analysis of free
MPA [22]. It removes proteins together with the fraction
of the drug that is bound to proteins. In this case, the
micropartition devices or microfilter kits are used, with
cutoff of around 30,000 D. It is essential that the sample is
not pretreated before this process, because the change of
pH can alter the protein binding of mycophenolic acid.
The prepared sample is usually diluted prior to injection
onto HPLC column. Larger injection volumes and less
dilution are commonly used in HPLC/UV methods,
whereas smaller volumes with larger dilutions are used for
mass spectrometric methods. Application of liquid-liquid
extraction (LLE)/SPE also allows for smaller injection
volumes. A wide variety of mobile phases have been
described for the analysis of MPA, although the primary
components of every mobile phase are water and an
organic solvent, either methanol or acetonitrile. Phosphoric
acid or phosphate buffer are often used in HPLC/UV
assays [13,14,17-21,23-25]. For LC/MS, the mobile phase
is usually acidified with acetic or formic acid [15,26-31].
The majority of the recently published methods use
gradients to decrease the runtime and increase selectivity.
The most commonly used type of stationery phase for
mycophenolic acid analysis is a reversed-phase C18
column, although reversed-phase C8 columns are also in
use. Various column lengths and particle sizes are used
across the published methods. Interested readers may find
more information in a recent review [32]. The column
length is usually between 150 and 250 mm, although
methods for shorter columns have also been published.
An increasing number of methods utilize on-line sample
clean-up techniques, performed using two columns, two
pumps, and a switching valve. Most methods employ the
cross-directional flow of the eluting phase; however, there
are also published methods that use the same flow
direction of the washing and eluting phases. Diagrams of
the switching valve connections for unidirectional and
cross-directional flow of eluting phase are shown in Figure
1. In this set-up, the main column for chromatographic
separation of MPA is a regular, full-length, reversed-phase
C18 or C8 column, and the washing column is a short
column of the same type, not exceeding 50 mm in length.
The washing column is used as an adsorbent material,
retaining the MPA for analysis while allowing the solvents
and impurities to be removed by the quick flow of the
washing phase. The activation of the switching valve
(switch from A to B position) ends the washing period and
starts the eluting period [15,27].
Ultraviolet Detection
The ultraviolet (UV) spectrum of mycophenolic acid is
shown in Figure 2 in the range from 220 to 360 nm. Three
major peaks of this spectrum are utilized in the published
methods: 215 nm (shown truncated), 254 nm, and 304 nm.
The latter peaks, despite their smaller intensity, provide the
advantage of less interference from other compounds of
human plasma.
Methods vary in terms of a compound used as an internal
standard, but most of the published methods utilize
carboxybutoxy ether of mycophenolic acid (MPAC). There
are also methods that do not use the internal standard at all.
 882

Mycophenolic Acid
Using a standard sample clean-up procedure, such as
protein precipitation, mycophenolic acid can be detected at
concentrations around 0.2 to 0.3 mg/L, whereas some
sample clean-up techniques allow for considerable
lowering of that limit. A short summary of calibration
ranges and lower limit of quantification values (LLOQ)
from several published methods are shown in Table 1.

Table 1: Method Information Summary for Selected HPLC/UV Methods

Reference Sample UV Peak Internal MPA* MPAG* AcMPAG*
(Chronologically) Prep [nm] Standard [mg/L] [mg/L] [mg/L]
13 LLE 215 & 304 Diazepam & 0.5-50 (no data)
indomethacin
21 Direct 215 None 0.1-20 (0.10)
injection
23 PP 215 Epilan D 0.25-15 (0.25) 3.4-218 (3.41) 0.20-15 (0.20)
14 PP 254 MPAC 0.5-20 (0.25) 5.0-200 (0.5) 2.5-100 (0.25)
17 SPE 306 Para-nitro-anilin 0.2-20 (0.2)
(PNA)
25 UF 304 MPAC 0.005-2.0 (0.005) free 1.0-150 (1.0) free
*Lower Limit of Quantitation (LLOQ) in brackets

Table 2: Method Information Summary for Selected HPLC/Fluorescence Methods

Reference Sample Prep Excitation & Emission [nm] Internal Standard MPA*
(Chronologically) [mg/L]
33 PP 310 & 430 None 0.2-20 (0.20)
34 PP 342 & 425 Naproxen 0.05-40 (0.05) total
0.005-1.0 (0.005) free
* Lower Limit of Quantitation (LLOQ) in brackets

Fluorescence Detection [27,36,37]. There are many chromatographic methods

As an alternative to ultraviolet detection, mycophenolic
acid can also be analyzed using HPLC with fluorescence
detector [33,34]. Since fluorescence characteristics of
chemical substances are generally more unique than
ultraviolet absorbance, the fluorescence methods are more
sensitive and allow for simpler sample pretreatment and
shorter runtime. However, for the same reason, these
methods do not allow for simultaneous analysis of MPA
and its metabolites. The fluorescence of MPA is highly pH
dependent, thus this method of detection requires strict
control of pH in the mobile phase, and the mobile phase
itself is of complex composition. Validation information
for these methods is summarized in Table 2 above.
Mass Spectrometric Detection
Mycophenolic acid can be analyzed with much greater
accuracy and precision using mass spectrometric (MS)
[29,35] or tandem mass spectrometric (MS/MS) assays
published for simultaneous determination of a whole panel
of immunosuppressants, where MPA and/or MPAG is just
one of multiple analytes [36,37]. Other methods allow for
simultaneous determination of MPA and both of its major
metabolites, MPAG and AcMPAG [27,28]. Virtually all of
the published methods utilize an electrospray ionization
source, either in negative or positive ionization mode.
Positive ionization mode requires an ion-pairing agent, and
most methods utilize ammonium acetate to produce NH4+
adducts. Na+ adducts are also used, although Na+ ions are
actually a contamination of the sample from glassware that
is used for sample preparation; therefore, this pairing ion is
less favorable. In the negative ionization mode, the
analytes minus a H+ ion are measured directly.
Table 3 summarizes MS parameters for several published
methods, and Figure 3 shows the most common MS/MS
fragments of the mycophenolic acid molecule.
 883

Mycophenolic Acid

Table 3: Method Information Summary for Selected HPLC/MS & HPLC/MS/MS Methods
Reference Sample Method Ionization Mode MS Masses or MS/MS Transitions
(Chronologically) Preparation (m/z)
29 PP MS Positive, MPA: 343.4
Na+ adduct IS (N-hexadecyl--D-glucopyranoside) n/d
38 PP MS/MS Negative MPA: 319.0 191.1
IS (indomethacin): 356.0 312.2
MPAG: 495.5 319.4
26 SPE MS/MS Positive, NH4+ MPA: 337.7 207.2
adduct IS (MPAC): 437.6 207.2
MPAG: 513.6 207.2
27 PP + on-line MS/MS Positive, NH4+ MPA: 338.1 207.1
cleanup adduct IS (MPAC): 438.0 303.0
MPAG & AcMPAG: 514.2 303.0

Table 4: Summary of Method Validation Information for Selected Mass Spectrometric Methods
Reference Runtime Linearity Range LOD* LLOQ Accuracy Imprecision Recovery
(Chronologically) [min] [mg/L] [mg/L] [mg/L] [%] [%] [%]
29 14 MPA: 0.01-1.0 0.001 0.021 9.8 13.2 80-85
38 MPA: 0.1-30 0.05 0.1 12.28 90-91
6
MPAG: 1-300 0.5 1 13.37 73-77
26 MPA: 0.1-16 99
7 <15
MPAG: 1-200 99
27 MPA: 0.05-30 0.05 3.0-4.7 98-100
5 MPAG: 2-400 2 97.5-103 5.4-7.3 101-103
AcMPAG: 0.025-15 0.025 6.3-13 98-103
*LOD, Lower Limit of Detection; , LLOQ, Lower Limit Of Quantitation; , Calibration range reported using quadratic fit.

The mass spectrometric methods are generally viewed as
requiring less chromatography because of the mass
selectivity of the detector instrument itself. However, in
the case of analysis of mycophenolic acid, a phenomenon
of in-source decomposition of MPAG back to MPA was
observed, especially with elevated source temperatures
[30]. An example of this phenomenon is shown in Figure
4. Table 4 above summarizes the available information
from method validation.
complex labeled with MPA is added. The activity of the
enzyme, directly proportional to the rate of formation of
NADH, is estimated by measuring the change in
absorption at a wavelength of 340 nm at two points in
time. After the first step, the antibodies present in the
solution that have not been bound to molecules of
mycophenolic acid bind to the MPA-labeled enzyme and
thus decrease the enzymatic activity, which allows for
calculation of MPA concentration.

Immunoassay Techniques The genetically engineered monoclonal antibody that is the

Enzyme-Multiplied Immunoassay Technique (EMIT)
An enzyme-multiplied immunoassay technique was
originally developed by Syva Corporation (Palo Alto, CA)
in 1972. The principle of the EMIT method is described in
full detail in Chapter 9, Principles for Competitive-Binding
Assays, of this book. An assay for mycophenolic acid was
later developed by Dade-Behring company [39].
In general, the method involves several steps, shown
schematically in Figure 5. In the first step, the sample is
mixed with the antibody, the enzyme substrate, and
nicotinamide adenine dinucleotide (NAD). In the second
step, the enzyme glucose-6-phosphate-dehydrogenase
subject of the referenced patent [39] is said to be capable
of distinguishing MPA from mycophenolate mofetil, the
ester pro-drug, and from the major metabolite, MPAG.
However, some cross-reactivity of this antibody was
observed with AcMPAG. Since AcMPAG exhibits similar
immunosuppressive potency to MPA in vitro [24], the
EMIT method may be perceived as allowing for estimation
of total immunosuppressive impact of MPA through
measurement of all the active moieties of MPA
simultaneously. This is sometimes presented as an
advantage of this method in advertising materials.
However, the EMIT results do not represent the true MPA
concentrations, and the level of bias for each individual
 884
Mycophenolic Acid
sample is unknown. A summary of several selected
publications comparing EMIT to either HPLC/UV or
MS/MS is shown in Table 5.
Table 5: Summary of Publications Comparing EMIT
to HPLC or MS Methods
Reference Average Bias Average Bias
Reference
Method of C0 [%] of AUC [%]
[24] UV 151.2%
[33] UV 11.1 15.2%
[40] UV 22% *
11.6%
[38] MS/MS 61.4 57.9%.
[31] MS/MS 18.7 26.8% 16.7% 22.5%
*
Value not directly reported in the paper but derived
from the data.
Total Mycophenolic Acid Assay on COBAS Integra
System
Recently a novel automated method for analysis of total
MPA was developed and approved by the U.S. Food and
Drug Administration, using Roche Diagnostics Cobas
Integra platform [41]. The method involves use of the
genetically engineered inosine-5-monophosphate
dehydrogenase (IMPDH) reagent, which is the direct target
enzyme of MPA.
The IMPDH catalyses the following reaction: IMP + NAD
+ H2O XMP + NADH + H+. First IMP binds to
IMPDH, which is followed by binding of NAD cofactor.
Then reduced cofactor NADH is released, and the product
xanthosine monophosphate (XMP) is released
subsequently. This reaction is the rate-limiting step in
guanine nucleotide synthesis, a critical step in the
proliferation of lymphocytes. Since mycophenolic acid
inhibits the IMPDH directly, this method relies in fact on
the measurement of direct immunosuppressive effect of
MPA. Therefore, there is no need for antibody or the
enzyme-labeled-MPA, as compared to the EMIT.
Similarly to the EMIT method, the rate of formation of
NADH is measured by monitoring the change in
absorption at a wavelength of 340 nm. Mycophenolic acid
binds to the active site of IMPDH uncompetitively and
inhibits the above reaction, thus the concentration of MPA
in a sample is inversely proportional to the absorbance of
NADH. The Cobas Integra assay for mycophenolic acid
uses six calibrators, and the measuring range is 0.4 to 15
mg/L of MPA. Comparison to the HPLC assay yielded the
following Passing/Bablok regression statistics for total
MPA: slope = 0.984, intercept = 0.096, r = 0.993 [42], or
slope between 1.01 and 1.10, intercept between 0.12 and
+0.068 mg/L and r > 0.99 from a multicenter trial [43].
This immunoassay method was validated in 4 independent
laboratories in Europe and the United States. Comparison
of patient results from one of the centers from this study is
shown graphically in Figure 6. The coefficient of variation
(CV) of the within-run imprecision of this assay measured
on spiked plasma samples was 1.3% to 4.4% for samples
below 1 mg/L of MPA, and 0.4% to 4.7% for samples
above 1 mg/L of MPA. Between-day imprecision was
1.1% to 9.4% CV and 0.0% to 6.7% CV for samples below
and above 1 mg/L of MPA, respectively. The lower limit
of quantification for this method was established to be
approximately 0.3 mg/L of MPA. Recovery ranged from
81% to 120% depending upon the sample and laboratory,
with mean approximately 100% [43]. This assay is also
capable of measuring free MPA concentrations in the
range 0 to 200 g/L, with CV for 25, 75, and 150 g/L
control samples below 5% [42].
Cloned-Enzyme Donor Immunoassay (CEDIA)
Another enzymatic immunoassay for mycophenolic acid,
based on the cloned-enzyme donor immunoassay (CEDIA)
principle, was developed by Microgenics Corporation for
use with regular chemistry analyzers. The CEDIA method
utilizes enzyme fragment complementation of the
genetically engineered Escherichia colis -galactosidase.
Two fragments of the enzyme, enzyme acceptor (EA) and
enzyme donor (ED), are capable of combining to form the
active enzyme [44].
In the mycophenolic acid assay, the ED is conjugated with
MPA. Similarly to the EMIT assay, the anti-MPA antibody
is introduced into the system and mixed with the sample.
The mycophenolic acid and the conjugate of ED-MPA
compete for the binding sites on the antibody. Whenever
the conjugate is bound to the antibody, it loses its ability to
combine with EA, and therefore no active enzyme is
formed, resulting in decreased substrate cleavage activity
in the sample. High enzyme activity results in a color
change, which is directly proportional to the content of
MPA. This assay uses only two calibrators, 0 and 10 mg/L
of MPA, and three QC samples, 1.1, 2.6, and 5.9 mg/L.
The CEDIA assay has been used for assay of cyclosporin,
tacrolimus, sirolimus, and everolimus for some time,
though it is a relatively new assay for mycophenolic acid.
As of June 2007, only one paper on MPA analysis utilizing
this method has been published [45]. The within-run
precision CV values did not exceed 5% for all QCs, while
between-run precision was within 6%.
Owing to the use of antibody, the CEDIA method is prone
to the same deficiency as the EMIT method, that is, cross-
reactivity with AcMPAG, and possibly also other
compounds in the sample. One of the studies found that the
degree of overestimation is dependent upon transplant
type. The bias for CEDIA versus HPLC/UV results
amounted to 44% for renal, 47% for lung, and 54% for
heart transplant patients [45], which is comparable to the
reported EMIT bias.
Reference and Preferred Methods
There is no formally established reference method of
 885
Mycophenolic Acid
analysis of MPA, but the HPLC/UV method is commonly
perceived as a gold-standard method. It has served as a
widely used analytical method in the phase III pivotal trials
of mycophenolate mofetil in renal transplant patients, as
well as in many other studies that included detailed
pharmacokinetic analysis of MPA. Despite the popularity
of the EMIT method, certain limitations, including
dynamic-range limitations and the mentioned positive bias
from MPA metabolites, have precluded this method from
being considered a gold-standard method. HPLC with
mass spectrometric detection is growing in popularity,
although it is not as widely used as the EMIT method
because of the high initial instrument cost. In addition,
mass spectrometric methods offer little advantage over
ultraviolet detection when only total MPA and MPAG
measurements are of concern.
Specimen
Mycophenolic acid is most commonly measured in plasma
or serum. Some MS/MS methods for analysis of multiple
immunosuppressant drugs involve MPA analysis in whole
blood; however, the standard of practice is to utilize
plasma or serum concentrations of MPA, and there is a
lack of reference pharmacokinetic information for whole-
blood MPA concentrations.
Samples should be collected and processed within 2 hours
following phlebotomy. Plasma is prepared from whole
blood by centrifugation. Plasma samples can be stored
frozen without any loss in either total or free MPA and
MPAG concentrations for at least one year at 24C. Due
to the instability of AcMPAG, it is recommended that the
samples are frozen at 80C and processed in the shortest
time possible whenever this metabolite is of concern.
Samples for which only the total level of analytes is of
interest can be acidified prior to freezing, which stabilizes
AcMPAG concentration for at least 3 months of storage at
24C [24]. However, when the free concentration and
free fraction of the analytes is of interest, acidification is
not recommended, except for a separate aliquot of the
primary sample, because it alters the binding between
proteins and mycophenolic acid.
Therapeutic Range and Pharmacokinetics
Mycophenolic acid acts by inhibiting inosine-5-
monophosphate dehydrogenase (IMPDH)the enzyme
essential in de novo guanosine nucleotide synthesis. T and
B lymphocytes critically depend on the de novo synthesis
of purines for their proliferative response, whereas other
types of cells can use salvage pathways for purine
production. Thus MPA has a selective effect on the
proliferation of lymphocytes. In addition, mycophenolic
acid suppresses antibody formation by B lymphocytes,
resulting in a potent immunosuppressive effect [46].
MPA is currently marketed in two forms: as
mycophenolate mofetil (MMF) by Roche, and as enteric-
coated mycophenolate sodium salt (Myfortic) by Novartis.
After oral administration, MMF is readily hydrolyzed to
MPA and rapidly absorbed, starting in the stomach.
Myfortic is mainly absorbed in the small intestine because
of the enteric coating that reduces MPA absorption in the
stomach. The median time to maximum MPA plasma
concentration is between 0.25 and 1.25 hours for MMF
and 1.5 and 2.75 hours for Myfortic [32]. MPA is
extensively bound to plasma albumin (around 97% to
99%). Only the remaining free fraction of MPA of about
1% to 3% is pharmacologically active in in-vitro test
systems [22].
MPA is metabolized in the liver, gut, and kidneys by
uridine-diphosphate glucuronyl transferase enzymes,
mainly to the pharmacologically inactive glucuronide
metabolite, MPAG. At steady state, MPAG can be present
in blood plasma at concentrations as much as 100-fold
higher than MPA. Two other metabolites, the acyl-
glucuronide (AcMPAG) and the glucoside (MPAGc), are
also formed in smaller quantities. The AcMPAG
metabolite was found in in-vitro test systems to be
pharmacologically active and of a potency similar to MPA
[47]. The metabolites are excreted mainly into urine and in
smaller quantities into bile. The excretion into bile allows
for the process of enterohepatic recycling of mycophenolic
acid, whereby the MPAG that enters the gastrointestinal
tract via bile is converted back to MPA by the hydrolytic
action of glucuronidase shed by enteric bacteria [48]. For
this reason, a second peak frequently appears in the MPA
concentration versus time plot, usually within 4 to 10
hours after drug administration. This also accounts for a
great between- and within-patient variability of the area
under the time-concentration curve (AUC) of MPA.
Leukopenia is the main undesired effect of over-
immunosuppression by mycophenolic acid, due to
bone-marrow suppression. Some other less severe but
more commonly occurring side effects of MPA include
gastrointestinal disturbances, mainly diarrhea, and
increased risk of viral infection, especially
cytomegalovirus [49].
The general variability in the pharmacokinetics of
mycophenolic acid and its relatively narrow therapeutic
window led to the recommendation of applying therapeutic
drug monitoring for assessment of the true exposure to this
drug and dose modification. Typically the total content of
MPA is used in monitoring; free MPA concentrations are
under investigation for their clinical relevance. The
therapeutic range of mycophenolic acid depends upon
whether there is concomitant use of other
immunosuppressants. Table 6 summarizes commonly used
regimens and their respective MPA therapeutic ranges:
 886
Mycophenolic Acid
Table 6: Therapeutic Ranges of MPA for Different Immunosuppressive Regimens [50]
Coadministered Immunosuppressant Target Range of MPA Trough
[mg/L]
Tacrolimus 1.9 4.0
Cyclosporine 1.0 3.5
The clinical utility of the 30 to 60 mg*h/L MPA AUC
target range in the above table was derived from
investigations in renal transplant patients who received
concomitant cyclosporine and corticosteroid
immunosuppression [50]. The efficacy (freedom from
acute early rejection) was confirmed in a recent
prospective concentration-control investigation [51]
comparing a Bayesian dose adjustment targeting an
estimated MPA AUC of 45 mgh/L with empiric dosing of
MMF in renal transplant patients who received MMF +
cyclosporine + corticosteroid and in a percentage of
patients, early exposure to monoclonal interleukin 2
receptor antibody therapy. Two prospective therapeutic
drug monitoring trials that compare active monitoring of
MPA estimated AUC (FDCC trial) or active monitoring of
MPA trough concentration with a control arm with no
active monitoring are under analysis and will hopefully,
together with the French Adaptation de Posologie du
MMF en Greffe Rnale (APOMYGRE) study, provide the
basis for further definition of therapeutic drug monitoring
strategies in contemporary clinical practice [52].
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[46] Ransom JT. Mechanism of action of

Figures
PUMP B PUMP A PUMP B PUMP A PUMP B PUMP A PUMP B PUMP A
(ELUTING) (WASHING) (ELUTING) (WASHING) (ELUTING) (WASHING) (ELUTING) (WASHING)

AUTO- AUTO- AUTO- AUTO-

SAMPLER SAMPLER SAMPLER SAMPLER
1 1 1 1
10 2 10 2 10 2 10 2

9 9
COLUMN

COLUMN

3 3
WASH

3 3
WASH

9 9
Waste Waste
A Waste B A Waste B
8 4 8 4 8 4 8 4
WASH WASH
COLUMN COLUMN
7 5 7 5 7 5 7 5
6 6 6 6

THERMOSTAT: 50C THERMOSTAT: 50C THERMOSTAT: 50C THERMOSTAT: 50C

ANALYTICAL COLUMN ANALYTICAL COLUMN ANALYTICAL COLUMN ANALYTICAL COLUMN

MS/MS MS/MS MS/MS MS/MS

Figure 1. Switching valve layout for across-directional (left) and uni-directional (right)
flow of mobile phase through working column.
 889

Mycophenolic Acid

14

12

10

mAU
6

220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
Wavelength [nm]

Figure 1: The UV spectrum of MPA, from [16]

275

193 207
Figure 3: Most common MS/MS
fragments of the mycophenolic acid
molecule.

NAD MPA PA S NAD

An M NADH
tib
od En
Antibody

zym
o dy

y
En

M Enzym
e
zy

PA e
m

Antib

e
Enzym
e
Antibody

Pr
D

S
NA

S
Enzym
e S
NAD
ody

NAD
Antib

dy
Antibody

e
bo E nzym NAD
MPA ti
An
Antibody
M
PA

NAD S
S

Figure 2: Schematic representation of EMIT principlebased on EMIT education materials from Dade-Behring.
 890

Mycophenolic Acid

50000

45000 MPA
MPAG peak
MPAC Figure 4: Magnified portion of MS/MS
MPAG
40000
MPAC (IS) peak chromatogram of a free MPA sample, showing
35000
additional MPA peak, that originated from in-
source decomposition of MPAG.
30000 MPA peak
[counts]

25000
MPAG decomposed
in-source to MPA
20000

15000

AcMPAG peak
10000

5000

0
4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6

Time [min]

30
Y = 1.06 X 0.03; R2 = 0.96

25
Integra 400 [mg/L of MPA]

20

15

10

0
0 5 10 15 20 25 30
HPLC [mg/L of MPA]

Figure 5: Comparison of total MPA concentration

measurements by Integra 400 assay versus HPLC.
 891

Mycophenolic Acid

List of Abbreviations Used in This Chapter

Acronym Explanation
AcMPAG Mycophenolic acid acyl-glucuronide
APOMYGRE Adaptation de Posologie du MMF en Greffe Rnale Study
AUC Area under the time-concentration curve
CAP College of American Pathologists
CAS Chemical Abstracts Service
CEDIA Cloned-enzyme donor immunoassay
CV Coefficient of variation
EA Enzyme acceptor
ED Enzyme donor
EMIT Enzyme-multiplied immunoassay technique
FDA U.S. Food and Drug Administration
FDCC Fixed Dose/Concentration Control study
HPLC High-performance liquid chromatography
IMP Inosine-5-monophosphate
IMPDH Inosine-5-monophosphate dehydrogenase (enzyme)
IUPAC International Union of Pure and Applied Chemistry
IS Internal standard
LC/MS Liquid chromatography/mass spectrometry
LLE Liquid-liquid extraction
LLOQ Lower limit of quantification
LOD Limit of detection
MMF Mycophenolate mofetil
MPA Mycophenolic acid
MPAG Mycophenolic acid glucuronide
MPAC Carboxybutoxy ether of mycophenolic acid
MS Mass spectrometer
MS/MS Tandem mass spectrometer
NAD Nicotinamide adenine dinucleotide
NADH Nicotinamide adenine dinucleotide - reduced
PP Protein precipitation
QC Quality control
SPE Solid-phase extraction
UF Ultrafiltration
UV Ultraviolet
XMP Xanthosine monophosphate
892
Myoglobin

Myoglobin
Alan H.B. Wu
Name: Myoglobin
Clinical significance: Refer to Chapter 36, Cardiac and Muscle Disease, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular mass: 17,500 D
Chemical class: Heme protein
Structure: See diagram below

i such as (1) spectrophotometry, (2) electrophoresis, (3)

Principles of Analysis and Current Usage
chromatography, and (4) ultracentrifugation have been
Myoglobin is a heme protein that is reddish-brown. A
blood or urine sample turns this color when myoglobin is
present at a concentration exceeding 1 g/L. The visual developed to more specifically demonstrate the presence
examination of urine samples from patients with muscle of myoglobin in body fluids but are used only rarely.
disorders has been used in the past as a rapid bedside
test for the presence of myoglobin. However, most In urine, myoglobin can be detected using the pad for
patients with muscle disease have far less myoglobin blood on a urinalysis dipstick test (Table 1, Method 1).
present in the urine. Many physicochemical methods The heme group from free hemoglobin and myoglobin
catalyses the oxidation of tetramethylbenzidine, an
i indicator that turns the dipstick pad green. The pad is
Myoglobin
read visually against a color chart to determine the
Previous and current authors of this method:
semiquantitative degree of heme/myoglobulinuria (1+,
First edition: Not done
2+, etc). The urine sample can be pretreated with the
Methods edition: I-Wen Chen
addition of saturated ammonium sulfate, which will
Second edition: Not updated
salt-out the hemoglobin. After centrifugation, the
Third edition: Not updated
supernatant of the residual sample is retested with a
Fourth edition: I-Wen Chen
dipstick for blood, and a positive reaction is due to
Fifth edition: Alan H.B. Wu
893
Myoglobin
myoglobin. However, this procedure does not
completely separate out hemoglobin from myoglobin,
therefore a false-positive result can be obtained in the
presence of significant hemoglobinuria [1]. Use of a
microconcentrator membrane offers a more complete
separation of myoglobin from hemoglobin and increased
specificity for myoglobin detection [2].
Qualitative and semiquantitative immunological
methods have also been used.
Counterimmunoelectrophoresis, with the simplicity of
the agar gel precipitin techniques and the speed of
electrophoresis, has been used to detect myoglobin in
both serum and urine (Table 1, Method 2) [3].
Hemagglutination-inhibition assays use myoglobin-
sensitized red blood cells (myoglobin chemically
attached to the membrane of the red blood cell).
Agglutination takes place when these red blood cells are
mixed with the antimyoglobin antibody (Table 1,
Method 3). When a sample containing myoglobin is
added, myoglobin competes with the red blood cell for
myoglobin-binding sites on the antibody, and
hemagglutination is inhibited [4].
Counterimmunoelectrophoresis is a qualitative
procedure and has a sensitivity of only 2 mg/L. It is thus
unsuitable for detection of acute myocardial infarction
(AMI).
A rapid, qualitative latex agglutination test for detection
of increased concentrations of myoglobin in serum has
been reported (Table 1, Method 4) [5]. In this test
procedure, any interfering rheumatoid factor present in
the sample is removed when 50 L of test serum is
mixed with 5 L of rheumatoid factor absorption
medium on a dark slide. About 50 L of a suspension of
latex particles with bound antimyoglobin antibodies is
then added and mixed with the serum by gentle tilting
for 5 min. Agglutination of the particles occurs in the
presence of myoglobin. A negative exponential
relationship has been found between the time of
agglutination and myoglobin concentrations. Myoglobin
concentration was considered to be greater than 400
mg/L if agglutination occurred within 1 min, 150 to 400
mg/L if agglutination time was between 1 and 2 min, 80
to 150 mg/L if agglutination time was between 2 and 3
min, and less than 80 mg/L (upper limit of reference
interval) if more than 3 min were required for
agglutination. Red blood cell agglutination techniques
are reported to be sensitive in the range of 50 to 100
mg/L [4]. A disadvantage of the technique is that some
sera containing a high concentration of rheumatoid
factors gave positive agglutination reactions. The latex
agglutination procedure is at best semiquantitative and is
somewhat subjective, requiring visual observation to
establish an end point. The sensitivity of the latex test
may not be adequate. The advantage of the latex test is
its simplicity.
A quantitative microcomplement fixation assay has been
developed to detect myoglobin in both the serum and
urine of patients with acute coronary syndrome or
myopathies [6] (Table 1, Method 5). This technique is
based on the consumption of a fixed amount of
hemolytic complement by myoglobin-antimyoglobin
complexes. The amount of myoglobin present in the
sample is inversely proportional to the extent of
hemolysis of the hemolysin-sensitized sheep cells used
as the indicator system. Quantitative microcomplement
fixation is an extremely time-consuming and laborious
technique. Few laboratories have mastered its intricacies,
and thus it is rarely used. In addition, its sensitivity of
300 mg/L is not adequate.
Currently, the determination of serum myoglobin for
diagnosis of AMI and other muscle disorders is
performed almost exclusively by immunoassay
techniques because of their high analytical sensitivity,
specificity, precision, and rapid turnaround time.
Radioimmunoassay (RIA) procedures have also been
described for quantitative measurement of serum
myoglobin (Table 1, Method 6) [7]. However, in clinical
laboratories, RIA has largely been replaced by
automated 2-site non-isotopic immunoassays ( Table 1,
Method 7). The capture antibody is linked to a bead or
paramagnetic particle. The detecting antibody is linked
to a tag that can produce a nephelometric, turbidimetric,
fluorometric, or chemiluminescence signal. Automated
immunoassay analyzers perform a separation between
bound and free labels to produce a quantitative result.
Qualitative and quantitative methods for serum
myoglobin have also been developed for point-of-care
(POC) use (Table 1, Method 8) [8,9]. One POC method
utilizes a self-calibrating immunoassay system whereby
whole blood is separated from plasma that reacts with
fluorescent antibody conjugates within a reaction
chamber [8]. Following an incubation step, the reaction
mixture flows down a detection lane by capillary action
and the myoglobinfluorescent antibody complex is
captured by a second antibody in a discrete measuring
zone. The intensity of the label (visual or fluorescence)
is related to myoglobin concentration. The procedure
takes approximately 15 min to complete. Given that
myoglobin measurements are most commonly used in
the context of diagnosis and exclusion of AMI, several
POC devices have combined myoglobin with other
cardiac markers such as CK-MB and cardiac troponin
into a single cartridge or slide [10,11]. International
guidelines such as those from the National Academy of
Clinical Biochemistry have suggested that results of
cardiac markers should be reported within 1 hour of
blood collection [12]. Other groups such as the
American Heart Association have suggested an optimal
turnaround time of less than 30 minutes [13]. These POC
devices are designed for use at the patient bedside or
emergency department satellite laboratories to produce
results that can meet these groups recommended 30- to
60-minute turnaround time goals for reporting test
results.
Reference and Preferred Methods
There are currently no reference methods or materials
for myoglobin in serum or urine. The preferred method
is a two-site sandwich immunoassay. Unfortunately,
these assays are not standardized to each other, and each
method has slightly different reference ranges.
894
Myoglobin
The chemical and physicochemical techniques to
measure myoglobin (e.g., spectrophotometry,
electrophoresis, chromatography, and
ultracentrifugation) were not capable of measuring the
submicrogram range of myoglobin frequently found in
certain muscle disorders, such as AMI or myositis, and
are thus not currently used. Immunoassays are more
sensitive than the above techniques. However, a
prerequisite of any of the immunochemical methods is
that the antisera be specific for myoglobin. Specific
antisera used in such methods should not cross-react
appreciably with hemoglobin or other constituents of
body fluids and should be capable of detecting
myoglobin to the submicrogram range.
A major issue for myoglobin measurements is the lack of
standardization among commercial manufacturers of
myoglobin assays. There has resulted in significant
proportional biases in myoglobin results between
manufacturers. The International Federation of Clinical
Chemistrys Committee on Standardization of Markers
of Cardiac Damage has begun a program to standardize
myoglobin measurements. Candidate secondary
reference materials from various manufacturers have
been evaluated [14]. Calibrating 12 different automated
testing platforms for myoglobin using a lyophilized
candidate material originating from human heart have
reduced the bias for a human serum pool from 32% to
13%. However, until there is an accepted reference
standard that has been adopted by all manufacturers,
complete standardization of myoglobin will not be
possible. The lack of a reference method for myoglobin
further complicates this issue. As myoglobin is a small
protein, it is likely that some mass-spectrometric method
will be needed.
Specimen
The preferred specimen is nonhemolyzed, nonlipemic
serum. One study found no significant difference
between heparin plasma and serum, although EDTA
plasma samples were significantly lower than serum
[15]. Another study found that different myoglobin
assays can show significant interference from different
anticoagulants [16]. Each method should be investigated
with respect to the differences observed between serum
and the use of various anticoagulants.
Specimens are stable at 4C for 1 week and for up to 4
weeks at 20C [17]. Urine samples should be collected
without preservative and assayed within 24 h of sample
collection. If the test is not performed immediately after
sample collection, the urine sample should be kept at
2C to 8C and centrifuged to remove any debris before
the assay. Myoglobin in urine is stable under alkaline
conditions (pH 9.0) for many days at 2C, 20C, and
70C; sodium hydroxide can be added if long-term
storage is needed [18]. The analyte is not stable under
acidic conditions (e.g., pH 5.5). Most commercial
methods for myoglobin are not cleared by the U.S. Food
and Drug Administration for use in urine, and
laboratories must validate this specimen type prior to
clinical use.
Interferences
The use of collection tubes with separator gels has been
reported to both increase and decrease results [16].
Bilirubin has been found not to interfere. Rheumatoid
factor (RF) has been found to affect results in some
assays, with a lack of concordance between
concentrations of RF and the magnitude of interference.
As with any 2-site immunoassays that use monoclonal
antibodies, the presence of human anti-mouse antibodies
can potentially cause a false-positive result.
Myoglobin Reference Intervals
Because myoglobin is the major protein in striated and
myocardial muscle and because muscle mass varies with
sex, age, and race, serum myoglobin levels may show
age, sex, and race dependence (Table 2). Results of
measurements of serum myoglobin concentrations in
292 ostensibly healthy subjects using a sensitive RIA
revealed significantly higher myoglobin concentrations
in males than in females for both black and white
subjects [19]. Myoglobin concentrations in black males
were significantly higher than those in white males,
whereas no racial differences were observed in the
female population. Myoglobin concentrations were
higher in the older population in all groups except that of
black males, who showed no age difference. Reference
intervals established with the use of various enzyme
immunoassay procedures showed that the 95% central
range of the normal distribution for males and females
varied significantly between different procedures. The
97.5th percentile value ranged from 67 to 86 g/L for
males and 50 to 63 g/L for females [16]. The age, sex,
and race dependence of myoglobin concentrations can be
partially eliminated by correction of the myoglobin
values for body mass using serum creatinine levels
(Table 2) [20].
Interpretation
Myoglobin is a relatively small globular protein
composed of a single polypeptide chain of 153 amino
acids and an iron-containing heme prosthetic group
identical to that of hemoglobin. It is found in skeletal
and cardiac muscle cells and is capable of reversibly
binding molecular oxygen and enhancing the rate of
oxygen diffusion through the muscle cell. Myoglobin is
an oxygen carrier in the cytoplasm and appears to be
responsible for the transport of oxygen from the muscle
cell membrane to the mitochondrion. Myoglobins from
skeletal and cardiac muscles are immunologically
identical and thus cannot be distinguished from each
other by immunological methods.
Myoglobin may leak from muscle tissue into the blood
circulation as a result of damage to skeletal or cardiac
muscle and subsequently may appear in the urine
because of its relatively low molecular mass.
Myoglobinemia and myoglobinuria have been used in
the diagnosis of myopathies and cardiomyopathies. A
pronounced increase in serum myoglobin is an early
quantitative indicator of AMI. It has been reported that
myoglobinemia is a more sensitive clinical indicator of
AMI than increased activities or concentrations of serum
creatine kinase-MB (CK-MB) isoenzyme, especially
895
Myoglobin
when blood samples are drawn in the early stage of the
disease (e.g., within 3 hours after onset) [21]. However,
because no assay can differentiate myocardial from
skeletal muscle myoglobin, these assays are less specific.
As such, myoglobin assays for diagnosis of AMI have
not been widely used in the United States but have been
used in some European countries [22]. Increased serum
myoglobin concentrations return to normal sooner than
total CK and the CK-MB isoenzyme in AMI. The faster
elimination of myoglobin from the blood may be
advantageous in the assessment of reinfarctions
occurring during the period of increased enzyme
activities. Recurrent increases of serum myoglobin are
thus suggestive of an extension of the original infarction.
Since myoglobin is also present in skeletal muscle and is
cleared almost exclusively through glomerular filtration,
increases of myoglobin will occur after acute muscle
trauma, in acute or chronic renal failure, severe
congestive heart failure, prolonged shock, and in patients
with myopathies of a variety of causes. Therefore, the
use of serum myoglobin levels to furnish early,
quantitative documentation of AMI should be limited to
patients who do not have these underlying or associated
problems and who are within the first 18 h of the clinical
onset of infarction. In addition, serum myoglobin levels
have been used in the evaluation of neuromuscular
diseases such as muscular dystrophy, muscular atrophy,
and polymyositis [21]. Percy et al. reported that the
combination of CK, hemopexin, and myoglobin
measurements provided significantly better detection of
Duchenne muscular dystrophy carriers than when these
tests were used alone or in the combination of CK and
hemopexin without myoglobin [23].
In studies conducted on myopathies in children [24],
serum myoglobin concentrations for subjects 0.6 to 20
years were lower than adults, so it is necessary to use
assays with a high sensitivity of 2 g/L or less. Mean
myoglobin concentrations of 17.1 and 12.5 g/L were
obtained from 37 males and 29 females, respectively,
and were statistically different. Racial differences were
not investigated. Investigations in children with muscle
diseases, however, have been rather disappointing [24].
Only 2 out of 14 patients studied had increased serum
myoglobin concentrations. The patients evaluated in this
study were relatively young, ranging from 0.3 to 16
years of age, and were presumably in the relatively early
stages of disease. This might have contributed to the low
rate of hypermyoglobinemia in this group of patients.
Edwards et al. also found that measurement of
myoglobin offered no advantage over CK for the
investigation of any aspect of Duchenne muscular
dystrophy [24]. However, serum myoglobin
measurements were found to be helpful in detection of
Duchenne muscular dystrophy carriers when used with
CK and hemopexin [25].
Myoglobin measurements are also used for diagnosis of
patients with rhabdomyolysis. At one time, the urinalysis
blood dipstick test was widely used to detect myoglobin
in urine. However, a more effective indicator is the
presence of very high activities and concentrations of
serum CK and myoglobin. Patients with rhabdomyolysis
and myoglobinemia are at risk for development of acute
renal failure [26]. Treatment of these patients with
diuretics and alkalinization has been shown to minimize
the risk of renal failure [27]. The aqueous stability of
myoglobin under basic pH conditions may facilitate its
clearance through the kidneys. Calculation of myoglobin
clearance has been suggested as a mechanism to predict
patients risk for renal failure with rhabdomyolysis
[18,28]. Patients with a high serum myoglobin and low
urinary clearance are at greatest risk. Some researchers
and clinicians have experimented with treating patients
with rhabdomyolysis by removing myoglobin using
veno-venous hemofiltration [29,30]. However, this has
not progressed beyond the experimental therapy stage.
Myoglobin Performance Goals
An assessment of the biological variation determines
analytical performance goals [31]. These studies are
conducted in healthy subjects and involve serial blood
collections. The biological variability for serum
myoglobin assays has been determined by Panteghini et
al. [32]. The analytical, (CVA), intraindividual (CVI),
and interindividual (CVG) variation were 6.9%, 11%,
and 14%, respectively. Based on these figures, the
analytical goals for imprecision at 1/2CVI was 5.6%.
The number of values needed to estimate the
homeostatic set point of an individual to within 5% was
25. The index of individuality (CVI/CVG) was 0.8. This
indicates that the use of population-based reference
intervals are appropriate for this analyte [31]. The
critical difference required for determining statistically
significant change in serial results (reference change
values [RCV]) was 35%. However, the RCV in the
Panteghini study was calculated using blood collected
over several weeks, and it is probably higher than the
expected RCV values that are calculated over several
hours, as would be applicable for use in patients
suspected of AMI. Studies have documented relatively
high imprecision and marked disagreement between the
various commercial immunoassay procedures available
for serum myoglobin [16]. Total CVs for specimens
within the normal reference interval ranged from 3.4% to
11%. Many of these assays do not meet goals established
by biological variation.
Bombardieri et al. [33] studied circadian variation of
serum myoglobin concentration in normal subjects and
in patients with polymyositis and found the highest
levels at 9 am and the lowest between 6 pm and
midnight in both groups. The percent differences
between the highest and lowest values could be as high
as 66%. Therefore it is important to take into account the
time of sample collection when myoglobin levels are to
be used to assess the degree of disease activity or to
monitor the response to treatment of muscle diseases
such as polymyositis. In these cases, it may be desirable
to have samples drawn for myoglobin at approximately
the same time each day.
896
Myoglobin
References
1 Adams EC. Differentiation of myoglobin and
hemoglobin in biological fluids. Ann Clin Lab
Sci 1971; 1: 208-21.
2 Kelner MJ, Alexander NM. Rapid separation
and identification of myoglobin and
hemoglobin in urine by centrifugation through a
microconcentrator membrane. Clin Chem 1985;
31: 112-4.
3 Hibrawi H, Garrison FD, Smith HJ. A
comparison of various agarose preparation in
counter-immunoelectrophoresis (CIE) system
for assaying urinary myoglobin. J Immunol
Methods 1977; 14: 59-63.
4 Cloonan MJ, Donald TG, Neale C, Wilcken
DEL. The detection of myoglobin in urine and
its application to the diagnosis of myocardial
infarction. Pathol 1976; 8: 313-20.
5 Ntorregaard-Hansen K, Hangaard J,
Ntorgaard-Pedersen B. A rapid latex
agglutination test for detection of elevated
levels of myoglobin in serum and its value in
the early diagnosis of acute myocardial
infarction. Scand J Clin Lab Invest 1984; 44:
99-103.
6 Kagen LJ, Scheidt S, Butt A. Serum myoglobin
in myocardial infarction: the staccato
phenomenon. Is acute myocardial infarction in
man an intermittent event? Am J Med 1977; 62:
86-92.
7 Stone MJ, Willerson JT, Gomez-Sanchez CE,
Weterman MR. Radioimmunoassay of
myoglobin human serum: results in patients
with acute myocardial infarction. J Clin Invest
1975; 56: 1334-9.
8 Muller-Bardorff M, Sylven C, Rasmanis G,
Jorgensen B, Collinson PO, Waldenhofer U, et
al. Evaluation of a point-of-care system for
quantitative determination of troponin T and
myoglobin. Clin Chem Lab Med 2000; 38: 567-
74.
9 Beneteau-Burnat B, Baudin B, Vaubourdolle
M. Evaluation of Stratus CS stat fluorimetric
analyser for measurement of cardiac markers
Troponin I (cTnI), creatine kinase MB (CK-
MB), and myoglobin. J Clin Lab Anal 2001;
15: 314-8.
10 Apple FS, Christenson RH, Valdes R Jr.,
Andriak AJ, Berg A, Duh SH, et al.
Simultaneous rapid measurement of whole
blood cardiac myoglobin, creatine kinase MB,
and cardiac troponin I by the Triage Cardiac
Panel for detection of myocardial infarction.
Clin Chem 1999; 45: 199-205.
11 Young GP, Murthi P, Levitt MA, Gawad Y.
Serial use of bedside CKMB/myoglobin device
to detect acute myocardial infarction in
emergency department chest pain patients. J
Emerg Med 1999; 17: 769-75.
12 Morrow DA, Cannon CP, Jesse RL, Newby
LK, Ravkilde J, Storrow AB, et al. National
Academy of Clinical Biochemistry Laboratory
Medicine Practice Guidelines: clinical
characteristics and utilization of biochemical
markers in acute coronary syndromes.
Circulation 2007; 115: e356-75.
13 Braunwald E, Antman EM, Beasley JW, Califf
RM, Cheitlin MD, Hochman JS et al.
ACC/AHA Guidelines for the Management of
Patients With Unstable Angina and NonST-
Segment Elevation Myocardial Infarction:
Executive Summary and Recommendations : A
Report of the American College of
Cardiology/American Heart Association Task
Force on Practice Guidelines. Circulation 2000;
102: 1193-1209.
14 Panteghini M, Linsinger T, Wu AHB, Dati F,
Apple FS, Christenson RH, et al.
Standardization of immunoassays for
measurement of myoglobin in serum. Phase I:
Evaluation of candidate secondary reference
materials. Clin Chim Acta 2004; 341: 65-72.
15 Pagani F, Bonetti G, Stefini F, Cuccia C,
Panteghini M. Serum and plasma samples for
ACS:Systems cardiac markers. Clin Chem
2000; 46: 1020-2.
16 Zaninotto M, Pagani F, Altinier S, Amboni P,
Bonora R, Dolci A. Multicenter evaluation of
five assays for myoglobin determination. Clin
Chem 2000; 46: 1631-7.
17 Carraro P, Plebani M, Varagnolo MC, Rossetti
M, Burlina A. A new immunoassay for the
measurement of myoglobin in serum. J Clin
Lab Anal 1994; 8: 70-5.
18 Wu AHB, Laios I, Green S, Gornet TG, Wong
SS, Parmaley L, et al. Immunoassays for serum
and urine myoglobin: myoglobin clearance
assessed as a risk factor for acute renal failure.
Clin Chem 1994; 40: 796-802.
19 Chen I-W, David R, Maxon HR, Sperling M,
Stein EA. Age-, sex-, and race-related
differences in myoglobin concentrations in the
serum of healthy persons. Clin Chem 1980; 26:
1864-80.
20 Maxon HR, Chen IW, Gerson M, Sperling M,
Stein EA. Diagnostic specificity of serum
myoglobin radioimmunoassay for acute
myocardial infarction is improved by using age-
, sex-, and race-specific reference intervals.
Clin Chem 1982; 28: 2179.
21 Askmark H, Osterman P, Roxin L, Venge P.
Radioimmunoassay of serum myoglobin in
neuromuscular diseases. J Neurol Neurosurg
Psychiatry 1981; 4: 68-72.
22 Roxin LE, Cullhed I, Groth T, Hallgren T,
Venge P. The value of serum myoglobin
determinations in the early diagnosis of acute
myocardial infarction. Acta Med Scand 1984;
215: 417-25.
23 Percy ME, Pichora GA, Chang LS, Manchester
KE, Andrews DF. Serum myoglobin in
Duchenne muscular dystrophy carrier detection:
a comparison with creatinine kinase and
hemopexin using logistic discrimination. Am J
Med Genet 1984; 18: 279-87.
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24 Chen IW, Sperling MI, Iannoccone S, Maxon
H, Gelfand M. A sensitive radioimmunoassay
for detection of serum myoglobin in children
for evaluation of myopathies. J Clin Chem Clin
Biochem 1981; 19: 636.
25 Edwards RJ, Rodeck CH, Watts DC. The
diagnostic value of plasma myoglobin levels in
the adult and fetus at risk for Duchenne
muscular dystrophy, J Neurol Sci 1984; 63:
173-82.
26 Hamilton RW, Hoplins MB, Shihabi ZK.
Myoglobinuria, hematoglobinuria, and acute
renal failure. Clin Chem 1989; 35: 1713-20.
27 Eneas JF, Schoefeld PY, Humphreys MH. The
effect of infusion of mannitol-sodium
bicarbonate on the clinical course of
myoglobinuria. Arch Intern Med 1979; 139:
801-5.
28 Laios I, Caruk R, Wu AHB. Myoglobin
clearance as an early indicator for
rhabdomyolysis-induced acute renal failure.
Ann Clin Lab Sci 1995; 25: 179-84.
29 Nicolau D, Feng YS, Wu AHB, Bernstein SP,
Nightingale CH. Myoglobin clearance during
continuous veno-venous hemofiltration with or
without dialysis. Int J Artif Organs 1998; 21:
205-9.
30 Amyot SL, Leblanc M, Thibeault Y, Geadah D,
Cardinal J. Myoglobin clearance and removal
during continuous venovenous hemofiltration.
Inten Care Med 1999; 25: 1169-72.
31 Fraser CG. Biological variation: from principles
to practice. Washington DC: American
Association for Clinical Chemistry Press, 2001.
32 Panteghini M, Pagani F. Biological variation of
mlyoglobin in serum. Clin Chem 1997; 43:
2435.
33 Bombardieri S, Clerico A, Riente L, Del Chicca
MG, Vitali C. Circadian variations of serum
myoglobin levels in normal subjects and
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898
Myoglobin

Table 1: Methods of Myoglobin Analysis

Method 1: Urinalysis dipstick analysis; qualitative
Principle of analysis: Oxidation of a dye by the heme group to form a color
Comments: Positive results also occur with hemoglobin. Ammonium sulfate precipitation followed by
centrifugation can separate hemoglobin from myoglobin, but incomplete separation can occur at high urine
hemoglobin concentrations causing a false-positive result for myoglobin.

Method 2: Counterimmunoelectrophoresis; qualitative

Principle of analysis: Serum or urine is placed in electric field. Anodically migrating myoglobin and cathodically
migrating antibody move through agarose toward each other. A precipitin band forms where they meet.
Comments: Rare; lacks sensitivity; convenient technique; rarely used

Method 3: Hemagglutination inhibition; semiquantitative

Principle of analysis: Red blood cells are coated with myoglobin; addition of antibodies to myoglobin causes red
blood cells to agglutinate. Presence of myoglobin inhibits red blood cell agglutination. Read visually.
Comments: Rare; lacks sensitivity; difficult quality control

Method 4: Latex agglutination; semiquantitative

Principle of analysis: Latex beads coated with antibody to myoglobin. Presence of myoglobin causes beads to
agglutinate. Read visually.
Comments: Common; lacks sensitivity; semiquantitative; individual reading variability; easy to perform. Has
been replaced by immunoassays.

Method 5: Complement fixation; quantitative

Principle of analysis: Hemolysin-sensitized sheep red blood cells compete with myoglobinantimyoglobin
complex for complement. Extent of RBC lysis is inverse of myoglobin concentration
Comments: Rare; lacks sensitivity; very difficult to perform consistently. Has been replaced by immunoassays.

Method 6: Radioimmunoassay (RIA); quantitative

Principle of analysis: Myoglobin competes for limited antibody with 125I-labeled myoglobin
Comments: Infrequently used. Sensitive; requires RIA facility. Has been replaced by automated non-isotopic
immunoassays or point-of-care devices.

Method 7: Immunoassay, quantitative

Principle of analysis: Myoglobin bound to capture antibody. Second labeled antibody (enzyme, fluorophore,
chemiluminescent tag) forms sandwich and enables detection.
Comments: Most frequently used technique; allows rapid turnaround of results

Method 8: Lateral flow point-of-care immunoassay testing devices

Principle of analysis: Sandwich non-isotopic immunoassay using passive capillary flow to immobilized
antibodies to myoglobin
Comments: Point-of-care testing device immunoassays are conducted on whole blood and enable rapid
turnaround time for reporting results.

Table 2: Race-, Sex-, and Age-Specific Reference Intervals for Serum Myoglobin [20]
Groups Myoglobin (g/L)
Race Sex Age (years) Mean Range*
White Male 50 32 2052
50 38 2071
Female 50 28 1842
50 32 1660
Black Male All 42 2087
Female 50 28 2038
50 31 1853
*Range includes at least 95% of the population with 90% confidence (log normal distribution).
899

Opiates

Opiates
Dennis E. Coyle

Name: Opiates
Clinical significance: click here
Molecular structure: Morphine, 7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol
Molecular formula: C17H19NO3
Molecular weight: 285.33 D
Merck Index: 6129

Structure:
Molecular structure: Heroin, diacetylmorphine,
7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol diacetate
Molecular formula: C21H23NO5
Molecular weight: 369.40 D
Merck Index: 2932

Structure:
Molecular structure: dihydromorphinone, hydromorphone,
4,5-epoxy-3-hydroxy-17-methylmorphinan-6-one
Molecular formula: C17H19NO3
Molecular weight: 285.33 D

C:\5th Edition Print Revised\Opiates_2009-07-02[1].doc

900
Opiates
Merck Index: 4714
Structure:
Principles of Analysis and Current Usage
Opioids are a class of drugs defined by their ability to
bind specifically to any of several subclasses of opioid
receptors to produce an agonistic action. Opiates are
commonly known as narcotic analgesics and derived
from gum opium, such as heroin, morphine, and
dihydromorphone. Synthetic compounds that have
opiate-like qualities include propoxyphene, methadone,
meperidine, and fentanyl. Opiates as well as many other
classes of drugs are subject to abuse. Because of this,
testing for the presence of opiates and determining the
concentrations in overdose patients are major
considerations of a toxic drug screen. This necessitates
the need for rapid qualitative assay methods capable of
detecting the opiates in urine at overdose levels in the
microgram-per-milliliter range.
Analyses of opiates present at therapeutic levels,
however, must measure concentrations in the low
nanogram-per-milliliter range.[1] This range requires
that the assay method used to measure therapeutic levels
have greater sensitivity than the methods used to
measure drug abuse. This requirement is of vital concern
because methods commonly used for drug screening,
such as thin-layer chromatography (TLC), adequate for
detecting drug overdose, lack the sensitivity to detect
this class of drugs when the drugs are present at
therapeutic levels.[2] Screening methods of the
immunoassay type are sensitive down to the picogram-
per-milliliter range. However, they lack specificity (are
cross-reactive) and therefore do not allow for
identification of the specific opiate involved.[3,4]
i before application to the TLC plate. Separation is
Opiates
Previous and current authors of this method:
First edition: Not done
Methods edition: Dennis Coyle
Second edition: Not done
Third edition: Not updated
Fourth edition: Dennis Coyle
Fifth edition: Not updated
The screening for opiates is further complicated by the
fact that the hepatic metabolism of the opiates allows for
the biotransformation of one opiate into another.[1]
Heroin serves as an example of this. Heroin is
metabolized into morphine and further into
hydromorphone. Identification of the particular opiate
present in a sample becomes dependent on the time
between the last dose (ingestion or administration) and
the collection of the sample. The opiates are metabolized
to form an inactive 3-O-glucuronide, which is present in
the plasma and urine in much higher concentrations than
the parent drug. This major metabolic route must be
considered during sample preparation, since the
glucuronide conjugate is not extracted under conditions
used for the parent opiate.
Qualitative Tests
Thin-layer chromatography is performed on urine
adjusted to a range of pH 10 to 11 and extracted into
25% ethanol in chloroform (v/v),[2] (method 1, Opiates
Methods Summary Table). The solvent is evaporated
and the residue taken up in a small amount of methanol
and applied to a thin-layer chromatography plate (silica).
After development of the plate using chloroform
methanolammonia (90:10:1) as the mobile phase, the
position of the opiate is visualized by use of long- or
short-wave ultraviolet radiation, iodine vapor, and
iodineiodoplatinate solution or ninhydrin solution. Rf
values and brown color are specific criteria for the
identification of the drug.
One can modify the TLC method by employing
Amberlite, an XAD-2 resin.[4] This polymeric
absorbent resin allows differential elution of the opiates
accomplished through adsorption (mainly through van
der Waals forces) of many water-soluble organic
compounds to the resin. This method also concentrates
the sample, allowing detection of the drug at lower
concentrations than with untreated samples.
Fluorescence can be employed to detect nonspecifically
the presence of opiates[5,6] (method 2, Opiates Methods
901
Opiates
Summary Table). Urine is extracted at a range of pH 8.5
to 9 into butanol/chloroform. The organic phase is then
back extracted into acid, and oxidation of the opiates is
accomplished with K3Fe(CN)6 (Opiates: Figure 1). A
fluorescent product is generated and is detected by a
fluorometer. The excitation maximum is 250 nm, and the
emission maximum is 440 nm.
Gas-liquid chromatography (GC) detection of the opiates
is performed by extraction of urine, adjusted to a basic
pH, with ethyl acetate[7,8] (method 3, Opiates Methods
Summary Table). The organic phase is then separated
and evaporated to dryness, and the residue is derivatized
with either trimethylsilylimidazole or heptafluorobutyric
anhydride. Separation and detection is accomplished
with an OV-series column with either a flame ionization
or electron-capture detector. The relative retention times
at specific column conditions allow presumptive
identification of the opiate. In some cases, an acid
hydrolysis step is employed before the extraction of the
urine.[2,7] This releases the opiate from the glucuronide
conjugate and therefore increases the amount of
extractable opiate. Enzymatic hydrolysis of the
glucuronide has also been used.[9]
The GC method may be used with mass spectrometry
(GC/MS).[10,11] The method is the same as described
for GC; however, the mass spectrometer is used for
detection. This increases sensitivity and allows more
accurate identification of the opiate, using the mass-
fragmentation pattern (method 4, Opiates Methods
Summary Table).[12]
High-performance liquid chromatography (HPLC) is
also useful in detection and identification of the
opiates[13,14] (method 5, Opiates Methods Summary
Table). Extraction of alkalinized urine is followed by
chromatography on a reversed-phase silica column
(C18) with 0.1 M NaH2PO4 in 25% CH3CN in water,
pH 4.8. The separated opiates are detected by
spectrometric analysis at 254 nm. A modification of this
method employs the use of the fluorescence method
described earlier.[6,7] After extraction from urine, the
opiate is oxidized using K3Fe(CN)6 to form a
fluorescent dimer. The resulting product is then
chromatographed on a silica column and detected using
a fluorescence detector.[14]
HPLC may also be used in combination with mass
spectrometry (HPLC-MS).[15] The method is the same
as that used for HPLC with the mass spectrometer used
as the detector. The HPLC-MS coupling, however, does
suffer from a lack of sensitivity. The mass spectrometer
in combination with the HPLC offers universality and
identification of the opiate (method 6, Opiates Methods
Summary Table).
The use of solid-phase extraction procedures have been
used for GC, GC-MS, HPLC, and HPLC-MS methods.
Samples are made alkaline and passed through a C18
reversed-phase column.[16,17] The opiates are then
eluted from the column with dichloromethane-acetone
(1:1)[16] or methanol.[17] Solid-phase extraction has
gained in popularity because it offers excellent
recoveries and yields the best sample purification.[17]
Immunoassays have also been employed for the
detection of opiates because of their high sensitivity and
ease of sample preparation. Most assays include a
competitive binding step involving competition between
the drug in the sample being analyzed and a labeled drug
used for detection. The specificity (lack of cross-
reactivity) of the assay method is dependent on the
antibody employed. Most antibody preparations exhibit a
broad range of cross-reactivity between the different
opiates.
The hemagglutination inhibition (HI) method utilizes
formaldehyde-stabilized red blood cells complexed with
a drugalbumin conjugate.[18] The red blood cells are
coated with the opiate. The test sample (urine) is added
to the coated red blood cells along with antibody
directed against an opiate, usually morphine. The free
opiate, morphine, or a cross-reactive opiate in the
sample, inhibits the agglutination (crosslinking) of the
red blood cells by the antibody. The nonagglutinated red
blood cells form a diffuse pellet, whereas the
agglutinated red cells settle and form a characteristic red
ring (method 7, Opiates Methods Summary Table).
The stable free radical-labeled assay technique (FRAT)
is a homogeneous competitive binding assay that uses a
spin-labeled drug (nitroxide radical attached to the
drug)[16] (method 9, Opiates Methods Summary
Table). The spin label is detected by the use of electron
spin resonance spectrometry (ESR). The FRAT is able to
measure the energy absorbed by the unpaired electrons
(spin-label radical) when they change from a ground to
an excited state. The spectrum is sharp when the drug is
free, and it broadens and flattens when the drug binds to
the antibody. The amount of spectrum sharpening
(increasing amplitude of the peaks) is proportional to the
amount of spin-labeled drug displaced or the amount of
drug in the sample.
The enzyme-multiplied immunoassay technique (EMIT)
is basically the same assay as an RIA method except that
an enzyme-labeled drug replaces the radioactive
label[11,19] (method 10, Opiates Methods Summary
Table). An enzyme (lysozyme or malate dehydrogenase)
is covalently bound to an opiate (usually morphine). The
drug in the sample proportionately displaces the enzyme-
labeled drug. The enzyme is inhibited when the complex
is bound to the antibody and is active when unbound.
The free enzymedrug complex is able to convert a
substrate into products, causing a shift in the absorbance
of the solution (increase or decrease). The wavelength is
340 nm for the dehydrogenase reaction and 450 nm for
the lysozyme assay. The change in the absorbance is
proportional to the amount of drug in the sample. An
advantage of this assay over RIA methods is that the
antibody-bound drug does not need to be removed
(method 10, Opiates Methods Summary Table).
902
Opiates
ELISA (enzyme-linked immunosorbent assays) have
become one of the most popular methods for assessment
of drug exposure (method 11, Opiates Methods
Summary Table). Antibodies raised against an opiate
are coated onto the surface of a polystyrene microtiter
plate. Blood or urine is added to the plate, along with an
opiateenzyme conjugate. Opiate in the sample and the
opiateenzyme complex compete for antibody binding
sites on the surface of the well. Following an incubation,
unbound drug is removed by washing and a colorimetric
reaction is used to measure how much drugenzyme
conjugate is bound to the microtiter well. The measured
absorbance is inversely proportional to the concentration
of drug in the sample.
Quantitative Assays
Methods used for the quantitation of the amount of
opiate present include GC, GC/MS, HPLC, and RIA
(methods 3 to 5 and 8, Opiates Methods Summary
Table).
The chromatographic methods (GC, GC/MS, and HPLC)
not only are able to quantitate the amount of drug but
can also identify the specific drug present in the sample.
The GC/MS technique is the only method in this
discussion that is able to identify unequivocably an
individual opiate. This is possible because of the
methods ability to generate a unique mass-
fragmentation pattern for each individual opiate. These
methods therefore allow quantitation of mixtures of
opiates resulting from the administration of more than
one opiate or from the biotransformation (metabolism)
of the opiate taken.
Radioimmunoassay (RIA) utilizes a radiolabeled drug
(3H, 14C, or 125I) in a heterogeneous competitive
binding assay. The displacement of the radiolabeled drug
from the antibody by the unlabeled drug in the sample is
measured by either scintillation spectrometry (3H and
14C) or a gamma-ray scintillation counter (125I). The
free, labeled drug, not complexed to the antibody, is
separated from the antibody-bound labeled drug by an
agent that precipitates the antibodydrug complex, such
as a second antibody (an anti-antibody), charcoal
dextran,[20] or ammonium sulfate.[21] The amount of
labeled, unbound drug is proportional to the amount of
drug in the sample (method 8, Opiates Methods
Summary Table).
Reference and Preferred Methods
Qualitative
The qualitative identification of an opiate in a toxic drug
screen requires, first, the determination of the presence
of an opiate and, second, its confirmation by a second
method. The confirmation may identify the specific
opiate. TLC has sufficient sensitivity for detection of
some urinary opiates in the overdose patient. Practical
limits of detection of TLC are usually in the 1 to 2
g/mL range.[22] Other chromatographic methods (that
is, GC, GC/MS, and HPLC) are able to detect nanogram
quantities of opiates when the extraction and
derivatization procedures are optimized. However, if the
methods require long periods of time for extraction and
derivatization of the sample, they are not suited for the
emergency situation. When time is not a factor, an
additional step of hydrolysis of the glucuronide
conjugate increases the sensitivity of detection.
The immunoassay techniques allow rapid detection of
the presence of an opiate. Because of their high
sensitivity and ability to be used without prior sample
treatment, these methods can be adapted for rapid and
easily automated analysis. Immunoassay methods most
frequently used include EMIT, ELISA, and CEDIA
technologies. The specificity of the assay is determined
by the nature of the antibody reagent used. For screening
purposes, a nonspecific antiserum is preferred, and one
that reacts with the glucuronide conjugate would have
maximal sensitivity. Of the immunoassay methods
discussed, EMIT is perhaps the best choice for a rapid
screening of samples. EMIT assays do not require
separation of the antibody-bound drug from the unbound
drug and can produce results within 20 min. ELISAs
have also become extremely popular because of their
ease of use and adaptability for use with blood or urine
specimens.[23] Unlike many of the other homogenous
immunoassays, ELISAs do not require pretreatment of
whole blood samples.
Confirmation of an opiate requires a different assay. The
chromatographic methods (TLC, HPLC, GC, HPLC/MS,
and GC/MS) are best suited for this type of analysis.
Thin-layer chromatography, which is readily available,
is usually employed and is adequate for many overdose
cases. For those cases in which EMIT is positive and
TLC is negative, a specific extraction of the opiate
followed by determination on a GC or GC/MS is usually
performed. The required instruments are generally
available in toxicology laboratories. GC/MS is the
method of choice because identification is verified by
the unique mass-fragmentation pattern of the separated
compound. This eliminates errors from any interfering
compounds with the same retention time as a particular
opiate present in the patient sample.
Quantitative
Methods used for quantitation of the opiate are GC,
GC/MS, HPLC, HPLC/MS, and RIA. The
chromatographic procedures are the only suitable
procedures for the analysis of mixtures of opiates,
especially if a quantitation of all pharmacologically
active opiates present in the sample is desired. Usually
for therapeutic monitoring only the opiate being
administered is assayed. Of the chromatographic
methods, GC/MS would be the method of choice, since
it has all of the advantages of the GC or HPLC methods
along with the ability to confirm the identification of the
separate opiates. The extraction method enables
selection of either a total opiate level (hydrolysis before
extraction measures levels of both conjugated and
unconjugated opiate) or an unconjugated opiate level
(extraction only, no hydrolysis). It must be noted that
acid hydrolysis will destroy some of the opiate (range of
4% to 15%). An enzymatic method (glucuronidase) does
not lead to a destruction of the opiate but requires more
903
Opiates
time, that is, 24 h for the enzymatic method compared to
1 h for the acid hydrolysis.[10]
When an individual opiate is being monitored for
therapeutic use, immunological procedures are
appropriate. In this circumstance EMIT and ELISA are
not quantitative. The RIA procedures have adequate
sensitivity. The type of antiserum used (antibody) must
be considered for determination of the specificity of the
assay method.[3,4] The specificity of the antibody is
determined by how the opiate is presented for antibody
formation. For example, morphine is too small a
molecule to elicit antibody formation. The morphine
molecule must be covalently coupled to a larger
molecule such as albumin. The site of the chemical
linkage of the morphine to the protein determines what
part of the molecule of morphine elicits a response by
the antibody-producing cell. Morphine conjugated to the
protein at the C-3 position (Opiates: Figure 1) elicits
antimorphine antibodies to the C-6 position and to the
nitrogen region between carbons 16 and 9. The antibody
therefore is sensitive to groups in this region. The 3-O-
glucuronide derivative of morphine cross-reacts with the
antibody and is detected as free morphine. This analysis
is valid if total morphine levels are to be measured. If,
however, pharmacologically active drug levels (amount
that would produce a response such as pain relief) are
needed, levels determined in this manner are unsuitable.
To remove interference by glucuronides, the sample can
be extracted into an organic phase and the dried residue
re-extracted into an aqueous solvent (saline or buffered
saline) before analysis. Another way to eliminate this
interference would be to use an antibody with specificity
for the C-3 position. This has been accomplished by use
of a morphine molecule conjugated to the carrier protein
through its C-6 position. This allows detection of
morphine without interference by the glucuronide and
without extraction of the sample. For a more detailed
examination of the effect of opiate structure on antibody
specificity, refer to the paper by Findlay, Butz, and
Jones.[9]
A comparison of EMIT, ELISA, RIA, HI, GC, GC/MS,
and HPLC detection levels is seen in Opiates Table:
Comparison of commonly used assay methods.
GC or GC/MS would provide the best overall method for
the determination of opiates. The method is the most
sensitive of the methods discussed and allows for both
identification and quantitation of the opiates. The
method also exhibits the lowest coefficient of variation
(Opiates Table: Comparison of commonly used assay
methods). The method does not, however, lend itself
well to automation and is not capable of rapid analysis
with large sample loads.
If separation is unnecessary and a specific drug is being
analyzed, as in the case of therapeutic monitoring, the
RIA procedure is the method of choice. This method is
able to handle large sample loads in a short period of
time (1 to 2 h).
Specimen
The specimen can be plasma, serum, urine, saliva, or
tissue extract. The blood collection tube may be of any
type. All glassware, however, must be silanized to
prevent adsorption of opiates to active sites. The samples
may be stored for 3 months at 20 C.
Interferences
One must be aware that the EMIT assay does result in a
positive test for codeine and dextromethorphan,
compounds present in nonprescription cold
remedies.[24,25] Cross reactivities for EMIT and
ELISA procedures are presented in Opiates Table:
Comparison of commonly used assay methods.
Consumption of foods that contain poppy seeds can
result in excretion of significant quantities of morphine
and codeine in urine. Detection of 6-acetylmorphine in
urine can help rule out poppy seed ingestion as a cause
of increased opiate excretion.
Opiates Therapeutic Range
The therapeutic blood levels for the opiates have not
been established. Since heroin has not been approved by
the FDA for clinical use, its therapeutic range has not
been investigated. Although hydromorphone is approved
for clinical use, its therapeutic range and basic
pharmacokinetic parameters have not yet been
established. The average peak plasma levels for
hydromorphone after a 2 mg intravenous or 4 mg oral
dose have been reported to be 31.6 ng/mL (5 min after
the dose; n = 6) and 22 ng/mL (1 h after the dose; n = 6)
respectively.[22] Morphine is the only opiate in which a
speculative therapeutic serum level has been reported. A
serum concentration of 50 ng/mL is considered the
minimum concentration of free morphine needed to
provide moderate analgesia.[26]
For purposes of screening for drugs of abuse, the two
most common cutoff concentrations for opiates are 300
ng/mL and 2000 ng/mL. SAMHSA raised its cutoff from
300 to 2000 ng/mL in an effort to eliminate opiate
(morphine) positive results from poppy-seed-containing
food items. Unfortunately, at the 2000 ng/mL cut-off
concentration, routine use of other opiate drugs such as
hydrocodone or oxycodone may not be detected.
Interpretation
Opiates are used as sedatives (premedicants), anesthetics
(narcotic nitrous oxidebalanced anesthetic technique),
and analgesics (for postoperative and chronic pain).
Rapid determinations for opiates have recently been
gaining more importance because of the advent of
narcotic antagonists (such as naloxone and nalorphine
hydrochloride), which allow quick reversal of the opioid
action. The undesirable side effects (such as nausea,
convulsions, respiratory depression, and coma) can be
quickly reversed by these antagonists.
Opiates have been demonstrated to affect levels of
pituitary hormones, though not to a clinically important
extent.[27] Because of the effects of morphine on
gastrointestinal motility, this drug has been used for
904
several diseases of the gastrointestinal tract such as
diarrheal disorders and colitis.
Opiate Performance Goals
Acceptable performance criteria (CLIA-88) for
qualitative measurement of opiates are based on 80%
participant consensus
.References
1 Brunk SF, Della M. Morphine metabolism in
man. Clin Pharmacol Ther 1974;16:51-7.
2 Mule SJ. Routine identification of drugs of
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fluorometry, thin-layer and gas-liquid
chromatography. J Chromatogr 1971;55:255-
66.
3 Van Vunakis H, Wasserman E, Levine L.
Specificities of antibodies to morphine. J
Pharmacol Exp Ther 1972;180:514-21.
4 Bastos ML, Jukofsky D, Mule SJ. Routine
identification of drugs in human urine. III.
Differential elution of the XAD-2 resin. J
Chromatogr 1973;81:93-8.
5 Kupferberg H, Burkhalter A, Way EL. A
sensitive fluorometric assay for morphine in
plasma and brain. J Pharmacol Exp Ther
1964;145:247-51,.
6 Sansur M, Baccafurim A, Morgenstern S.
Automated fluorometric method for the
determination of morphine in urine.J Assoc Off
Anal Chem 1972;55:880-7
7 Yeh SY, McQuinn RL. GLC determination of
heroin and its metabolites in human urine. J
Pharm Sci 1975;64:1237-9.
8 Nicolau G, Vanhear G., Kaul B, Davidow B.
Determination of morphine by electron capture
gas-liquid chromatography. Clin Chem
1977;23:1640-3.
9 Findlay JA, Butz RF, Jones EC. Relationship
between immunogen structure and antisera
specificity in the narcotic alkaloid series. Clin
Chem 1981;27:1524-37.
10 Ebbighausen WO, Mowat J, Vestergaard P,
Kline NS: Stable isotope method for the assay
of codeine and morphine by gas
chromatography-mass spectrometry:
feasibility study. Adv Biochem
Psychopharmacol 1973;7:135-46.
11 Van der Slooten E.P, Van der Helm HJ.
Comparison of the EMIT (enzyme multiplied
immunoassay technique) opiate assay and a
gas-chromatographic-mass-spectrometric
determination of morphine and codeine in urine.
Clin Chem 1976;22:1100-11.
12 Wasels R, Bellvill F. Gas chromatographic-
mass spectrometric procedures used for the
identification and determination of morphine,
codeine and 6-monoacetylmorphine.
Chromatogr 1994;A:674:225.
13 Wu CY, Wittick JJ. Separation of five major
alkaloids in gum opium and quantitation of
morphine, codeine and thebaine by isocratic
Opiates
reverse phase high performance liquid
chromatography. Anal Chem 1977;49:359-63.
14 Jane I, Taylor JF. Characterization and
quantitation of morphine in urine using high
pressure liquid chromatography with
fluorescence detection. J Chromatogr
1975;109:37-42.
15 Pacifici R, Pichini S, Altieri I, Caionna A, Passa
AR, Zuccaro P. High-performance liquid
chromatographicelectroscopy mass
spectrometric determination of morphine and its
3- and 6-glucuronides: application to
pharmacokinetic studies. J Chromatogr
1995;B:664:326,.
16 Fehn J, Megges G. Detection of 06-mono-
acetylmorphine in urine by GC/MS as evidence
for heroin use. J Anal Toxicol 1985;9:134.
17 Bermejo AM, Ramos I, Fernandez P, Lopez-
Rivadulla M, Cruz ., Chiarotti M, Fucci N,
Marsilli R. Morphine determination by gas
chromatography/mass spectroscopy in human
vitreous humor and comparison with
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1992;16:372.
18 Vanzetti G, Casani M, Vatente D. Detection of
morphine in urine by hemagglutination
inhibition with use of lyophilized reagents. Clin
Chem 1983;29:1376-9.
19 Rubenstein KE, Schneider RS, Ultman EF.
Homogeneous enzyme immunoassay: a new
immunochemical technique. Biochem Biophys
Res Commun 1972;47:846-51.
20 Honigberg TL, Stewart JT, Brown WJ, et al.:
Radioimmunoassay of hydromorphone. J Anal
Toxicol 1977;1:70.
21 Honigberg TL, Stewart JT, Jackson, C.
Radioimmunoassay procedure for
hydromorphone and hydrocodone in human
plasma. J Pharm Sci 1980;69:1171-3.
22 Rowley GL, Rubenstein KE, Huisjen J, Ullman
EF. Mechanism by which antibodies inhibit
hapten-malate dehydrogenase conjugates, an
enzyme immunoassay for morphine. J Biol
Chem 1975;250:3759-66.
23 Kerrigan S, Phillips WH. Comparison of
a ELISAs for opiates, methamphetamine, cocaine
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and cannaboids in whole blood and urine Clin
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24 Mule SJ, Bastos ML, Jukofsky D. Evaluation of
immunoassay methods for the detection of
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25 Mule SJ, Whitlock E, Jukofsky D.
Radioimmunoassay of drugs subject to abuse:
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26 Berkowitz BA, Ngai SH, Yang JC, Hempstead
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905

Opiates

27 Schick R, Schusdziarra V. Physiological, of exogenous and endogenous opiates. Clin

pathophysiological and pharmacological aspects Physiol Bioche. 1985;3:43-60.

Tables
Methods for Opiate Analysis
Method 1: Thin-layer chromatography (TLC)
Principle: Extract opiate; chromatograph; observe Rf factor (relative mobility) and color reaction presumptive
for opiate
Usage: Blood, plasma, serum, urine, tissue
Comments: Qualitative for high concentrations; can separate mixtures
Method 2: Fluorescence spectroscopy
Principle: Extract opiate; oxidize with K3Fe(CN)6; fluorescence measured
Usage: Blood, plasma, serum, urine
Comments: Nonspecific for opiates; qualitative
Method 3: Gas chromatography (GC)
Principle: Extract opiate; derivatize (such as trimethylsilylimidazole); chromatograph; retention time identifies
opiate
Usage: Blood, plasma, serum, urine, tissue, saliva, hair, tissues, vitreous humor
Comments: Qualitative and quantitative; resolves mixtures; only a few drugs are known to interfere
Method 4: Gas chromatography-mass spectrometry (GC/MS)
Principle: Steps same as in 3; mass spectrometer identifies opiates and helps resolve complex mixtures
Usage: Blood, plasma, serum, urine, tissue, saliva, hair tissues, vitreous humor
Comments: Qualitative and quantitative; better identification of opiates than with GC (method 3)
Method 5: High-performance liquid chromatography (HPLC)
Principle: Extract opiates; may or may not be derivatized (derivitization as in 2); retention time identifies opiate
Usage: Blood, plasma, serum, urine, tissue, saliva
Comments: Qualitative and quantitative; resolves mixtures; as efficient as GC but run time may be longer
Method 6: High-performance liquid chromatographymass spectrometry (HPLC/MS)
Principle: Steps as in Method 5; mass spectrometer ideitifies opiates and helps resolve complex mixtures
Usage: Blood, plasma, serum, urine
Comments: Qualitative and quantitative; resolves mixtures; run time may be longer than for GC/MS;
identification of opiates
Method 7: Hemagglutination inhibition (HI)
Principle: Opiate bound to fixed red blood cells competes with opiate bound in sample for reaction with
agglutinating antibodies; inhibition of agglutination indicates presence of opiates
Usage: Urine, serum, plasma
Comments: Very sensitive but nonspecific; used qualitatively; may be used quantitatively if opiate is known; no
extraction necessary
Method 8: Radioimmunoassay (RIA)
Principle: Radiolabeled opiate competes with opiate in sample for reaction with limited amount of antibody;
competitive binding assay
Usage: Urine, serum, plasma, tissue extract, saliva, breast milk
Comments: High sensitivity; qualitative and quantitative; may distinguish between glucuronide conjugate and
unconjugated opiate; no extraction necessary
Method 9: Free radical-labeled assay technique (FRAT)
Principle: Uses stable free-radical label (nitroxide) in competitive binding assay
Usage: Urine, serum, plasma, saliva
Comments: Qualitative and quantitative; commercially available
Method 10: Enzyme-multiplied immunoassay technique (EMIT)
Principle: Uses enzyme label in competitive binding assay
Usage: Urine
Comments: Qualitative; commonly used technique; no need to separate bound from unbound labeled drug
Method 11: Enzyme-linked immunosorbent assay (ELISA)
Principle: Drug in sample and drugenzyme conjugate compete for solid phase antibody
Usage: Serum, plasma, urine
Comments: Qualitative; frequently used; no need to separate bound from unbound labeled drug
906

Opiates

Comparison of Commonly Used Assay Methods for Preparation of Various Assay Tubes for
Serum Opiates Radioimmunoassay Procedure (Amounts are in
GC microliters)
Sample size (mL of plasma): 1 Method 1: Nonspecific binding (NSB)
Sensitivity: Electron capture: 0.1 ng/mL Blank serum: 100
Coefficient of variation: 2% Working buffer (WB): 400
Assay time: 1.5 to 2 h Labeled drug working solutions (LDWS):
reference: [8] 100
GC/MS Working antiserum solution (WAS):
Sample size (mL of plasma): 1 Standards:
Sensitivity: 0.5 ng/mL Sample:
Coefficient of variation: 5% Dextran-coated charcoal (DCC): 100
Assay time: 1.5 to 2 h Method 2: 100% binding
reference: [11] Blank serum: 100
HPLC Working buffer (WB): 300
Sample size (mL of plasma): 1 Labeled drug working solutions (LDWS):
Sensitivity: Fluorometric: 4 ng/mL 100
Coefficient of variation: 6% Working antiserum solution (WAS): 100
Assay time: 1.5 to 2 h Standards:
reference: [14] Sample:
HPLC/MS Dextran-coated charcoal (DCC): 100
Sample size (mL of plasma): 1 Method 3: Standards
Sensitivity: 10 ng/mL Blank serum:
Coefficient of variation: 6.9% Working buffer (WB): 300
Assay time: 1.5 to 2 h Labeled drug working solutions (LDWS):
reference: [14] 100
EMIT Working antiserum solution (WAS): 100
Sample size (mL of plasma): 0.05 Standards: 100
Sensitivity: 100 ng/mL Sample:
Coefficient of variation: 7% Dextran-coated charcoal (DCC): 100
Cross-reactivity: Other opiates, 3-O- Method 4: Test samples
glucuronide conjugate, dextromethorphan Blank serum:
Assay time: 20 min Working buffer (WB): 300
reference: [11] Labeled drug working solutions (LDWS):
ELISA 100
Sample size (mL of plasma): 0.010 Working antiserum solution (WAS): 100
Sensitivity: 100 ng/mL Standards:
Coefficient of variation: 5% (blood) and 8% Sample: 100
(urine) Dextran-coated charcoal (DCC): 100
Cross-reactivity: Other opiates
Assay time: 1 h
reference: [23]
RIA
Sample size (mL of plasma): 0.2
Sensitivity: 2.5 ng/mL
Coefficient of variation: 9%
Cross-reactivity: Other opiates,
dextromethorphan
Assay time: 2 h
reference: [21]
Hemagglutation assay
Sample size (mL of plasma): 0.1 (urine)
Sensitivity: 200 ng/mL
Coefficient of variation: Qualitative
Cross-reactivity: Other opiates, 3-O-
glucuronide conjugate, dextromethorphan
Assay time: 1 h
reference: [18]
907

Opiates

5. Working buffer (WB). 0.05 M phosphate

buffer, pH 7.4 with 0.1% gelatin and 0.6% NaCl. Place
Figures 1.0 g of gelatin in a 400 mL Erlenmeyer flask with 200
mL of distilled water. Heat the flask with stirring until
Opiates: Figure 1 the gelatin is completely dissolved. Allow the solution to
cool, and transfer to a 1 L volumetric flask. Add 1.31 g
of NaH2PO4, 10.87 g of Na2HPO4, 6.00 g of NaCl, and
700 mL of distilled water. Mix to dissolve salts, and
adjust pH to 7.4 with H3PO4. Bring volume to mark
with distilled water, and mix. Stable 1 week at 4 C.
6. Dextran-coated charcoal (DCC). Suspend 2.5
g of charcoal in 100 mL of distilled water. Add 0.25 g of
dextran to 100 mL of suspended charcoal. Mix overnight
Reaction for formation of pseudomorphine. (From at 4 C. Centrifuge the solution at 2000 g for 15 min.
Kupferberg, H., et al.: J. Pharmacol. Exp. Ther. 145:247- Remove the supernatant and suspend the dextran-coated
251, 1964.) charcoal in 400 mL of working buffer.
7. [7,8-3H(N)]-dihydromorphine. Stable at 20
C in the dark under N2 for 1 year. Check purity by TLC
Opiates: Figure 2 every 3 months according to manufacturers
recommended method.
8. [N-Methyl-3H]-morphine. (NEN) NET-653
(10 to 20 Ci/mmol). Prepare as described above for
dihydromorphine.
9. Diacetyl [I-3H(N)]-morphine (heroin).
10. Labeled-drug working solution (LDWS).
Dilute 20 L of dihydromorphine or heroin in 20 mL of
working buffer (0.25 Ci/mL). Dilute 16.7 L of
morphine in 20 mL of working buffer (0.25 Ci/mL).
11. Morphine, hydromorphone, and heroin.
Obtain pure salt form from manufacturer. Stable 1 year
if kept dry and under inert atmosphere (N2) at room
temperature.
12. Calibration standards. Dissolve 11 mg of
opiate in 50 mL of distilled water. This is solution A.
Dilute 100 L of solution A to 10 mL with drug-free
serum or plasma (solution 1). Dilute 50 L of solution A
to 100 mL with serum or plasma (solution 2). Store
Typical radioimmunoassay calibration curve for solutions 1 and 2 at 20 C in 1 mL aliquots. Stable for
hydromorphinone over concentration range of 2.5 to 25 about 3 months. Prepare the following solutions fresh
ng/mL. each day for a calibration curve:

Volume of Volume of Final standard

Solution solution working buffer concentration
Procedure: Radioimmunoassay no. (L) (L) (ng/mL)
The opiates contained in the sample are incubated with 2 250 9750 2.5
radiolabeled morphine, antibody, and phosphate buffer 1 25 9975 5.0
at room temperature. The unbound morphine is removed 1 75 9925 15.0
1 100 9900 20.0
by the addition of dextran-coated charcoal. An aliquot of 1 125 9875
the supernatant is then removed and placed in a 25.0
scintillation vial with scintillation fluid and counted by
use of a scintillation spectrometer. The percentage of 13. Antiserum. Obtain lyophilized antiserum to
labeled opiate bound to antibody is inversely morphine directed toward the 3-hydroxy and nitrogen
proportional to the logarithm of the drug concentration. moieties between positions C-9 and C-16. The
lyophilized serum is stable 1 year at 20C. Cross-
Reagents reactivity with hydromorphone, 78%; hydrocodone,
1. Gelatin, calf skin. 60 Bloom, Type IV. 59%; heroin, 100%; monoacetylmorphine; 100%; and
2. Charcoal. Stable indefinitely. morphine-3-glucuronide, 4.8%. Each vial contains
3. Dextran T 70. enough antiserum for 200 tubes.
4. Scintillation fluid. Cocktail designed for 14. Working antiserum solution (WAS).
aqueous samples (able to absorb 0.7 mL of water into 10 Reconstitute according to manufacturers instructions.
mL of cocktail). Dilute 0.6 mL of antiserum to 6 mL with working buffer
(60 samples). Stable for 1 week at 4 C.
908
Opiates
Assay
Equipment: Liquid scintillation counter, a
refrigerated centrifuge capable of generating 2000 g or
greater, 20 mL glass scintillation vials, and a liquid
scintillation counting cocktail designed for aqueous
samples (able to absorb 0.7 mL of water into 10 mL of
cocktail).
1. To 10 75 mm labeled plastic or glass
disposable tubes, add 0.1 mL of the appropriate
standard or sample. See Opiates Table:
Preparation of various assay tubes for
radioimmunoassay procedure for addition of
sample and reagents to tubes.
2. To prepare a nonspecific binding (NSB) or
blank tube, add 0.1 mL of blank plasma, 0.1 mL
of labeled drug solution, and 0.4 mL of working
buffer. To prepare a 100% binding (100%B)
tube, or zero-dose tube, add 0.1 mL of blank
plasma, 0.1 mL of labeled drug solution, 0.1
mL of working antiserum solution, and 0.3 mL
of working buffer.
3. To each standard and sample tube, add 0.1 mL
of labeled drug working solution (55,550 cpm),
0.1 mL of working antiserum solution, and 0.3
mL of working buffer. Incubate all tubes at
room temperature for 50 min and at 4 C for 10
min.
4. Add 0.1 mL of 4 C dextran-coated charcoal
suspension to each tube. Incubate at 4 C for 10
min.
5. Centrifuge at 2000 g at 4 C for 20 min. Place
0.2 mL of supernatant into 20 mL scintillation
vial. To each scintillation vial, add 0.5 mL of
distilled water and 10 mL of counting cocktail.
Cap, and mix vials. Count for 10 min in
scintillation counter optimized for tritium
counting.
Calculations
Calculate percent binding of standards and
samples by the following equation:
Percent binding = Counts for sample - NSB counts
100
Counts for 100% binding - NSB
Plot log standard percent binding versus log
concentration. Fit data using linear regression analysis
(Opiates: Figure 2). From slope (a) and intercept (b),
calculate concentration (X, in ng/mL) by the following
equation:
log X = (log y - b)/a or X = 10(log y - b)/a
For hydromorphone, as an example, the effective assay
range is 2.5 to 20 ng/mL. The percentage of radiolabeled
drug bound to the antiserum in the absence of unlabeled
drug is 30.0% + 5.4% (B0). The 50% displacement point
(B/B0)50 occurs at 7.2 ng/mL.
Procedure: Opiates, Urine: Confirmation by SIM-
MSD (GC/MS)
Principle
Opiates are first hydrolyzed and then are
isolated from urine by liquidliquid extraction,
followed by acetylation derivatization and
analysis in the SIM mode by GC/MS to
generate qualitative findings.
Specimen of Choice
Urine (5 mL); spot aliquot. May be transported
at room temperature. Store at 28 C prior to
testing.
Indications
To confirm a presumptively positive "Opiates"
immunoassay screen test result for the presence
of codeine, morphine, and/or hydromorphone.
Reagents and Materials:
A. Toxi-A Extraction Tubes (Toxi-Lab, Inc.)
B. 16 100 mm (10 mL) glass tubes
C. 5 ml volumetric glass pipet
D. 100 L Eppendorf pipet
E. Glass Pasteur pipets
F. 5 mL glass "Reacti-vials" (Pierce Co.)
G. Ethyl acetate (Chromatoquality)
H. Glusulase (Sigma). Store at 4 C. Stable
as per stated shelf life.
I. Sodium acetate (1.1 M), pH 5.2
Dissolve 37.38 g sodium acetate in 5.5
mL of glacial acetic acid and dilute to 250
mL with H2O. Store at room
temperature. Stable for 1 year.
J. Pyridine (Chromatoquality)
K. Acetic anhydride (Chromatoquality)
L. Urine Positive Control
(1) Reconstitute two lyophilized vials
with 25 mL of deionized water each.
(2) Allow to stand 30 min, then transfer
contents of both vials to a 500 mL
volumetric flask, rinsing the vials
several times with deionized water
and transferring washes to flask as
well.
(3) Dilute to mark with deionized
water.
(4) Aliquot 5 mL fractions into plastic
tubes and store at -20 C. Stable
for 1 year.
M. Negative Control
A urine pool previously testing below 10
ng/mL by FPIA for opiates and negative
for opiates by GC/MS. 5 mL aliquots
stored at -20 C. Stable for 1 year.
N. Stock Internal Standard (100 mg/mL)
Deuterated D3 morphine. Store at 28 C.
Stable as per stated shelf life.
909
Opiates
O. Working Internal Standard (10 ng/l). To
a 12 75 glass tube, add 300 mL of stock
internal standard and dilute with 2.7 mL
of deionized water. Store at 4 C. Stable
1 week.
P. Calibration Standard (DASC Calibrator).
"GC-MS Calibration Standard."
Instrument and Parameters
A. Hewlett-Packard 5972/5970 MSD GCMS
and Chem Station.
B. Heating block
60 C
C. Centrifuge
Procedure
With each run include a calibration standard
and positive and negative controls, all
processed in singlecate. Process as with subject
specimens, including hydrolysis.
A. To a 10 mL glass test tube, add 5 mL
urine, 1 mL of 1.1 M sodium acetate
buffer, and 100 L of Glusulase.
B. Incubate at 60 C for 5 h.
C. To a 10 mL glass tube, add 5 mL of
hydrolysate and 100 L of working
internal standard. Vortex mix.
D. Transfer the hydrolysate/internal standard
mix to the fill line of a Toxi-A tube.
E. Extract for 5 min. Centrifuge for 2 min.
Using a disposable glass Pasteur pipet,
transfer the top layer to a Reacti-Vial.
F. Evaporate to dryness at 60 C under
nitrogen.
G. Add 20 L of pyridine and 20 L of
acetic anhydride. Vortex, cap, and heat at
60 C for 20 min.
H. Uncap and evaporate to dryness.
I. Recap the vials.
J. Reconstitute the sample in 50 L of ethyl
acetate (immediately before injecting)
vortex and inject 1 L into method
"OPIATES."
NOTE: The following injection sequence
should be utilized:
Calibration standard
Positive control
Negative control
Patient/subject
Calculations and Quality Control
NOTE: If using 5972 MSD refer to Addendum
III (Sequencing).
A. Calibration
1. From Top Level, choose method.
2. Press Load and Run Method.
3. Choose the Method of Interest.
4. Update the data file name, i.e.,
c:\HPCHEM\1\DATA\1118\POSCOC.
5. Press Run Control.
6. Inject calibrator.
7. At the end of the run, an
uncalibrated report will print. To
calibrate, go to DATA ANALYSIS,
Main Panel, and load the calibration
data file specified above.
8. Choose Calibrate and Update
Levels.
9. Choose Recalibrate Level 1 Update
Levels Update Qualifying ions
update Response factors.
10. Press OK, Exit.
11. To print the report, press Quantitate
and Calculate Report.
12. Make sure retention times,
calibration values and ion ratios
have been updated. If not, update
the levels again.
13. Subsequent injections, i.e., controls
and patient material, will print
calibrated results.
B. Changing Retention Time Windows
1. Choose calibrate within the Data
analysis header.
2. Choose Edit Compounts
3. Highlight the compound to be
updated and choose "View."
4. Update the retention time and OK
the selection.
5. Save the changes to the compound
and Exit.
6. Recalibrate, if necessary.
7. Reprint the report.
C. Manual Calculation
1. List retention times of drug of
interest for both control and
patient/subject sample.
2. Calculate ion ratios for drugs of
interest using the following:
Codeine
Ratio in % =Individual 229, 282, or 341 ion area 100%
Sum of 229 + 282 + 341 ion areas
Hydromorphone
Ratio in % =Individual 228, 285 or 327 ion area 100%
Sum of 228, 285, and 327 ion areas
Morphine
Ratio in % = Individual 310, 327 or 369 ion area 100%
Sum of 310, 327 and 369 ion areas
910

Opiates

3. Calculate the concentration of the searched. The sample may

drug of interest using the following: then be considered negative
Codeine concentration in ng/mL = if these actions prove so.
Ratio of 327 ion area/330 ion area for Control or patient sample 500 Retention times may
Ratio of 327 ion area/330 ion area for calibration standard shift from day to
day, or even from
Hydromorphone Concentration in ng/ml = run to run.
Ratio of 327 ion area/330 ion area for Control or Patient sample 500
Interpretation and Notes
Ratio of 327 ion area/330 ion area for calibration standard

Morphine A. A positive finding indicates drug usage,

Same as for hydromorphone using but cannot be used to interpret
morphine areas. impairment. All positive results are noted
4. Compare control and as "positive," with no quantitative ng/mL
patient/subject's retention time, ion report.
ratios, and concentration to that of B. Sensitivity300 ng/mL; negatives
the calibration standard. A positive reported as "none detected."
sample will include: C. SpecificityNo known interfering
A retention time match 1% of standard compounds.
Three ion ratios matching within 20% D. Recovery80%.
of standard
Addendum I: GC-MS Calibration Standard
A calculated concentration equal to or
above the threshold cutoff of 300 ng/mL.
1. Prepare the following stock standards (a) through
D. Result Acceptance/Rejection
(d).
Acceptance:
NOTE: Prepare fresh for each new lot.
Retention times match within 1%.
(a) Phencyclidine (PCP)
Results in ng/mL units must exceed documented cutoff
To a 10 mL volumetric flask add 11.4 mg of
levels of 300 ng/mL.
PCPHCl. Dissolve and dilute to mark with
Ion ratios must match within 20%. methanol.
If multiple drug peaks are being analyzed the retention
(b) 9-Carboxy-11-nor--9-THC (cannabinoid
times must be unique for each of
metabolite) (THC).
the different drugs. Example:
Make sure that the target software (c) Benzoylecgonine (BE)
has properly assigned different To a 10 mL volumetric flask, add 12.5 mg of
retention times to the different benzoylecgonine tetrahydrate. Dissolve and
"opiates". Codeine, dilute to mark with methanol.
Hydromorphone, and Morphine (d) Into a 10 mL volumetric flask add:
must have unique retention times on
the calibration run. Drug Amounts (mg)
Rejection and Corrective Action: Amphetamine sulfate 13.7
Results below documented cutoffs Methamphetamine HCl 12.3
which meet all other acceptance Desmethyldiazepam 10
criteria, should be reported as
Temazepam 10
negative or unconfirmed.
Codeine 10
When ion ratios do not match the
range stated on the report, several Morphine sulfate 11.8
options are available: Amobarbital 10
(a) Reinject the patient/subject Butabital 10
sample. Secobarbital 10
(b) Reinject the calibration Pentobarbital 10
standard in the cal mode and Phenobarbital 10
reassess the patient ion Phentermine HCl 12.35
ratios.
Hydromorphone HCl 11.2
(c) Calculate the ion ratios for
the calibrator and the patient
Dilute to the mark with methanol.
in question manually.
(d) If these steps do not rectify
the problem, the sample
may be injected into a scan
program and library
911

Opiates

2. Pool Spiking (b) Transfer aliquots into 16 100 polyethylene

(a) To a 2 L volumetric flask, add 200 mL of tubes
negative urine pool. (c) Cap and freeze at -20C in racks marked
(b) To the flask add: with the respective lot number
200 L (0.2 mL) of standard 1(a)
2000 L (2.0 mL) of standard 1(b)* ADDENDUM III Sequencing
600 L (0.6 mL) of standard 1(c)
1000 L l (1.0 mL) of standard 1(d) 1. Click on the "Sequence" header and choose
*For standard 1(b), THC, transfer the "Load."
contents from two commercial vials, rinsing 2. Choose the proper sequence for the mode of
each vial three times with deionized water, operation required, i.e., choose the THC
and transferring the washings to the flask. sequence for running THC assays.
(c) Mix flask with constant Note: The sequence tables are simply
swirling while diluting to mark with deionized water. templates and can be modified as you
(d) Transfer contents of flask to see fit. Remember that multiple
wide-mouth flask for aliquoting. methods can be run within a
sequence. (If THC sequence is
3. Target concentrations
chosen, you may run any method
THC, DRUGS1, COCAINE, or any
Drug Concentration (ng/mL) combination.)
3. Choose "Edit Sample Log Table." The Sample
Amobarbital 500 Log Table appears.
Butalbital 500 4. Fill out the Sample Log Table. Use one line per
Pentobarbital 500 injection. The injections will be made
Secobarbital 500 sequentially in the order they are listed in the
table. To set up multiple injections from one vial,
Phenobarbital 500
make a separate line in the Sample Log Table for
THC metabolite 100 each injection .
BE metabolite 300 5. For each injection, select the sample type
Morphine 500 (Sample, Blank, or Calibration)
Codeine 500 Note: When a calibration is specified the
Hydromorphone 500 calibration LEVEL identifier must
Phencyclidine 100 be specified. For our purpose type
Amphetamine 500 in a 1. Choose REPLACE for Upd
Methamphetamine 500 RF, Upd RT, and Upd Ql.
Phentermine 500 6. Enter the position of the vial in the autosampler
tray.
4. Lot testing and verification 7. Assign a data file name (sample number or
description).
(a) Assign sequential lot number to the pool.
8. Type in the name of the method to use to analyze
(b) Submit pool for testing/verification prior to
the sample or type a question mark (?) in the
use, including:
field to display a list of available methods.
TDx FPIA analysis for each of the
9. Type in an identifier for the sample injection.
individual drugs (PCP, THC, BE,
(Optional)
amphetamines, opiates,
benzodiazepines, and barbiturates) 10. Once the Sample Log Table is complete, it may
be printed by choosing "Print Brief Format"
EMIT analyses for the individual
within SEQUENCE.
drugs
11. Save the Sample Log Table.
Specific GC-MS quantitative
analyses run in parallel with current 12. Choose "Load and Run Sequence."
lot of DASC calibrator a. Click on "Full Method" under Method
(c) Acceptable analyses include: Sections to Run.
TDx FPIA positive results for all drug classes b. Update the Data File Directory to reflect
the date of the run, i.e.,
EMIT positive results for all drug classes
C:\hpchem\1\data\0715\ .
GCMS quantitation within 10% of target value
c. Click on "Run Sequence" to start the run.
Acceptable calculated GC-MS control data
5. Aliquoting and storage
(a) Aliquot approximately two-thirds of the total
pool in 5 mL fractions and one third of the
pool in 10 mL fractions
912
Organic Acid Screening
Organic Acid Screening
Kevin Carpenter i
Clinical Interpretation Chapter 52, Diseases of Genetic Origin, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Principles of Analysis and Current Usage
Organic acid analysis has become a key component in
the diagnosis of inherited metabolic disease. The term
organic acids covers a wide range of low-molecular-
weight carboxylic acids, including those derived from
intermediary metabolism of amino acids, carbohydrates,
lipids, and biogenic amines. Although methods such as
capillary electrophoresis [1] and tandem mass
spectrometry [2] are used, the commonest methods in
use are based on gas chromatography [3].
The principle steps of organic acid analysis are (1)
isolation of the organic acids from physiological fluids,
(2) formation of volatile derivatives, (3)chromatographic
separation using capillary gas chromatography, and (4)
detection of the compounds, now most commonly
achieved using benchtop mass selective detectors (mass
spectrometers).
Before extraction, urine samples may be treated with an
oximating agent such as ethoxylamine hydrochloride
that preserves ketoacids. Without oximation, a
significant proportion of ketoacids are converted to their
corresponding 2-hydroxyacid. In practice, organic acid
analysis with or without oximation still allows
identification of abnormal patterns and appropriate
interpretation of the findings. A recent review of
analytical methods used to screen urine organic acids by
participants in the European Research Network for the
evaluation and improvement of screening, Diagnosis,
and treatment of Inherited Metabolic disorders external
quality assessment schemes (ERNDIM-QAP) found just
over half used oximation [4].
Extraction of organic acids from urine is most
commonly performed using solvent extraction from
acidified, salt-saturated urine. It is most convenient to
dilute the urine to a fixed concentration of creatinine
prior to extraction to control for concentration of the
sample. Addition of one or more internal standards will
serve to control the extraction process and allow for a
reproducible qualitative scoring of abnormal compounds
(although true quantitation requires calibration and
ideally a stable, isotope-labeled internal standard for
each compound). Solvents used are typically ethyl
acetate, sometimes in combination with diethyl ether.
Solid phase extraction using silicic acid [5] or ion
exchange [6] columns have been used but do not retain
some of the neutral compounds which may be seen using
solvent extraction and are of diagnostic value (e.g.,
glycerol).
i
Organic acid screening
New method
Fifth edition: Kevin Carpenter
Following removal of the solvent by evaporation, the
concentrated residue containing the organic acids needs
to be derivatized to allow separation by gas
chromatography. Since the technique was first described,
the majority of workers have used trimethylsylyl (TMS)
esters, and almost all published data for retention times
and mass spectra are for TMS esters. The derivatization
is easily and rapidly performed using N,O-
bis(trimethylsilyl)trifluoroacetamide with 1% trimethyl
chlorosilane (BSTFA/1% TMCS). Care must be taken to
ensure no water remains in the extract, because this will
impair derivatization. Problems of formation of multiple
TMS esters for compounds with more than one site of
derivatization may make quantitation difficult but in
practice does not cause problems with diagnosis.
Separation of the derivatized organic acids requires
capillary chromatography with temperature
programming. Typically the oven temperature will start
in the 80C to 100C range, and temperature gradients of
4C to 6C per minute are employed to selectively elute
the compounds. The rate chosen will depend upon the
throughput requirements of the laboratory, but there is
little to be gained from very slow gradients, especially
when mass selective detectors can distinguish co-eluting
peaks. The column used will typically be 30 meters in
length with an internal diameter of 0.2 to 0.5 mm.
Stationary phases are a matter of personal choice, and
retention characteristics are published for many
compounds of interest on OV1, OV5, and OV17 (or
equivalent) columns. To allow for variations in
temperature programming, column length, and carrier-
gas flow rates, retention data is quoted compared to
alkanes of different chain lengths run under the same
conditions. For example, a compound eluting midway
between the C14 and C16 alkanes would have a
retention index of 15, and this would be fixed for any
given stationary phase.
Detection and data acquisition is best performed using a
mass selective detector (mass spectrometer). Although
flame ionization detection can be used, mass
spectrometry allows positive identification of the peaks,
identification of co-eluting compounds, and the ability to
detect very small amounts of pathognomonic compounds
hidden in the baseline. Data acquisition is not started
until the solvent peak has returned to baseline, after
which the mass spectrometer is operated in full scan
mode over a mass range of 50-500m/z, with a scan cycle
time of around 0.5 seconds, allowing reliable peak
913
Organic Acid Screening
reconstruction in Total Ion Current mode and the ability
to search for key compounds by using extracted ion
searches. Comparison of mass spectra obtained with
library entries, in conjunction with retention time, is
used to identify peaks. Several commercial libraries are
available for purchase, but most centers measuring
urinary organic acids build their own in-house libraries
based upon experience and availability of samples from
patients with organic acidurias. Most modern software
systems allow the construction of a quantitation
database which allows the software to identify and label
peaks based on their retention time and the presence of a
number of ions in the appropriate proportions.
Reference and Preferred Methods
There is no formal reference method for organic acid
screening.
The American College of Medical Genetics publishes
Standards and Guidelines for Clinical Genetics
Laboratories [7], the 2006 edition of which states the
requirements for organic acid analysis under the
following headings:
Extraction:
Internal standards added to sample of fixed
creatinine concentration.
Solvent extraction.
Oximation performed either routinely or under
specific circumstances.
Derivatization:
TMS esters formed using BSTFA/TCMS.
GC/MS Analysis:
Capillary column separation
Detection by quadrupole or ion trap mass
spectrometer.
Computer library of mass spectra for confirmation
of peak identities.
Calibration:
Reporting of quantitative data without the
availability of a reference standard should be
reduced to a minimum.
Isotope-dilution, selected ion mass spectrometry
should be used for more accurate analyte
quantification.
Chromatogram Analysis:
Identification of peaks relies on mass spectra.
Quality Control:
Tuning of mass spectrometer should be checked on
daily basis.
The internal standards in each specimen serve as a
QC check for each specimen.
Reporting:
Diagnostic specificity of acute versus asymptomatic
conditions.
Identification of all relevant organic acids listed.
Abnormal results require a detailed interpretation,
including significance, differential diagnosis,
recommendations for additional biochemical testing,
and in vitro confirmatory studies.
Specimen
Organic acids are well cleared by the kidney, and urine
is the near-universal sample of choice for analysis.
Samples should be stored and transported at or below
20C. Under such conditions, organic acids are stable
for many years. The volume required will be dependent
on the concentration of the urine (see procedure below).
An important precursor to the analytical process is
obtaining a specimen most likely to be informative for
diagnosis of inherited metabolic disease. Many
conditions may present in an episodic manner, and
diagnostic metabolites may only be detectable during
clinical crises. For this reason, samples should always be
sought during an acute exacerbation of the condition.
The absence of pathological findings in samples
collected at times other than during acute exacerbations
cannot be taken to exclude these conditions.
Interferences
Since the organic acid method is designed to identify a
broad range of excreted compounds, many exogenous
substances (or their metabolites) will also be detected.
This includes many drugs and food additives, but in
practice, the technique requires experienced
interpretation, part of which is the identification of such
compounds and knowledge of their origin. A small
number of urine samples appear to contain unknown
substances that inhibit effective extraction of some
compounds. Careful monitoring of internal standard
responses should allow identification of potentially poor
extraction, and a request for a repeat sample should be
made.
Interpretation
The potential number of disorders detectable by organic
acid analysis stands at over 30. Interpretation of the
profile obtained is a highly skilled job requiring
experience of what to expect in normal and disease
states. For this reason, organic acid analysis is usually
performed in specialist centers where the required
experience is concentrated. An excellent source of
information on interpretation of findings has been
published by Kumps et al. [8].
Organic Acid Performance Goals
The primary performance goal of organic acid analysis is
the correct identification of metabolites and
interpretation of their significance. For the majority of
disorders, the abnormalities are gross, and identification
is not a problem. However, several key marker
metabolites are excreted in trace amounts, and it is
useful to know the sensitivity of the method on a day-to-
day basis. Regular analysis of a spiked urine with
addition of hexanoylglycine, octanedioylglycine, 4-
hydroxybutyrate, and orotic acid at levels typical of mild
disease (5, 10, 100, and 15 mol/mmol creatinine,
respectively) will ensure adequate sensitivity is being
914
Organic Acid Screening
achieved and flag when column change or MS ion
source cleaning is required.
Appropriate interpretation of the profiles obtained is
critical and as mentioned previously requires experience
and skill normally only available in specialist centers.
Participation in an external quality assurance program
which incorporates interpretation (e.g., ERDIM-QAP;
http:// www.erndimqa.nl is an essential component of
ensuring performance goals are met.
References
1 Garcia A, Barbas C, Aguilar R, Castro M.
Capillary electrophoresis for rapid profiling of
organic acidurias. Clin Chem 1998; 44: 1905-
1911.
2 Pitt JJ, Eggington M, Kahler SG.
Comprehensive screening of urine samples for
inborn errors of metabolism by electrospray
tandem mass spectrometry. Clin Chem 2002;
48: 1970-1980.
3 Tanaka K, West-Dull A, Hine DG, Lynn TB,
Lowe T. Gas-chromatographic method of
analysis for urinary organic acids. II.
Description of the procedure, and its application
to diagnosis of patients with organic acidurias.
Clin Chem 1980; 26: 1847-1853.
4 Bonham JR. Personal Communication. 2007-
07-17.
5 Hoffmann G, Aramaki S, Blum-Hoffmann E,
Nyhan WL, Sweetman L. Quantitative analysis
for organic acids in biological samples: batch
isolation followed by gas chromatographic-
mass spectrometric analysis. Clin Chem 1989;
35:5 87-595.
6 Thompson JA, Markey SP. Quantitative
metabolic profiling of urinary organic acids by
gas chromatography-mass spectrometry:
comparison of isolation methods. Anal Chem
1975; 47: 1313-1321.
7 American College of Medical Genetics.
Standards and Guidelines for Clinical Genetics
Laboratories 2006 Edition. Internet page 2006
[cited 2007 Aug 9];Available from: URL:
http://www.acmg.net/Pages/ACMG_Activities/s
tds-2002/f.htm
8 Kumps A, Duez P, Mardens Y. Metabolic,
Nutritional, Iatrogenic, and Artifactual Sources
of Urinary Organic Acids: A Comprehensive
Table. Clin Chem 2002; 48: 708-717.
Procedure: Gas Chromatography/Mass
Spectrometry
Principle
Using ethyl acetate, organic acids are extracted from
acidified, salt-saturated urine which has been diluted to a
fixed creatinine concentration. The solvent is removed
by evaporation and the sample derivatized to form
trimethylsylyl esters. Derivatized organic acids are
separated by capillary gas chromatography and detected
by mass selective detector.
Reagents
1. BSTFA/1% TMCS (N,O-
bis(trimethylsilyl)trifluoroacetamide with 1%
trimethyl chlorosilane)
2. Heptadecanoic acid 20 mmol/L in ethanol
(internal standard). Weigh out 0.54g of
heptadecanoic acid, and dissolve in 100 mL of
ethanol.
3. 1,3 15N orotic acid 1 mmol/L (internal
standard). Weigh out 15.8 mg of 1,315N orotic
acid, and dissolve in 500 L 1M NaOH.
Transfer to a 100 mL volumetric flask with
multiple washings and make up to volume with
deionized water.
4. Hydrochloric acid 6M.
5. Sodium chloride. Analar
6. Ethyl acetate. Analar.
Assay
Equipment:
A suitable GC-MS system capable of
operation in electron impact ionization;
full scan mode is required. The gas
chromatograph is fitted with a 5%
phenylmethyl-silicone capillary column,
25 mm 0.20 mm, 0.33 m film
thickness.
Glass tubes, 16 100 mm with PTFE
lined screw cap.
Fortuna Optifix dispenser containing ethyl
acetate and set at 2 mL.
Pasteur capillary pipettes, glass,
disposable.
Supply of dry air.
Heating block set at +60C.
Pearce Reacti-therm heating block with
Reacti-vap head set to +37C.
Centrifuge.
Vortex mixer.
Estimate urine creatinine concentration, and dilute urine
to creatinine of 1.0 mmol/L with deionized water. For
samples with initial creatinine less than 1.0 mmol/L,
dilute to 0.5 mmol/L and use 2 mL of sample and 4 mL
of ethyl acetate to extract (see below).
1. In a screw-capped glass tube containing
approximately 0.5 g of sodium chloride, add 10
L of each of the two internal standards,
heptadecanoate and 1,3 15N orotic acid, and 1
mL of diluted urine.
2. Acidify with 50 L of 6M HCl and add 2 mL of
ethyl acetate.
3. The tubes are vortexed for 2 min and
centrifuged for 5 min at 2000 g.
4. Transfer the upper solvent layer to a second
tube, being careful not to carry any aqueous
phase across.
5. The solvent is evaporated to dryness under a
stream of air at 37C.
6. The residue is reconstituted with 50 L of
BSTFA/TCMS, and the tubes incubated at 60C
for 30 min.
915
Organic Acid Screening
7. GC/MS parameters:
The mass selective detector is used in
the electron impact ionization mode
at 70 eV.
Helium is utilized as a carrier gas.
The injection port temperature 240C.
Initial column temperature 80C, held
for 1 minute then ramped at 6C/min
to 250C, ramped at 20C/min to
270C, with a final hold of 2.5 min.
Interface temperature 280C.
Split injection ratio of 1:15 and
injection volume 2 L.
Full scan data is obtained and peaks identified on the
basis of retention time and mass spectra and qualitatively
scored by comparison of total ion response to the
internal standards. Key marker compounds such as
acylglycines, 4-hydroxybutyrate, orotate, and 3-
hydroxyglutarate, which may be critical in small
quantities, may be sought using extracted ion
chromatograms over narrow time windows looking for
the presence of unique ions present in the mass spectra
of the compound of interest.
916
Osmolality
Osmolality
Goce Dimeski
Name: Osmolality
Clinical significance: Refer to Chapter 30, Renal Function, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
i
Principles of Analysis and Current Usage
Osmolality is a colligative property of solutions that
depends on the number of dissolved particles present in
the solution. The three types of solutes most often
encountered in biological fluids are electrolytes, organic
molecules, and colloids. In healthy persons, the principle
contributors to the measured osmolality are Na+, Cl, and
bicarbonate. In persons with diabetes or renal failure,
glucose and urea may also contribute significantly to the
measured osmolality. As the number of dissolved
particles increases, the freezing point and vapor pressure
of a solution are decreased, and the osmotic pressure and
boiling point are increased [1]. In dilute solutions, there
is a linear change in the colligative properties as the
solute concentration increases. It is important to realize
that it is not the mass concentration but the molal
concentration (that is, moles per kilogram of solvent)
that is the basis of colligative properties. For example, in
biological fluids the concentration of salts and low-
molecular-mass organic compounds (such as glucose
and urea) affect osmolality far more than albumin, which
is present in a large mass amount but because of its large
molecular mass is present at low molal concentrations.
The molar concentration of plasma albumin, when
present at 40 g/L, is approximately 0.58 mmol/L. In
contrast, the molar concentration of plasma NaCl, when
present at 5.8 g/L, is approximately 150 mmol/L.
Normal plasma is approximately 93% water, with the
remaining 7% being solids (proteins, lipids, etc). The
electrolytes are dissolved in the water component. Since
there are 2 osmotically active components in NaCl (Na+
and Cl), the osmotic contribution is 0.93 2, or 1.86
times the concentration of Na+ (2 or 1.86 accounting for
Na+ and Cl, with Cl being the major accompanying
anion).
The most frequently measured colligative property used
to estimate fluid osmolality is freezing-point depression.
When 1 mole of solute is added to 1 kg of water, the
freezing point is depressed by 1.858C. The laboratory
osmometer actually employs a sensitive heat thermistor
to measure the heat released by the freezing fluid and
relates it to the freezing point of the fluid.
i
Osmolality
Previous and current authors of this method:
First edition: Lawrence A. Kaplan
Methods edition: Lawrence A. Kaplan
Second edition: Lawrence A. Kaplan
Third edition: Steven C. Kazmierczak, Lawrence A.
Kaplan
Fourth edition: Lawrence A. Kaplan
Fifth edition: Goce Dimeski
An infrequently measured parameter is vapor-pressure
depression. The vapor-pressure osmometer actually
measures the dew-point depression of the vapor, that is,
the vapor in equilibrium with the solution being
measured. The more dissolved particles present
(increased osmolality), the lower the vapor pressure of
the aqueous component of the solution. An important
exception to this is found when the solute itself is a
volatile substance.
Formulae for Calculating Osmolality
A stat procedure frequently used to estimate the
osmolality of a fluid is the calculated osmolality. To
calculate serum osmolality, one sums up the molar
concentrations of those substances which contribute
most to the measured osmolalitynamely, sodium,
glucose, and urea. The contribution of chloride and
bicarbonate is handled by multiplying the sodium
concentration by a variable factor as discussed below
[2].
Although there are many equations used to calculate
osmolality, the simplest and most frequently used is:
Eq. 1 [3]
Calculated mOsm/L H2O = 2 [Na+] + glucose (mmol/L)
+ urea (mmol/L)
or
Calculated mOsm/L H2O = 2 [Na+] +
glucose (mg/dL) + BUN (mg/dL
18 2.8
where sodium concentration is expressed in
milliequivalents per liter (mEq/L) or mmol/L, and the
concentrations of glucose and urea or blood urea
nitrogen (BUN) are divided by their respective formula
equivalent weights, 180 and 28, to convert these mass
concentrations into millimoles per liter (mmol/L) [4].
This formula is proposed to balance simplicity and ease
of calculation with the goal of obtaining results that
closely compare to a measured osmolality. Other more
accurate formulas that have been suggested include the
following two:
Eq. 2 [5]
Calculated mOsm/L H2O = 1.86 [Na+] + glucose
(mmol/L) + urea (mmol/L) + 9
or
Calculated mOsm/L H2O = 1.86 [Na+] +
glucose (mg/dL) + BUN (mg/dL) + 9
18 2.8
917
Osmolality
Eq. 3 [6]
Calculated mOsm/L H2O =
1.86 [Na+] + 1.38 [K+] + glucose (mmol/L) + urea
(mmol/L) + 7.45
or
Calculated mOsm/L H2O =
1.89 [Na+] + 1.38 [K+] + 1.08[glucose (mg/dL)] +
18
1.03[BUN (mg/dL)] + 7.45
2.8
Rasouli and Kalantari [7] have suggested formula 3 for
computer calculations on automated analyzers. There are
many other formulas suggested for calculation of
osmolality [2,6,8,9,10,11]. It is important to note that
although osmolality should be expressed as milliosmoles
per kilogram (mOsm/kg) of water, it is most frequently
calculated and reported as milliosmoles per liter
(mOsm/L) of water. In dilute solution like plasma at
normal temperature and pressure, the osmolality and
osmolarity are effectively the same. The slight error
introduced due to the difference between these two
expressions has little clinical significance [5]. The 9 or
7.45 mOsm/kg is added to represent the contribution of
other osmotically active substances in plasma such as
K+, Ca2+, Mg2+, and their unmeasured anions and
proteins.
The calculation of urine osmolality is less accurate than
plasma [12]. The commonest formula is Urine
osmolality = 2 (Na+ + K+) + Urea (mmol/L). Other
formulae may include in the calculation the glucose that
may be present in some urine samples, namely from
diabetic patients. The presence of glucose, ammonium,
or foreign substances such as mannitol will result in
underestimation of the calculated urine osmolality [13].
Fecal osmolality is infrequently requested in
investigations of chronic idiopathic diarrhea. Calculated
fecal osmolality should be used in preference to
measured osmolality, which increases after defecation
due to continued bacterial fermentation [14]. The Na+
and K+ are measured in the fecal water, and their sum is
doubled to account for anions: Calculated fecal
osmolality = 2 (Na+ + K+).
A 2007 College of American Pathologists (CAP) quality
assurance survey report indicated that the vast majority
(97.9%) of laboratories used a freezing-point depression
osmometer. It should be pointed out that nearly all
automated analyzers now determine a calculated serum
osmolality, or it can be calculated by laboratory
information systems. Thus the most frequently reported
serum osmolality is a calculated serum osmolality.
Reference and Preferred Methods
Although there is no reference method for osmometry,
the freezing-point depression method is considered the
de-facto reference method.
It has been pointed out that the two types, vapor-pressure
and freezing-point osmometers, differ in their ability to
respond to volatile solutes that may be clinically
encountered [15,16]. The contribution to serum
osmolality of several commonly seen alcohols (e.g.,
ethanol, methanol, and isopropanol) is directly
proportional to their molar concentrations when
osmolality is measured by freezing-point depression
osmometry. However, vapor-pressure osmometers do
not detect these volatile substances, because volatile
solutes contribute to the total vapor pressure present
above the solution, though they decrease the vapor
pressure of the water portion of the solution [17].
Therefore, the decreased vapor pressure of water is
counterbalanced by the increase in vapor pressure caused
by the presence of a volatile substance. As the
concentration of the volatile substance increases in
aqueous solutions, the vapor pressure above the solution
actually increases, giving falsely low osmolality results.
Apart from the problem encountered with volatile
solutes, the osmolality of most specimens measured by
freezing-point depression correlates well with the
osmolality measured by vapor-pressure depression. In
practice, vapor-pressure osmometers are seldom used.
Because of its greater sturdiness and precision, most
laboratories use the freezing-point osmometer.
The calculated osmolality is not meant to provide a
definitive result but only one sufficient for most acute
emergency service work. Used as such, the calculated
osmolality can provide clinicians with valuable
information with no extra analysis time. The error
introduced by calculation of the serum osmolality as
mOsm/L instead of mOsm/kg can be clinically
significant only if the percentage of water in the sample
(approximately 93%) changes significantly. Thus highly
lipemic samples or samples with large amounts of
protein (as from multiple-myeloma patients) can have an
inaccurate calculated osmolality. The laboratory might
not wish to report a calculated osmolality on such
specimens or alternatively might affix a comment to the
results warning the physician of a possible error. The
calculated osmolality will also give falsely low results in
the presence of volatile solutes. Additionally, the
inclusion of K+, Ca2+, or Mg2+ concentrations in
formulas has not resulted in a more accurate calculated
osmolality.
Specimen
Blood drawn without the use of any anticoagulant is the
preferred sample. The serum should be removed from
the clotted blood cells as rapidly as possible. Well-
centrifuged urine or other body fluids are also acceptable
samples. Random urine samples can vary considerably
in the concentration of analytes and are rarely useful
clinically. Therefore an aliquot of a 24-h urine collection
is the preferred specimen for the measurement of urine
osmolality.
Any fluid left uncovered will have an increased
osmolality because of evaporation and concentration of
sample solutes. Serum osmolality has been reported [18]
to be stable for 3 days at room temperature, 14 days at
7C, 14 days at 21C, and as long as 56 days at
918
Osmolality
78C. Urine osmolality may be stable at 4C for up to
24 h [19]. Urine samples should be centrifuged to
remove particulate matter.
Fecal specimens requested for osmolality estimation are
useful in diagnosis of watery diarrhea. Samples require
centrifugation, and the supernatant should be used for
the Na+ and K+ measurement. Dilution of solid fecal
samples in water is not recommended [14].
Interferences
As previously mentioned above, volatile substances such
as alcohols cause a significant bias in osmolality
measured using vapor-pressure depression osmometers.
Use of citrate as an anticoagulant has been found to
significantly increase measured osmolality [20].
Hemolysis, even when present in gross amounts, has
been found not to interfere with osmolality measured by
use of freezing-point depression [21].
Any particulate matter, microclots in serum/plasma,
particulate matter in urine, feces, or other fluid types are
capable of altering the freezing or vaporizing process,
thus leading to inaccuracies. Therefore, all samples
should be free of such particulate matter and may require
centrifugation prior to analysis.
Osmolality Reference Interval
The reference interval for serum osmolality in a healthy
person, as measured using a freezing-point depression
osmometer, is 280 to 295 mOsm/kg [22]. The
approximate contribution from osmotically active
substances is as follows: Na+ 140; Cl 105; HCO3 27;
K+ 4; Glucose 5; Urea 5; Ca2+ 2; Mg2+ 1; HPO42 1;
protein 1 and organic compounds ~5. Individuals living
at high altitude (i.e., > 3800 m) have been found to have
significantly lower serum osmolality compared to
individuals living at sea level [23].
Urine osmolality can range from 50 to 1000 mOsm/kg
[13,24].
Interpretation
Osmolality measurements are most frequently used to
help determine whether a patient is in a hyperosmolal
state. Such states can be seen in renal failure
(hyperuremia), diabetes (hyperglycemia), and disease
states in which there is water loss in excess of solute
loss. In the latter clinical states, the degree of
hyperosmolality parallels the degree of hypernatremia.
Hyperosmolality due to urea is far less dangerous than
when due to glucose. Urea freely diffuses across cell
membranes, resulting in concentration equilibrium.
Glucose does not diffuse across cell membranes and
causes water movement out of cells. Hyperosmolality
due to hyperglycemia is often associated with acute
illness, shock, and renal failure, including deficit in body
potassium.
The use of the osmolal gap (the difference between
calculated and measured osmolality) to screen for the
presence of exogenous substances can be very useful in
an emergency room situation. It must be pointed out that
the osmolal gap may depend less on the equation
employed than on the population tested. For example, in
patients with increased unmeasured solutes, such as
patients in circulatory shock, renal failure, in very low
birth weight neonates, hyperosmolal nonketotic coma,
alcoholic ketoacidosis, diabetic acidosis (due to increase
in ketone concentration), or in patients with multi-organ
failure (due to increased cellular permeability with
leakage of amino acids and other cellular components),
there will be a larger difference between calculated and
measured osmolalities [11]. An osmolal gap > 10
mOsm/kg has been applied clinically as indicating the
presence of other osmotically active compounds, but
because this is dependent on the laboratory equipment
and the equation chosen, institutions should calculate
their own reference range for the osmolal gap.
The osmolal gap has been suggested as a rapid test for
the presence of ingested volatiles, especially alcohols
[6,11,25,26,27,28]. Alcohols have a significant effect on
the serum osmolality because of their low molecular
weights. There is a very good correlation between the
alcohol concentration measured enzymatically and that
determined by calculation [29]. The most common cause
of coexisting hyperosmolality and coma is ethanol
ingestion [29]. If an increased osmolal gap is present, a
screen for blood ethanol should be performed
immediately. If the screen is negative, a screen for other
toxic substances (e.g., ethanol, methanol, isopropanol,
acetone, ethylene glycol) that can increase serum
osmolality should be run. Additionally, if indirect ISE
methods are used for measuring Na+ and Cl in samples
with hyperlipidemia or hyperproteinemia, Na+ and Cl
results will be decreased (pseudohyponatremia and
pseudohypochloremia); the reverse occurs with
hypoproteinemia [30,31], which will translate as an
inaccurate osmolal gap. Low serum osmolality can be
seen in cases of overhydration, hyponatremia, and cases
of inappropriate secretion of antidiuretic hormone. The
osmolal gap should be used with caution and must be
used in conjunction with clinical information.
Urine osmolality varies in response to plasma osmolality
and sodium regulation. The appropriate physiological
response is that urine osmolality should be high when
plasma osmolality is high and vice versa. Urine
osmolality measurements are performed as a test of the
kidneys concentration ability. After fluid restriction for
12 to 14 h, a patient with normal renal function should
be able to concentrate urine to approximately 800
mOsm/kg. Loss of this function is seen early in renal
failure, and a concentration ability of only 400 mOsm/kg
would indicate severe renal dysfunction [32,33].
Urine osmolal gap has been proposed to serve as a
surrogate for urine ammonium in cases of
hyperchloremic acidosis. The urine osmolal gap formula
suggested and used by Kirschbaum et al. (Osmolal gap=
0.5 x( urine osmolality 2 x[Na+ + K+] + Urea
[mmol/L]) obtained the following correlation against
measured ammonium: y = 2.17x + 10, and r2 of 0.6. Any
gap between the measured and calculated largely
919
Osmolality

represents ammonium. In summary, the derivation of the
urine osmolal gap is very cumbersome in that many
analytes are required to be measured, yet it is far easier
on modern analyzers to directly measure urinary
ammonium.
The calculated fecal osmolalities subtracted from the
measured plasma osmolality (or if plasma osmolality is
not measured, use 290 mOsm/kg for approximation) is
used to work out the osmotic gap. A value of < 50
mOsm/kg is proposed as a suitable cut off to
differentiate osmotic from secretory diarrhea. In osmotic
diarrhea, the osmotic gap is > 50 mOsm/kg, and this may
be due to ingestion of magnesium (e.g., magnesium-
containing laxative abuse) or malabsorbed
carbohydrates. In secretory diarrhea, the value is <
50mOsm/kg, and this is associated with colonic
stimulant laxatives, hormonal causes (e.g., tumors
producing vasoactive intestinal peptide), and some
gastrointestinal infections (e.g., cholera) [14].
Osmolality Performance Goals
Based on intra- and interindividual variation of serum
osmolality, as measured by freezing depression in
healthy adults, the coefficient of variation was found to
be less than 1% [33]. However, review of the 2007 CAP
data shows serum osmolality, as measured by freezing-
point depression osmometers, has an interlaboratory
coefficient of variation of 1.1% to 1.5% for osmolality
within the normal reference interval. Vapor-pressure
osmometers have worse precision at all levels of
osmolality2.3% to 2.8% coefficient of variation within
the normal reference interval. It is clear that osmolality
measurements by either method do not meet the goal of
achieving the desirable CV of < 1%.
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Procedure: Osmolality by Freezing-Point Depression
Principle
The sample or standard is supercooled below its freezing
point and then agitated by a rapidly vibrating probe. This
induces rapid crystallization of the sample, and it begins
to freeze. Heat of fusion is released by the crystallization
and is accurately measured by a thermistor probe present
in the solution. The released heat is related to the
freezing-point depression (1 mOsm of solute depresses
the freezing point by 0.001858C), which is converted to
osmolality.
Reagent
Osmolality standards can be purchased from instrument
manufacturers or other commercial sources. Calibrators
are used to standardize the instrument for either serum or
urine osmolalities.
A relatively simple procedure for preparing osmolality
standards has been described [34]. Reagent-grade NaCl
is heated to 200C overnight to drive off any water
present in the crystals. After it cools, the desired amount
of NaCl is weighed out (see below) and added to 1 kg of
class I distilled water. The most convenient way to do
this is to fill a 1-L class A volumetric flask with water at
20C to the mark. Add exactly 1.8 mL of additional
water, and then add the salt to dissolve.
Desired osmolality Grams of NaCl per kilograms
(mOsm/kg) of H2O
100 3.094
300 9.476
500 15.93
1000 32.12
The standard should be stored in clean polyethylene or
borosilicate glass bottles. Always pour the standards into
clean test tubes before use. When stored properly in
closed containers, the standards will be stable for 3 to 6
months at room temperature.
921
Oximetry
Oximetry
Goce Dimeski i
Name: Oximetry, hemoximetry, CO-oximetry
Clinical significance: Refer to Chapter 40, Hemoglobin; Chapter 29, Acid-Base Control and Acid-
Base Disorders; and Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry: Theory,
Analysis, Correlation.
Principles of Analysis and Current Usage
Oximeters (or hemoximeters) are specialized
spectrophotometers used to determine the oxygen
saturation and hence the amount of oxygen bound to
hemoglobin. Depending on the type of oximeter, the
analytes that can be measured are total hemoglobin,
hemoglobin fractions or derivatives, and the oxygen
saturation. The hemoglobin fractions are of clinical
interest in assessment of oxygenation. The different
hemoglobin fractions absorb light at different and
specific wavelengths in the visible region (520 to 622
nm, Table 1) and thus permit use of spectrophotometry
to determine their concentrations. CO-oximetry is a term
derived from the technical ability to measure
simultaneously the concentration of the hemoglobin
fractions: carboxyhemoglobin (COHb, FCOHb),
oxyhemoglobin (O2Hb, FO2Hb), deoxyhemoglobin
(HHb, FHHb), methemoglobin (metHb or Hi, FMetHb),
and sulfhemoglobin (SHb). In turn, the sum of all
fractions is the total hemoglobin concentration (ctHb).
The oxygen saturation (sO2, SaO2) is determined from
the O2Hb and HHb. The term CO-oximeter originates
from the first commercially available oximeter, the IL
282 CO-oximeter produced by Instrumentation
Laboratory (IL) in 1979.
The Hb fractions are divided into functional fractions
(O2Hb, HHb), which are capable of reversibly binding
O2, and dysfunctional fractions (COHb, MetHb, SHb),
which are incapable of reversibly combining with O2
under physiological conditions. Increased concentrations
of dysfunction hemoglobins result in a decreased
capacity to carry oxygen and deliver it to the tissues,
increasing the likelihood of cellular hypoxia.
Measurement of the Hb fractions is important because
they may be direct contributors to the clinical picture.
This section presents methods for determination of the
hemoglobin derivatives: O2Hb, HHb, COHb and CO,
metHb, SHb, and oxygen saturation (sO2) in blood.
Hemoglobin
Hemoglobin is a tetrameric protein, and the usual form
in adults comprises two and two chains and is called
hemoglobin A (Hb A). Hemoglobin in the fetus and
newborn is mainly fetal Hb (Hb F) (50% to 100 %) [1],
and it has a slightly different absorbance spectrum to Hb
A (Figure 2). After birth, Hb A gradually replaces Hb F,
and at around 3 months of age, the Hb F will usually be
< 10% of total hemoglobin. The oxygen dissociationi
curve with Hb F is shifted to the left when compared to
HbA.
Oxyhemoglobin and Deoxyhemoglobin
Oxyhemoglobin is the main Hb fraction, composing
94% to 98% of total Hb in arterial blood. It is the
oxygenated fraction of hemoglobin, and fully saturated it
can bind four oxygen molecules (1 gm of fully saturated
Hb (O2Hb) binds 1.39 mL of O2).
Hemoglobin without oxygen is referred to as
deoxyhemoglobin. Other names that have been used are
deoxyHb, desaturated Hb or reduced Hb.
Deoxyhemoglobins main role is as a buffer for
hydrogen ions. The functional fractions (O2Hb and HHb)
are also used to determine the oxygen saturation (sO2).
Carboxyhemoglobin (Carbon Monoxide)
Carboxyhemoglobin is formed when carbon monoxide
(CO) binds to hemoglobin. CO gas can easily displace
O2 from Hb, owing to its greater (~220 times greater)
affinity for Hb. CO binds even more tightly to Hb F,
making infants particularly vulnerable to its effects [2].
There are two sources of CO: exogenous and
endogenous. The exogenous sources include tobacco
smoke, malfunctioning furnaces and gas dryers,
combustion of motor fuels (68%), burning buildings, and
industrial processes (12%). The endogenous (human)
source is a by-product of heme catabolism, resulting in a
background carboxyhemoglobin saturation of 0.4 to 0.7
% in healthy subjects [3].
Persons who have inspired carbon monoxide in chronic
or acute doses usually are seen as medical emergencies.
Thus a means to measure the extent of carbon monoxide
exposure rapidly, either through measurement of carbon
monoxide concentration in blood or through
measurement of the fraction of hemoglobin that is
converted to carboxyhemoglobin, is needed.
These measurements help the clinician ascertain (1)
whether COHb is present in clinically significant or
toxic amounts and (2) what treatment modality can be
used to treat the patient effectively. CO or COHb can
lead to tissue hypoxia, acidosis, or effects on the central
i
Oximetry
New method:
Fifth edition: Goce Dimeski
922
Oximetry
nervous system but does not produce cyanosis.
Treatment of the CO-poisoned patient begins with
supplemental oxygen for the hypoxia and to accelerate
elimination of CO from the body. The half-life of COHb
is 5 to 6 hours [4].
Methemoglobin
MetHb is hemoglobin that has the ferrous ion (Fe2+)
oxidized to the ferric (Fe3+) state, hence it is unable to
bind oxygen. MetHb is formed by either hereditary or
acquired means. Some metHb forms naturally in RBCs
and can amount to 1% to 2% of total Hb [5]. In normal
persons, the Fe3+ is reconverted to Fe2+ by the action of
NADH metHb (cytochrome b5) reductase. The
hereditary form is due to mutation leading to NADH
metHb reductase deficiency, or to amino acid
substitution in the alpha or beta globin chain, resulting in
hemoglobin M (Hb M). Life expectancy with hereditary
metHb is significantly shortened in most individuals.
The acquired methemoglobinemia has a fairly acute
onset and is a result of exposure to agents such as
nitrites, nitrates, sulfonamides, aniline dyes, acetanilid,
phenacetin, local anesthetics (benzocaines, lignocaine,
etc), dapsone etc [4,5]. The other acquired form is after
prolonged exposure of blood to air, with the O2Hb being
oxidized to form metHb.
Persons who develop methemoglobinemia classically
present with hypoxia and cyanosis. The measurement of
metHb is important for prompt medical intervention
either by removing the causative agent or commencing
treatment with metHb reducing agents, namely
methylene blue or, less effectively, with ascorbic acid.
Patients with cytochrome b5 reductase deficiency
respond to methylene blue therapy but not those with Hb
M disease. The additional therapies for such patients
may include blood transfusions and hemodialysis [6].
These patients do not respond well to oxygen therapy.
The half-life of metHb is ~ 55 minutes [4].
Sulfhemoglobin
Sulfhemoglobin is formed by the linkage of sulfur to
hemoglobin. Sulfhemoglobin appears in some
individuals rather than metHb after exposure to
sulfonamides, phenacetin, acetanilid, and trinitrotoluene.
Other drugs or compounds that have been implicated in
causing elevated SHb are dapsone, metoclopramide, and
H2S gas [7]. The sulfur binding to hemoglobin is stable
and irreversible. Because sulfhemoglobin stays in the
circulation until the red blood cells complete their life
cycle; it causes significant and prolonged hypoxia and
cyanosis. There is no known treatment for elevated SHb
levels other than discontinuation of the implicated agent.
Transfusion may be necessary in severe cases.
Oximeters
Oximeters belong to one of the two major classes: pulse
oximeters or CO-oximeters.
1. Pulse Oximeters
The first pulse oximeter was developed in 1974 by a
Japanese firm and was based on the work of Takuo
Aoyagi [8]. The spectrum of light transmitted through
the skin is picked up to determine the sO2. Pulse
oximeters are capable of providing a noninvasive
estimate of tissue oxygen saturation via the measurement
of the differential absorption at two different light
wavelengths (660 and 940 nm) by oxygenated and
deoxygenated hemoglobin [9,10,11]. Deoxyhemoglobin
absorbs more energy at 660 nm, whereas O2Hb absorbs
more energy at 940 nm. Pulse oximeter probes consist of
a photodetector and two light-emitting diodes. One diode
emits at 660 nm (red band of the spectrum) and the
second at 940 nm (infrared band of the spectrum). The
photodiodes are continuously switched on and off
several hundred times per second to record the
absorption of O2Hb and HHb during pulsatile and
nonpulsatile flow.
It is the ratio of the absorbance at the two wavelengths
from which the oxygen saturation is determined. These
instruments are calibrated from data on healthy
individuals who had simultaneous CO-oximetry
measurement of arterial oxygen saturation ranging from
75% to 100%. Pulse oximeters are not reliable when the
saturation is < 75% [9]. A ratio of absorbance (660/940
nm) of 0.43 corresponds to 100% oxygen saturation, and
a ratio of 3.4 corresponds to 0% oxygen saturation. In
the absence of dysfunctional hemoglobins, a ratio of 1.0
corresponds to an oxygen saturation of 85%. Pulse
oximeters do not measure the dysfunctional hemoglobins
(COHb and MetHb). Elevated levels of dysfunctional
hemoglobins will result in false-normal sO2 [10]. Pulse
oximeters are not affected by the difference in the
absorption spectra of Hb A and Hb F [12]. A major
technological advance in pulse oximetry is the new pulse
oximeter, Masimo Rad-57 (Masimo Inc), capable of
measuring MetHb and COHb using 8 wavelengths [11].
2. CO-oximeters
Laboratories that measure hemoglobin fractions perform
this as a stat procedure using benchtop blood-gas
analyzers equipped with CO-oximeter modules. CO-
oximeters are divided into discrete wavelengths or
continuous spectrum, which may be with or without
(slide technology) hemolysis.
CO-oximeters offer major advantages in that they
require an extremely short time of analysis (~1 min) and
little or no sample preparation, measure the total Hb and
the fractions simultaneously, and require minimal
technical skills.
a. Discrete Wavelengths CO-oximeters
The earliest CO-oximeter, IL 282, utilizes an aspirated
arterial whole-blood sample (125 L) to analyze the
various fractions of hemoglobin. It consists of a
hemolyzer unit (e.g., ultrasound device) to hemolyze the
cells, a lamp (e.g., Tl-Ne hollow-cathode lamp) to
provide the light source, a lens system to focus the light
onto four interference filters to provide monochromatic
light of specific wavelengths (535.0, 585.2, 594.5, and
626.6 nm), a photodetector to detect the amount of light
absorbed by the different Hb fractions, and a readout
device. The sum of the absorbances of the fractions is
used to compute the total hemoglobin, and the fractions
923
Oximetry
are expressed as a percentage of total hemoglobin.
Calculations are performed automatically by a built-in
microprocessor from absorbance measurements and
molar absorptivity constants. The concentrations of each
fraction are summated to yield a total hemoglobin
concentration. The proportions of each hemoglobin
speciesO2Hb, metHb, and COHbare displayed.
Calibration of the output of the instrument is
accomplished daily using a dye solution. SHb, measured
in the Evelyn and Molloy method [13], can be detected
but not quantitated in the IL 282 CO-oximeter. When
SHb is present, the instrument will display a positive
result for metHb and a negative result for COHb.
Presence of Hb F and SHb may interfere with
quantitative measurements. A dye (amaranth) is used as
a calibrator.
The next phase in the development of the discrete
wavelength CO-oximeters was in the mid-1980s, with
the major change being an increase in the number of
wavelengths (6 or 7 wavelengths in general; but some,
such as the AVL 912, used as many as 17 wavelengths),
including a correction for lipid interference (wavelength
reading at ~ 670nm), and some even included correction
for bilirubin interference (wavelength reading ~ 488
nm). Although the Hb fractions have specific
wavelength peaks, the wavelength selection between the
different analyzers is variable. Even though wavelengths
used are provided, manufacturers do not explain in detail
the exact mathematical calculations involved in deriving
the results [14]. CO-oximeters are either (1) hemolyzing
types, with samples being hemolyzed either
ultrasonically (e.g., AVL Omni 6, CC270, IL682,
OSM3) or chemically (e.g., IL482), or (2) non-
hemolyzing types (e.g., AVOXimeters 1000 and 4000,
IL Synthesis 35) to make measurement on unhemolyzed
blood [15].
Early CO-oximeters lacked accuracy at low
concentrations (<5%) for COHb when compared to a GC
method and also differed in the degree of interference
from Hb F, temperature, bilirubin, SHb, and medical
dyes (e.g., methylene blue with absorbance peak at 550
to 570 nm) [16]. The other feature differing among the
CO-oximeters was that not all were capable of providing
quantitated SHb. Some only showed its presence when it
was above certain levels such as > 1.5% (e.g.,
Radiometer ABL 700).
b. Continuous Spectrum CO-oximeters
The most recent CO-oximeters measure a continuous
spectrum on a sample. Multiple readings are obtained, a
spectrum is constructed, and it is compared to a model
or typical data set. By pattern recognition, it can
compensate for any interference from turbidity,
therapeutic dyes, Hb F, or other factors. The usual
spectral range used is between 450 and 700 nm. The
available CO-oximeters are either hemolyzing or non-
hemolyzing types. Examples of systems using sample
hemolysis are the Radiometer 700 & 800 series
analyzers, which measure 128 different readings
between 478 and 672 nm to construct the continuous
spectrum [17]. The GEM (IL) 4000 analyzer takes 1400
readings from 450 to 700 nm to construct its continuous
spectrum [18]. An example of the non-hemolyzing type
is the Siemens (Bayer) Rapidlab 1265 analyzer, which
utilizes a slide cell technique. It measures the
absorbance readings at 47 wavelengths on whole blood
to construct its continuous spectrum. The sample
chamber has a sliding cell design that opens and closes
(narrowing the pathway to create a narrower sample
path) to allow for sample absorbance measurement. The
sample from the slide cell continues to be used for
gases and electrolyte measurements [19].
Oxygen Saturation (sO2)
The sO2 provides an immediate estimate of whether
additional oxygen can be carried by Hb. Oxygen
saturation can be accurately calculated by the use of
Equation 1 [20], directly determined from the measured
O2Hb and HHb fractions (Equation 1), estimated
(Equations 2,3,4), or determined by pulse oximetry.
Equation 1:
It is often referred to as the fractional sO2 (SaO2) because
it is determined from the two hemoglobin fractions. This
is the most accurate method for sO2 estimation, since it
is a direct measure of the parameters that affect sO2. CO-
oximetry is considered the gold standard for oxygen
saturation measurement.
Estimated oxygen saturation does not account for
variations in 2,3 DPG levels, nor the presence of
dysfunctional hemoglobins (COHb, MetHb). These
equations assume no presence of dysfunctional
hemoglobins. If any of these conditions change, the
estimated sO2 will be inaccurate. Oxygen saturation can
be calculated from the measured parameters PO2 and pH,
on the basis of standard oxygen-dissociation curves [21].
Equation 2: sO2 = [(PO2 + 150 x PO2)-1 x 23 400] +1-1
The Severinghaus equation assumes the following
parameters are normal: pH = 7.40, PCO2 = 40 mm Hg
and 2,3-diphosphoglycerate = 5 mmol/L and no presence
of dysfunctional Hbs. If any of these conditions change,
the sO2 will be inaccurate.
Equation 3: The Radiometer ABL uses the following
Hill equation to determine oxygen saturation from the
measured pH and O2:
in which
924
Oximetry
Found in the equation is a Bohr factor of 0.48 and a
standardized P50 of 26.6 mm Hg.
Equation 4: The Siemens (formerly Bayer) analyzers use
the relationship described by Kelman [22] and Thomas
[23].
N = pO2 10[0.48(pH(37) -7.4) 0.0013BE(B)]
Pulse oximetry is a third method for sO2 estimation. The
saturation calculated from pulse oximetry is a functional
saturation, SpO2. It is the percentage of HbO2 compared
with the sum of HbO2 and HHb only. Because these
systems do not measure COHb and metHb, the SpO2 is
falsely elevated in the presence of dysfunctional
hemoglobins. In the presence of normal SpO2 with
cyanosis, CO-oximetry must be used to determine the
saturation status and presence of any dysfunctional
hemoglobin.
Reference and Preferred Methods
The spectrophotometric cyanmethemoglobin method is
the reference method for total hemoglobin estimation in
whole blood [24]. There are no stated reference methods
for hemoglobin fractions or oxygen saturation
estimation, but currently available co-oximeters, either
stand-alone or part of blood-gas analyzers, provide
acceptable methods for their estimation.
Specimen
CO-oximetry has become the method of choice for
measurement of the different hemoglobin fractions.
Samples are whole blood anticoagulated with heparin
(reduced heparin syringes contain ~ 7 IU/mL, balanced
heparin syringes contain ~ 35 or more IU/mL) or less
frequently, EDTA (1.5 mg/mL). With the ease of use
and rapid analysis of samples on blood gas analyzers
equipped with CO-oximetry or stand-alone CO-
oximeters, prolonged sample storage requirements are
diminished. Samples collected in this manner are stable
at least 5 days at ambient temperatures or at 4C [21].
Although significant loss of CO can occur during
storage, freezing of the sample can help maintain CO
concentrations [25]. Some reports suggest that samples
can be stabilized by the addition of sodium dithionite
[26]. For maintaining optimal stability, anticoagulated
blood should be sealed in vials with a minimum of air
space and stored frozen or at least at 3C prior to
analysis [27]. COHb has been found to be stable even in
postmortem blood samples stored in Vacutainer tubes for
up to 2 years at 3C with or without preservative [28].
Interferences
Spectrophotometric methods
Turbidity due to proteins, lipids, and cellular matter is a
potential problem with spectrophotometric methods.
CO-oximeters
Spuriously high COHb values may be encountered in the
presence of HBF or with increased oxygen saturation of
fetal blood when older CO-oximeter models are used
[29]. In addition, the presence of other hemoglobins such
as SHb complicates these methods.
Discrete wavelength CO-oximeters were prone to
interferences from HbF, SHb, bilirubin, and medical
dyes. For example, the discrete CO-oximeter on the
Radiometer ABL 625 was unable to measure Hb M
methemoglobin, unlike the continuous type used on the
735 [4]. The IL-482 CO-oximeter was able to detect
SHb indirectly by indicating a spuriously high metHb
value and a negative value for COHb [15,30]. The likely
reason for SHb interference is that early CO-oximeters
measured metHb absorbance at one wavelength, 630 nm.
SHb has an absorbance peak at ~ 620 nm, which
overlaps to 630 nm, which would have been reported as
metHb [10].
Significant bilirubin interference (1.2% COHb per 496
mol/L bilirubin) was found with the CCD250 CO-
oximeter [31].
Perflubron emulsion (Oxident), a non-hemoglobin,
oxygen-carrying blood substitute, was shown to interfere
with hemolyzing, discrete-type CO-oximeters but not
with non-hemolyzing types. The effects were variable,
but in general, it decreased the O2Hb, increased COHb
and MetHb, or produced turbidity errors [15].
In one such system (an earlier model Chiron [Bayer] 800
that used 10 wavelengths to calculate the various
fractions) the presence of HbF produced falsely elevated
metHb values in the clinically significant range, > 5%
[32]. This was overcome in the later version of the same
model, with the introduction of 40 wavelengths [33].
Pulse Oximeters
Pulse oximetry cannot distinguish the different types of
hemoglobins such as COHb and metHb [34]. With
increasing COHb or metHb, the reported oxygen
saturation remains > 90%. MetHb absorbance at 660 nm
is like that of HHb, and its absorbance at 940 nm is
markedly greater than that of HHb or O2Hb. As a result,
metHb will contribute to the perceived absorbances of
both HHb and O2Hb in a pulse oximeter. In fact, in the
presence of 100% metHb, the ratio is 1% or 85% oxygen
saturation. At ~ 30% metHb, the ratio reaches a plateau,
and the apparent oxygen saturation becomes stable at ~
85% [10]. COHb absorbs approximately the same
amount of 660 nm light as does O2Hb [35].
Other factors that can lead to limitations with pulse
oximeters are sensitivity to motion, decreased peripheral
perfusion, improper probe placement, bright ambient
light, tattoos, and dark nail polish [36,37,38,39]. Dark
skin has been found to cause overestimation in SpO2
during hypoxia in dark-skinned individuals [40].
925
Oximetry

Reference Intervals bind more tightly to hemoglobin than oxygen does, it

Oxyhemoglobin and Deoxyhemoglobin
The O2Hb concentration in an arterial sample is 94% to
98%, and 1% to 2% for HHb. Cyanosis develops when
the level of HHb reaches 5g/dL (~30 %) [41].
Carboxyhemoglobin (COHb)
Nonsmokers not otherwise exposed to external sources
of CO have 1% to 2% COHb because of endogenous
production. Smokers have been found to have as little as
2.1% and as much as 10% COHb, depending on
individual rates of tobacco consumption. Reference
intervals have been discussed by Allen [42] in more
detail.
also interferes with the release of the oxygen already
bound to hemoglobin, because it causes a leftward shift
of the oxyhemoglobin dissociation curve.
Chronic and acute exposures are judged safe or unsafe
by the resulting fraction of COHb that results. The
current standard for maximum exposure in the United
States allows a maximum concentration of 50 ppm over
8 hours [44]. Concentrations of 200 ppm in inhaled air
are considered dangerous, but the lower limit of a safe
exposure is difficult to determine; in some persons, a 3%
blood concentration of COHb results in impaired
alertness and psychomotor performance.

Reference Intervals for COHb in Blood [41] The concentration of COHb should be considered only a
guide to diagnosis and does not always correlate closely
Population Range Mean S.D.
with clinical findings. Correlation of blood COHb levels
(n) (%) (%) (%)
with patient outcome is weak. This is because CO
Navy hospital patients persists in tissues long after COHb levels have returned
Nonsmokers to normal.
34 0.421.50 0.85 0.26
Smokers Therapy for carbon monoxide poisoning involves
34 1.538.47 3.97 1.88 reoxygenation of the blood with oxygen therapy. The
Navy divers half-life of CO in blood in a person breathing room air is
Nonsmokers 5 hours, 20 min. With a tight-fitting oxygen mask and
17 Not given 0.88 0.21 100% O2, the half-life is reduced to 1 hour, 20 min.
Smokers
19 Not given 4.66 1.68 Methemoglobin
In normal individuals, a small amount of the hemoglobin
Levels of 10% to 20% generally cause headache and in erythrocytes is oxidized to methemoglobin, and
fatigue, whereas values from 20% to 30% are typically methemoglobin concentration remains < 2% [6]. The
associated with severe headache, nausea, vomiting, majority of cases of methemoglobinemia are acquired
dizziness, blurred vision, and fainting. Coma and and mild, and therapy is focused on removal of the
convulsions can occur when COHb levels get above causative toxin. Healthy individuals with levels up to
40%. Respiratory failure and death are typically seen 15% tend to be asymptomatic. Levels between 15% and
when levels exceed 70%. 30% lead to varying symptoms such as hypoxia and
cyanosis. Levels of 30% to 50% cause dyspnea,
Methemoglobin headache, fatigue and dizziness. Breathlessness,
Levels of 1% to 2% can occur naturally in RBCs [5]. metabolic acidosis, CNS depression, confusion, and
coma are quite likely to follow levels > 50%, whereas
Sulfhemoglobin levels > 70% are fatal [4].
Concentrations > 1% should be regarded as abnormal.
Sulfhemoglobin
Interpretation SHb is an uncommon cause of cyanosis. Unlike metHb,
Carbon monoxide is a combustible, nonirritating gas that SHb has few adverse clinical consequences and can
is odorless and tasteless. Because it is a by-product of resolve spontaneously with the physiological turnover of
incomplete combustion, it is usually produced in areas red blood cells. SHb concentration of > 5 g/L (>3.0%)
where humans can be exposed to it. When carbon can produce detectable cyanosis [45]. However, it has
monoxide is inhaled, it is readily absorbed into the blood been observed that levels as low as 2g/L have produced
and binds avidly to hemoglobin. Affinity constants for dark complexion and discolored blood [46].
the COHb complex are several hundred times greater Concentrations > 1% should be regarded as abnormal. It
(220 to 250) than those for oxyhemoglobin [12,43]. The is not clear, but it has been reported that levels of 20% to
increased affinity constant is the result of an increase in 60% may be benign in some patients [7].
the absolute affinity of CO for Hb. Thus in the following
reaction: Performance Goals
k1 A review of the 2007 blood oximetry proficiency
CO + Hb HbCO surveys of the College of American Pathologists shows
k2 similar precisions for the vast majority of analyzers
included in the survey. The O2Hb coefficients of
k1 is one tenth the k1 for the binding of O2 to Hb, but k2 variation (CVs) are higher at the low end (O2Hb ~ 41 %)
is 2400-fold less than that for the dissociation of O2 < 2.5% and decrease down to ~ 1% with values at the
from oxyhemoglobin. Not only does carbon monoxide high end (O2Hb ~ 95 %). The COHb CVs are higher at
926
Oximetry

the low end (COHB ~ 5 %), where ~ 85 % of analyzers oximeters that use overdetermined systems.
have CVs < 15 %, and with the remaining analyzers, the Clin Chem 1997;43:189-190.
CVs are as high as ~ 27%. For the higher end (COHB ~ 15 Shephard RJ. Carbon Monoxide, the Silent
37 %), the CVs for all the analyzers are < 4 %. The Killer. Springfield, IL: Charles C Thomas;
proficiency samples do not contain suitable levels of 1983:44-67.
MetHb to properly test the analyzers capability. The 16 Mahoney JJ, Vremen HJ, Stevenson DK, Van
concentration of MetHb in all the samples is < 1 %. Kessel AL. Measurement of
carboxyhemoglobin and total hemoglobin by
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6 Baraka AS, Ayoub CM, Yazbeck-Karam V, 23 Thomas LJ. Algorithm for selected blood acid-
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7 Wu C, Kenny MA. A case of sulfhemoglobin (International Committee for the
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31 Vreman HJ, Stevenson DK.
Carboxyhemoglobin determined in neonatal
blood with a CO-oximeter unaffected by fetal
hemoglobin. Clin Chem 1994;40:1522-1527.
32 Lynch PL, Bruns SE, Boyd JC, Savory J.
Chiron 800 system CO-oximeter module
overestimates methemoglobin concentration in
neonatal samples containing fetal hemoglobin.
Clin Chem 1998;44:1569-70.
33 Keijzer M H. Comment on the overestimation
of methemoglobin concentrations in neonatal
samples with the Chiron 800 system CO-
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conditions of poor perfusion. Anesthesia
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38 Hinkelbein J, Genzwuerker HV, Sogl R, Fiedler
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39 Severinhaus JW, Naifeh KH, Koh SO. Errors in
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40 Bickler PE, Feiner JR, Severinghaus JW.
Effects of skin pigmentation on pulse oximeter
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41 Grace RF. Pulse oximetry: gold standard or
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48 Van Slyke DD, Hiller A, Weisiger JR, Cruz
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normal human blood. J Biol Chem
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49 Conway EJ. Microdiffusion Analyses and
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Determination of carbon monoxide in blood by
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51 Ostrander CR, Cohen RS, Hopper AO, Cowan
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determinations of blood carboxyhemoglobin
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52 Vreman HJ, Kwang LK, Stevenson DK. Carbon
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53 Zwart A, Buursma A, van Kampen EJ,
Oeseburg B, van der Ploeg PH, Zijlstra WG. A
multi-wavelength spectrophotometric method
for the simultaneous determination of five
haemoglobin derivatives. J Clin Chem Clin
Biochem 1981;19:457-463.
54 Zwart A, Buursma A, Oeseburg B, Zijlstra WG.
Determination of hemoglobin derivatives with
the IL 282 CO-oximeter as compared with a
manual spectrophotometric five-wavelength
method. Clin Chem 1981;27:1903-1907.
55 Zijlstra WG, Buursma A, Meeuwsen-van der
Roest WP. Absorption spectra of human fetal
and adult oxyhemoglobin, de-oxyhemoglobin,
carboxyhemoglobin, and methemoglobin. Clin
Chem 1991;37:1633-8.
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saturation of hemoglobin, percent
carboxyhemoglobin and methemoglobin and
concentrations of total hemoglobin and oxygen
in blood of man, dog, and baboon. Clin Chem
1980;26:1304-1308.
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WG. Multicomponent analysis of hemoglobin
derivatives with reversed-optics
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58 Zwart A, Buursma A, van Kampen EJ, Zijlstra
WG. Results of routine determination of
clinically significant hemoglobin derivatives by
multicomponent analysis. Clin Chem
1986;32:972-8.
59 Blanchette E, Tagilaferro D, Hotaling T,
Uretsky LS. Improvements in robustness of the
Evelyn and Molloy
methemoglobin/sulfhemoglobin manual assay.
Clin Chem 2006;52(Suppl A):191.
60 Biochemical aspects of hematology. Chapter
46. In: Tietz Textbook of Clinical Chemistry.
3rd ed. Philadelphia: Saunders; 1987:1677.
928
Oximetry

61 Rodkey FL, Hill TA, Pitts LL, Robertson RF. 63 Rand RN. Practical spectrophotometric
Spectrophotometric measurement of standards. Clin Chem 1969;15:839-863.
carboxyhemoglobin and methemoglobin in 64 Frings CS, Broussard LA. Calibration and
blood. Clin Chem 1979;25:1388-1393. monitoring of spectrometers and
62 Beutler E, West C. Simplified determination of spectrophotometers. Clin Chem 1979;25:1013-
carboxyhemoglobin. Clin Chem 1984;30:871- 1017.
874.

Table 1: Absorption Peaks of Hemoglobin Fractions

Fraction Wavelength (nm)
O2Hb 542, 577
HHb 431, 556
COHb 538, 568
MetHb 500, 630
SHb 620

Table 2: Matrix of Millimolar Absorptivities

Millimolar Absorptivities Expressed in cm-1mmol-11 (10-2m2mol-1; Lineic Absorbances)

(nm) HHb O2Hb COHb metHb S Hb

500 4.09 5.05 5.35 9.04 7.20
569 11.27 11.27 14.27 4.10 8.10
577 9.40 15.37 10.00 4.10 8.10
620 1.23 0.24 0.33 3.35 20.80
760 0.43 0.13 0.03 0.24 1.04

Reproduced with permission from Zwart A et al. Determination of hemoglobin derivatives with the IL 282 CO-oximeter as
compared with a manual spectrophotometric five-wavelength method. Clin Chem 1981;27:1903.

Figure 1: Absorption spectra of Hemoglobin Fractions

Absorption spectra of reduced hemoglobin (HHb), carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb), methemoglobin
(MetHb), sulfhemoglobin (SHb), and lipid.
Reproduced with permission from Widdop B. Analysis of carbon monoxide. Ann Clin Biochem 2002;39:378-391.
929
Oximetry

Figure 2: Absorption spectra of adult and fetal hemoglobin

Light absorption spectra of the common derivatives of HbA and HbF in the visible range. Left panel: O2Hb (1) and HHb
(2). Right panel: MetHb (1) and COHb (20. The absorptivity is expressed in Lmmol-1 cm-1.
Reproduced with permission from Zijlstra WG, Buursma A, Meeuwsen-van der Roest WP. Absorption spectra of human
fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin and methemoglobin. Clin Chem 1991;37:1633-8.

Historical Methods lactic, or hydrochloric, which acts to release CO (and

1. Carbon Monoxide (CO)
a. Comparative Color Test
Individuals with CO poisoning often have blood that is
cherry red in appearance. However, lack of a cherry-red
appearance does not exclude poisoning. One
modification of this simple visual inspection test is to
compare 0.5 mL of blood diluted with 10 mL of 10
mmol/L ammonia with that of normal blood treated in
the same manner. A pink coloring indicates the presence
of COHb. However, blood from patients with cyanide
poisoning may also cause a pink coloration.
CO2) to the atmosphere inside the closed dish. In the
inner chamber of the dish is a solution of palladium
chloride. The PdCl2 is reduced by the CO to metallic
palladium as the CO diffuses through the atmosphere in
the dish and settles into the inner section of the dish.
After allowing time for CO to react as completely as
possible, an aliquot of inner fluid is removed, and the
remaining palladium chloride is titrated with I, or
measured photometrically at 278 nm. The amount of
palladium chloride remaining is inversely proportional to
the CO content of the sample. The method is most useful
when the CO concentrations are at levels of 10% or
more.

Gas chromatography can also be used to measure CO

b. Carbon Monoxide (CO) Measurements
CO is present in blood both as dissolved CO and as
COHb. Direct analysis procedures measure total CO by
releasing the CO from COHb and from solution and then
determining the amount of CO by one of several
measurement techniques: manometry, microdiffusion,
gas chromatography, or infrared analysis.
Manometric techniques use the Van Slyke apparatus
[47,48]. Other gases present, such as oxygen, nitrogen,
and carbon dioxide, are chemically removed, and then
the carbon monoxide is liberated by the addition of
acidified ferricyanide, and the volume percent of CO is
measured.
A Conway diffusion method [49] employs a dish
comprising two concentric chambers. In the outer
chamber, sample is mixed with an acid, usually sulfuric,
that has been liberated in a closed system. The column
packing frequently used is Molecular Sieve 5A, which is
used to separate and analyze many gas mixtures. In
many cases, a thermal conductivity detector is used to
monitor the column effluent. Calibration can be
accomplished by use of a gas containing a known
amount of carbon monoxide, or use of blood that has
been tonometered to contain a known percentage of
carboxyhemoglobin [50]. In the gas chromatographic
method of Collinson et al. [50] as modified by Ostrander
et al. [51] and Vreman et al. [52], blood is collected in
capillary tubes that have been coated internally with
heparin and saponin. The anticoagulated blood is mixed
with potassium ferricyanide (K3Fe[CN]6, 100 g/L) in a
vial, and hemoglobin-bound carbon monoxide is
liberated into the headspace of the vial. The vials air
space is sampled with a needle through the septum cap
and injected into the gas chromatograph. Air, the carrier
930
Oximetry
gas (50 mL/min), must be purified by passage through a
catalytic converter and a moisture trap, which uses
magnesium perchlorate prior to usage. Although the
method is very precise and accurate, it is too slow and
labor intensive to be used in emergent situations.
c. CO Reference and Preferred Methods
The modified GC method [51,52] of Collinson et al [50]
can detect as little as 0.1 nL of CO in 2 L of blood.
Reproducibility of the method at 1.80 mL of CO/L is
0.04 mL of CO/L. Linearity of the method extends to
9.36 mL of CO.
Gas chromatography can be used to measure the total
CO content in blood regardless of binding state; in
addition, gas chromatography requires only a small
sample volume. Because it measures total CO, pre-
measurement steps are simplified, and CO measurement
in other tissues is possible. The disadvantage of gas
chromatography as a routine tool for CO analysis in the
clinical chemistry laboratory is that it is a relatively
complex and slow procedure compared to
spectrophotometry. However, the GC method is highly
accurate and can serve as a reference method.
The spectrophotometric method of Zwart et al. [53,54]
compares favorably with the four-wavelength method
when SHb is not present. There are three major sources
of error in this technique. Even the introduction of a
small amount of oxygen in the detergent solution can
cause significant error in the analysis. Use of literature
values of molar absorptivities in an uncalibrated
spectrophotometer can bias the results. In addition, the
presence of other hemoglobin moieties may make the
method inaccurate.
In blood samples containing fetal hemoglobin,
spuriously high COHb values were encountered by
Zwart et al. [55] using co-oximetry techniques. These
values increased with the increased oxygen saturation of
fetal blood. However, in the absence of fetal blood, the
instrument compared well with the reference method
cited here. Zwart and co-workers [53] obtained the
regression equation for the IL-282 CO-oximeter as
follows:
% CO Hb (IL-282) = 1.03 (%CO Hb [ref.
method]) 0.05
for n = 20. The range of %Hb CO was 10 to 80. Similar
correlation was obtained with gas chromatography [56].
Precision of measurements was determined to be 0.4%,
expressed as the maximum range of differences of
triplicate measurements. Precision of the total
hemoglobin measurement of a single sample expressed
as the standard deviation was 0.8 g/L at a mean of 141.6
g/L, n = 20.
2. Hemoglobin Fractions
a. Blood Color Test
In a cyanosed patient, the differentiation of HHb from
MetHb can be done by observing the blood color change
over time. Simply put a couple of blood drops on white
filter paper and look for color change. Unlike the
brightening of the original dark red HHb color with
exposure to air, the chocolate brown color of metHb
does not change [4]. The only problem is that this does
not distinguish metHb from SHb, which if present, also
gives blood a dark brown appearance.
b. Photometric Methods
Measurements of the proportion of hemoglobin fractions
in blood are usually made photometrically, employing
various wavelengths at which the hemoglobin species in
blood absorb light. Because the absorption spectra
overlap, it is usually necessary to make two or more
measurements and then solve simultaneous equations.
Spectra of various hemoglobin species are shown in
Figure 1. Binary mixtures can be analyzed with
measurements at two wavelengths. Since one normally
encounters oxyhemoglobin (O2Hb), deoxyhemoglobin
(Hb), and COHb in blood to be analyzed for COHb,
methods have been developed to convert these samples
into two-component systems. This is accomplished by
the addition of sodium dithionite (Na2S2O4) to blood,
which converts O2Hb to HHb. The HHb and COHb are
then measured at 420 and 432 nm, respectively. The
presence of other hemoglobins, such as Hb F or SHb,
complicates these methods. To cope with the presence of
these hemoglobins, one can increase the number of
measurements, and the resulting simultaneous equations
can be solved to quantitate these other Hb species.
Availability of microprocessors or computers simplifies
solution of the equations, but one is also required to
standardize the multicomponent systems to determine
the constants used in the equations. Some of the tedium
of performing these tasks can be avoided by purchase of
dedicated systems that are programmed to perform the
calculations and have been standardized at the factory.
These topics have been previously reviewed [42,44].
Zwart and co-workers [53] have developed a
spectrophotometric method that allows the measurement
of hemoglobin derivatives in a simple hemolysate in the
presence of all hemoglobins: O2Hb, metHb, HHb, and
SHb. Absorbances of the hemolysate must be measured
at each of five wavelengths, since a five-component
mixture requires five simultaneous equations of the
following general type to be constructed from Beers
law:
Anm = a1(nm)bC1 + a2(nm)bC2 ...
+ a5(nm)bC5
where Anm is the absorbance at wavelength nm, a is the
molar absorptivity of the numbered species at
wavelength nm, b is the pathlength of the cuvette used,
and C is the concentration of the numbered species.
Zwart [53] used wavelengths that allow measurement of
the absorbances at a wavelength maximum wherever
possible: Hb, 760 nm; O2Hb, 577 nm; COHb, 569 nm;
metHb, 500 nm; SHb, 620 nm. Molar absorptivities at
these wavelengths were derived from standards and are
931
Oximetry
given in Table 2: Millimolar Absorptivities. Because of
the large differences in a, two pathlengths are required to
keep observed absorbances within the range of most
instruments without the need for dilution. At 620 and
760 nm, b = 0.200 cm, but at 500, 569, and 577 nm, b =
0.007 cm.
The hemolysate is prepared when one mixes heparinized
whole blood in a syringe with a 5% solution of a
nonionic detergent, nonoxynol (Sterox). One filters the
cellular debris from the hemolysate by expelling the
syringe contents through a cotton fiber filter.
Absorbances are measured in a narrow bandpass
spectrophotometer (<1 nm) against a water blank. Blood
samples with various proportions of hemoglobin species
are prepared and used for calibration.
Zwart et al. [57,58] next used a multiwavelength method
for multicomponent analysis of Hb derivatives. The
lithium heparin whole-blood sample was hemolyzed
using100 mL/L solution of Nonidet-P40. The absorption
spectrum was determined between 480 and 650 nm and
absorbance measurements taken in increments of 2 nm
(86 absorbance readings) with a diode array
spectrophotometer. The concentrations of the individual
components are calculated using the spectrophotometer
computer.
1. Methemoglobin and Sulfhemoglobin Method
Measurement is based on the original Evelyn and
Molloy [12] reference method with modifications as
suggested by Blanchette et al. [59] of the Teitz [60]
textbook method.
Principle:
MetHb has an absorbance peak at 530 to 535 nm. The
addition of cyanide converts metHb to cyanmetHb. The
decrease in absorbance is proportional to the metHb
concentration. SHb has an absorbance peak at 618 to 622
nm. SHb is not converted by cyanide to cyanmetHb so
there will be no change in absorbance.
Reagents
1. Potassium phosphate buffer, 0.15 mol/L, pH 6.6:
Dissolve 17.1 g of K2HPO43H2O (or 13.2 g anhydrous)
in 500 mL of distilled water and 10.2 g of KH2PO4 in
500 mL of distilled water. Transfer 100 mL of each the
two solutions to a new beaker. Adjust the pH to 6.6
using the K2HPO4 solution.
2. KCN solution: Dissolve 500 mg of KCN in ~ 7 mL
of water, and dilute to 10 mL.
3. K3F(CN)6 solution: Dissolve 2.0 g of in ~ 7.0 mL
distilled water, and dilute to 10 mL.
Assay
Equipment: Spectrophotometer, narrow bandpass (<1
nm), that has been calibrated and checked regularly for
wavelength accuracy, photometric accuracy, and
linearity and for the presence of stray radiant energy.
1. Label a cuvette C1, and add to it 1.5 mL of
phosphate buffer and 1.5 mL of water. Zero the
spectrophotometer at 620 and 630 nm.
2. Obtain a fresh whole-blood sample (EDTA or
lithium heparin).
Aliquot 0.2 mL of blood into a tube containing
7 mL of distilled water. Mix by gentle
inversion. Wait 5 to 10 minutes. Add 4.0 mL of
K2HPO4 buffer. Mix the specimen, and
centrifuge at 4000 rpm for 10 min to remove
cell stroma and unhemolyzed cells.
3. Transfer 3.0 mL of clear supernatant from step
2 into a cuvette labeled C2,
measure the absorbance at 630 nm, and
designate this value A1.
4. Transfer 1.0 mL of clear supernatant from step
2 into a cuvette labeled C3, and add 1.0 mL of
K2HPO4 buffer and 1.0 mL of water. Mix by
gentle inversion.
5. Add 0.1 mL of KCN to cuvettes C2 and C3.
Cover with Parafilm, and invert several times to
mix thoroughly. Allow to stand for 5 minutes.
6. Measure the absorbance of cuvette C2 at 630
nm (A2) and 620 nm (A3) against a blank
cuvette C1.
7. Add 0.1 mL of K3F(CN)6 solution to cuvettes
C1 and C3. Cover with Parafilm, and invert
several times to mix thoroughly. Allow to stand
for 30 minutes.
8. Set spectrophotometer to 540 nm and adjust to
zero absorbance with cuvette C1. Measure
absorbance of C3 (A4).
Calculations
Total Hb (g/dL) = F1 A4
MetHb (g/dL) = F2 (A1-A2)
SHb g/dL) = 80 F3 (A3-F4(A1-A2)-3 F5 A4)
MetHb % = [(MetHb (g/dL)/total Hb g/dL)] 100
SHb % = [(SHb (g/dL)/total Hb g/dL)] 100
Where F1=35.1, F2=33.6, F3=0.64 and F4=0.300 and
F5=0.022
2. Carboxyhemoglobin Manual Spectrophotometry
Method
In the selection of a general method for
carboxyhemoglobin measurements, however,
considerations of accuracy and simplicity predominated
over versatility. The preferred method presented below
is based on Rodkey et al. [61] as modified by Beutler
and West [62].
Principle
Hemoglobin in blood is mixed with sodium hydrosulfite
(Na2S2O4), which reacts with oxyhemoglobin and
methemoglobin to produce reduced hemoglobin, thereby
converting a multicomponent system into a two-
component system: carboxyhemoglobin and reduced
hemoglobin. Absorbance measurements are made at two
wavelengths, 420 and 432 nm, using a narrow bandpass
spectrophotometer, which if properly calibrated, allows
one to employ literature values for molar absorptivities
932
Oximetry

of reduced Hb and Hb CO in the calculations using reagent-grade water. Bring volume to 1 L, and mix well.
Beers law. Prepare pH 6.85 buffer by mixing equal amounts of each
stock buffer. Check pH of solution and adjust to pH 6.85
At each wavelength used in the measurement step, with 0.1 M HCl or NaOH. This solution, reagent 1, is
Beers law gives the absorbance as follows: stable for 2 months at 4C to 8C. Check weekly for
bacterial growth.
A420 = (aHb420[1 x] + aHb CO420[x])bC 2. Hemolyzing solution. Buffer, reagent 1,
1 diluted one part to 9 parts of distilled water. Prepare and
A432 = (aHb432[1 x] + aHb CO432[x])bC use as needed from buffer stock.
3. Hb CO diluting solution. To 25 mg of sodium
2
hydrosulfite, add 20 mL of buffer, reagent 1 (see above).
Prepare this solution with minimum introduction of air.
where a is the molar absorptivity of each species at each
In a procedure described by Rodkey [61], a test tube is
wavelength, b is the cuvette pathlength in cm, C is the
filled to the top with buffer containing glass beads for
molar concentration of hemoglobin iron in solution, and
agitation. Solid sodium hydrosulfite is added to the full
x is the fraction of the total amount of hemoglobin that is
tube, and the tube is covered and inverted to dissolve the
present as carboxyhemoglobin. Note that (1 x)
sodium hydrosulfite. This reagent must be prepared
represents the fraction of the hemoglobin present as
immediately before use.
reduced hemoglobin.
Assay
Combining equations 1 and 2 and solving for x
Equipment: Spectrophotometer, narrow
yields the following:
bandpass (<1 nm), that has been calibrated and checked
regularly for wavelength accuracy, photometric
accuracy, and linearity and for the presence of stray
3
radiant energy. Procedures and tools for these quality
x = ___________(A432)(aHb420)
assurance measures are given by Rand [63] and Frings
(A420)(aHb432)___________ [64].
(A420[aHb CO432 aHb432]) 1. Add 25 L of whole anticoagulated blood to 3
(A432[aHb CO420 aHb420]) mL of hemolyzing solution in a small test tube.
Mix two or three times by inversion. Let stand
about 5 min to ensure complete lysis of cells.
Rodkey [61] has published carefully derived molar
2. In a 1-cm glass cuvette, mix 0.2 mL of the
absorptivity values (a) required to solve the above
hemolysate from step 1 and 2.3 mL of the Hb
equation. The values given below are derived from
CO diluting solution. Cover with Parafilm, and
human hemoglobins:
invert to mix. Let stand 10 to 60 min, no longer.
3. Read the absorbances of the sample at 420 and
Wavelength aHb aHb CO
432 nm against a matched 1-cm glass cuvette
420 0.988 1.970
containing Hb CO diluting solution. Record
432 1.317 1.237
A420 and A432.
The user must verify the literature values of molar Calculations
absorptivities on the spectrophotometer used for analysis 1. Calculate Ar = A420/A432 for each unknown.
and should regularly check wavelength calibration, 2. Using equation 4, calculate the fraction of Hb
photometric accuracy and linearity, absence of stray CO, using literature values of the constants as
radiant energy, and cuvette pathlengths. follows: [61]
Equation 3 has been simplified by Beutler [62] to the F1 = 1.3330, F2 = 0.4787, F3 = 1.9939,
following: after confirming that they are valid to use in
%x = __(1 [Ar][F1])100%__ your laboratory procedure. Verify as often as
Ar(F2 F1) F3 + 1 the spectrophotometer is subjected to source-
lamp changes or electronic repair.
where Ar = A420/A432 Calibration and Controls
1. Frequent confirmation of the reference intervals
F1 = aHb432/aHb420
for smokers and nonsmokers is helpful to
F2 = aHb CO432/aHb420 ensure accuracy.
F3 = aHb CO420/aHb420 2. Determination of molar absorptivity ratios can
%x = percentage of Hb CO. be accomplished as described by Beutler
[62].
Reagents a. Prepare a hemolysate using 6 mL of
1. KH2PO4/K2HPO4 buffer, 0.1 mol/L, pH the hemolysate reagent and 50 L of
whole blood from a nonsmoking
6.85. Weigh 13.6 g of KH2PO4 (anhydrous), and
person with no known recent exposure
dissolve in approximately 800 mL of reagent-grade to other sources of carbon monoxide.
water. Bring volume to 1 L and mix well. Weigh 17.4 g b. Using a class A volumetric pipet, add
of K2HPO4, and dissolve in approximately 800 mL of 2 mL of the hemolysate to each of two
933
Oximetry

class A 25-mL volumetric flasks. F2 = AHb CO432/AHb420;

Accuracy in pipetting is crucial to the F3 = AHb CO420/AHb420.
accuracy of the determination of these
molar absorptivities. Calibration of the
3. Measurement of Carboxyhemoglobin in Breath
pipets and flasks is recommended.
Sensors have been developed that enable measurement
c. In a well-ventilated hood and with
of carbon monoxide in air or expired breath samples.
frequent swirling and stirring, allow
With this technique, CO is oxidized at the anode, with
carbon monoxide gas to pass through
resultant generation of an electrical signal as follows:
one flask while compressed air is
passed through the other. Use a bright CO + H2O CO2 + 2H+ + 2 e-
light to dissociate any traces of carbon
monoxide from the hemoglobin in the Oxygen in the air is reduced at the cathode:
air flask.
d. Bring each flask to volume with Hb 2O + 8H+ + 8e- 4H O
2 2
CO diluting solution, and mix well.
Immediately measure the absorbance The current that is generated in this system can be
(A) at 432 and 420 nm for each diluted displayed as parts per million (ppm) or as a percentage
hemolysate, using matched 1-cm glass of COHb saturation if measured in breath. This
cuvettes and blanking against Hb CO technique offers the advantages of being noninvasive
diluting solution. and relatively inexpensive, and it requires little training
e. Calculate molar absorptivity factors for use [3].
(F) from the ratio of observed
absorbance values as follows:
F1 = AHb432/AHb420;
934
Parathyroid Hormone (PTH, Parathyrin)
Parathyroid Hormone (PTH, Parathyrin)
Susan Vickery and Edmund J Lamb
Name: Parathyroid hormone, parathyrin, parathormone, PTH
Clinical significance: Refer to Chapter 33, Bone Disease, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular mass: 9500 D
Merck Index: 6898
Chemical class: Polypeptide
i Different antibodies were available that bound to
Principles of Analysis and Current Usage
Parathyroid hormone (PTH) is a single polypeptide
consisting of 84 amino acids. Intact PTH (1-84) is
secreted by the parathyroid glands in response to low
circulating (ionized) calcium concentration. The most
important role of PTH is to increase blood calcium
concentration by increasing bone resorption, increasing
the tubular reabsorption of calcium in the kidney, and
increasing the production of 1,25-
dihydroxycholecalciferol (vitamin D), which
subsequently increases absorption of calcium from the
gut [1]. PTH determination is important in the diagnosis
and management of primary hyperparathyroidism
(oversecretion of PTH from the parathyroid glands),
secondary hyperparathyroidism (e.g., due to kidney
disease), vitamin D deficiency, and other hyper- and
hypocalcemic states.
The complete amino acid sequence of human PTH has
been determined [2]. Parathyroid cells synthesize a pre-
pro-hormone, consisting of 115 amino acids, which is
cleaved immediately after its synthesis to give the 90
amino acid compound pro-PTH [3]. The removal of six
amino acids from the carboxyl terminus of the molecule
yields the final glandular product PTH with residues 1-
84. Classical biological activity, mediated via interaction
with the PTH receptor PTH1R, resides in the N-terminus
of the molecule. C-terminal (CPTH) fragments are also
directly secreted by the parathyroid gland. Within the
liver and kidney, PTH (1-84) is cleaved into N-terminal
and CPTH fragments. Circulating PTH is a mixture of
PTH (1-84) and CPTH fragments [4]; this raises
problems when immunological techniques are employed
for the measurement of this hormone.
Berson et al. described the first radioimmunoassay (RIA)
for the determination of PTH in 1963 [5]. The assay
utilized a single antibody obtained from polyclonal
antisera raised against partially pure non-human PTH.
i
Parathyroid hormone (PTH)
Previous and current authors of this method:
First edition: Not done
Methods book: Helmut W. Minne, Reinhart Ziegler
Second edition: Not done
Third edition: Not done
Fourth edition: Helmut W. Minne, Reinhard Ziegler
Fifth edition: Susan Vickery, Edmund J. Lamb
epitopes either in the mid-region or the C-terminus of
PTH. Highly pure isotopically labeled intact PTH would
then competitively displace bound sample PTH [6]. Such
assays were insensitive, owing to the poor cross-
reactivity between sample PTH and the non-human
antibody [7,8].
It then became apparent that CPTH fragments secreted
by the parathyroid glands and produced during the
metabolism of PTH were being measured in addition to
intact PTH (1-84) [9]. CPTH fragments, are found in
varying amounts in all patients but accumulate in
patients with renal disease. Circulating concentrations of
CPTH fragments accounted for up to 95% of the
measured PTH in patients with end-stage renal disease
(ESRD), arising from increased glandular secretion of
CPTH fragments, increased metabolism of intact PTH,
and reduced glomerular filtration [8,10]. For patients
with renal disease, PTH concentrations were
overestimated and unreliable. Attempts were made to
use an antibody that could recognize an epitope in the N-
terminal region of PTH, which the CPTH fragments lack
[11]. However, the poor sensitivity of these assays
limited their clinical use.
First-generation RIAs have been replaced with
immunometric assays that detect PTH using two
different antibodies targeted at two distinct epitopes in
the PTH molecule. Typically, the first antibody,
immobilized to a solid phase, binds to a region near the
C-terminus and removes PTH from the serum. A second
labeled antibody then binds to the N-terminus of the
immobilized PTH [12]. Only full-length PTH (1-84) is
capable of binding both antibodies, so CPTH fragments
are not measured. These assays have been termed intact
PTH assays. They are highly sensitive, reasonably
specific, readily automated, and can be used with a
variety of labels: radiolabeled isotopes,
chemiluminescent, biotinylated, or enzyme-linked labels
[13]. Such second-generation PTH assays have been in
routine clinical use for more than a decade.
In the mid-1990s, a Canadian research group discovered
that another molecular form of PTH was being detected
by the second-generation assays [14,16]. In high-
performance liquid chromatography, this molecule co-
chromatographs with synthetic PTH (7-84). The
discovery that the second-generation intact PTH assays
measure other PTH molecules has lead to the
935
Parathyroid Hormone (PTH, Parathyrin)
development of specific PTH (1-84) assays, referred to
here as third-generation assays. Third-generation assays
are defined by their ability to measure whole, intact PTH
(1-84) without cross-reacting with PTH (7-84), now
thought to be a major C-terminal circulatory form that
cross-reacts with second-generation assays. Exploitation
of the difference between the two N-terminal regions has
provided an antibody that exclusively detects PTH (1-
84); polyclonal antibodies are targeted to epitopes within
the first four amino acids from the N-terminal [17].
Third-generation assays at present are expensive, and
very few are automated [18].
With the introduction of third-generation PTH assays,
numerous studies have been undertaken to compare
second- and third-generation assays. Recent studies have
shown that third-generation assays are more sensitive
(>23%) for detecting increased plasma PTH in primary
hyperparathyroidism than first- or second-generation
assays [19,20]. In contrast, a study comparing a third-
generation Bio-PTH (1-84) assay with a second-
generation intact PTH assay concluded a good
correlation between the two assays, with both providing
similar clinical information [21].
Reference and Preferred Methods
A reference preparation of purified human PTH for PTH
immunoassays (WHO 79/500) was prepared in 1981
[22]. However, its content is formally defined in
International Units (0.1 IU/ampoule), whereas PTH
measurements tend to be expressed in mass or molar
units. Some manufacturers do claim traceability to
79/500, but its nominal mass concentration is probably
inaccurate and does not provide a sound basis for
conversion to the use of mass units for reporting PTH.
Further, stocks of 79/500 are becoming depleted. A
newer preparation based on recombinant PTH and
defined in mass units has been prepared by the National
Institute of Biological Standards and Controls (NIBSC
95/646; http://www.nibsc.ac.uk/documents/ifu/95-
646.pdf) but has not been widely adopted to date.
There is currently no reference method for PTH
measurement.
Second-generation immunometric intact PTH assays are
currently used in most clinical laboratories. The
distinguishing feature between these assays is their mode
of detection; there is a clear decline in the use of
radiolabeled isotopes, where chemiluminescence (using
acridinium esters) is now the preferred choice [7]. Most
commercial assays are now fully automated. Of the 257
participants in the United Kingdom National External
Quality Assessment Scheme (UK NEQAS) for plasma
PTH in March 2007, all of which used second-
generation assays, the major reagent manufacturers were
Roche Diagnostics Ltd (39%, Modular and Elecsys
platforms), Diagnostic Product Corporation (30%),
Siemens Medical Solutions Diagnostics (18%, Advia
Centaur platform), and Abbott Diagnostics (5%).
Specimen
EDTA plasma is the preferred sample; PTH in EDTA
whole blood is stable for up to 48 h at room temperature
prior to separation (centrifugation >1000g for 15 to 20
min) [23]. This is of benefit in clinical situations where
rapid separation of samples may be hard to achieve.
Once separated, EDTA plasma should be frozen at
20C (for up to 3 months) prior to analysis. Recurrent
freeze/thaw cycles are not recommended.
Serum specimens collected into plain tubes can also be
used, providing samples are transported rapidly to the
laboratory, allowed to clot at room temperature, and then
separated and frozen without delay (1). To facilitate
interpretation, serum calcium, albumin (to allow
correction of calcium concentration), and renal function
(i.e., creatinine and eGFR) should always be measured at
the same time as PTH concentration.
Interferences
Unless a third-generation assay solely measuring PTH
(1-84) is used, CPTH fragments will be detected in
addition to PTH (1-84); this is particularly applicable to
renal disease patients. The instability of PTH in serum
can result in spuriously decreased values in samples that
are not analyzed immediately following collection or
immediately frozen. One manufacturer has reported that
samples from patients routinely receiving high dose
biotin therapy may show falsely decreased results. As
with all other immunoassay procedures, the presence of
heterophilic antibodies could have the potential to cause
interference in PTH measurements. Following good
laboratory practice, grossly hemolyzed, icteric, or
lipemic samples should not be used.
PTH Reference Interval
The upper limit of normal for PTH in healthy individuals
is dependent on the assay used, for example:
First-generation PTH RIA:
10 to 65 ng/L, Nichols Advantage Chemiluminescence
Intact Parathyroid Hormone
Second-generation PTH immunometric assays:
11 to 79 ng/L, Intact PTH assay; Siemens Medical
Solutions Diagnostics
Third-generation PTH immunometric assay:
5 to 39 ng/L, Whole PTH (1-84) Specific Scantibodies
Laboratory, Inc.
To convert ng/L to pmol/L, multiply by 0.106.
The lack of international standardization makes direct
comparisons between assay systems difficult.
Laboratories must use a reference range appropriate for
their assay. A further difficulty in defining reference
intervals for PTH is the high prevalence of (a) occult
primary hyperparathyroidism and (b) vitamin D
deficiency in the reference populations studied.
No significant gender- or race-related differences have
been noted. Plasma concentrations during the first few
936
Parathyroid Hormone (PTH, Parathyrin)
days of life are lower when compared with those in older
children [24]. For older children, however, PTH
concentrations are lower and in a narrower range when
compared with those in adults [25]. In adults, a
significant increase in concentration has been noted with
aging. One study found that compared with women
under 40 years of age, women in their fifth through
seventh decades of life had PTH concentrations that
were 24%, 36%, and 47% higher, respectively [26]. A
diurnal variation, with highest values occurring in the
early to mid-midmorning hours, has been described by
some but not all investigators.
Interpretation
PTH measurement is used in the differential diagnosis of
hypercalcemia.
As second-generation PTH immunometric assays are
routinely used, the question of whether CPTH fragments
(primarily PTH [7-84]) detected by these assays possess
biological activity needs to be addressed. It has been
proposed that PTH (7-84) is an antagonist to PTH (1-84)
in inhibiting the turnover of bone and preventing an
increase in blood calcium concentration [27]. In primary
hyperparathyroidism and vitamin D deficiency, PTH (7-
84) accounts for less than 10% of intact PTH. However,
accumulation of PTH (7-84) has been shown in patients
with chronic kidney disease [28]. In this scenario, an
increased PTH concentration measured by a second-
generation assay may be indicative of bone disease, but
if a significant amount of PTH (7-84) is present
inhibiting PTH (1-84)s action, bone disease is less
likely. It can therefore be argued that specifically
measuring PTH (1-84) is of merit.
Like first-generation assays, second-generation assays
overestimate PTH (1-84) concentrations, and may be
providing a worse picture of bone turnover for patients
with renal disease than is the case [27]. The Nichols,
Diagnostic System Laboratories, and Instars second-
generation PTH assays have all been found to
overestimate PTH (1-84) concentrations [16]. However,
the clinical implications of third-generation assays are
controversial. A number of studies have concluded that
no additional diagnostic information is obtained from
third-generation assaysfor example, Scantibodies
Whole PTH (1-84) Specific assay [29,30]. Perhaps of
greater clinical relevance is determining both PTH (1-
84) and (7-84) concentrations and their ratio in those
patients with known renal bone disease [18].
PTH Performance Goals
Second-generation PTH assays do not agree with each
other, and this is confirmed by external quality
assessment data. Such differences can be attributed to
the lack of standardization, variation in recovery of PTH
(1-84), and differences in the amount of cross-reactivity
with PTH (7-84). Data from UK NEQAS has estimated
between 69% and 102% cross-reactivity with PTH (7-
84) for all commercially available second-generation
assays [31]. The between-all method coefficient of
variation (CV) was 34% in the March 2007 UK NEQAS
for plasma PTH. However, within-method CVs for
Siemens Medical Solutions Diagnostics (5.2%), Roche
Diagnostics Ltd (5.7%), Abbott Diagnostics (6.4%), and
Diagnostics Product Corporation (10.6%) were
acceptable. Reported intraindividual variation for PTH is
25.9% in healthy individuals [32]. Assuming a typical
performance goal of 50% of the biological variation, this
is achieved by most modern PTH assays. Within-
laboratory CVs for the Siemens Medical Solutions
Diagnostics Intact PTH assay are typically 10.2%, 5.9%,
and 4.3% for plasma PTH concentrations of 36, 232 and
855 ng/L, respectively (Department of Clinical
Biochemistry, East Kent Hospitals NHS Trust, personal
communication).
References
1. Ashby JP, Newman DJ, Gow SM. Clinical
application of intact parathyroid hormone
assays. Ann Clin Biochem 1997;34:588-98.
2. Brewer HB, Jr., Fairwell T, Ronan R, Sizemore
GW, Arnaud CD. Human parathyroid hormone:
amino-acid sequence of the amino-terminal
residues 1-34. Proc Natl Acad Sci USA
1972;69:3585-8.
3. Niall HD, Sauer RT, Jacobs JW, Keutmann HT,
Segre GV, O'Riordan JL, et al.. The amino-acid
sequence of the amino-terminal 37 residues of
human parathyroid hormone. Proc Natl Acad
Sci USA 1974;71:384-8.
4. Murray TM, Rao LG, Divieti P, Bringhurst FR.
Parathyroid hormone secretion and action:
evidence for discrete receptors for the carboxyl-
terminal region and related biological actions of
carboxyl- terminal ligands. Endocr Rev
2005;26:78-113.
5. Berson SA, Yalow RS, Aurbach GD.
Immunoassay of bovine and human parathyroid
hormone. Proc Natl Acad Sci USA
1963;49:613-7.
6. Goodman WG, Salusky IB, Juppner H. New
lessons from old assays: parathyroid its
receptors, and the potential biological hormone
(PTH), relevance of PTH fragments. Nephrol
Dial Transplant 2002;17:1731-6.
7. Carter AB, Howanitz PJ. Intraoperative testing
for parathyroid hormone - A comprehensive
review of the use of the assay and the relevant
literature. Arch Pathol Lab Med
2003;127:1424-42.
8. Segre GV. Advances in Techniques For
Measurement of Parathyroid-Hormone -
Current Applications in Clinical Medicine and
Directions For Future-Research. Trends
Endocrinol Metab 1990;1:243-7.
9. Brossard JH, Whittom S, Lepage R. Carboxyl-
terminal fragments of parathyroid hormone are
not secreted preferentially in primary
hyperparathyroidism as they are in other
hypercalcemic conditions. J Clin Endocrinol
Metab 1993;77:413-9.
10. Nguyen-Yamamoto L, Rousseau L, Brossard
JH, Lepage R, Gao P, Cantor T, et al. Origin of
parathyroid hormone (PTH) fragments detected
937
Parathyroid Hormone (PTH, Parathyrin)
by intact-PTH assays. Eur J Endocrinol
2002;147:123-31.
11. Martin KJ, Hruska K, Freitag JJ. Clinical utility
of radioimmunoassays for parathyroid
hormone. Min Electrolyte Metab 1980;3:283-
90.
12. Nussbaum SR, Zahradnik RJ, Lavigne JR,
Brennan GL, Nozawa-Ung K, Kim LY, et al.
Highly sensitive two-site immunoradiometric
assay of parathyrin, and its clinical utility in
evaluating patients with hypercalcemia. Clin
Chem 1987;33:1364-7.
13. Martin KJ, Akhtar I, Gonzalez EA. Parathyroid
hormone: New assays, new receptors. Semin
Nephrol 2004;24:3-9.
14. Brossard JH, Lepage R, Cardinal H, Roy L,
Rousseau L, Dorais C, et al.. Influence of
glomerular filtration rate on non-(1-84)
parathyroid hormone (PTH) detected by intact
PTH assays. Clin Chem 2000;46:697-703.
15. D'Amour P, Brossard JH, Rousseau L, Nguyen-
Yamamoto L, Nassif E, Lazure C, et al.
Structure of non-(1-84) PTH fragments secreted
by parathyroid glands in primary and secondary
hyperparathyroidism. Kidney Int 2005;68:998-
1007.
16. Lepage R, Roy L, Brossard JH, Rousseau L,
Dorais C, Lazure C, et al. A non-(I-84)
circulating parathyroid hormone (PTH)
fragment interferes significantly with intact
PTH commercial assay measurements in uremic
samples. Clin Chem 1998;44:805-9.
17. Gao P, Scheibel S, D'Amour P, John MR, Rao
SD, Schmidt-Gayk H, et al. Development of a
novel immunoradiometric assay exclusively for
biologically active whole parathyroid hormone
1-84: Implications for improvement of accurate
assessment of parathyroid function. J Bone
Miner Res 2001;16:605-14.
18. Blumsohn A, Al Hadari A. Parathyroid
hormone: what are we measuring and does it
matter? Ann Clin Biochem 2002;39:169-72.
19. Silverberg SJ, Gao P, Brown I, Logerfo P,
Cantor TL, Bilezikian JP. Clinical utility of an
immunoradiometric assay for parathyroid
hormone (1-84) in primary
hyperparathyroidism. J Clin Endocrinol Metab
2003;88:4725-30.
20. Yamashita H, Gao P, Noguchi S, Uchino S,
Watanabe S, Ogawa T, et al.. Comparison of
intact and whole parathyroid hormone levels
after parathyroidectomy between patients with
primary and secondary hyperparathyroidism. J
Bone Miner Res 2003;18:S100-S.
21. Inaba M, Nakatsuka K, Imanishi Y, Watanabe
M, Mamiya Y, Ishimura E, et al.. Technical and
clinical characterization of the Bio-PTH (1-84)
immunochemiluminometric assay and
comparison with a second- generation assay for
parathyroid hormone. Clin Chem 2004;50:385-
90.
22. Zanelli JM, Gaines-Das RE. The first
international reference preparation of human
parathyroid hormone for immunoassay:
characterization and calibration by international
collaborative study. J Clin Endocrinol Metab
1983;57:462-9.
23. Teal TK, Reed M, Stevens PE, Lamb EJ.
Stability of parathyroid hormone ex vivo in
haemodialysis patients. Ann Clin Biochem
2003;40:191-3.
24. Mayne PD, Kovar IZ. Calcium and phosphorus
metabolism in the premature infant. Ann Clin
Biochem 1991;28 :131-42.
25. Cioffi M, Corradino M, Gazzerro P, Vietri MT,
Di Macchia C, Contursi A, et al.. Serum
concentrations of intact parathyroid hormone in
healthy children. Clin Chem 2000;46:863-4.
26. Kotowicz MA, Melton LJ, 3rd, Cedel SL,
O'Fallon WM, Riggs BL. Effect of age on
variables relating to calcium and phosphorus
metabolism in women. J Bone Miner Res
1990;5:345-52.
27. Malluche HH, Mawad H, Trueba D, Monier-
Faugere MC. Parathyroid hormone assays -
evolution and revolutions in the care of dialysis
patients. Clin Nephrol 2003;59:313-8.
28. Brossard JH, Cloutier M, Roy L, Lepage R,
Gascon-Barre M, D'Amour P. Accumulation of
a non-(1-84) molecular form of parathyroid
hormone (PTH) detected by intact PTH assay in
renal failure: importance in the interpretation of
PTH values. J Clin Endocrinol Metab
1996;81:3923-9.
29. Godber IM, Parker CR, Lawson N, Hitch T,
Porter CJ, Roe SD, et al. Comparison of intact
and whole molecule parathyroid hormone
assays in patients with histologically confirmed
post-renal transplant osteodystrophy. Ann Clin
Biochem 2002;39:314-7.
30. Goodman WG. New assays for parathyroid
hormone (PTH) and the relevance of PTH
fragments in renal failure. Kidney Int
2003;64:S120-S4.
31. Lamb EJ, Vickery S, Ellis AR. Parathyroid
hormone, kidney disease, evidence and
guidelines. Ann Clin Biochem 2007;44:1-4.
32. Ankrah-Tetteh T, Wijeratne S, Swaminathan R.
Intra individual variation in serum thyroid
hormones, parathyroid hormone, and insulin
like growth factor-1 (IGF-1). Ann Clin Biochem
2007;in press.
938
Parathyroid Hormone (PTH, Parathyrin)

Table 1
Method 1: First Generation PTH Radioimmunoassay
Principles of analysis:
PTH (both intact PTH (1-84) and fragments) competes with 125I-labelled fragments for binding to
antibodies to specific fragments or the intact PTH molecule. Antibodies used will bind either to the C-
terminal, mid-molecule or to the N-terminal region of PTH. Separation of bound and free ligand is
achieved by use of dextran-coated charcoal or a second antibody.

Method 2: Second Generation Intact PTH Immunometric Assays

Principles of analysis:
A signal (e.g. chemiluminescent) polyclonal antibody targeted to the N-terminal region of PTH and a
second (e.g. biotinylated) polyclonal antibody targeted to the C-terminal region of PTH detects PTH (1-
84) and some truncated PTH fragments. The second polyclonal or capture antibody is typically coated
to latex particles or bound to the wells of microtiter plates. Unbound protein is removed by a series of
washing steps. The concentration of antibody-bound PTH is directly proportional to the signal
produced (e.g. relative light units (RLUs)).

Method 3: Third Generation Bioactive/Whole PTH Immunometric assay

Principles of analysis:
A signal (e.g. chemiluminescent) polyclonal antibody targeted to amino acids 1-4 of the N-terminal of
PTH and a second (e.g. biotinylated) polyclonal antibody to the C-terminal of PTH ensures that only
intact PTH (1-84) is detected. The second polyclonal or capture antibody is typically coated to latex
particles or bound to the wells of microtiter plates. Unbound protein is removed by a series of washing
steps. The concentration of antibody-bound PTH is directly proportional to the signal produced (e.g.
RLUs).
939
Phenylalanine
Phenylalanine
Theodore Dashman, Helen K. Berry
Name: Phenylalanine, -amino--phenylpropionic acid
Clinical significance:
Molecular formula: C9H11NO2
Molecular weight: 165.2 D
Merck Index: 7150
Chemical class: amino acid
Structure:
Principles of Analysis and Current Usage
i tyrosine uses the N(O)-trifluoroacetyl methyl esters
The determination of phenylalanine in serum is most
frequently used to confirm the diagnosis of
hyperphenylalaninaemias, that is, phenyl-ketonuria and
disorders of tetrahydrobiopterin metabolism, and to
follow changes in phenylalanine blood levels in persons
undergoing therapy for phenylketonuria. One method for
measurement of phenylalanine is automatic ion-
exchange chromatography using an amino acid analyzer
(Table 1, method 1). The single-column method of Piez
and Morris forms the basis for operation of most amino
acid analyzers [1]. The ion-exchange resins are highly
cross-linked sulfonated styrene copoly-mers that have an
affinity for both the ionic and nonionic portion of the
amino acid molecule [2]. Amino acids are eluted from
the resin when the column temperature is increased and
buffers of different pH and ionic strength are used. The
column effluent is reacted with ninhydrin and the
produced blue reaction product is quantified
spectrophotometrically at 570 nm.
Gas-chromatographic procedures are used for separation
of amino acids, including phenylalanine, in
physiological mixtures (Table 1, method 2). A number
of derivatizing agents have been used for the gas
chromatography of amino acids. Trimethylsilyl
derivatives are frequently used [3], as are derivatives
prepared with N,N-bis(trimethyl-silyl)trifluoroacetamide
(BSTFA) [4]. A method specific for phenylalanine and
i
Phenylalanine
Previous and current authors of this method:
First edition: Helen K. Berry
Methods edition: Helen K. Berry
Second edition: Not updated
Third edition: Not updated
Fourth edition: Theodore Dashman, Helen K. Berry
Fifth edition: Not updated
prepared by reaction of the amino acid mixture with
acetonitrile and trifluoroacetic anhydride, followed by
methylation using diazomethane [5]. One can use a
variety of column packings. Conditions of gas flow and
column and oven temperatures are adjusted for the
particular derivative. Detection is by flame ionization or
mass spectrometry with selective ion monitoring.
Reversed-phase high performance liquid
chromatography has been developed for rapid analysis of
plasma amino acids (Table 1, method 3). The most
common practice is to derivatize the amino acids with
one of several reagents, such as, dabsy lchloride, phenyl-
isothiocyanate, or 9-fluorenylmethyl chloro-formate, but
the most popular compound is o-phthala-adehyde [6].
The advantages of these methods are high precision,
optimal identification of primary and secondary amino
acids, and economic mobile phase [7].
The Guthrie microbiological inhibition assay is used for
semiquantitative measurement of phenylalanine in
blood, particularly in newborn screening programs [8].
The test is based on the inhibition of growth of Bacillus
subtilis by -2-thienylalanine and the ability of
phenylalanine to overcome this inhibition (Table 1,
method 4).
The fluorometric method of McCaman and Robins [9-
13] is preferred for routine use in the clinical laboratory
from the standpoint of simplicity, sensitivity, and cost.
The procedure is based on the enhanced fluorescence
produced when phenylalanine condenses with ninhydrin
in the presence of the dipeptide L-leucyl-L-alanine
(Table 1, method 5). The reaction is carried out at pH
5.9. The fluorescent compound is stabilized by addition
of an alkaline copper tartrate reagent. The fluorescence
produced by excitation at 365 to 390 nm is measured at
489 to 515 nm.
940
Phenylalanine
Techniques for quantitating both phenylalanine and
tyrosine by tandem mass spectrometry (MS/MS) have
also been described [14]. Studies have shown that
calculation of the molar ratio of phenylalanine to
tyrosine can allow detection of affected newborns under
24 h of age without increasing the rate of false positive
test results. In addition to phenylketonuria, MS/MS can
also detect other aminoacidopathies, including maple
syrup urine disease and homocystinuria. The
disadvantages to MS/MS include the initial high cost of
equipment and the need for sample derivatization. One
way around this is employ stable isotope dilution
reversed-phase LS-MS [15].
Reference and Preferred Methods
The amino acid analyzer yields accurate and precise
measurements of phenylalanine. From derivatization to
producing a report takes 2 to 3 h and is expensive in
terms of both cost of equipment and cost of reagents.
Gas chromatography of amino acids from physiological
fluids requires a preliminary separation of amino acids
from other substances that may react with the
derivatizing agents. Although gas chromatography has
excellent sensitivity and a short analysis time and the
apparatus is less expensive than an automatic amino acid
analyzer, the problems involved in preparation of the
specimen and preparation of the derivative make it
undesirable for routine work.
Although the fluorometric method yields less accurate
results than the amino acid analyzer, it has the advantage
of being able to analyze large numbers of samples
rapidly with a fair degree of precision. However,
reaction of amino acids other than phenylalanine may
yield a nonspecific fluorescence that results in
overestimation of phenylalanine concentrations in
specimens from normal persons.
In a cooperative study, values obtained by several
laboratories using the fluorometric procedure were
compared with values measured in the same specimens
by the amino acid analyzer [16]. At the lowest
phenylalanine level tested, 16 mg/L, the mean values
from the fluorometric procedure were significantly
higher than control values measured on the analyzer. At
the phenylalanine concentration of 36 mg/L, higher
values were obtained by the fluorometric procedure,
except in instances in which a serum blank (ninhydrin
without peptide) was used to correct for nonspecific
fluorescence. At concentrations of phenylalanine above
50 mg/L, none of the mean values from fluorometric
procedures was significantly different from the values
measured by the amino acid analyzer. Dilution of the
specimen when one is measuring phenylalanine in
patients with phenylketonuria reduces the error from
nonspecific fluores-cence. However, when
phenylalanine concentrations are very low in a
nonphenylketonuric patient, the fluorometric procedure
may cause overestimation of the serum phenylalanine
content, and a phenylalanine deficiency may be falsely
undiagnosed. For general screening purposes, the
fluorometric method with a blank correction is rapid and
accurate and thus is the recommended procedure.
Use of MS/MS can diagnose phenylketonuria with much
lower false positive rates (approximately 1/100)
compared with fluorometry. Also, in addition to other
aminoacidopathies, MS/MS can also screen for organic
acid disorders and disorders of fat metabolism. Because
of the low false-positive rates, this method is an efficient
way to screen for large numbers of disorders while
minimizing the cost of follow-up.
Specimen
Either serum or plasma may be used. The nature of the
anticoagulant does not affect the measurement. The
specimens are stable at 4 to 8 C for about a week.
Blood dried on filter paper is used for regional screening
programs and is stable for mailing and handling at room
temperature. Ideally, specimens should not be collected
until 48 h following birth. Approximately 2 days worth
of protein intake is usually required to result in the
accumulation in an abnormal concentration of amino
acids in affected infants.
Interferences
The most important limitation of the ion-exchange
chromatography method is the presence in the sample of
compounds with primary amino acid moieties. These
compounds react with ninhydrin and may coelute with a
plasma amino acid. These compounds include a variety
of drugs, such as ampicillin. For specimens analyzed
using the Guthrie procedure, antibiotics or other
substances that inhibit bacterial growth must not be
present. Otherwise, false negative results may occur.
Premature infants may show false positive results
because immature hepatic function may cause increased
amino acid concentrations in serum.
Phenylalanine Reference Interval
Serum phenylalanine values measured in several studies
of normal persons were, in milligrams per liter: 15 + 3
[13], 15.5 + 3.4 [17] and 21 + 5 [18]. For comparison,
mean phenylalanine concentration in 330 specimens
from infants and children measured by the amino acid
analyzer was 12 mg/L, with values ranging from 8 to 16
mg/L [19]. One recent study that compared the
fluorometric assay with MS/MS found a cutoff of 4.3
mg/dL for the flouorometric procedure compared with
3.0 mg/dL for MS/MS [14]. The difference in cutoff
values between fluorometry and MS/MS is probably due
to fluorescent interference which raises the concentration
of phenylalanine.
941
Phenylalanine

Phenylalanine Concentration in Some Genetic Defects

Blood phenylalanine
Defect concentration (mg/L) mmol/L
Classical phenylketonuria (phenylalanine hydroxylase deficiency)
Untreated 300 1.81
Treated, good control 3080 0.1820.484
Variant phenylketonuria (partial phenylalanine
hydroxylase deficiency) untreated 40200 0.2421.21
Hyperphenylalaninemia 40300 0.2421.816
Dihydropteridine reductase deficiency[20] 160530 0.969-3.208
Biopterin synthetase deficiency[20] 210490 1.2712.966
Normal 9 0.055
942
Phenylalanine

Interpretation 13 McCaman MW, Robins E: Fluorimetric method

Phenylalanine concentrations greater than 40 mg/L are for the determination of phenylalanine in serum.
rarely seen except in patients who have a genetic defect J Lab Clin Med 1962;59:885-90
in phenylalanine metabolism. Ranges reported in such 14 Chace DH, Sherwin JE, Hillman SL, Lorey F,
patients are shown in the preceding [20,21]. In a few Cunningham GC. Use of phenylalanine-to-
rare disorders, galactosemia, hereditary fructose tyrosine ratio deter-mined by tandem mass
intolerance, hereditary tyrosinemia, and transient spectrometry to improve newborn screening for
tyrosinemia of the newborn, abnormal tyrosine phenylketonuria of early discharge specimens
metabolism may cause a secondary elevation of collected in the first 24 hours. Clin Chem
phenylalanine [21]. 1998;44:2405-9
15 Tuchman M, McCann MT. Phenyl-alanine and
References tyrosine quantification by stable isotope dilution
1 Piez KA, Morris L. An automatic procedure for liquid chromato-graphy-mass spectrometry
the automatic analysis of amino acids. Anal from filter paper blood spots. Clin Chem 1999;
Biochem 1960; 1:187-201 45:571-3
2 Benson JV, Patterson JA. Accelerated 16 Ambrose JA. Report on a cooperative study of
chromatographic analysis of amino acids various fluorometric procedures and the Guthrie
commonly found in physiological fluids on a bacterial inhibition assay in the determination
spherical resin of specific design, Anal of hyperphenylalaninemia, Centers for Disease
Biochem 1965;13:265-80 Control, Health Services and Mental Health
3 Blau K. Biomedical applications of gas Administration, Public Health Service, Atlanta,
chromatography 1968 vol. 2, New York Plenum 1972, U.S. Government Printing Office.
Press. 17 Wong PWK, OFlynn ME, Inouye T.
4 Gehrke CW, Leimer K. Trimethylsilylation of Micromethods for measuring phenylalanine and
amino acids derivatization and chromatography. tyrosine in serum. Clin Chem 1964; 10:1098-
J Chromatogr 1971;57:219-38 104
5 Zagalak MJ, Curtius HC, Leimbacher W, 18 Hsia DY, Berman JL, Slatis HM. Screening
Redweik U. Quantitation of deuterated and non- newborn infants for phenyl-ketonuria. JAMA
deuterated phenylalanine and tyrosine in human 1964; 188:203-6
plasma using the selective ion monitoring 19 Berry HK, Porter LJ. Newborn screening for
method with combined gas chromatography phenylketonuria. Pediatrics 1982;70:505-6
mass spectrometry: application to the in vivo
measurement of phenylalanine-4- 20 Danks DM, Bartholome K, Clayton BE, Curtius
monooxygenase activity. J Chromatogr 1977; H, Grbe H, Kaufman S, Leeming R, et al.
142:523-31 Malignant hyper-phenylalaninemiacurrent
6 Teerlink T, van Leeuwen PAM, Houlijk A, status (June 1977). J Inherited Metab Dis 1978;
Guthrie R, Susi A. Plasma amino acid 1:49-53
determined by liquid chromatography within 17 21 Berry HK. The diagnosis of phenylketonuria.
minutes. Clin Chem 1994; 40:245-9 Am J Dis Child 1981; 135:211-3
7 Worthen HG, Lui H. Automatic pre-column
derivitization and reversed phase high
performance liquid chromatography of primary
and secondary amino acids in plasma with
photo-diode array and fluorescence detection. J.
Liq Chromatogro 1992; 15:3323-41
8 Guthrie R, Susi A. A simple phenyl-alanine
method for detecting phenyl-ketonuria in large
populations of newborn infants. Pediatrics 1963
;32:338-43
9 Bambach K, Burkey R. Micro-determination of
lead by dithizone. Indust Eng Chem Anal Ed
1942 ;14:904-7
10 Cholak J, Hubbard D, Burkey, R.
Microdetermination of lead in biological
material with dithizone at high pH. Anal Chem
1948; 20:671-2
11 Cholak J. Analytical methods for deter-
mination of lead. Arch Environ Health
1964;8:222-31
12 Kopito L, Schwachman H: Measurement of
lead in blood, urine and scalp hair by atomic
absorption spectrometry. Stand Methods Clin
Chem. 1972;7:151-62
943
Phenylalanine

Tables Method 3: High performance liquid chromatography

Table 1: Phenylalanine Methods Summary
Method 1: Automatic column chromatography
Principle of analysis: Cation-exchange
chromatography with detection of amino acids
by ninhydrin
Reaction: Triketohydrindene hydrate
(ninhydrin) + RCH(NH2)CO2 yields
diketohydrindylidenediketohydrindamine
(Reuhlmans purple)
Comments: Serum, plasma, urine, or other
physiological fluids; most accurate and
preferred
Method 2: Gas chromatography
Principle of analysis: Separation of
derivatized amino acids on glass columns;
packing selected depends on derivative;
detection by flame ionization
Reaction: Formation of N(O)-
trimethylsilylamino acid esters or of amino acid
alkyl ester followed by acylation; alkyl groups
methyl through pentyl have been used
Comments: Serum or plasma; requires
removal of interfering substances; not widely
used for routine analysis; most valuable when
using stable iosotopes
Principle of analysis: Precolumn
derivitization of amino acids, separation by
reversed-phase chromatography; fluorescence
detection
Reaction: Formation of an amino acid O-
phthala-dehyde derivative in the presence of a
sulhydryl reagent
Usage: Plasma
Comments: Derivatization may take up to 1 h
and chromatography up to 2 h
Method 4: Guthrie microbiological assay
Principle of analysis: Inhibition of growth of
Bacillus subtilis by -2-thienylalanine;
inhibition overcome by phenylalanine
Comments: Dried blood spots collected on
filter paper; routinely used for newborn infant
screening; semiquantitative
Method 5: Fluorometric
Principle of analysis: Conversion of
phenylalanine to fluorescent derivative
Reaction: Mechanism of reaction of
phenylalanine with ninhydrin to yield
fluorescent compound is not known
Comments: Serum, plasma, blood eluted from
filter paper; recommended for routine use; has
been automated

Reaction Conditions for Fluorometric Analysis of Phenylalanine

Condition Requirements
Temperature 80 C
pH 5.9
Final concentration of reagents Succinate buffer: 352 mmol/L
(before dilution with copper reagent)* Ninhydrin: 7.0 mmol/L
L-Leucyl-L-alanine: 0.59 mmol/L
Fraction of sample volume (protein- 0.0625
free supernatant)
Sample volume 100 L
Sample Protein-free supernatant of serum/plasma or protein-
free supernatant of diluted serum/plasma
methanol eluate of dried blood spot may be used
Time of reaction 25 min
Wavelengths Excitation: 365390 nm
Emission: 489515 nm
Linearity With most fluorometers the reaction is linear between
5 and 80 mg/L; the range can be extended up to 800
mg/L using the dilutions specified
Precision: mean (& CV) mg/L mg/L mg/L
16 (11.6%) 19 (12.8%)
55 (11.8%) 35 (7.1%)
110 (6.3%) 140 (10.5%) 167 (4.7%)
*Volume of copper reagent can vary from 3.0 to 4.0 mL.
**From Berry, H.K., and Porter, L.J.: Newborn screening for phenylketonuria, Pediatrics 70:505-506, 1982.
From Sigma Chemical Co., St. Louis, Mo., Technical Bulletin No 60F.
From:H.K. Berry's laboratory, Children's Hospital; Cincinnati,OH.
944
Phenylalanine
Example of Phenylalanine Concentration Calculation*
Sample g per assay mL of serum per = g per mL
size (mL) (from curve) assay
0.010 0.133 0.000833
0.020 0.264 0.00167
0.040 0.551 0.00333
*Average = 161 g/mL 1000 mL/L = 161 mg/L
1000 g/mg
Procedure: Fluorometric Determination of
Phenylalanine
(Reaction Conditions for Phenylalanine Analysis Table)
Principle
The chemical basis of the method is the specifically
enhanced fluorescence of phenylalanine after reaction
with ninhydrin in the presence of a peptide, L-leucyl-L-
alanine. A reagent blank is included to correct for
nonspecific fluorescence. This procedure is available as
a kit from Sigma Chemical Co., St. Louis, MO.
Reagents
All reagents are prepared in deionized water unless
otherwise stated.
1. Succinate buffer, 0.6 mol/L, pH 5.9. Dissolve
16.2 g of Na2C4H4O46H2O (disodium succinate
hexahydrate) in 90 mL of water. Adjust pH with 1 M
HCl. Dilute to 100 mL. Store at 0 to 5 C. Stable for 2
to 4 months.
2. Ninhydrin, 30 mmol/L (5.34 g/L). Dissolve
534 mg of ninhydrin in approximately 80 mL of water
and bring to a final volume of 100 mL. Store in a brown
bottle at 0 to 5 C. Stable for 7 to 10 days.
3. L-Leucyl L-alanine, 5 mmol/L (1095
mg/L). Prepare volumetrically by dissolving 109.5 mg
in water and dilute to a final volume of 100 mL. Store
frozen at 20 C in aliquots of 1 mL. Stable for 4 to 6
months.
4. Ninhydrinpeptide reagent. Prepare daily by
combining 5 volumes of succinate buffer reagent with 2
volumes of ninhydrin reagent and 1 volume of peptide
reagent.
5. Copper reagent
Stock solution. Na2CO3, 151 mmol/L; CuSO4, 2.40
mmol/L; sodium potassium tartrate, 2.51 mmol/L.
1. 16.0 g of Na2CO3 (anhydrous sodium
carbonate)
2. 0.650 g of NaKC4H4O66H2O (sodium
potassium tartrate hexahydrate)
3. 0.600 g of CuSO45H2O (copper sulfate
pentahydrate)
Dissolve each in about 300 mL of water; mix together in
order listed; dilute to 1 L. Store at room temperature.
Stable for 6 to 8 months.
Working reagents. For daily use dilute 1
volume of stock with 9 volumes of water.
serum
160
158
165
6. Trichloroacetic acid (TCA), 0.6 mol/L (98
g/L). Prepare volumetrically; 0.3 mol/L, 1:1 dilution of
0.6 mol/L. Stable 6 months at refrigerated temperature.
7. Phenylalanine standard solution
Stock solution, 200 mg/L (1.21 mmol/L). Dissolve 20
mg of L-phenylalanine in water and dilute to a final
volume of 100 mL. Store frozen. Stable for 1 to 2
months.
Working standard solution, 20 mg/L (0.121 mmol/L).
Combine 1 mL of stock solution, 4 mL of water, and 5
mL of TCA 0.6 mol/L to prepare phenylalanine solution
containing 20 mg/L in TCA 0.3 mol/L. Store frozen.
Stable for 2 to 4 weeks.
Assay
Equipment: Heating bath set at 80 C (ethylene glycol
produces more uniform temperature than water), a
centrifuge, and a fluorometer. One of the following is
acceptable: Turner Model 110 or 111 Fluorometer with
General Purpose Lamp; filters: Primary 7-60 (Turner
110-611); Secondary 2A (Turner 110-816) or 8 (Turner
110-817) + 65A (Turner 110-825); Farrand Model A
Fluorometer, filters as above; Aminco Bowman
Spectrophotofluorometer, activation 365 nm and
emission 515 nm.
1. Preparation of protein-free supernatant
For analysis of specimens from normal
persons: Combine 1 volume (usually 0.100 mL)
of serum or plasma with 1 volume of TCA 0.6
mol/L.
For analysis of specimens from untreated
patients with phenylketonuria: Dilute 1 volume
of serum with 9 volumes of water; combine 1
volume of diluted serum with 1 volume of TCA
0.6 mol/L.
For analysis of specimens from phenylketonuric
patients under treatment: Dilute 1 volume of
serum with 2 or 5 volumes of water (depending
on expected degree of phenylalaninemia);
combine 1 volume of diluted serum with 1
volume of TCA 0.6 mol/L.
The serum-TCA solutions are mixed well and
allowed to stand at room temperature for 10
minutes. The tubes are centrifuged at 3000 rpm
(approximately 1000 g) for 10 minutes. The
protein-free supernatant is used for analysis.
2. Standard curve. Pipet 0.010, 0.020, 0.030, and
0.040 mL of phenylalanine working standard,
945
Phenylalanine

corresponding to 0.2, 0.4, 0.6, and 0.8 g per Example

assay, into small test tubes. Adjust total volume Serum diluted 1:6 for preparation of protein-free
to 0.040 mL by addition of TCA 0.3 mol/L. supernatant:
3. Sample. Prepare three tubes for each specimen; 0.020 mL of serum + 0.100 mL of water +
use 0.010, 0.020, and 0.040 mL of protein-free 0.120 mL of TCA (0.6 mol/L) =
supernatant; bring to final volume to 0.04 mL 0.240 mL total volume
with TCA 0.3 mol/L.
4. Prepare a reagent blank by pipetting 0.040 mL Calculated volume of serum per assay:
of TCA 0.3 mol/L (in duplicate). __0.020 mL of serum__ 0.010 mL (sample
size) = 0.000833 mL
5. Add 0.60 mL of ninhydrinpeptide mixture to
0.240 mL (total volume)
each standard, sample, and blank tube. Mix
thoroughly.
One calculates the concentration of phenylalanine by
6. Place in heating bath at 80 C for exactly 25 dividing the micrograms of phenylalanine in each tube
min; remove; cool in tap water for 5 minutes. (from the standard curve) by the volume of serum per
7. Add 4 mL of dilute copper reagent, mix, and assay as shown in Phenylalanine Table: Example of
allow to stand for 10 min. Read in fluorometer. phenylalanine concentration calculation.
Calculation
The fluorescence of each standard minus the Phenylalanine Reference Interval
fluorescence of the reagent blank is plotted versus the
respective phenylalanine concentration. The
concentration of phenylalanine in the unknown specimen
is obtained from interpolation of the standard curve.
Average the results of three determinations for each
unknown. Dilute if the fluorescence is greater than the
highest value on the standard curve.
946
Phenytoin
Phenytoin
Gus Koerbin
Name: Phenytoin
Clinical significance: Refer to Chapter 47, Nervous System, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular formula: C15H12N2O2
Molecular weight: 252.26 D
Merck Index: 7130
Chemical class: Hydantoin
Structure:
i
Principles of Analysis and Current Usage
Phenytoin is an anticonvulsant drug which can be useful
in the treatment of epilepsy. The primary site of action
appears to be the motor cortex, where spread of seizure
activity is inhibited by possibly promoting sodium efflux
from neurons. Phenytoin tends to stabilize the threshold
against hyperexcitability caused by excessive stimulation
or environmental changes capable of reducing
membrane sodium gradient. Phenytoin reduces the
maximal activity of brainstem centers responsible for the
tonic phase of tonic-clonic (grand mal) seizures.
Most anticonvulsant drugs have specific ultraviolet
spectral characteristics (Figure 1) that were exploited in
the initial attempts to monitor the therapeutic levels of
these drugs [1,2]. Phenytoin was extracted into an
organic solvent and reextracted into aqueous solution,
and the absorbance of the solution was recorded (Table
1, Method 1). By use of the appropriate solvents,
phenytoin could be extracted with relative specificity.
By recording the difference spectra of the extract at two
pH values, one could also increase the specificity. The
difference spectra were obtained by use of a dual-beam
spectrophotometer and placement of one solution into
the reference compartment and the other into the usual
test compartment.
i
Phenytoin
Previous and current authors of this method:
First edition: Steven J. Soldin
Methods edition: Steven J. Soldin
Second edition: Steven J. Soldin
Third edition: Paul Salm, Paul J. Taylor, Julia M.
Potter
Fourth edition: Not updated
Fifth edition: Gus Koerbin
A second method for measuring phenytoin involved
extraction and separation by thin-layer chromatography
[3,4]. Quantitation was achieved by either ultraviolet
scanning of the plate or by elution of the drug from the
plate and recording of its ultraviolet absorption (Table 1,
Method 2).
Gas chromatographic techniques (GC) were for many
years the primary technique used to analyze phenytoin
and other antiepileptic drugs in biological fluids (Table
1, Method 3). In a review by Rambec and Meijer [5],
flame ionization detection (FID) or the more sensitive
nitrogen-selective detection (NSD) systems were the
most widely used. NSD may have considerable
advantage over FID in terms of specificity and
sensitivity, since almost all antiepileptics except valproic
acid contain nitrogen. However, NSD requires more
frequent and careful maintenance. Approximately half of
the gas chromatographic methods described by Rambec
and Meijer do not prepare derivatives of the
anticonvulsant drugs [6-9]. However, of those
procedures that use derivatization, methylation is most
commonly used [5]. In the past, column support
materials most frequently used were acid-washed
silanized materials (e.g., Gas Chrom-Q, Chromosorb-W,
and Supelcoport). Nowadays fused-silica capillary
columns are preferred because of their high temperature
stability and inertness, which not only removes the need
for derivatization but offers high sensitivity, selectivity,
and speed [10]. In the method described by Volmut et al.
[11], six antiepileptic drugs may be analyzed
simultaneously by capillary gas chromatography.
High-performance liquid chromatography assays
(HPLC) require some form of sample preparation before
separation on a column (Table 1, Method 4). Sample
preparation usually involves either organic solvent
extraction of the drugs or protein precipitation. Nearly
all methods use reverse-phase chromatography with
ultraviolet absorption spectrometry for detection [12-20]
947
Phenytoin
Immunological procedures (Table 1, Methods 5a-5j)
developed for the measurement of phenytoin include
radioimmunoassay (RIA), enzyme immunoassay (EIA),
fluorometric enzyme immunoassay (FEIA), fluorescence
immunoassay (FIA), fluorescence polarization
immunoassay (FPIA), nephelometric inhibition
immunoassay (NIA), cloned-enzyme donor
immunoassay (CEDIA), particle-enhanced turbidimetric
immunoassay (PETINIA) and dry-film multilayer
immunoassay (DFMI).
The RIA procedures (Table 1, Method 5a) involve the
standard competitive-binding procedure in which there is
competition between the labeled drug with that from the
sample for a limited amount of antibody [21].
The introduction of the enzyme-multiplied immunoassay
technique (EMIT) into the clinical chemistry laboratory
in the 1970s made possible the routine monitoring of not
only phenytoin but all the routinely prescribed
anticonvulsant drugs (Table 1, Method 5b). This
homogeneous enzyme immunoassay is based on the
competitive protein-binding technique, with an enzyme
as the label and an antibody as the binding protein.
When the enzyme-labeled drug becomes bound to an
antibody to the drug, the activity of the enzyme is
reduced. Free drug in the sample competes with the
enzyme-labeled drug for binding to the antibody,
decreasing the amount of antibody-bound labeled drug,
and thereby decreases the antibody-induced inactivation
of the enzyme. The enzyme activity then correlates with
the concentration of the free drug.
Fluorometric enzyme immunoassay (FEIA) is a
competitive-binding method that is based on the radial
partition immunoassay principle that combines solid-
phase immunological techniques and radial
chromatography (Table 1, Method 5c) [22]. In FEIA,
phenytoin in the sample is premixed with enzyme-
labeled conjugate and spotted onto a glass-fiber filter
paper which contains immobilized antibody at the
analysis site. These two constituents then compete for
antibody binding sites. After a suitable incubation
period, unbound label is removed from the center of the
reaction zone through radial elution by applying a wash
solution. This solution contains the substrate for the
enzyme that initiates enzymatic activity simultaneously
with the wash. The enzymatic rate of the bound fraction
at the center of the reaction zone is quantified by front-
surface fluorescence and is inversely proportional to the
concentration of drug present in the sample [23].
Competitive protein binding has also been used to
measure phenytoin in serum or plasma [24] (Table 1,
Method 5d). With this method, the drug is labeled with a
derivative of the fluorogenic enzyme substrate
umbelliferyl--D-galactoside. This fluorogenic drug
reagent (FDR) remains nonfluorescent under the
conditions of the assay until hydrolysis catalyzed by -
galactosidase yields the fluorescent product. When
antibody to the anticonvulsant drug reacts with the FDR,
the FDR is prevented from acting as a substrate for -
galactoside. Competitive binding reactions are set up
with a constant amount of FDR, a limiting amount of
antibody, and a patient sample containing the drug, as
follows:
1. FDR + antibody + anticonvulsant
2. AntibodyFDR + antibodyanticonvulsant + FDR
+ anticonvulsant
3. FDR -galactosidase umbelliferone + galactose
The drug in the sample competes with the FDR for the
limited number of antibody-binding sites. The FDR not
bound to antibody is hydrolyzed by -galactosidase to
produce the fluorescent product. Therefore, the
fluorescence produced is proportional to the
concentration of the drug in the sample.
Fluorescence polarization immunoassay (FPIA) uses the
principle of competitive binding assay and measures
binding of tracer directly by fluorescence polarization
(Table 1, Method 5e). The fluorescent tracer consists of
fluorescein covalently attached to the drug being
analyzed. When excited with polarized light, this tracer
will emit light with a degree of polarization that is
inversely proportional to its rate of rotation; the more
slowly the tracer molecule rotates, the greater the
polarization signal of the emitted light. When a specific
antibody binds to the tracer, its rotation is slowed, and
the emitted light increases in polarization. It is a
competitive-binding assay in which a specific
fluorescein-labeled drug competes with endogenous drug
from a patient sample for a limited amount of antibody.
The amount of bound, labeled drug is inversely
proportional to the drug in the patient sample. The
intensity of the polarized fluorescence is proportional to
the amount of antibody-bound, labeled anticonvulsant.
The greater the polarized fluorescence, the smaller the
amount of drug in the patient sample.
Another method for the measurement of phenytoin
employs a labeled prosthetic group. In this assay, a
prosthetic group necessary for the activity of the enzyme
is covalently linked to the drug to be measured: for
example, phenytoin linked to flavine adenine
dinucleotide (FAD) (Table 1, Method 5f). The FAD is
necessary for the enzyme apoglucose oxidase to function
[25]. If a limited amount of antibody to the drug is
present, the drug will bind to the antibody. In the
presence of drug in the patient sample, less FAD-labeled
drug will bind to the antibody, making more FAD
available to bind to the apoprotein. The enzyme is then
activated, and the amount of activity is proportional to
the amount of drug in the patient sample. The enzyme
converts glucose and O2 to gluconolactone and H2O2.
The color reaction is generated by use of peroxidase and
a dye substrate, 3,3, 5,5-tetramethylbenzidine, which
when oxidized, forms a blue-colored product. In this
assay, the patient serum or plasma sample is first diluted
with distilled water, and a specific volume of the
prepared specimen is placed on the reagent area of the
strip. The strip is then placed in the instrument, a solid-
phase reflectance photometer. The strip color is at 740
948
Phenytoin
nm, and the results are calculated from a calibration
curve.
A rate nephelometric inhibition immunoassay is also
commonly used (Table 1, Method 5g). In this assay,
phenytoin is covalently linked to a protein. Drug from
the patients sample competes with this protein-bound
drug for binding to an antibody. The rate of change of
light scatter is measured by the instrument [26].
A homogeneous immunoassay technique, cloned-
enzyme donor immunoassay (CEDIA), is a unique
method that utilizes genetically engineered -
galactosidase enzyme fragments (Table 1, Method 5h).
The CEDIA principle is based on the use of two inactive
-galactosidase fragments which have been constructed
using recombinant DNA techniques. One of these is
designated the enzyme donor (ED), a small amino-
terminal peptide, and the other is the enzyme acceptor
(EA), a larger protein that has residues deleted near the
amino terminus [27]. The EA and ED each on their own
show no enzymatic activity; however, active -
galactosidase forms when the fragments recombine
spontaneously. The formation of this active enzyme can
be measured spectrophotometrically by the hydrolysis of
a chromogenic substrate such as o-nitrophenyl--D-
galactopyranoside. A ligand molecule such as a drug or
hormone can be covalently linked to the ED peptide,
forming an ED-ligand conjugate, such that the enzyme
complementation reaction is not affected. Adding a
ligand-specific antibody to the system will block or
inhibit spontaneous assembly of the enzyme by binding
to the ED-ligand conjugate and therefore decrease the
amount of enzyme formed. Active enzyme formation is
influenced by the concentration of antibody available for
binding to the ED-ligand conjugate. In turn, the
concentration of available antibody is dependent on the
concentration of the analyte in the sample that can
competitively bind to the antibody [28]. The amount of
enzyme created and the rate of substrate hydrolysis is
directly proportional to the sample analyte concentration.
Another homogeneous immunoassay is the particle-
enhanced turbidimetric immunoassay (PETINIA), where
small latex particles are coated with antibody, antigen, or
hapten (Table 1, Method 5i). This phenytoin assay is a
non-isotopic competitive-binding immunoassay based on
the inhibition of agglutination of latex particles coated
with covalently-bound phenytoin by a monoclonal
antibody against phenytoin. The degree of inhibition of
the latex phenytoin particle agglutination is proportional
to the amount of drug in the patients sample. The rate of
agglutination is monitored at 340 nm by measuring the
change in turbidity after the particle-bound phenytoin,
antibody, and patients sample are mixed.
Another procedure is based on a combination of
multilayer film technology and competitive
immunoassay (Table 1, Method 5j). [29]. A coated
multilayer film chip is encased in a plastic test module.
The serum sample is applied to the topcoat layer in the
test module. This layer contains buffer components,
surfactants, and other reagents (i.e., antibody and labeled
hapten drug), as well as acting as a filter to prevent
protein interference with the immunoreaction. The
sample travels from the topcoat layer through an iron-
oxide screen to the signal layer. The iron oxide serves to
block excitation of the label outside the signal layer. The
signal layer contains a monoclonal antibody specific for
the drug being assayed, conjugated with a fluorescent-
labeled hapten in an agarose matrix coated onto a clear,
polyester-film base. The sample antigen displaces the
labeled antigen from the binding sites of the antibodies.
The unbound, labeled antigen then diffuses into the
upper layers where it cannot be measured. Equilibrium
occurs within 3 to 5 min, after which the fluorescent
signal from the remaining bound conjugate is measured
from the bottom of the test module by front-surface
fluorimetry. The fluorescent intensity measured is
inversely proportional to the antigen concentration in the
serum sample.
Reference and Preferred Methods
There is no reference method for phenytoin. The ideal
analytical procedure for anticonvulsant drugs including
phenytoin should be as follows:
1. Both accurate and precise, affording the reliable
quantitation of the antiepileptic drugs.
2. Technically easy, so that little effort is required in
training to perform the task.
3. Rapid, to minimize delay in reporting results to
clinician. Many requests for drug quantitation
arise as a result of problems in patient
management.
4. Good sensitivity and specificity
Unfortunately, no method meets all these requirements,
and the decision on which procedure to adopt depends
largely on the laboratory conditions and on personal
preferences of those involved.
Spectrophotometric methods were initially used for the
measurement of phenytoin concentrations in body fluids
[1,2]. Although differential extraction procedures were
developed to overcome interference by other substances,
particularly drugs, the specificity of these and other
wet-chemistry procedures is questionable, and the
possibility of drug interferences still exists. For these
reasons, alternative modes of analysis were developed,
employing either chromatographic or immunological
procedures.
Thin-layer chromatography procedures have been
described that semiquantitate or quantitate many
anticonvulsant drugs [3,4]. This procedure, because of
both its lack of specificity and semiquantitative nature, is
not a very popular mode of analysis.
Gas chromatography (GC) affords reliable analysis for
phenytoin analysis on microscale samples. The
procedure is easily automated and also allows
simultaneous determination of other anticonvulsive
drugs. This is an obvious advantage, since many patients
with epilepsy are on multiple drug regimens. Although
steadily losing ground to both high-performance liquid
chromatography (HPLC) and some of the competitive
binding assays, GC is useful in laboratories with a large
949
Phenytoin
workload. Disadvantages of GC include the
requirements of specialized equipment and considerable
expertise.
The adoption of HPLC for therapeutic drug monitoring
has been one of the most significant advances in the
clinical laboratory. The early spectrophotometric
methods, thin-layer chromatography, and gas
chromatography have been largely replaced by HPLC.
Some advantages of HPLC over earlier
techniques:
1. Analyte volatility and thermal stability, so
essential for gas chromatography, are not
required for this type of chromatography.
2. Relatively little sample workup is required
before analysis.
3. Characteristically, methods involving HPLC
require only a short analysis time.
4. Derivatization of the analyte is not required.
5. For laboratories with a large workload, the cost
per analysis is very low because reagent costs
are low.
6. HPLC affords good sensitivity.
7. HPLC methods are readily automated. Routine
analyses therefore require a minimum of
technician time, a major factor in arriving at the
low cost per analysis.
8. HPLC makes possible the simultaneous
analysis of most anticonvulsant drugs in a
microsample when required.
Some limitations of HPLC in drug analysis
are as follows:
1. Spectrophotometric, fluorometric, and
amperometric detectors are commonly used.
Therefore the analyte in question must either
absorb light, fluoresce, or be electrochemically
active.
2. Equipment is expensive. Investment of capital
is only warranted if the laboratory has a
substantial workload.
Phenytoin can be determined by radioimmunoassay [21].
Although extremely sensitive, these techniques have the
disadvantages of a relatively long analysis time. RIA
requires the availability of a liquid or gamma-ray
scintillation counter and availability and stability of
suitable antisera.
FPIA and immunoturbidimetric immunoassay have in
the main become the assays of choice for the clinical
laboratory. The 2007 College of American Pathologists
(CAP) Participant Summary Report showed that more
than 80% of participant laboratories used one of these
methods.
Specimen
Plasma or serum may be used for the gas
chromatographic or high-performance liquid
chromatographic methods. Serum is the preferred
specimen for the immunoassays. Sera can be stored at
room temperature for several hours. If frozen at 20C,
samples are stable for at least 1 year. Saliva may also be
used. The saliva concentration provides an estimate of
the free drug concentration for phenytoin.
Interferences
Icteric or hemolyzed specimens have less effect on the
chromatographic methods than on immunoassays.
Therapeutic Reference Interval
The therapeutic reference interval for phenytoin is 10 to
20 mg/L (40 to 80 mol/L).
Interpretation
Therapeutic drug monitoring of anticonvulsants is the
best established of any medical therapy. The prevention
of any single seizure is important because each episode
is potentially a life-threatening event. Epilepsy is a
general term for a group of disorders in which there are
recurrent seizures. The causes of epilepsy are numerous.
In many patients, a particular pathological brain lesion is
not identified (i.e., the seizures are idiopathic). The
major known causes of epilepsy include head injury,
cerebrovascular accidents (strokes), tumors (both
primary and secondary), infections (meningitis and
encephalitis), electrolyte disturbances, and alcohol and
other drugs. The description of a seizure determines the
category or type of epilepsy (Table 2). Such definition is
important; different forms of epilepsy respond to
different anticonvulsant drugs. It should not be
overlooked that the treatment of some secondary
seizures (e.g., hypoglycemia, electrolyte disturbances,
fever) is primarily the treatment and correction of the
underlying problem.
The major drugs currently used in the treatment of
epilepsy can be divided into three groups with regard to
their mechanisms of action [11,12,30]. All of them alter
neuronal conductance of ions, particularly sodium and
calcium. Phenytoin inhibits high-frequency, repetitive
neuronal firing, as does carbamazepine. Phenobarbital
and valproic acid enhance the synaptic inhibition
associated with gamma-aminobutyric acid (GABA), as
do the benzodiazepines (e.g., diazepam and
clonazepam). Ethosuximide alters calcium currents in
the thalamus. In the past few years, the practice of
monotherapy with anticonvulsant drugs has become
increasingly accepted as desirable, and a concerted
attempt should be made to control an individual patients
seizures with a single drug rather than multiple
anticonvulsants. Patients on monotherapy tend to have a
lower incidence of side effects and drug interactions, as
well as better compliance overall. However, if it
becomes clear that in an individual patient, control is not
obtained with a single agent, then combination therapy is
utilized. The combination will comprise drugs from
groups with different mechanisms of action.
The wide acceptance of the role of therapeutic drug
monitoring of anticonvulsants is due not only to the
importance of good clinical control of epilepsy, but to
the widely varying pharmacokinetics of the drugs
between individuals. The major pharmacokinetic
parameters of phenytoin are summarized in Table 3. The
timing of the sample to be assayed in relation to the dose
950
Phenytoin
is important. The most appropriate sample for routine
monitoring is the trough concentration, immediately
prior to the next dose of the drug. When introducing a
drug or adjusting the dose, it is not generally useful to
measure serum concentrations until the patient has
reached steady state. From a practical point of view, the
steady state may be considered as being approached at a
time interval equivalent to five times the elimination
half-life; for phenytoin, this is more than 5 days.
In most patients maintained at a steady dosage, stable
serum phenytoin levels are achieved, but there may be
wide inter-patient variability in levels with equivalent
dosages. Patients with unusually low levels may be
noncompliant or hypermetabolizers of phenytoin.
Unusually high levels may result from liver disease,
congenital enzyme deficiency or drug interactions which
result in metabolic interference.
The plasma therapeutic reference intervals shown are
total concentrations. If a drug is highly protein bound, a
decrease in binding protein may potentially increase the
unbound moiety of drug. If in addition, there is rate-
limited clearance of that drug, then the unbound moiety
will increase, and there will be a high likelihood of
clinical toxicity. Such is the case with phenytoin. At the
same time, the total plasma concentration may remain
within the reference interval. In these circumstances,
some practitioners would also measure the free
phenytoin concentration, utilizing equilibrium dialysis or
ultracentrifugation.
Anticonvulsant Drugs Performance Goals
Survey data from the 2007 CAP Participant Summary
Report show imprecision values (% coefficient of
variation) for phenytoin to range from 3.6% to 8.5% in
samples with drug concentrations within the therapeutic
range. Acceptable Clinical Laboratory Improvement
Amendments performance criteria (CLIA 88) for
measurement of phenytoin require that laboratories be
accurate to within 25% of the peer-group mean.
Imprecision for free phenytoin ranges between 10% and
15% at values of 1.85 mg/L with the majority of assays
undertaken by FPIA.
References
1. Plaa GL, Hine CH. A method for the
simultaneous determination of phenobarbital
and diphenylhydantoin in blood. J Lab Clin
Med 1956;47:649-57.
2. Svensmark, O, Kristensen, P. Determination of
diphenylhydantoin and phenobarbital in small
amounts of serum. J Lab Clin Med
1963;61:501-7.
3. Pippenger CE. Scott JE, Gillen HW. Thin-layer
chromatography of anticonvulsant drugs. Clin
Chem 1969;15:255-60.
4. Huisman JW. The estimation of some important
anticonvulsant drugs in serum. Clin Chim Acta
1966;13:323-8.
5. Rambeck B, Meijer JW. Gas chromatographic
methods for the determination of antiepileptic
drugs: a systematic review. Ther Drug Monit
1980;2:385-96.
6. Meijer JWA. Simultaneous quantitative
determination of anti-epileptic drugs, including
carbamazepine, in body fluids. Epilepsia
1971;12:341-52.
7. Gardner-Thorpe C, Parsonage MJH, Smethurst
PF, Toothill C. A comprehensive gas
chromatographic scheme for the estimation of
antiepileptic drugs. Clin Chim Acta
1972;36:223-30.
8. Toseland PA, Albani M, Gauchel FD. Organic
nitrogen selective detector used in gas-
chromatographic determination of some
anticonvulsant and barbiturate drugs in plasma
and tissues. Clin Chem 1975;21:98-103.
9. Rambeck B, Meijer JW. Comprehensive
method for the determination of anti-epileptic
drugs using a novel extraction technique and a
fully automated gas chromatograph.
Arzneimittelforschung 1979;29:99-103.
10. Plotczyk LL. Application of fused-silica
capillary gas chromatography to the analysis of
underivatized drugs. J Chromatogr
1982;240:349-60.
11. Volmut J, Matisova E, Pham Thi H.
Simultaneous determination of six antiepileptic
drugs by capillary gas chromatography. J
Chromatogr 1990;527:428-35.
12. Meatherall R, Ford D. Isocratic liquid
chromatographic determination of theophylline,
acetaminophen, chloramphenicol, caffeine,
anticonvulsants, and barbiturates in serum. Ther
Drug Monit 1988;10:101-15.
13. Riedmann M, Rambeck B, Meijer JW.
Quantitative simultaneous determination of
eight common antiepileptic drugs and
metabolites by liquid chromatography. Ther
Drug Monit 1981;3:397-413.
14. Soldin SJ, Hill JG. Rapid micromethod for
measuring anticonvulsant drugs in serum by
high-performance liquid chromatography. Clin
Chem 1976;22:856-9.
15. Christofides JA, Fry DE. Measurement of
anticonvulsants in serum by reverse-phase ion-
pair liquid chromatography. Clin Chem
1980;26:499-501.
16. Kabra PM, Koo HY, Marton LJ. Simultaneous
liquid-chromatographic determination of 12
common sedatives and hypnotics in serum. Clin
Chem 1978;24:657-62.
17. Kabra PM, Stafford BE, Marton, LJ.
Simultaneous measurement of phenobarbital,
phenytoin, primidone, ethosuximide, and
carbamazepine in serum by high-pressure liquid
chromatography. Clin Chem 1977;23:1284-8.
18. Ou CN, Rognerud CL. Simultaneous
measurement of ethosuximide, primidone,
phenobarbital, phenytoin, carbamazepine, and
their bioactive metabolites by liquid
chromatography. Clin Chem 1984;30:1667-70.
19. Adams RF, Vandemark FL. Simultaneous high-
pressure liquid-chromatographic determination
951
Phenytoin

of some anticonvulsants in serum. Clin Chem 25. Tybach R. Adaptation of prosthetic-group,

1976;22:25-31.
20. Szabo GK, Browne TR. Improved isocratic
liquid-chromatographic simultaneous
measurement of phenytoin, phenobarbital,
primidone, carbamazepine, ethosuximide, and
N-desmethylmethsuximide in serum. Clin
Chem 1982;28:100-4.
21. Cook CE, Christensen HD, Amerson EW.
Radioimmunoassay of anticonvulsant drugs:
phenytoin, phenobarbital, and primidone. In:
Kellaway P, Petersen IS, eds. Quantitative
Analytical Studies in Epilepsy. New York:
Raven; 1976.
22. Price CP, Newman DJ. Radial partition
immunoassay. In: Price CP, Newman DJ, eds.
Principles and Practice of Immunoassay. New
York: Stockton; 1991.
23. Giegel JL, Brotherton MM, Cronin P,
DAquino M, Evans S, Heller ZH, Knight WS,
Krishnan K, Sheiman M. Radial partition
immunoassay. Clin Chem 1982;28:1894-8.
24. Wong RC, Burd JF, Carrico RJ, Buckler RT,
Thoma J, Boguslaski RC. Substrate-labeled
fluorescent immunoassay for phenytoin in
human serum. Clin Chem 1979;25:686-91.
labeled, homogeneous immunoassay to reagent
strip format. Clin Chem 1981;27:1499-504.
26. Nishikawa T, Kubo H, Saito M. Competitive
nephelometric immunoassay method for
antiepileptic drugs in patient blood. J Immunol
Methods 1979;29:85-9.
27. Henderson DR, Friedman SB, Harris JD,
Manning WB, Zoccoli MA. CEDIA, a new
homogeneous immunoassay system. Clin Chem
1986;32:1637-41.
28. Engel WD, Khanna PL. CEDIA in-vitro
diagnostics with a novel homogeneous
immunoassay technique. J Immunol Methods
1992;150:99-102.
29. Jandreski M.A, Shah JC, Gardincius J, Bermes
EW. Clinical evaluation of five therapeutic
drugs using dry-film multilayer technology on
the OPUS immunoassay system. J Clin Lab
Anal 1991;5:415-21.
30. Albers GA, Peroutka SJ. Neurologic disorders.
In: Melmon KL, Morrelli HF, Hoffman BB,
Nierenberg DW, eds. Melmon and Morrellis
Clinical Pharmacology: Basic Principles in
Therapeutics. 3rd ed. New York: McGraw-Hill;
1992.

Tables
Table 1: Phenytoin Methods Summary
Method 1: Extraction and ultraviolet spectroscopy
Principle of analysis: Anticonvulsant extracted free of other ultraviolet compounds; ultraviolet spectrum or the
ultraviolet difference spectra are specific for the drug.
Comments: Interference with drugs and endogenous compounds
Method 2: Thin-layer chromatography (TLC)
Principle of analysis: Extracted drug separated by TLC and its Rf noted; drug quantitatively eluted and
quantified by ultraviolet spectroscopy
Comments: Technically difficult to achieve reproducibility
Method 3: Gas chromatography (GC)
Principle of analysis: C18 solid phase extraction; capillary column; flame ionization detection
Comments: Can resolve phenytoin and other anticonvulsants simultaneously; requires simple extraction
Method 4: High-performance liquid chromatography (HPLC)
Principle of analysis: Organic solvent extraction of drugs or protein precipitation of sample; anticonvulsants are
separated by reversed-phase chromatography and monitored by ultraviolet spectroscopy
Comments: Can resolve phenytoin and other anticonvulsants except valproate simultaneously; commonly used
Method 5: Competitive-binding assays
a. Radioimmunoassay (RIA)
Principle of analysis: Competitive binding of radioactive ligand; radio-labeled hapten competes with unknown
for antibody binding site
Comments: Problems of radioactive usage; slower than other immunoassays
b. Enzyme-multiplied immunoassay technique (EMIT)
Principle of analysis: Competitive binding with drug attached to enzyme
Comments: In common use
c. Fluorometric enzyme immunoassay (FEIA)
Principle of analysis: Combination of competitive binding with enzyme-labeled conjugate for antibody binding
site (on a solid-phase matrix) and radial chromatography
d. Substrate-labeled fluorescence immunoassay (SLFIA)
Principle of analysis: Competitive-binding substrate label; substrate competes with unknown antigen
for antibody binding site
Comments: Fluorescence interferes; not widely used
e. Fluorescence polarization immunoassay (FPIA)
952
Phenytoin

Table 1: Phenytoin Methods Summary (Cont)

Principle of analysis: Competitive binding with fluorescein-labeled drug; fluorescent drug competes with unknown for
antibody site
Comments: In common use (Abbott systems)
f. Ames test strip
Principle of analysis: Antibody binding to drug labeled with prosthetic group prevents glucose oxidase activity;
competition with endogenous drug allows prosthetic group to activate enzyme; glucose oxidase is coupled to
colorimetric reaction
Comments: Designed for inexpensive instrument; reflectance spectrophotometer; rapid test
g. Rate nephelometric inhibition immunoassay
Principle of analysis: Competitive binding for hapten; hapten-protein competes with unknown for antibody
binding site
Comments: Requires specialized instrument
h. Cloned-enzyme donor immunoassay (CEDIA)
Principle of analysis: Competitive binding with enzyme donor (ED)ligand conjugate for Ab binding site;
endogenous drug modulates enzymatic activity by influencing free ED-ligand conjugate available for
complementation
Comments: Can use routine clinical chemistry analyzers (e.g., Roche systems)
i. Particle-enhanced turbidimetric immunoassay (PETINIA)
Principle of analysis: A non-isotopic competitive-binding immunoassay based on the inhibition of agglutination
of latex particles. Antigen is coupled to latex particles, and the antibody remains in the solution phase; inhibition
of agglutination formation
Comments: Can use routine clinical chemistry analyzers (e.g., Siemens Dimension family of analyzers)
j. Dry-film multilayer immunoassay (OPUS)
Principle of analysis: Combination of competitive binding with a fluorescent-labeled hapten in an agarose
matrix and multilayer film technology; labeled antigen competes with sample antigen for antibody binding site
Comments: Requires specialized instrument; not widely used.

Table 2: Classification of Epileptic Seizures*

Type and Characteristics
I. Partial (focal, local). Electroencephalography (EEG) may reveal localized abnormality over seizure focus.
A. Simple partial: consciousness is not impaired. Includes Jacksonian motor epilepsy (confined to a single
muscle group or limb).
B. Complex partial: consciousness is impaired. Most commonly arise in the temporal lobe. Include many
different symptoms and behavior patterns.
C. Secondarily generalized: both (A) and (B) may spread to involve both hemispheres, with convulsions of
all limbs and loss of consciousness.
II. Generalized seizures. Simultaneous initiation of epileptic activity in both hemispheres.
Absence: Brief impairment of consciousness. Motor activity may not occur.
A. Myoclonic: isolated muscle jerks
B. Clonic: rhythmic contractions, loss of consciousness
C. Tonic-clonic (grand mal): major convulsions, loss of consciousness,
autonomic activity, and prolonged depression of central nervous system
function

* Modified from Commission on Classification and Terminology of the Internation League against Epilepsy. Epilepsia
1981;22:489-501.

Table 3: Pharmacokinetic properties.

Anticonvulsant: Phenytoin
Volume of distribution (L/kg): 0.40.8
Percent protein binding: 8893
Elimination half-life (hr): 726
Comments: Dose-dependent kinetics with saturable hepatic metabolism; induction as for phenobarbital; protein
binding important: NB hypoalbuminemia (e.g., renal disease)
953
Phenytoin

Figures
Figure 1

Anticonvulsant Drugs. Ultraviolet absorption spectra of 5 drugs in acetonitrile. Solid line, carbamazepine (10 g/mL); thick
dashed line, ethosuximide (100 g/mL); dashed and dotted line, phenytoin (100 g/mL); regular dashed line,
phenobarbital (100 g/mL); dotted line, primidone, (100 g/mL)

Figure 2

Chromatogram of a serum sample spiked 10 g/mL phenytoin (PT), with internal standard (CA) at 20 g/mL. The serum
and column were acidified prior to the extraction with 0.5 M HCl. Injected volume, 2 L of extract.

peak height relative to an internal standard. Tolybarb is

Procedure: High-Performance Liquid used as an internal standard.
Chromatography Analysis of Anticonvulsant Drugs
Specimen
Principle Serum or plasma.
Phenytoin is separated by reversed-phase liquid
chromatography and quantitated by measurement of Reagents and Materials
954
Phenytoin
1. HPLC-grade acetonitrile, methanol,
isopropanol and chloroform can be purchased
from Fisher Scientific (Winnipeg, Manitoba,
Canada).
2. Standards. The pure drug can be purchased
from Sigma Chemical Co., St. Louis, MO
(phenytoin).
3. Phosphate buffer.
a. 10 mmol/L potassium phosphate
buffer (pH 6.3) is prepared by
dissolving 2.72 g of KH2PO4 in 1900
mL of distilled water. The pH is
adjusted to 6.3 with about 2 mL of 1 M
NaOH, then the volume is brought to 2
L.
b. 1 mmol/L potassium phosphate
buffer (pH 6.8) is prepared by
dissolving 13.6 g of KH2PO4 in 90
mL of distilled water. The pH is
adjusted to 6.8 with about 3 mL of 10
M NaOH, then the volume is brought
to 100 mL.
4. Stock internal standards are prepared by
dissolving 30 mg of tolybarb in 20 mL of
methanol.
5. Solvent extraction solution is prepared by
combining 500 L of the stock internal standard
solution with 500 mL of 5% isopropanol in
chloroform.
6. Mobile phase is prepared by combining 1300
mL of phosphate buffer (pH 6.3) with 350 mL
of methanol and 350 mL of acetonitrile. The
mixture is degassed by stirring in a magnetic
mixer for 30 min, then filtered by suction
through a 0.45-m pore filter (Millipore Corp.,
Bedford, MA). The apparent pH of the mobile
phase is 7.0.
Assay
1. Equipment. The liquid chromatographic
system consists of a Series 2 pump, a model
LC-75 variable wavelength detector, an R-100
strip chart recorder (all from PerkinElmer
Corp., Norwalk, CT), and a model 7105 injector
(Rheodyne Inc., Cotati, CA). A 5-m particle
size, 250 4.6 mm Supelcosil LC-1 analytical
column and a 5-m particle size, 20 4.6 mm
Supelguard column (Supelco, Bellefonte, PA)
are used to effect the drug separations.
2. Sample preparation.
a. Add 100 L of 1 mmol/L phosphate buffer
(pH 6.8) and 1.5 mL of solvent extracting
solution (containing internal standard) to 100
L of serum or plasma.
b. Vortex mix the sample for 30 sec and
centrifuge briefly to separate the two phases.
c. Aspirate the aqueous phase (top layer) to
waste, and transfer the organic phase (lower
layer) to a clean tube.
d. The organic layer is evaporated at room
temperature with a dried air stream. The
samples are removed from the air stream on
reaching dryness.
e. Reconstitute the residue with 100 L of
mobile phase.
3. Chromatographic parameters.
a. Flow rate 2.0 mL/min.
b. Wavelength 204 nm.
c. Sensitivity 0.08 AUFS(absorbance units at
full scale).
d. Column temperature ambient.
e. Injection volume 4 L.
f. Run time 7 min.
A typical chromatogram is shown in the Figure 2:
Calculations
In each sample, peak heights (PH) of each drug
are compared to the internal standard from which peak
height ratios (PHR) are calculated. Each unknown
sample PHR is compared to a calibrator PHR of known
concentration. The calculation of drug concentration is
as follows.
[DRUG] = [CAL] PH of sample/ PH of internal
standard
PH of calibrator/ PH of internal
standard
where [DRUG] = drug concentration (mg/L)
[CAL] = calibrator concentration (mg/L)
Procedure: Antiepileptic Drugs Including Valproate
by Capillary Gas Chromatography
Principle
Phenytoin is extracted from acidified serum
samples by a simple solid-phase (C18) extraction
process. Prior to evaporation, basic methanol is added to
the eluate from extraction column to convert the acid
drug to its potassium salts in order to reduce the loss of
volatile drug. The residue is then redissolved in acidic
methanol, and 2 L is injected into the gas chromatograph
equipped with a split capillary inlet system and a flame
ionization detector.
Specimen
Serum.
Reagents and Materials
1. Phenytoin (SPOFA, Prague, Czechoslovakia
and VEB Arzneimittelwerk, Dresden,
Germany).
2. Stock standard solution of the drug is
prepared in analytical reagent-grade methanol
to a concentration of 1 g/L.
3. Stock internal standard is prepared by
dissolving 40 mg of MPPH [5-(4-
methylphenyl)-5-phenylhydantoin] in 20 mL of
methanol.
4. 0.5 M HCl solution in water. Dilute 4.2 mL of
concentrated HCl to 100 mL with H2O.
955
5. 0.05 M KOH in methanol. Add 0.281 g of
KOH to 80 mL of methanol, and bring to 100
mL with methanol.
6. 0.05 M HCl in methanol. Dilute 42 L of
concentrated HCl to 100 mL of methanol.
Assay
Equipment: A HP-5790A gas chromatograph
(Hewlett-Packard, Avondale, PA) equipped with a split
injection port (ratio 1:20) using an empty glass liner and
flame ionization detection. A fused-silica capillary
column packed with cross-linked
phenylmethylsilicone gum phase HP-5 (Hewlett-
Packard), 25 m 0.20 mm ID, 0.33 m film thickness is
used to separate the drug of interest.
Sample preparation
1. Add 100 L of 0.5 M HCl and 10 L of internal
standard solution (MPPH) to 500 L of serum,
and thoroughly vortex mix.
2. Precondition the extraction column (a 2-mL
polyethylene syringe packed with 200 mg of
Silipor C18) with a 1-mL wash of methanol
followed by a 1-mL wash of 0.5 M HCl.
3. Pour mixture (from step 1) onto the column,
and allow the mixture to flow through. Then
rinse the column with two 1-mL volumes of
H2O, and dry by applying a vacuum.
4. The drug is then eluted from the column with 1
mL of methanol. A 50-L aliquot of 0.05 M
KOH is added to the methanol solution. The
solvent is then evaporated under nitrogen at
40C in a heating block. The residue is
reconstituted in 50 L of 0.05 M HCl in
methanol.
5. Chromatographic parameters:
a. Injection temperature 240C
b. Detector temperature 300C
c. Dual ramp column temperature
program (60C to 250C)
d. Nitrogen inlet pressure 100 kPa
e. Injection volume 2 L
Calculation
1. A single-point calibration:
Peak Height Ratio (PHR) = Peak Height of Drug
Peak Height of IS
[Drug] = PHR of Drug [Standard]
PHR of Standard
where: IS = Internal Standard
[Drug] = concentration of drug (mg/L)
[Standard] = concentration of standard (mg/L)
Free Phenytoin
Abbott TDx/FLx (Abbott Laboratories TDx and
TDx/FLx Operations Manual.)
I. Principle
The concentration of free, or unbound, phenytoin
in samples can be determined using fluorescence
Phenytoin
polarization immunoassay. After a filtration step, a
clear supernatant containing the drug is analyzed.
Unlabeled drug in the sample competes with the
tracer labeled with fluorescein for limited antibody
sites. The tracer, when excited by linearly
polarized light, emits fluorescence with a degree of
polarization inversely related to its rate of rotation.
If the patient sample contains high concentrations
of the drug, the polarization value is low. If the
patient sample contains a low concentration of the
drug, the polarization value is high.
5%
II. Specimens of Choice
Serum (2 mL) drawn in a red-top Vacutainer tube
without separator gel. (NOTE: Gel tubes are not
recommended because of their potential to
decrease drug concentration during storage. This
is negated somewhat by always aliquoting serum
into a separate pour-off tube immediately.)
Recommended drawing time is the trough level,
immediately prior to the next scheduled dose of
drug, or during the acute phase of toxicity.
Specimens may be transported at RT. Separate
serum, and store at RT for up to 24 hr until time of
analysis or prepare ultrafiltrate (Section VI.A),
then freeze at 20C until time of testing.
Grossly hemolyzed, lipemic, or icteric specimens
are unacceptable samples for routine analysis.
III. Indications
For the monitoring of phenytoin for therapeutic
drug monitoring (TDM) purposes, as an aid in
setting the dosing regimen, and to assess toxicity
when total phenytoin drug analysis does not
provide adequate information.
IV. Reagents and Material
A. Bio-Rad Control Materials (Level 1 and 2)
Store at 4C. Stable as labeled
B. 100-L and 1-mL Eppendorf pipets
C. Amicon CentriFree Micropartition Filters
(Amicon)
D. Sample Cartridges (Abbott)
E. Sample Cuvettes (Abbott)
F. TDx Reagent Pack Adapter for FLx
G. Calibration and Routine Carousels (Abbott)
H. Buffer (Abbott); store at room temperature
The following Abbott Laboratories Phenytoin
Assay Kit Materials:
I. Phenytoin Reagent Pack* (Batch Assay)
J. Free Phenytoin Calibrators*
*Store at 4C. Stable for 1 month after opening.
V. Instrument and parameters.
Abbott TDx/FLx; however, any of the automated drug
analyzers which have a linear range to 0.5 g/mL are
acceptable
956
Phenytoin
VI. Procedure
Each routine run will generally consist of QC,
along with patients processed in singlicate.
Controls and patients require Amicon filtering;
calibrators do not.
A. Pretreatment (for control and patient
specimens)
When conducting ultrafiltration prior to
measurement of free phenytoin, check that
the room temperature at that time does not
exceed 25C. If the temperature is > 25C,
contact the area supervisor. If the sample is
ultrafiltered at room temperature of 30C to
37C, protein binding will be affected, and a
different reference range may need to be
applied:
12 g/mL at 25C
1.53 g/mL at 37C
1. Free phenytoin calibrators, controls,
and patient specimens should be mixed
by gentle inversion. Avoid foaming; if
this should occur, allow sample to sit
until foam dissipates.
2. Number an Amicon filter for each
specimen to be tested, and place in a
suitable rack. A maximum of 20
specimens can be run in one batch.
3. Pipet 1 mL of control or patient
sample to be assayed into its
corresponding Amicon filter.
4. Cap the Amicon filter sample
reservoir.
5. Place filter into a fixed-angle
centrifuge, and centrifuge samples for
25 min.
6. After centrifugation, transfer 100 L
of ultrafiltrate into appropriate sample
wells, and place in a sample carousel.
B. Calibration (no Amicon filtering required)
1. Performing the calibration:
Assays should be calibrated whenever:
A new assay is introduced
An assay activation (new reagent pool) is
issued
The memory circuit board (Board #2) is
replaced
Assay control values fall outside the acceptable
range specified in the specific assay section and
the control is not suspect.
Assays may require recalibration (when indicated
by unacceptable control data) whenever:
A new lot number of reagent is used
A new lot of buffer is used
Any dispense component is replaced
Any instrument calibration procedure is performed
A wide variation in room temperature is
experienced
The TDx/FLx System stores a calibration curve for
each assay. The random-access calibration curves
are separate and distinct from the calibration
curves stored for the batch assays. Therefore,
prior to running an assay, the instrument must be
calibrated for that assay in the mode of operation
being run.
C. Sensitivity: 0.5 g/mL; report negatives as < 0.5
g/mL
Notes and Interpretations
A. Optimal therapeutic concentrations of free
phenytoin will generally be between 1 and 2
g/mL. Frequency and severity of toxic
effects due to drug therapy will increase as
the free phenytoin level exceeds 3.0 g/mL.
Results may be reported up to 10 g/mL.
B. Specificity: no known interfering substances
 957
Phosphorus and Phosphate
Phosphorus and Phosphate
Steven C. Kazmierczak
Name: Phosphorus, inorganic phosphorus, phosphate
Clinical significance: Refer to Chapter 33, Bone Disease, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Molecular mass: Phosphate 97.00, 96.00 D (phosphorus at 30.98)
Chemical class: Inorganic anions (P is nonmetallic group 5 element)
Forms: H2PO4, HPO42
Principles of Analysis and Current Usage
Elemental phosphorus does not exist to any appreciable
extent in the body, and so methods have been directed
toward analysis of the two phosphate anions, which
interchange rapidly depending on the pH. i
The monovalent and divalent anion forms are present in
serum at about equal concentrations in acidosis, in a ratio
of 1:9 in alkalosis, in a ratio of 1:4 at pH 7.4, and in a ratio
of 100:1 in a pH 4.5 urine. Thus it is impossible to say
with any certainty what the molecular mass of inorganic
phosphate is. Therefore, the units traditionally chosen
have been milligrams or millimoles of phosphorus (in a
volume) but never milliequivalents of phosphate, since that
would change rapidly with the charge. Because
phosphorus exists in the body as phosphate, and because
that is the form measured, the two terms are used
interchangeably.
The oldest and still most commonly used methods are
based on the reaction of phosphate ions with molybdate to
form complex structures such as ammonium
phosphomolybdate; (NH4)3[P(Mo3O10)4]. One can
measure phosphomolybdate complexes directly or convert
them to molybdenum blue using a wide variety of reducing
agents (Table 1, Methods 1 to 6). Some of the earlier
methods required heating or produced colored complexes
that were unstable. Molybdenum blue is a complex
heteropolymer of unknown structure whose absorbance is
usually measured at 660 nm (Figure 1A). Approximately
20% of laboratories reporting in College of American
Pathologists (CAP) proficiency surveys use a
phosphomolybdate reduction assay.
Kutter and Cohen used stannous chloride stock reagent to
produce a more intense color with phosphomolybdic acid
(Table 1, Method 1) [1]. Hurst stabilized the dilute
stannous chloride working reagent by combining it with
i First Edition
E. Christis Farrell
Methods E. Christis Farrell
Second Edition E. Christis Farrell
Third Edition Steven C. Kazmierczak
Fourth Edition Steven C. Kazmierczak
hydrazine sulfate [2] (Table 1, Method 2). This also
permitted rapid color development at room temperature.
Methods were developed for automated analyzers based on
the work of Hurst and Kraml [3], in which the
concentration of stannous chloride is doubled to increase
linearity.
Taussky and Shorr [4] used ferrous ammonium sulfate as a
reducing agent (Table 1, Method 3). The merits and
drawbacks of the use of these different reducing reactions
have been reviewed by Martinek [5].
Simonsen et al. [6] showed that phosphate can be
measured using the 340-nm absorbance of the unreduced
phosphorusmolybdate complex (Table 1, Methods 4 and
5). This made the assay faster and simpler and improved
reagent stability. It also produced three to four times more
absorbance change than the molybdenum blue reductions.
Close to 80% of laboratories use this technique today (see
Table 2).
Enzymatic methods have been developed to measure
serum inorganic phosphate (Table 1, Method 6). A cited
advantage of this technique is the stability of organic
phosphate compounds in the neutral range, where most
enzymes function. A number of enzyme-based methods
have been described. One early assay proposed by Schultz
et al. [7] used a coupled enzyme system of glycogen
phosphorylase, phosphoglucomutase, and glucose-6-
phosphate dehydrogenase. The amount of phosphate
present was determined by measurement of the NADPH
formed. A coupled enzymatic method has also been
marketed [8]. The steps of the enzymatic reaction involve
formation of ribose-1-phosphate and hypoxanthine from
inorganic phosphate and inosine, oxidation of
hypoxanthine to uric acid and H2O2, and formation of a
chromogen-using peroxidase and a chromogenic substrate.
Ghetta et al. [9] recently described the use of
polynucleotide phosphorylase, an enzyme that catalyzes
phosphorolysis of polynucleotides, with release of
nucleotide diphosphates. In the presence of polyadenylic
acid, phosphate is converted into ADP by this enzyme.
ADP then reacts with phosphoenolpyruvate in a pyruvate
kinasecatalyzed reaction, giving rise to adenosine 5-
triphosphate and pyruvate. Finally, pyruvate oxidizes
 958
Phosphorus and Phosphate
NADH through the action of L-lactate dehydrogenase, with
a concomitant decrease in absorbance at 340 nm. Only
approximately 2% of laboratories utilize enzymatic
phosphate methods.
Reference and Preferred Methods
There is no reference method for serum phosphorus.
The majority of laboratories today perform analysis of
phosphate by employing direct analysis of the phosphorus-
molybdate complex. There are two factors that complicate
what seems to be a simple, straightforward adaptation of
this reaction to an automated system. The first factor is that
aqueous standards react considerably more slowly than
protein-based standards and may not attain the end-point at
the same time as serum unknowns [10]. Albumin can be
added to the standards to increase the reaction rate, but the
albumin may also contain phosphate. The second factor is
the unusual influence of pH; acidity is necessary, but as the
pH decreases to 1.0, the reaction is substantially slowed,
and as it is increased beyond 2.0, spontaneous reduction of
molybdate takes place [10,11].
In the automated methods, a serum blank is needed
because of the presence of a great number of 340-nm
absorbing compounds and drugs. The blank should also
contain surfactant, to clear serum turbidity, and sulfuric
acid, to correct for the effect of pH on the ultraviolet
absorption of many compounds.
Additional problems encountered with the
phosphomolybdate-reduction type of assay include
instability of the reducing reagent. This is especially true
for the stannous chloride reagent, which also shows poor
adherence to Beers law [12]. Stabilization of SnCl2 with
hydrazine sulfate has been used to overcome this
difficulty.
Crouch and Malmstadt studied the kinetics of the overall
molybdenum blue reaction and found that the rate was first
order with respect to phosphate but that the velocity was
highly sensitive to changes in pH and molybdate
concentration [13]. It has also been noted using the ANS
procedure that at higher pH readings of 2 or 3, nonspecific
reduction of the molybdate reagent takes place [14].
The reaction conditions of these reduction assays are
strong enough to hydrolyze glucose phosphate, creatine
phosphate, and other organic phosphates, creating a
positive error. Experience with the enzymatic methods
shows these to be less affected by organic phosphate
compounds [15].
Specimen
Serum is preferred by some because it has been suggested
that phosphate concentration in plasma samples are
approximately 0.2 to 0.3 mg/dL lower than in serum [16].
The release of intracellular phosphate during clotting
accounts for this difference. However, others have found
no significant difference between serum and plasma [17].
No significant difference has been seen in concentrations
from specimens collected into plastic and those collected
into glass serum separator tubes [18]. Storage of whole-
blood specimens at room or refrigerated temperatures for
prolonged periods will result in increased phosphate
concentrations. Prompt separation of serum or plasma
following collection is necessary. Separated serum or
plasma is stable for 4 days at room temperature, 7 days at
4C and indefinitely at 20C [19].
Serum values will be lower after meals, and it is best that
the patient be fasting before the sample is drawn.
Phosphate will also be lower during the menstrual period.
A diurnal variation has been found, with a minimum
occurring at 8:00 am and highest around 2:00 am [20].
Intravenous glucose and fructose also physiologically
lower phosphate.
Urine may contain larger quantities of organic phosphates,
which can decompose on exposure to elevated
temperatures. When acidified with HCl, urine phosphate is
stable for more than 6 months [12]. Urine samples are
usually diluted 1:10 with normal saline before analysis.
Interferences
Compounds such as citrate, fluoride, oxalate, tartrate, and
EDTA can form complexes with molybdate and decrease
color development. Heparin is the preferred anticoagulant.
Leakage of phosphate from cells falsely increases serum or
plasma concentrations. Whereas losses from erythrocytes
produce a positive influence, hemoglobin will produce a
strong negative photometric influence. For these reasons,
the preferred methods of phosphate analysis require correct
blanking of the individual sera. Use of 340- and 380-nm
filters and 340-nm monitoring with blanking is optimum.
Icteric sera should be properly blanked. Blanking with use
of bichromatic analysis with 340- and 380-nm wavelengths
is helpful. Lipemic sera, whose absorbance is higher at the
shorter wavelengths, can produce a positive bias with some
methods.
Phosphate Reference Intervals
Reference intervals for serum phosphate are age dependent
and also method dependent, as illustrated below. Mean
concentrations in neonates are approximately 2.0 mmol/L
higher than adults [21]. Concentrations of phosphate show
a gradual decline with age. Healthy elderly individuals
show values approximately 0.2 mmol/L lower when
compared with individuals < 60 years of age [22]. Age-
related changes are more pronounced in older males as
compared with females. Decreases in phosphate in older
men may be related to decreased tubular reabsorption due
to higher parathyroid hormone (PTH) concentrations [23].
Serum phosphate concentrations show a decrease
 959
Phosphorus and Phosphate

following food intake, as a result of phosphorylation of

glucose and metabolism [24]. Factors that may cause an
increase in serum phosphate include exercise and
pregnancy.

Serum Phosphorus Urine

Method mg/dL (mmol/L) g/24 hr
Molybdenum blue methods
Henry TCA Filtrate 12
In children 4.07.0 (1.292.26) 0.50.8
In adults 2.54.8 (0.811.55) 0.31.3
Unreduced phosphomolybdate
methods
Gilford 340 nm rate, adults 2.54.8 (0.811.55)
Abbott 340 nm kinetic, adults 2.04.6 (0.651.49)

Urinary phosphate excretion is influenced by a variety of Phosphorus Performance Goals

factors, including muscle mass, renal function, patient age,
and time of day. A diurnal variation in urine phosphate
excretion has been noted, with the highest output occurring
in the afternoon. No significant gender-related differences
have been reported.
Interpretation
Phosphorus and calcium metabolism are intertwined. In
healthy persons, as serum calcium levels rise, those of
phosphorus fall, under the influence of PTH. Control of
phosphorus levels is in part accomplished by regulation of
renal excretion. A more detailed description of the levels
of phosphorus in disorders of bone disease is presented in
Chapter 33 of Kaplan and Pesces Clinical Chemistry:
Theory, Analysis, Correlation, 5th edition. In addition to
being present in plasma, phosphorus is a major
intracellular anion. A significant portion of both plasma
and intracellular phosphorus is in the form of
organophosphate compounds such as glucose-6-phosphate
and 2,3-diphosphoglycerate. Methods of measurement of
serum phosphate usually attempt to measure only the
inorganic phosphate. However, fairly rapid fluctuations in
serum inorganic phosphate can occur because the serum
inorganic phosphate concentration is influenced by
carbohydrate metabolism. Part of the metabolic processing
of carbohydrates is the formation of organic phosphate 3-
and 6-carbon intermediates in the cell cytoplasm. This
causes a phosphate shift from plasma into the cell.
In diabetes, severe loss of phosphate is possible, since
carbohydrate metabolism is deranged, and phosphate tends
to pass from the cell into extracellular fluid and then into
plasma. It is then extracted and excreted by the kidney.
Another example of the interrelationship between
phosphorus and carbohydrates can be found in the use of
insulin therapy to treat diabetic ketoacidosis. There can be
a large migration of phosphorus from extracellular to
intracellular space accompanying the passage of glucose
into cells because of the formation of 3- and 6-carbon
phosphate intermediates.
Acceptable performance criteria for measurement of
phosphate requires that laboratories be accurate to within
the greater of 0.3 mg/dL or 10.7% of the peer-group
mean (Clinical Laboratory Improvement Amendments of
1988 [CLIA 88]). Precision goals based on biovariability
data indicate a maximum coefficient of variation (CV) of
4.3% for serum phosphate measurements and 13.2% for
urine [25]. Data from proficiency testing surveys indicate
laboratories using the phosphomolybdate method for
phosphorus meet this requirement for precision.
Intraindividual variation for serum phosphate measured
over periods greater than 1 month has been reported as
ranging from approximately 2% to 7% [26].
References
1 Kuttner T, Cohen HR. Microcolorimetric studies.
I. A molybdic acid stannous chloride reagent: the
microestimation of phosphate and calcium in pus,
plasma, and spinal fluid. J Biol Chem
1927;75:517-531.
2 Hurst RO. The determination of nucleotide
phosphorus with stannous chloride-hydrazine
sulphate reagent. Can J Biochem 1964;42:287-
292.
3 Kraml M. A semi-automated determination of
phospholipids. Clin Chim Acta 1966;13:442-448.
4 Taussky HH, Shorr J. Microcolorimetric method
for determination of inorganic phosphorus. J Biol
Chem 1953;202:675-685.
5 Martinek RG. Review of methods for determining
inorganic phosphorus in biological fluid. Am J
Med Technol 1970;32:337-345.
6 Simonsen DG, Wertman M, Westover LM, Mehl
JW. The determination of serum phosphate by the
molybdivanadate method. J Biol Chem
1946;166:747-755.
7 Schultz DW, Passanneau JV, Lowry OH. An
enzymatic method for the measurement of
inorganic phosphate. Anal Biochem 1967;19;300-
 960
Phosphorus and Phosphate
314.
8 Adam A, Boulanger J, Azzouzi M, Ers P.
Colorimetric versus enzymatic determination of
serum phosphorus. Clin Chem 1984;30:1724-
1725.
9 Ghetta A, Matus-Ortega M, Garcia-Mena J, Deho
G, Tortora P, Regonesi ME. Polynucleotide
phosphorylase-based photometric assay for
inorganic phosphate. Anal Biochem
2004;327:209-214.
10 Michelsen OB. Photometric determination of
phosphorus as molybdovanadophosphoric acid.
Anal Chem 1957;29:60-62.
11 Daly JA, Ertinghausen G. Direct method for
determining inorganic phosphorus in serum with
Centrifichem. Clin Chem 1972;18:263-265.
12 Henry RJ. Clinical Chemistry: Principles and
Technics. 2nd ed. New York: Harper & Row;
1974:726.
13 Crouch SR, Malmstadt HV. A mechanistic
investigation of molybdenum blue method for the
determination of phosphate. Anal Chem
1967;39:1084-1089.
14 Tietz N. Fundamentals of Clinical Chemistry. 2nd
ed. Philadelphia: Saunders; 1976:915.
15 Zoppi F, Giachetti M. More on enzymatic assay
of inorganic phosphorus [letter]. Clin Chem
1985;31:1242.
16 Carothers JE, Kurtz NM, Lemann J. Error
introduced by specimen handling before
determination of inorganic phosphate
concentrations in plasma and serum. Clin Chem
1976;22:1909-1912.
17 Miles RR, Roberts RF, Putnam AR, Roberts WL.
Comparison of serum and heparinized plasma
samples for measurement of chemistry analytes
[letter]. Clin Chem 2004;50:1704-1706.
18 Hill BN, Laessig RH, Koch DD, Hassemer DJ.
Comparison of plastic versus glass serum
separator (SST) blood-drawing tubes for common
clinical chemistry determinations. Clin Chem
1992;38:1474-1478.
19 Winsten S. Collection and preservation of
specimens. Stand Methods Clin Chem 1965;5:1-
17.
20 Calvo MS, Eastell R, Offord KP. Circadian
variation in ionized calcium and intact parathyroid
hormone: evidence for gender differences in
calcium homeostasis. J Clin Endocrinol Metab
1991;72:69-76.
21 Evans SE, Durbin GM. Aspects of the
physiological and pathological background to
neonatal clinical chemistry. Ann Clin Biochem
1983;20193-207.
22 Thompson SP, While DA, Hosking DJ, Wilton
TJ, Pawley E. Changes in osteocalcin after
femoral neck fracture. Ann Clin Biochem
1989;26:487-491.
23 Tietz NW, Shuey DF, Wekstein DR. Laboratory
values in fit aging individualssexagenarians
through centenarians. Clin Chem 1992;38:1167-
1185.
24 Garb S. Clinical Guide to Undesirable Drug
Interactions and Drug Interferences. New York:
Springer; 1972.
25 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernandez A, Jimenez CV et al. Current
databases on biological variation. Pros, cons, and
progress. Scand J Cin Lab Invest 1999;59:491-
500.
26 Young DS. Effects of Preanalytical Variables on
Clinical Laboratory Tests. Washington, DC:
AACC Press; 1997.
961
Phosphorus and Phosphate

Tables
Table 1: Methods of Phosphorus Analysis
Method 1: SnCl2*
Principle of analysis: Reduction of phosphomolybdate to molybdenum blue
Comments: Early continuous flow; replaced by improved methods because of reagent instability, poor linearity, and
lack of precision
Method 2: SnCl2 + Hydrazine*
Principle of analysis: Reduction of phosphomolybdate to molybdenum blue
Comments: Continuous flow; good accuracy, limited linearity, better reagent stability than method 4
Method 3: NH4FeSO4*
Principle of analysis: Reduction of phosphomolybdate to molybdenum blue
Comments: Discrete analyzers; not so sensitive as ultraviolet methods
Method 4: No reduction*
Principle of analysis: 340 nm, monitoring
Comments: Discrete and continuous flow; good accuracy, precision, sensitivity
Method 5: No reduction*
Principle of analysis: Bichromatic
Comments: Some discrete analyzers; biochromatic (340 and 380 nm) about one-half sensitivity of 340 nm; blanking
problems with hemolysis and lipemia
Method 6: Enzymatic
Principle of analysis:
_PNP
HPO42 + inosine hypoxanthine + ribose-1-phosphate
_
Hypoxanthine + 2H2O + 2O2 XOD uric acid + 2H2O2
_
H2O2 + chromogenic substrate peroxidase reddish purple complex
(Amaximum, 555 nm)
Comments: Discrete analyzers; new assay
*General principle: PO4 + H2SO4 + (NH4)6Mo7O24 4H2O Mo-PO4 complex
Mo/PO4 complex + reducing agent heteropolymeric blue complex
PNP, Purine nucleoside phosphorylase (EC 2.4.2.1); XOD, xanthine oxidase (EC 1.2.3.2); chromogenic substrate is
N-ethyl-N-(3-methylphenyl)-N-acetylethylenediamine.
962
Phosphorus and Phosphate

Table 2: Reaction Conditions for Serum Phosphate Analysis

Condition: Sample volume
Nonreduction at 340 nm*: 3 L
Reduction method: 10 L
Condition: Fraction of sample volume
Nonreduction at 340 nm*: 0.013
Reduction method: 0.014
Condition: Final concentration of reagents
Nonreduction at 340 nm*:
Ammonium molybdate: 2.25 mmol/L
Sulfuric acid: 750 mmol/L, pH 1.55
Tween 80: 0.45 mL/L
Reduction method:
Sodium acetate: 55.7 mmol/L
Acetic acid: 328 mmol/L
Sodium metabisulfate: 37.2 mmol/L
p-Methylammonium phenol sulfate: 11.0 mmol/L
Ammonium paramolybdate 7 H2O: 587 mol/L
H2SO4: 16.2 mmol/L
CU2+: as catalyst
Condition: Temperature
Nonreduction at 340 nm*: 37C
Reduction method: 30C
Condition: Wavelength
Nonreduction at 340 nm*: 340 nm
Reduction method: 620 nm
Condition: Reaction
Nonreduction at 340 nm*: End point with serum blank
Reduction method: Two-point kinetic
Condition: Time
Nonreduction at 340 nm*: 200 sec
Reduction method: 300 sec
Condition: Precision (X, %CV)
Nonreduction at 340 nm*: 25 mg/L, 9.2% CV; 78.7 mg/L, 5.3% CV
Reduction method: 24.9 mg/L, 7.2% CV; 77.3 mg/L, 4.8% CV
Condition: Linearity
Nonreduction at 340 nm*: 80 mg/L
Reduction method: 150 mg/L
X, Mean; CV, coefficient of variation.
*As analyzed on COBAS analyzer (Roche Inc., Nutley, NJ), using reagent obtained from Gilford Diagnostics,
Oberlin, Ohio.
As analyzed on CentrifiChem Analyzer (Baker Instruments, Dallas, Texas), using reagent from Smith-Kline-
Beckman (Sunnyvale, CA).
From College of American Pathologists quality assurance survey data for nonreduction and reduction method
groups (overall).
963
Phosphorus and Phosphate

Figure
Figure 1

A, Absorption spectra of reduced phosphomolybdate complex. Dashed line, Reagent versus H2O blank; dotted-
dashed line, serum and reagent versus H2O blank; solid line, serum and reagent versus reagent blank. Reagent used
metabisulfite as reducing agent. Serum standard contained 50 mg/L of inorganic phosphate.
B, Absorption spectra of unreduced phosphomolybdate complex. Curve a, Reagent versus H2O blank; Curve b,
hemolyzed serum versus reagent blank; Curve c, lipemic serum versus reagent blank; Curve d, icteric serum versus
reagent blank; Curve e, 80 mg/L of standard versus reagent blank. In b to e, 20 L of serum and 2.0 mL of reagent
were mixed. The final reaction was performed at pH 1.55. In this determination, hemolysis was defined as deep red
color, lipemia as creamy serum, and icteric as a bilirubin content of 24 mg/L.
964
Metanephrines, Plasma Free
Metanephrines, Plasma Free
Brett McWhinney i
Name: Metanephrines: normetanephrine (NM), 3-O-methyl-norepinephrine;
metanephrine (M), 3-O-methylepinephrine
Clinical significance: Refer to Chapter 51, Adrenal Hormones and Hypertension, in the 5th edition
of Clinical Chemistry: Theory, Analysis, and Correlation
Molecular formula: NM, C9H13NO3; M, C10H15NO3
Molecular mass: NM, 183.20 D; M, 197.23 D
Merck Index: NM, 6549; M, 5777
Chemical class: O-Methylated catechol derivative
Structure:
Principles of Analysis and Current Usage
Recently, Eisenhofer and colleagues have proposed that
plasma free metanephrines (metanephrine [M] and
normetanephrine [NM]) may be a superior analytical
tool for the investigation of the patient with a diagnosis
of pheochromocytoma [1,2].
There are theoretical reasons why plasma metanephrines
may be superior diagnostically, including that tumor
tissue has a high activity of COMT, an enzyme that
converts catecholamines to metanephrines, thus causing
selective synthesis of metanephrines in tumor tissue
[3,4,5].
Plasma concentrations of total metanephrines (i.e., free
plus conjugated metanephrines) are 20- to 30-fold higher
than free metanephrines. They therefore reflect largely
the concentration of conjugated metanephrines.
Considerable amounts of free metanephrines are
produced in adrenal medullary or pheochromocytoma
tumor tissue. Sulfate-conjugated metanephrines are
formed predominately in gastrointestinal tissue, owing to
the high concentrations of monoamine-preferring
sulfotransferase [6].
Plasma free metanephrines are present in pg/mL
concentrations, and as a result, many of the urinary
metanephrine assays do not have the sensitivity required.
In the early 1990s, Eisenhofer developed an HPLC assay
using coulometric detection to measure plasma free
metanephrines. The plasma metanephrines were
extracted from plasma and their concentration
determined by reverse-phase isocratic high-performance
liquid chromatography coupled with electrochemical
detection (HPLC-ED).
The extraction procedure serves to isolate and
concentrate the low pg/mL concentrations of free
metanephrines in plasma into a purified low-volume
form appropriate for injection onto the HPLC apparatus.
Extractions are performed by ion-exchange
chromatography, using AccuCAT silica solid-phase
cation exchange columns. Extraction columns are first
washed with the eluting solution (10% ammonium
i
Plasma Free Metanephrines
New method
Fifth edition: Brett McWhinney
hydroxide/methanol [1:3]) to remove any impurities that
might otherwise contaminate the final elution. Columns
are then activated by washing with a solution of 1%
KOH in methanol to bring up the negative charges on
the column matrix. Residual 1% KOH in methanol is
washed off the column with water, after which the
columns are loaded with sample. Cationic species in
samples of plasma are retained on the column, while the
bulk of the sample is eluted to waste. Addition of 1 mL
of water prior to loading of the columns with samples
helps to retard positively charged metanephrines on the
column, increasing their recovery from less than 30% to
typically more than 70%. Subsequent washing of
columns with acetic acid followed by a slightly basic
solution of ammonium phosphate, water, and methanol
serves to further purify the finally eluted sample.
Glass tubes are placed under the columns, and a solution
of 10% ammonium hydroxide/methanol solution (1:3) is
passed through the columns to disrupt ion bonding of
analyte to column matrix and the elute collected and
concentrated using nitrogen evaporation at 80C. The
metanephrines, unlike catecholamines, are stable in the
highly basic ammonium hydroxide/methanol solution,
which is fully evaporated away under nitrogen. The
residue is resuspended in a minimum quantity of mobile
phase ready for injection onto the HPLC system.
The HPLC-EC method relies on separation of the
analytes of interest on an HPLC column followed by
their ordered elution off the column and post-column
detection by way of the current generated from the
oxidation or reduction of the eluting analytes. The triple
electrode system allows the removal of acetaminophen at
the first reducing electrode. The second measuring
electrode is therefore free from interference. This is an
improvement on the original Eisenhofer method, where
acetaminophen interfered with NM quantitation.
Enzymatic immunoassays based on microtiter plate
technology have recently been developed for the
determination of plasma free NM and M. However,
compared to HPLC assays, they are very susceptible to
965
Metanephrines, Plasma Free

cross-reactivity and artifacts caused by non-specific
binding. There is also very poor agreement between
results obtained from different immunoassay
manufacturers. These assays also lack an internal
standard to monitor recovery through the extraction
process.
The heparinized blood sample should be centrifuged and
separated within 2 hours of blood collection. Each
aliquot should be at least 2 mL, placed into plastic tubes
clearly marked with the date and patient ID and frozen
immediately (e.g., on dry ice). The sample should be
stored at 20C or colder [8].

Recently, liquid chromatography with tandem mass Interferences

spectrometry (LC-MS/MS) methods have been The method of Eisenhofer suffers from interference by
developed. Sample preparation still involves off-line acetaminophen [9,10], a frequently prescribed analgesic
solid-phase cleanup prior to injection on to the LC- drug. Modifications to the original assay with changes to
MS/MS. Further on-line cleanup occurs using reverse- the extraction procedure and detector settings have now
phase columns and switching valves prior to the elution removed this problem. Medication-associated false-
into the MS/MS [7]. positive results can occur with phenoxybenzamine and
tricyclic antidepressants, producing levels 1.9- to 2.6-
Reference and Preferred Methods fold higher results compared with patients on no
There is no reference method for either plasma M or medications [11].
NM. The HPLC technique is the most popular because it
can separate M and NM quickly (less than 20 min) with Metanephrines Reference Interval
almost no interference from any drug or natural Using the original Eisenhofer method, the following
biochemical. The sensitivity of the HPLC methods with concentrations of NM and M were found in 178
electrochemical detection is in the picomole range, with normotensive individuals, including 100 men and 78
between-run coefficients of variation (CVs) reported to women, ages ranging from 18 to 72 yrs [12].
be less than 10%. Because the HPLC analysis provides
highly specific measurements of both M and NM, and Reference Ranges (Adjusted for Gender or Age)
HPLC equipment is rapidly becoming routine equipment Reference Ranges (Not Adjusted for Gender or Age)
in many clinical chemistry laboratories, it is the Analyte Geometric Upper
Geometric Upper Lower
Lower
recommended method for the analysis of plasma free Analyte Mean
Mean Limit Limit
Limit Limit
metanephrines. Normetanephrine (NMN)
(pg/mL)
Normetanephrine (NMN)45 112 18
Mobile phase eluting from the outlet of the analytical (nmol/L)
age < 38 yr (pg/mL) 0.25
40 0.61
95 0.10
17
column and containing the analytes of interest passes Metanephrine (MN)
age < 38 yr (nmol/L) 0.22 0.52 0.09
through a triple electrode system, consisting of a series (pg/mL)
age > 38 yr (pg/mL 2751 61
125 1221
Model 5021 conditioning cell, followed by a Model (nmol/L)
age > 38 yr (nmol/L)0.140.28 0.31
0.68 0.060.11
5011 analytical cell. Both cells are connected to an ESA Metanephrine (MN)
Model 5100A Coulochem detector. The first electrode in Male (pg/mL) 29 62 21
the Model 5021 conditioning cell is set to a potential of Male (nmol/L) 0.1 0.31 0.11
about +0.41 V; the second and third electrodes are set to Female (pg/mL) 24 55 11
potentials of +0.01 and about 0.35V respectively. Female (nmol/L) 0.12 0.28 0.06
Because of variations between cells, the potential of a
new cell may require adjustment according to Reference ranges above are adjusted for a highly
voltamograms that should be generated when cells are significant influence (P<0.001) of gender on plasma
first installed. Coulometric oxidation at the first concentrations of metanephrine and total metanephrine
electrode serves to screen out irreversibly oxidized and a highly significant influence of age on plasma NM
contaminating species. The second electrode reduces and total NM.
compounds that may otherwise interfere with subsequent
detection by a reducing potential at the third analytical The development of newer assays has also resulted in the
electrode. The hydroxyl group on metanephrines is establishment of other reference ranges. The upper limits
normally in the reduced state, but the compounds are of the reference ranges were 165 pg/mL for NM and 100
reversibly oxidized and reduced so that measurement by pg/mL for M [6,7,13,14].
reduction is possible after the compounds are oxidized at
the first electrode in the conditioning cell. These values are higher than those established by
Eisenhofer. It has been proposed that the differences in
the upper limit may be attributable to variations in the
Specimen specimen collection procedures [15].
It is preferable that the sample be obtained in the
morning after an overnight fast. The sample should be Slight to moderate elevation of NMN (112 to 400
collected into an evacuated glass collection tube pg/mL) or MN (61 to 236 pg/mL) can be seen in false-
containing heparin (in any form) as an anticoagulant positive patients, and this maybe due to hypertension or
(e.g., a green-top Vacutainer tube). It is preferable, but medications. Additional biochemical tests can provide
not essential, to draw the sample without a tourniquet further information for firmly establishing a diagnosis.
[7]. Conclusive diagnosis of pheochromocytoma in most
patients can be achieved by coupling the clonidine
966
Metanephrines, Plasma Free
suppression test with measurements of plasma NMN
[11].
Interpretation
Testing for the catecholamines and their metabolites is
important in the diagnosis of the cause and extent of
certain neurological and endocrinological disorders such
as orthostatic hypotension and pheochromocytoma.
Pheochromocytoma is an uncommon but important
cause of hypertension. Its secretory products
catecholamines and their metabolic productsare
powerful pressor agents, and severe hypertension and
stroke may be the sequelae if the condition is not
identified and treated [16,17].
There is substantial literature which suggests that many
cases of pheochromocytoma are not picked up during
life [18].
To date, diagnostic strategies for pheochromocytoma
have depended primarily on the measurement of
catecholamines and their metabolites in a 24-hour timed
urine collection. These collections are inconvenient for
patients, and inaccurate collections are not uncommon.
There is disagreement as to the testing protocol that
should be followed, because each urine catecholamine
fraction will occasionally be normal in a person with
significant clinical disease [19]. The secretory products
of pheochromocytoma tissue may also be produced to
excess or accumulate in conditions such as stress, renal
failure, and certain drug therapies [20]. This is
potentially a particular problem in the acute hospital
setting with seriously ill, stressed patients [3,21].
Plasma catecholamines have been advocated as a
diagnostic tool, but this has not proved popular, firstly
because of false negatives resulting from overlap
between some pheochromocytomas and the upper limit
of the normal range, and secondly because of frequent
false-positive results due to sympathoadrenal
overactivity in other pathophysiological states and
inadequately prepared patients [11,22].
Eisenhofer has presented compelling evidence that
plasma metanephrines may be superior to both urine
catecholamine fractions and plasma catecholamines
because of tumor-specific synthesis of metanephrines
[5,23]. These theoretical grounds appear to be borne out
by clinical application [1,11,24]. Therefore, a negative
test for plasma free metanephrines means that a
pheochromocytoma is highly unlikely, so no other tests
are necessary [6].
Plasma free metanephrines provide a very good initial
diagnostic test for exclusion of pheochromocytoma.
Interindividual variations of plasma free metanephrines
are lower, and plasma half-lives are longer than those of
catecholamines [1].
Comparison of biochemical tests or combinations of
tests has shown that plasma free metanephrines provide
the best test to diagnose pheochromocytoma. The
reasons include:
1. Plasma free metanephrines are produced
continuously by metabolism of catecholamines
within pheochromocytoma cells; this is in
contrast to the episodic secretion of
catecholamines.
2. Stress causes large increases in catecholamine
release, whereas plasma free metanephrines
remain relatively unaffected.
3. Urine markers such as HMMA and
metanephrines are different metabolites from
free metanephrines and are produced in
different parts of the body by metabolic
processes not directly related to the tumor [6].
4 The conjugated urine metanephrines are
produced outside of tumor tissue, and HMMA
(VMA) is produced predominantly in the liver.
The clonidine suppression test was first introduced to
distinguish patients with pheochromocytoma from those
with falsely positive results. Clonidine suppresses
norepinephrine release by sympathetic nerves.
Therefore, a decrease in plasma norepinephrine after
clonidine administration suggests the elevated result was
due to sympathetic activation, and a lack of decrease
suggests a pheochromocytoma. Compelling evidence has
shown that the coupling of the clonidine suppression test
with measurements of plasma NM and M can give a
conclusive diagnosis of pheochromocytoma [25].
Metanephrines Performance Goals
For M measured in plasma, intra- and interassay CVs of
7.0% and 12%, respectively, have been reported at a
concentration of 153 pmol/L measured by HPLC. For
NM at 671 pmol/L measured in plasma by HPLC, intra-
and interassay CVs of 4.7% and 12%, respectively, have
been reported [10]. The HPLC method for the analysis
of plasma free NM and M has been found to be highly
precise in the low normal and high abnormal values
relevant for the screening and diagnosis of
pheochromocytoma.
References
1 Raber W, Raffesberg W, Bischof M, Scheuba
C, Niederle B, Gasic S et al. Diagnostic efficacy
of unconjugated plasma metanephrines for
detection of pheochromocytoma. Arch Intern
Med 2000;160:2957-2963.
2 Eisenhofer G. Biochemical diagnosis of
pheochromocytoma: is it time to switch to
plasma free metanephrines? J Clin Endocrinol
Metab 2003;88:550-552.
3 Goldstein DS, Eisenhofer G, Kopin IJ. Sources
and significance of plasma levels of catechols
and their metabolites in humans. J Pharm Exp
Ther 2003;305:800-811.
4 Eisenhofer G, Walther MM, Huynh T, Sheng-
Tinget Li, Bornstein SR, Vortmeyer A et al.
Pheochromocytomas in von Hippel-Lindau
syndrome and multiple endocrine neoplasia
type 2 display distinct biochemical and clinical
967
Metanephrines, Plasma Free
phenotypes. J Clin Endocrinol Metab
2001;86:1999-2008.
5 Eisenhofer G, Keiser H, Friberg P, Mezey E,
Huynh TT, Hiremagalur B et al. Plasma
metanephrines are markers of
pheochromocytoma produced by catechol-O-
methyltransferase within tumours. J Clin
Endocrinol Metab 1998;83:2175-2185.
6 Eisenhofer G. Free or total metanephrines for
diagnosis of phaeochromocytoma: what is the
difference? Clin Chem 2001;47:988-989.
7 Lagerstedt SA, OKane DJ, Singh RJ.
Measurement of plasma free metanephrine and
normetanephrine by liquid chromatography-
tandem mass spectrometry for diagnosis of
phaeochromocytoma. Clin Chem 2004;50:603-
611.
8 Willemsen JJ, Sweep CJG, Lenders JWM, Alec
Ross H. Stability of plasma free metanephrines
during collection and storage as assessed by an
optimized HPLC method with electrochemical
detection. Clin Chem 2003;49:1951-1953.
9 Roden M, Raffesberg W, Raber W, Niederle B,
Waldhausl W, Gasic SL. Quantification of
unconjugated metanephrines in human plasma
without interference by acetaminophen. Clin
Chem 2001;47:1061-1067.
10 Lenders JW, Eisenhofer G, Armando I, Keiser
HR, Goldstein DS, Kopin IJ. Determination of
metanephrines in plasma by liquid
chromatography with electrochemical detection.
Clin Chem 1993;39:97-103.
11 Eisenhofer G, Goldstein DS, Walther MM,
Friberg P, Lenders JWM, Keiser HR.
Biochemical diagnosis of pheochromocytoma:
how to distinguish true- from false-positive test
results. J Clin Endocrinol Metab 2003;88:2656-
2666.
12 Lenders JWM, Pacak K, Eisenhofer G. New
advances in the biochemical diagnosis of
pheochromocytoma, moving beyond
catecholamines. Ann NY Acad Sci
2002;970:27-40.
13 Heider EC, Davis BG, Frank EL.
Nonparametric determination of reference
intervals for plasma metanephrine and
normetanephrine. Clin Chem 2004;50:2381-
2384.
14 Sawka AM, Jaeschke, Singh RJ, Young WF. A
Comparison of biochemical tests for
pheochromocytoma: measurement of
fractionated plasma metanephrines compared
with the combination of 24-hour urinary
metanephrines and catecholamines. J Clin
Endocrinol Metab 2003;88:553-558.
15 Grouzmann E, Fathi M, Gillet M, de Torrente
A, Cavadas C, Brunner H et al. Disappearance
rate of catecholamines, total metanephrines, and
neuropeptide Y from the plasma of patients
after resection of pheochromocytoma. Clin
Chem 2001;47:1075-1082.
16 Lenders JWM, Eisenhofer, Mannelli M, Pacak
K. Phaeochromocytoma. Lancet 2005;366:665-
675.
17 Eisenhofer G, Goldstein DS, Sullivan P, Csako
G, Brouwers FM, Lai EW et al. Biochemical
and clinical manifestations of dopamine-
producing paragangliomas: utility of plasma
methoxytyramine. J Clin Endocrinol Metab
2005;90:2068-2075.
18 McNeil AR, Blok BH, Koelmeyer TD, Burke
MP, Hilton JM. Phaeochromocytoma
discovered during coronial autopsies in Sydney,
Melbourne, and Auckland. Aust N Z J Med
2000;30:648-652.
19 Lenders JWM, Pacal K, Walther MM, Lineham
WM, Mannelli M, Friberg P et al. Biochemical
diagnosis of pheochromocytoma. JAMA
2002;287:1427-1434.
20 Eisenhofer G, Huysmans F, Pacak K,
MacClellan MW, Sweep F, Lenders JWM.
Plasma metanephrines in renal failure. Kidney
Int 2005;67:668-677.
21 Raber W, Raffesberg W, Waldhausl W, Gasic
S, Roden M. Exercise induces excessive
normetanephrine responses in hypertensive
diabetic patients. Eur J Clin Invest
2003;33:480-487.
22 Pacak K, Ilias I, Adams KT, Eisenhofer G.
Biochemical diagnosis, localization and
management of pheochromocytoma: focus on
multiple endocrine neoplasia type 2 in relation
to other hereditary and sporadic forms of the
tumour. J Int Med 2005;257:60-68.
23 Eisenhofer G, Rundquist B, Aneman A, Froberg
P, Dakak N, Kopin IJ et al. Regional release
and removal of catecholamines and
extraneuronal metabolism to metanephrines. J
Clin Endocrinol Metab 1995;80:3009-3017.
24 Eisenhofer G, Lenders JWM, Goldstein DS,
Mannelli M, Csako G, McClellan MW et al.
Pheochromocytoma catecholamine phenotypes
and prediction of tumor size and location by use
of plasma free metanephrines. Clin Chem
2005;51:735-744.
25 Goldstein DS, Eisenhofer G, Flynn JA, Wand
G, Pacak K. Diagnosis and localization of
pheochromocytoma. Hypertension
2004;43:907-910.
968
Metanephrines, Plasma Free

Methods of Analysis of Plasma Free Metanephrines

Method 1: High-performance liquid chromatography mass spectrometry
Principle of analysis: Metanephrines are extracted off-line using solid-phase extraction followed by
on-line sample cleanup.
Comments: Expensive capital equipment; rare; good sensitivity and specificity; fairly labor intensive
Method 2: High-performance liquid chromatography
Principle of analysis: Free metanephrines are purified by mixed-mode ion exchange solid-phase
extraction chromatography. Separation into NM and M fractions by reversed-phase HPLC with
detection by electrochemical detectors.
Comments: Common; excellent sensitivity and very good specificity; reasonably rapid; preferred
method
Method 3: Immunoassay (RIA or ELISA)
Principle of analysis: Antibodies produced against N-acetylated derivatives of metanephrine or
normetanephrine by coupling to bovine serum albumin as hapten
Comments: Requires overnight incubation; lack of internal standard

phosphoric acid. Bring volume to 1000 mL with

Procedure: Plasma Free Metanephrines by
High-Performance Liquid Chromatography
(HPLC)
Brett C. McWhinney
Principle
The plasma free metanephrines are extracted from
plasma and their concentration determined by
reverse-phase isocratic high-performance liquid
chromatography coupled with electrochemical
detection.
The extraction procedure isolates and concentrates
the low picomolar concentrations of free
metanephrines in plasma into a purified low-
volume form appropriate for injection onto the
HPLC apparatus. Extractions are performed by
reverse-phase ion-exchange chromatography using
silica solid-phase octyl + benzyl sulfonic acid
cation-exchange columns. Hydrophobic/cationic
species in samples of plasma are retained on the
column, while the bulk of the sample is eluted to
waste. Subsequent washings of the columns
removes compounds that were only retained by
either hydrophobic or ion-exchange interaction.
The metanephrines are eluted and concentrated
using nitrogen evaporation at 80C. The
metanephrines, unlike catecholamines, are stable in
the highly basic elution reagent. The residue is
resuspended in a small volume of mobile phase and
injected onto the HPLC system.
REAGENTS AND EQUIPMENT
Reagents
1. 100 mM Sodium Phosphate, pH 6.0
Dissolve 14.2 g disodium phosphate in 800 mL
deionized water. Adjust the pH to 6.0 with
deionized water.
2. 10 mM Ammonium Phosphate, pH 8.5
Dissolve 2.3 g monobasic ammonium phosphate
(MW = 115.03) in 800 mL deionized water.
Adjust the pH to 8.5 with 5% ammonium
hydroxide (5 mL ammonium hydroxide in 95 mL
deionized water). Bring volume to 1000 mL with
deionized water, and add 0.1 g EDTA. Store
refrigerated for up to 6 months. This is a 20 mM
ammonium phosphate solution. Prepare 10 mM
solution by mixing 100 mL deionized water with
100 mL of the 20 mM solution.
3. 5 % Ammonium Hydroxide/95%
Methanol Solution
Add 5 mL concentrated ammonium hydroxide
solution and 95 mL HPLC-grade methanol.
Prepare this solution fresh immediately before
extraction. Sufficient for 24 samples. Store in a
flask and cover to minimize contact with air
while in use. Expiry: 3 hours.
5. 0.1 M Acetic Acid
Dilute 6 mL of glacial acid with deionized water
to a total volume of 1 L. Store at room
temperature for up to 1 year.
6. Mobile Phase
(a) Mobile Phase Stock Solution
Add 78 g of sodium dihydrogen orthophosphate
(NaH2PO4.2H2O), 1.28 g of 1-octane sulfonic acid,
and 100 mg EDTA to a clean 1-L flask. Make up
to 1 L with deionized water, and stir thoroughly to
dissolve.
Store at 4C for up to 6 months.
(b) Mobile Phase for HPLC-EC Assay of
Metanephrines
Add 200 mL of mobile-phase stock solution and
150 mL of HPLC-grade acetonitrile to a clean 2-
L flask. Make up to 1950 mL with deionized
water, and adjust the pH with HPLC grade
phosphoric acid, with stirring, to pH 3.25. Make
up to a final volume of 2 L with deionized water,
and filter through a 0.45 m type HV (Millipore)
969
Metanephrines, Plasma Free
filter under vacuum. Decant to a clean bottle, and
pump through HPLC system.
Store at 4C for up to 2 months
(depending on background current).
NOTE: During equilibration of a new column with
mobile phase, always run mobile phase at 1
mL/min for at least 12 hours while recycling and
before the injection of standards. Adjust pH and
acetonitrile concentration as required.
7. Standard Solutions
(A) Plasma Metadrenaline Internal
Standard: 4-Hydroxy-3-
Methoxybenzylamine (HMBA)
Plasma Metanephrine Stock Internal
Standard (500 M HMBA)
Weigh out 0.10 g of 4-hydroxy-3-
methoxybenzylamine. Dissolve in 100 mL of 0.05
M HCl. Store frozen for up to 6 months.
Plasma Metanephrine Intermediate Internal
Standard (10 M HMBA)
Dilute plasma metanephrine stock internal
standard 1:50 with 0.05 M HCl. Store at 4C.
Plasma Metanephrine Internal Standard
(40 nM HMBA)
Dilute plasma metanephrine intermediate
internal standard 1:250 with 0.2 M acetic acid.
Store at 4C.
(B) Aqueous Metanephrine Standards
Stock Standards (500 M)
(a) Metanephrine Stock Standard.
Dissolve 116.8 mg of metanephrine in 1000
mL 0.05 M HCl. Store at 20C.
(b) Normetanephrine Stock
Standard.
Dissolve 109.8 mg of
Normetanephrine in 1000 mL 0.05 M
HCl. Store at 20C.
Plasma Metanephrine Intermediate
Standards (10 M)
Dilute Metanephrine and
Normetanephrine stock standards
1:50 with 0.1 M acetic acid. Store at
4C.
(C) Calibration Standard (Plasma Metanephrine
Standard 80 nmol/L)
(a) Combine Metanephrine and Normetanephrine
into one calibration standard (80 nmol/L).
Add 2 mL of each intermediate standard,
and make up to 250 mL with 0.1 M acetic
acid
(b) Store in 2.5-mL aliquots at 20C.
(D) External Metanephrine Standard
Working Standard For HPLC-EC
Calibration
The standard mix (2400 pmol/L) is
prepared by mixing together 300 L of the
Calibration Standard (Plasma
Metanephrine STD 80nmol/L), 500 L of
Plasma Metanephrine Internal Standard
(50 nmol/L HMBA in acetic acid) and
400 L of Mobile Phase. Inject 100 L
on HPLC column.
NOTE: When calibrating HPLC-EC,
always compare peak heights of the new
working standard solution with those of
that used for the previous assay. If peaks
areas differ by more than 5%, make up
new working standard solution, and use
solution that compares most favorably
with the other or the standard from the
previous HPLC run. Note also, however,
that new detector cells or mobile-phase
conditions or extended time between
assays may give rise to different detector
responses to injection of identical
standards.
Equipment
1. LABORATORY APPARATUS
a. Solid-phase extraction (SPE)
cartridges
Clean screen columns 10 cc/200 mg
UCT Catalog no. ZSDAU020 (50
columns/box)
b. Waters 24-port extraction
manifold attached to a vacuum pump
2. HPLC SYSTEM
a. Pump
Waters pump, Model 616.
Flow rate usually 1.0 mL/minute
b. Autosampler
Waters 717 plus autosampler
with heater/cooler module
attached
c. Column
Phenomenex stainless steel
Luna C18 (2) columns, 4.6 mm
150 mm with 5-m packing.
Part No. 00F-4252-E0.
Immediately preceding the
analytical column is an
integrated Phenomenex
Security Guard holder
containing a C18 insert. Part
No. AJ0-4287.
d. Data Manager
970
Metanephrines, Plasma Free

Waters Empower Build No. 3. Activate columns with 1 mL 100

2154, Chromatography mM sodium phosphate buffer, pH
Manager 6.0, in methanol under gravity. A
e. Detector light vacuum (1 to 5 mmHg) may be
ESA Coulochem II 5200A used to start the flow, but once it has
Coulometric Detector (ECD) commenced, release any vacuum
with conditioning and present. Again, do not allow columns
analytical cells dedicated to to dry out.
Plasma Metanephrine Assay.
Settings as shown in table Sample Extraction Procedure
below:
1. Remove standards, QC, and patient
ECD SETTINGS plasma samples from 20C freezer,
Guard Cell and thaw at room temperature. Spin
Cell Potential +450 mV thawed plasma at 3000 g for 10 min
Channel 1 to pellet fibrin and other particulate
Cell Potential +10 mV matter.
Full Scale Range 100 nA 2. To the standard tube, add 30 L of
Filter 5 sec Plasma Metanephrine Standard (80
nmol/L) and 2 mL of water.
Signal Output Voltage 1V
Baseline Offset 0% 3. To the appropriate 10-mL conical
Channel 2 plastic tubes, add 1 mL of QC and 1
mL of patient plasma (or 1 mL of
Cell Potential -350 mV
water for the blank).
Full Scale Range 100 nA
Filter 5 sec
4. Add 1 mL of deionized water to all
tubes, followed by 50 L of Plasma
Signal Output Voltage 1V
Metanephrine Internal Standard (50
Baseline Offset 0% nM HMBA).
N.B. These values must be determined for
5. Cap and vortex the tubes for 15 sec,
and centrifuge at 3000 rpm for
each cell and will vary depending on the age
10min. (It is essential that samples
of cells and mobile phase.
are free of particulate matter for their
efficient passage through extraction
columns).
Standards, controls, and specimens are 6. Add the samples to their appropriate
treated similarly. The extracts are stable extraction column. Pass the whole
when stored at 4C for up to 4 days. solution slowly through the column
under gravity. (Elution time should
Once step 1 of the Column Preparation be between 5 and 10 min.)
Procedure has started, begin step 1 of the 7. Wash columns with 3 mL water.
Sample Extraction Procedure so that the 8. Wash columns with 1 mL 100 mM
samples are ready for extraction when acetic acid under gravity. When flow
the last step of the Column Preparation is finished, dry the column out by
procedure is finished. increasing the vacuum to 20 mmHg
for 3 minutes.
Column Preparation Procedure
9. Wash columns with 2 mL 10 mM
1. Wash extraction columns with 3 mL ammonium phosphate buffer pH 8.5
of methanol under gravity. A light under gravity. A light vacuum (1 to 5
vacuum (1 to 5 mmHg) may be used mmHg) may be used to start the
to start the flow and draw through flow, but once it has commenced,
approximately 1 mL, then release release any vacuum present.
any vacuum present. Do not allow 10. Wash columns with 2 mL deionized
columns to dry out. water under gravity. A light vacuum
2. Wash columns with 2 mL deionized (1 to 5 mmHg) may be used to start
water under gravity. A light vacuum the flow, but once it has commenced,
(1 to 5 mmHg) may be used to start release any vacuum present. Elute all
the flow, but once it has commenced, water.
release any vacuum present. Again, 11. Wash columns with 3.0 mL of 100%
do not allow columns to dry out. methanol. A light vacuum (1 to 5
971
Metanephrines, Plasma Free
mmHg) may be used to start the
flow, but once it has commenced,
release any vacuum present. Allow
methanol to drip through the
columns. Apply full vacuum to
remove any residual methanol.
12. Place labeled glass culture tubes (13
100 mm) into a rack, and position
under the needles.
13. Elute samples with 3 mL of 5%
NH4OH/methanol under gravity. A
light vacuum (1 to 5 mmHg) may be
used to start the flow, but once it has
commenced, release any vacuum
present. Finally, use a vacuum to
elute all the NH4OH/methanol.
14. Take glass tubes to the heating block
set at 80C, and dry the sample down
completely using a strong stream of
nitrogen (approximately 20 min).
15. Dissolve the residue in 120 L of
mobile phase, vortex for 30 sec, and
centrifuge the tubes at 2000 rpm for
5 min.
16. Transfer the samples to limited-
volume vials, and inject 100 L onto
the HPLC.
After extraction, samples are stable at
4C for at least 4 days.
System Settings Summary
Injection volume: 100 L
Flow rate: 1.0 mL/min
Run time: 40 min
Recycle mobile phase: yes
Retention time for internal standard (IS)
must be 17 to 19 minutes. Total run time is
~40 minutes
CALCULATIONS
The Empower Chromatography Data
Manager measures the peak heights,
calculates the ratios of the analytes of
interest to the IS, and constructs a
standard curve of this ratio versus
concentration in pmol/L. The
concentrations of the unknowns are then
determined from this standard curve,
using their calculated peak height ratios.
Check the internal standard peak heights.
Variations of up to 20 % are to be
expected, but low recoveries of the
internal standard suggest some problem
with the sample clean-up, and the sample
should be re-assayed.
For manually calculating the concentration
of an analyte, use the following formula:
PH UNK PH ISSTD
Concentration pmoles / L Std CONC
PH STD PH IS UNK
PH=peak height of UNK or STD
UNK = analyte of interest in unknown
STD = analyte of interest in standard
IS = internal standard
972
Metanephrines, Plasma Free

450.00
IS
400.00

350.00

300.00

250.00 NM

200.00 M
mV 150.00

100.00

50.00

0.00

-50.00

-100.00

-150.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

Minutes
Figure 1: Ion-pair high-performance liquid chromatography (HPLC) analysis of a normal plasma sample by method
described in text: electrochemical detector (ECD) response versus elution time. Normetanephrine (NM) 15 minutes,
metanephrine (M) 20.5 minutes, and internal standard (IS) 28 minutes.
973
Metanephrines, Plasma Free

A
1000.00

900.00

800.00

700.00

600.00

mV 500.00
NM
400.00 M IS
300.00

200.00

100.00

0.00

-100.00

-200.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Minutes

Figure 2: Ion-pair high-performance liquid chromatography (HPLC) analysis of a normal plasma sample spiked with 100
mg/L acetaminophen by method described in text: electrochemical detector (ECD) response versus elution time.
Acetaminophen (A) 12minutes, normetanephrine (NM) 14 minutes, metanephrine (M) 18 minutes and internal standard
(IS) 25 minutes.
974
Porphobilinogen Screening and Quantitation

Porphobilinogen (PBG) Screening and Quantitation

Enrico Rossi

Name: Porphobilinogen, PBG, 5-(aminomethyl)-4-(carboxymethyl)-1H-

pyrrole-3-propionic acid
Clinical significance: see Interpretation page 4, line 6, left column.
Molecular formula: C10H14N2O4
Molecular mass: 226.23 D
Merck Index: 7469
Chemical class: Pyrrole

Structure:
Refer to Chapter 39, Iron, Porphyrin, and Bilirubin Metabolism, in the 5th edition of Clinical Chemistry: Theory, Analysis,
Correlation.

i extracted into the chloroform layer. Color that remains in

Principles of Analysis and Current Usage
Porphobilinogen (PBG) is a water-soluble monopyrrole
precursor of heme found in high concentrations in the
urine of persons suffering from one of the acute
porphyrias. PBG levels are highest during the acute
phases of acute intermittent porphyria (AIP), hereditary
coproporphyria, or variegate porphyria.
Production of this abnormal metabolite was the most
notable feature of the first recorded case of drug-induced
acute porphyria. It occurred in 1889 in a woman who,
after ingestion of the drug sulfonmethane (Sulfonal),
excreted red urine and died [1]. In 1931, Sachs
discovered that although the metabolite produced a color
reaction with Ehrlichs aldehyde (or diazo) reagent, it
was different from urobilinogens [2].
For many years, the standard test for clinical detection of
PBG was the test introduced by Watson and Schwartz in
1941 [3]. The method is qualitative and indicates the
presence or absence of PBG. The basis of the procedure
is the reaction between PBG and Ehrlichs aldehyde
reagent (p-dimethylaminobenzaldehyde in strong acid)
to form a red condensation compound. Sodium acetate is
added to the reagent to increase the pH. This maximizes
the color produced by urobilinogen and renders it
preferentially soluble in chloroform. Thus if color is
produced during the first step of the reaction, a
chloroform extraction is carried out. The colored
products of urobilinogen and other compounds are then
i that indole compounds which inhibit the condensation
PBG Screening and Quantitation
Previous and current authors of this method:
First edition: Not done
Methods edition: Michael D.D. McNeely, Robert S.
Franco, Tony Kraft, and William E.
Schreiber
Second edition: Not updated
Third edition: Not updated
Fourth edition: Michael D.D. McNeely
Fifth edition: Enrico Rossi
the supernatant aqueous phase is usually caused by PBG;
this can be confirmed following further extraction with
butanol [4].
Problems With the Watson-Schwartz Screening Test
The Watson-Schwartz screening test has several
shortcomings and is no longer recommended. The chief
drawback is lack of adequate sensitivity, a problem
especially evident in dark-colored urine samples [5].
Pigments usually present in urine, such as urobilinogen,
indole, and indican, not only interfere with the color
reaction, they can also lead to spurious color
development, resulting in lack of specificity [6]. Even a
PBG concentration of 50 to 100 mol/L (11 to 22 mg/L),
well above the reference range of < 9 mol/L (<2 mg/L),
may not be detected [7].
Samples from acute porphyria patients with current
symptoms usually have PBG concentrations >100
mol/L (<22 mg/L), but there is a risk that a patient with
porphyria may be missed. It is well known that diagnosis
should not be made on clinical grounds alone, and the
laboratory carries the responsibility for successful
detection of PBG.
A further difficulty is an apparent decrease in sensitivity
of the Watson-Schwartz screening test with aging of the
urine sample. This has been observed even in urine
samples tested on the same day of collection. The
reasons are not well described, but it has been suggested
reaction with Ehrlichs aldehyde reagent form on
standing [8].
A simpler method is the Hoesch test, which uses
Ehrlichs aldehyde reagent without sodium acetate. No
extractions are required. However, this test is also not
recommended; it has the same shortcomings as the
Watson-Schwartz test. Positive results have been
observed with indoles, indole-3-acetic acid, alpha-
975
Porphobilinogen Screening and Quantitation
methyldopa, phenazopyridine hydrochloride, and end-
stage alcoholic malnutrition [9]. A popular
misconception is that strip tests for urinary urobilinogen
that employ Ehrlichs aldehyde reagent will routinely
change color in the presence of PBG. Thus it has been
suggested that a negative strip test will rule out acute
intermittent porphyria (AIP). This is inappropriate, since
PBG will not reliably change the test strips color.
Recommended Porphobilinogen Screening Method
More recently, a sensitive and specific screening test has
been described which uses a syringe prepacked with an
ion-exchange resin. As originally described by Buttery et
al., [10] the test used anion-exchange resin in test tubes;
however, it is now available commercially with the resin
supplied in more convenient, disposable plastic syringes
and has been formally evaluated (Thermo Electron
Corporation, Louisville, CO) [11]. The urine sample is
adsorbed onto the resin, washed and eluted through a
series of procedures involving the addition and removal
of a 5-micron filter to the syringe and a final color
formation with Ehrlichs reagent. Visual assessment of
color formation is accomplished with use of a color chart
or a dye color calibrator.
Quantitative Porphobilinogen Assays
The earliest quantitative assays for PBG depended on
reaction with Ehrlichs reagent (p-
dimethylaminobenzaldehyde [DMAB]) to form a red
compound (Table 1, Method 1). The reaction has been
described as occurring in two phases [12]. In the first,
the rapidly occurring step, porphobilinogen (or any
pyrrole with an unsubstituted alpha hydrogen) reacts
under strongly acidic conditions to form a red
condensation product whose absorbance maximum is at
555 nm (PBG Quantitation: Figure 1 and Reaction 1). In
the second, the slower step, the red compound reacts
with an additional pyrrole molecule to form a colorless
dipyrrylphenylmethane product (Porphobilinogen
Quantitation: Reaction 2).
The rate and extent of each reaction is dependent on the
reaction conditions. Using Ehrlichs reagent prepared
with HCl, there is an increase in the rate of the second
reaction, especially at high concentrations of pyrrole,
resulting in decreased linearity of the reaction [13].
Replacing the HCl by perchloric acid (HClO4, 2-4 N),
Mauzerall and Granick increased the linearity of the
reaction and stabilized the red condensation product so
that the molar absorptivity of the reaction product was
almost twice that of the reaction product formed when
employing HCl [13].
The methods employing Ehrlichs reagent are
distinguished by the procedure used to separate PBG
from the interfering compounds that are present in urine
(Table 1).
In the quantitative method of Mauzerall and Granick
(Table 1, Method 1a), PBG is adsorbed onto Dowex 2
resin, and the resin is washed to remove other urinary
constituents. The PBG is then eluted from the column,
with essentially quantitative recovery [13], and the
eluate is reacted with Ehrlichs reagent. At very high
urinary concentrations of PBG, diluted urine can be
analyzed directly by use of Ehrlichs reagent, with
minimal interference from interfering compounds.
A more rapid procedure [14] employs batch adsorption
of PBG onto Dowex 2 resin rather than employing
column chromatography (Table 1, Method 1b). By
adjustment of the diluted urine pH to about 10 to 11,
maximal adsorption of PBG to the resin is achieved.
After several water washings of the resin and
centrifugation, the PBG is quantitatively eluted by
multiple extractions with 1 N acetic acid.
High-performance liquid chromatography (HPLC) can
also be used to separate and quantitate PBG (Table 1,
Method 2) [15]. The difficulty that arises is maintaining
HPLC equipment and reagents in readiness for a
relatively infrequent test which is usually not batched,
because the result is required urgently. Either a
combination of a strong anion-exchange stationary-phase
column and a strong cation-exchange stationary-phase
column or a single C18 reversed-phase column is used to
separate PBG from -aminolevulinic acid. Analysis time
is under 15 min, and no extraction of urine is necessary
with analysis being performed directly on diluted urine.
Reference and Preferred Methods
The reference method for PBG in urine is stable-isotope
dilution liquid chromatography-tandem mass
spectrometry (LC-MS/MS), first reported in 2001 [16].
The procedure is briefly described as Method 3 in Table
1. It requires 1 mL of urine and uses 5-(aminoethyl)-4-
(carboxymethyl)-1H-2,4-[13C]PBG (2.75 g) as the
internal standard. As expected from a reference method,
this technique shows greater analytical specificity and
improved clinical sensitivity and specificity compared to
other available methods.
For the first time, mild elevations (13 to 66 mol/L, 3-15
mg/L) of PBG could be reliably detected and the true
reference range for PBG established. The quantitative
Mauzerall and Granick method obtained a PBG
reference range of < 15 mol/day (<3 mg/day), whereas
LC-MS/MS gives a much lower and probably more
correct range of < 2.2 mol/day (<0.5 mg/day).
Porphobilinogen Screening Method
The preferred screening method uses an anion-exchange
resin, as first described by Mauzerall and Granick [13]
for their quantitative technique. Adaptation of the
quantitative resin method into a rapid method suitable
for screening took place in stages. The originally
described method required the addition of resin to a test
tube, and the subsequent washing and elution steps
caused delays because of the need for several
centrifugation steps to remove supernatant fluids from
the mobile resin bed in the bottom of the tube [10].
Blake et al. [17] described several useful modifications:
The anion-exchange resin is purchased from
Bio-Rad in the required acetate form and
immobilized in a syringe.
976
Porphobilinogen Screening and Quantitation
A range of color standards based on methyl red
solutions allow the result to be reported semi-
quantitatively as not detected, positive +,
positive ++ and positive +++.
Using a syringe with an attached filter
decreases the time required to approximately 10
minutes.
These modifications have been incorporated into a
commercial urine PBG kit available from Thermo
Electron Corporation, Louisville, Colorado
(www.thermo.com/clinicalchemistry). This test is more
sensitive and specific than the Watson-Schwartz method,
and an evaluation of the commercial kit has been
published [11].
The College of American Pathologists (CAP) external
quality control scheme does not include porphyrins or
PBG. However, the scheme administered by the Royal
College of Pathologists of Australasia (RCPA) in
Australia has circulated urine samples for PBG
screening. Over a 4-year period from 2003 to 2006, the
proportion of laboratories persisting with the outmoded
Watson-Schwartz or Hoesch screening methods declined
from 23% to 10%. During the same period, enrolled
laboratories using the Watson-Schwartz or Hoesch
screening methods obtained a sensitivity of 92%
compared to 98% for those using the recommended
anion-exchange resin screening method. The specificity
for all methods was 100%.
Porphobilinogen Quantitation Method
The preferred method for quantitation of PBG is one of
those that utilizes an ion-exchange resin for isolation of
PBG with subsequent spectrophotometric quantitation
using DMAB. The Mauzerall and Granick method [13]
has been widely used in clinical laboratories and has
stood the test of time to become a literature citation
classic. The use of column chromatography to isolate
PBG should not be a problem in most clinical
laboratories.
Routine analysis of urines should employ either the
column or batch chromatographic method to isolate PBG
from interfering compounds such as urea and ascorbic
acid [18]. If a sample is known to contain very high
concentrations of PBG (tenfold higher than normal),
direct analysis of diluted urine can be performed
satisfactorily; results will be only 5% too high [14]. Both
methods report quantitative recovery of PBG from the
ion-exchange resin and an assay sensitivity of 0.44 mol
(0.1 mg) of PBG (4.4 mol/mL or 1 mg/mL of urine).
The batch method presented below also incorporates
measurement of absorbance at 2 wavelengths to allow
estimation of the contribution of interfering compounds
that are not removed by the ion-exchange procedure
[14]. Using the batch method, it is possible to process
several samples simultaneously in about 1 h. This is
particularly useful when several members of a family are
examined at the same time.
Specimen
For screening, a freshly voided urine specimen collected
during or immediately after an attack of acute abdominal
pain is recommended. The assay should be conducted
within several hours to prevent the degeneration of PBG
or the formation of an inhibitor to the reaction with
Ehrlichs reagent [19]. After several days at room
temperature, and especially if exposed to light, colorless
PBG polymerizes to form porphyrins and other pigments
that darken the urine. At alkaline pH (pH 9), PBG is
stable at 4C for 1 to 2 weeks. If the assay is to be
delayed for a longer time, an aliquot of a well-mixed
urine collection may be frozen at 20C for several
months [20].
Twenty-four-hour urine specimens for quantitation of
PBG are not recommended. They delay diagnosis and
increase the risk of losses during the collection period. A
random urine reference range obtained with the
Mauzerall and Granick quantitative method of < 1.5
mol/mmol creatinine has been determined [17].
Interferences
Inappropriate specimen preservation and storage is the
most important aspect affecting accurate measurement of
PBG.
Stored acidified and exposed to light at room
temperature, urines containing PBG would be expected
to rapidly lose it, usually leading to significant darkening
of the urine sample and interference with both the
Watson-Schwartz and Hoesch screening methods.
Pigments normally present in urine, such as
urobilinogen, indole, and indican, are also thought to
cause increased interference to these screening methods
with increasing storage time [8]. The preferred anion-
exchange resin screening method has a decisive
advantage in removing these interfering pigments prior
to the color reaction [11].
Porphobilinogen Reference Interval
The reference interval for a random urine is < 2 mg/L
(<9 mol/L), but a creatinine-corrected result is
preferred. The recommended reference interval of < 1.5
mol/mmol creatinine is based on the 95th centile for
290 random urines [17].
The older literature quotes reference intervals based on
24-hour urine collections, which are not recommended,
since they delay diagnosis and increase the risk of losses.
Moore and Labb [14] reported a reference interval of
5.7 3.5 mol/day (1.3 0.8 mg/day). Determination of
reference intervals with the LC-MS/MS method gives a
lower PBG excretion of the order of 0 to 2.2 mol/day (0
to 0.5 mg/day). Reference intervals obtained by the
established colorimetric methods [13] probably
overestimated PBG excretion because of incomplete
removal of interfering substances from the sample before
complexation with Ehrlich reagent [16]. PBG excretion
in children is equivalent to that of adults when the values
are corrected for body weight.
977
Porphobilinogen Screening and Quantitation
Interpretation
Urinary PBG concentrations are greatly increased in
acute attacks of acute intermittent porphyria (AIP),
hereditary coproporphyria, and variegate porphyria. In
AIP, levels up to 440 mol/L (100 mg/L) of PBG may
be found. AIP, the most commonly seen acute porphyria
in the United States and northern Europe, most
frequently is accompanied by acute abdominal pain,
constipation, and vomiting. AIP is almost exclusively
described only after puberty, most likely resulting from
the increase in sex hormones, which are known to
exacerbate symptoms of porphyria. Acute attacks
become progressively less likely with advancing age,
especially after menopause in women. Any positive
screening test result should be followed up by a
quantitative PBG and urinary porphyrin assay.
During acute porphyric attacks, urinary PBG
concentrations will be very high (>133 mol/day [30
mg/day]) and can be detected by most qualitative
screening tests for PBG [9]. Between acute episodes,
urinary PBG excretion for some patients will be too low
to detect with the screening procedure, even though
urinary PBG is above normal. Other patients may have
completely normal PBG excretion between acute
episodes, although the more sensitive LC-MS method
often detects elevated PBG in cases where the screening
procedures return a negative result.
In acute intermittent porphyria, there is a decrease in the
enzyme uroporphyrinogen I synthase (URO-S), also
known as hydroxymethylbilane synthase. This enzyme is
responsible for converting PBG to uroporphyrinogen I in
the synthesis of heme. The assay of this enzyme allows
the diagnosis of acute intermittent porphyria to be made
on a portion of the subset of patients with normal PBG
levels during the latent period of the disease, although
there is an area of overlap between the lower end of the
normal range and the upper end of ranges for AIP
patients [21]. The enzyme assay is most useful in family
studies, where the enzyme activity of the proband is
known [22].
References
1 Magnussen CR. Acute intermittent porphyria:
clinical and selected research aspects, Ann Int
Med 1975;83:851-864.
2 Sachs P. Ein Fall von akuter Porphyrie mit
hochgradiger Muskelatrophie. Klin Wochenschr
1931;10:1123-1125.
3 Watson CJ, Schwartz S. Simple test for urinary
porphobilinogen. Proc Soc Exp Biol Med
1941;47:393-394.
4 Schwartz S, Keprios M, Schmid R. Experimental
porphyria: type produced by lead,
phenylhydrazine and light. Proc Soc Exp Biol
Med 1952;79:463-468.
5 Schreiber WE, Jamani A, Pudek MR. Screening
tests for porphobilinogen are insensitive. The
problem and its solution. Am J Clin Pathol
1989;92:644-9.
6 Buttery JE, Carrera AM, Pannall PR. Analytical
sensitivity and specificity of two screening
methods for urinary porphobilinogen. Ann Clin
Biochem. 1990;27:165-6.
7 Buttery JE, Carrera AM, Pannall PR. Reliability
of the porphobilinogen screening assay.
Pathology 1990;22:197-8.
8 Watson CJ, Taddeini L, Bossenmaier I. Present
Status of the Ehrlich Aldehyde Reaction For
Urinary Porphobilinogen. JAMA. 1964;190:501-
4.
9 Pierach CA, Cardinal R, Bossenmaier I, Watson
CJ. Comparison of the Hoesch and the Watson-
Schwartz tests for urinary porphobilinogen. Clin
Chem 1977;23:1666-1668.
10 Buttery JE, Chamberlain BR, Beng CG. A
sensitive method of screening for urinary
porphobilinogen. Clin Chem 1989;35:2311-2.
11 Deacon AC, Peters TJ. Identification of acute
porphyria: evaluation of a commercial screening
test for urinary porphobilinogen. Ann Clin
Biochem 1998;35:726-732.
12 Treibs A, Herrmann E. Pyrrole dyes IV: the
Ehrlich reaction of pyrroles and indoles. Hoppe-
Seylers Z Physiol Chem 1955;299:168-185.
13 Mauzerall D, Granick S. The occurrence and
determination of delta-aminolevulinic acid and
porphobilinogen in urine. J Biol Chem
1956;219:435-446.
14 Moore DJ, Labb RF. A quantitative assay for
urinary porphobilinogen. Clin Chem
1964;10:1105.
15 Lim CK, Rideout JM, Samson DM.
Determination of delta-aminolaevulinic acid and
porphobilinogen by high-performance liquid
chromatography. J Chromatogr 1979;185:605-
611.
16 Ford RE, Magera MJ, Kloke KM, Chezick PA,
Fauq A, McConnell JP. Quantitative
measurement of porphobilinogen in urine by
stable-isotope dilution liquid chromatography-
tandem mass spectrometry. Clin. Chem
2001;47:1627-1632.
17 Blake D, Poulos V and Rossi E. Diagnosis of
porphyria: recommended methods for peripheral
laboratories. Clin Biochem Revs 1992;13[suppl
1]:S1-24.
18 Pounty FTG. Acute porphyria: some properties of
porphobilinogen. Biochem J 1945;39:446-451.
19 Watson CJ, Bossenmaier I, Cardinal R. Acute
intermittent porphyria. JAMA 1961;175:1087-
1091.
20 Bossenmaier I, Cardinal R. Stability of -
aminolevulinic acid and porphobilinogen in urine
under varying conditions. Clin Chem
1968;14:610-614.
21 Bottomley SS, Bonkowsky HL, Kreimer-
Birnbaum M. The diagnosis of acute intermittent
porphyria: usefulness and limitations of the
erythrocyte uroporphyrinogen I synthase assay.
Am J Clin Pathol 1981;76:133-139.
22 Astrup EG. Family studies on the activity of
uroporphyrinogen I synthase in diagnosis of acute
intermittent porphyria. Clin Sci Mol Med
1978;54:251-256.
978
Porphobilinogen Screening and Quantitation

Tables
Table 1: Methods for Urinary Porphobilinogen (PBG) Quantitation
Method 1: Ion-exchange
a. Column
b. Batch
Principle of analysis: Partially purified PBG quantitated after reaction with Ehrlichs reagent
a. PBG separated from interfering compounds by column chromatography
b. PBG separated from interfering compounds by batch ion-exchange chromatography
Comments:
a. Common; standard method
b. Common; may be more suitable for clinical laboratory

Method 2: High-performance liquid chromatography (HPLC)

Principle of analysis: PBG separated by HPLC and quantitated spectrophotometrically at 240 nm
Comments: Rare; requires special equipment, but should be very specific

Method 3: Liquid chromatographytandem mass spectrometry

Principle of analysis: Following solid-phase extraction of PBG, LC-MS/MS analysis performed. PBG and
internal standard monitored through their own precursor and product-ion settings
Comments: Method exhibits greater analytical specificity and improved clinical sensitivity and specificity as
compared with other methods. Requires solid-phase extraction of urine to remove urine components that suppress
the ion signal.

Reaction Conditions for Porphobilinogen (PBG) Quantitation by Reaction with Ehrlichs Reagent
Sample: Ion-exchange chromatography extract of urine
Temperature: Ambient temperature
Sample volume: 1 mL urine; ion-exchange chromatography column eluate, 2 mL
Fraction of sample volume: 0.5 (Ehrlichs reaction)
Final concentration of reagents:
HCl: 1.46 mol/L
Acetic acid: 6.6 mol/L
p-Dimethylaminobenzaldehyde (DMBA): 67 mmol/L
Time of reaction: 6 to 8 min
Linearity: Up to at least 100 g/mL
Sensitivity: 0.1 g (1 g/mL of urine)

Figures

Figure 1: Porphobilinogen Quantitation.

Absorbance spectrum of red condensation product produced by reaction of PBG and Ehrlichs reagent. Dashed line,
Ehrlichs reagent versus water; dotted-dashed line, Ehrlichs reagent and PBG versus water; solid line, Ehrlichs reagent
and PBG versus Ehrlichs reagent.
979
Porphobilinogen Screening and Quantitation
Porphobilinogen Quantitation: Reaction 1
Porphobilinogen Quantitation: Reaction 2
Screening Procedure: Resin Assay for
Porphobilinogen
Principle
This screening test is more sensitive and specific than
the Watson-Schwartz test it replaces [17]. The anion-
exchange resin is prepacked in a syringe. The urine
sample is adsorbed onto the resin, washed and eluted
through a series of procedures involving the addition and
removal of a 5-micron filter and a final color formation
with Ehrlichs reagent. Visual assessment of color
formation is accomplished with use of a dye color
calibrator for the in-house method given below or a color
chart for the commercial version available from Thermo
Electron Corporation, Louisville, Colorado;
www.thermo.com/clinicalchemistry.
Reagents
1. Ehrlichs aldehyde reagent (18.8 mmol/L in
7.2 M HCl). Combine 700 mg of reagent-grade p-
dimethylaminobenzaldehyde, 150 mL of concentrated
HCl, and 100 mL of deionized water. The solution
should be colorless or very light yellow. Brown or
brownish-red material should not be used. Store in
amber bottle at room temperature. It will remain stable
for months. Discard if discolored.
2. Anion-exchange resin (AG1-X2 200-400 mesh,
acetate form BioRad, Richmond CA). Mix 100 g resin
with 100 mL distilled water. While mixing, draw up 2
mL aliquots into 2 mL syringes and cap with a rubber
plug. Store ready for use.
3. Acetic acid (1 mol/L). To 17 volumes of
distilled water, add 1 volume of glacial acetic acid.
4. Ammonia solution. To 3 volumes of distilled
water add 1 volume of concentrated (33%) ammonia
solution.
5. 5-Micron filters (Argyle, St Louis, MO).
6. Methyl red color standards. For the stock
solution dissolve 33.3 mg of methyl red sodium salt in
3.3 mL concentrated hydrochloric acid and 25 mL of
distilled water, then make up to 200 mL with distilled
water. For the working solution, dilute the stock solution
with 0.1 mol/L hydrochloric acid as shown below.
Standard Dilution Absorbance
Range
25 mol/L 1:200 0.10-0.15
50 mol/L 1:100 0.25-0.30
100 mol/L 1:50 0.50-0.60
5. Control urine. A fresh, otherwise normal,
urine is run. Commercially available urine controls
usually contain no detectable PBG and can be used as
negative controls. A positive control can be prepared by
addition of PBG (Sigma Chemical Co., St. Louis, MO;
no. P1134) to a final concentration of 75 mol/L, 16.97
mg/L. The spiked pool should then be frozen at 20C in
2 mL aliquots. It is stable for up to 6 months.
980
Porphobilinogen Screening and Quantitation
Assay
1. Check urine pH is > 6; if not, add one drop of
ammonia solution.
2. Remove the syringe cap and fit filter securely to
the syringe. Expel the water in the syringe,
remove the filter, and draw up 1 mL of urine
using the graduations on the syringe. To help
with mixing, introduce an air bubble into the
syringe. Replace the filter, and mix the resin
and urine for 10 seconds.
3. Expel the urine to waste. Draw up 1 mL of
water and an air bubble and mix for 10 seconds.
4. Expel the water to waste. Draw up 1 mL of 1
mol/L acetic acid and an air bubble and mix for
10 seconds.
5. Expel the acetic acid into a clear tube
containing 1 mL of Ehrlichs reagent and mix.
Wait 3 minutes and then compare the color of
the solution to the methyl red solutions against
a white background.
6. Report as follows:
Not detected
PBG concentration <25 mol/L +
PBG concentration 25-50 mol/L ++
PBG concentration 50-100 mol/L +++
PBG concentration >100 mol/L ++++
Notes
1. This resin-based method overcomes the major
drawback of the Watson-Schwartz screening method,
namely lack of adequate sensitivity, a problem especially
evident in dark-colored urine samples. Pigments usually
present in urine such as urobilinogen, indole, and indican
not only interfere with the color reaction, they can also
lead to spurious color development, resulting in lack of
specificity.
Procedure: Quantitative Porphobilinogen Assay
Using Ehrlichs Reaction [14]
Principle
PBG Quantitation Table: Reaction conditions with
Ehrlichs reagent.
PBG and other pyrroles condense in an acid medium
with p-dimethylaminobenzaldehyde (DMAB) to form a
colored product. There is evidence [13,14] that further
reactions occur, resulting in fading of the color with
time. For this reason, all measurements should be made
at a consistent time, between 6 and 8 min after the
addition of DMAB. The DMAB is contained in a
modified Ehrlichs reagent that contains acetic acid to
increase the color intensity.
Reagents
1. Ehrlichs reagent (modified): (p-
dimethylaminobenzaldehyde [DMAB] 134 mmol/L;
HCl, 2.92 mol/L; acetic acid, 13.2 mol/L). Dissolve 2 g
of DMAB in 25 mL of concentrated HCl, and add 75 mL
of glacial acetic acid. Store refrigerated in the dark up to
several months [1]. If the clear reagent becomes yellow,
it should be discarded.
2. Sodium acetate, 100 g/L (1.2 mol/L). Place
100 g of sodium acetate in a liter volumetric flask, and
add approximately 900 mL of distilled water to dissolve
salt. Add distilled water to mark, mix well, and store at
room temperature. Stable for 6 months.
3. Dowex 2-X8 resin (200 to 400 mesh) acetate
form. The resin may be prepared in large batches (100 to
200 g), since it is stable for at least several months.
Suspend the resin in 4 volumes of sodium acetate
solution to convert it to the acetate form. Mix well, and
allow all but the very fine particles to settle. Decant and
discard the supernatant. Wash the resin about eight times
with 4 volumes of distilled water. These washes can be
facilitated by brief centrifugation to pack the resin. A
stock resin suspension is prepared by the addition of 1
volume of distilled water to 1 volume of washed, packed
resin. The resin will settle quickly and should be
completely suspended before use.
4. 1 M acetic acid (1 mol/L). Take 5.7 mL of
glacial acetic acid, and dilute with distilled water to 100
mL in a volumetric flask. Mix thoroughly, and store at
room temperature. Stable for 6 to 12 months.
5. Ammonium hydroxide, concentrated (28%
NH3).
Assay
Equipment: Narrow band-width (10 nm)
spectrophotometer, general-purpose centrifuge,
12 mL graduated centrifuge tube.
1. Place 2 mL of thoroughly resuspended resin
into a 12 mL centrifuge tube, and centrifuge
sufficiently to pack the resin (1 min at several
hundred g). Remove the supernatant, and add 1
mL of urine onto the packed resin followed by
0.1 mL of concentrated ammonium hydroxide.
Resuspend thoroughly, centrifuge, and discard
supernatant. The PBG is bound to the resin.
2. Wash the resin four times with 5 mL aliquots of
distilled water. The resin is packed after each
wash by centrifugation. Remove the final
supernatant.
3. Add 2 mL of 1 M acetic acid to the packed
resin, and resuspend thoroughly. Pack the resin
by centrifugation, and save the supernatant,
which contains PBG. Elute three more times
with 2 mL aliquots of 1 M acetic acid, and
combine all supernatants together.
4. Adjust the total volume of pooled supernatants
to 10 mL with 1 M acetic acid, and mix well. If
necessary, centrifuge to remove any resin that
contaminates the supernatants.
5. Pipet 2 mL of the pooled eluates into a clean
tube, and add 2 mL of Ehrlichs reagent to each
tube at 2-min intervals. Allow the color of each
tube to develop for 6 mins at ambient
temperature, and read the absorbance.
6. Read the absorbance (A) (1 cm cuvette) at 525
nm and 555 nm using a blank consisting of 2
mL of 1 M acetic acid and 2 mL of Ehrlichs
reagent.
Calculation
981
Porphobilinogen Screening and Quantitation
where A555 = absorbance at 555 nm of unknown; 10 =
dilution factor for urine;
0.114 = A555 of 1 g/mL of PBG when reacted
with modified Ehrlichs reagent
24-h excretion (mg/24 h) =g of PBG/mL Total urine
volume (mL) (1 mg/1000 g)
Notes
1. An extinction coefficient of 6.2 104 Lmol-
1cm-1 has been described for the reaction
product [14]. The factor of 0.114 was derived
from a molar absorptivity of approximately 2.6
104 Lmol-1cm-1, but it should be verified
for each spectrophotometer [14]. For this
purpose, preweighed vials of PBG are
commercially available (Sigma Chemical Co.,
St. Louis, MO). The PBG should be dissolved
in 1 M acetic acid and assayed in the reaction
described above at several concentrations. One
can verify the entire procedure by adding a
known, relatively high (for example, 20
mg/mL) amount of PBG to normal urine and
performing the assay.
2. The red condensation product that is measured
in this assay has a strong absorption peak at 555
nm and a weaker peak at 525 nm. Most
pigments that cause interference absorb more
strongly at the lower wavelength. Therefore the
ratio A525/A555 should be close to 0.83. Urines
giving a value > 1 for this ratio should not be
reported as containing elevated levels of PBG.
This is frequently the case when the A555 is
only slightly elevated. Under these
circumstances, the assay should be repeated
with a fresh specimen.
3. It is essential that the water washes of the resin
after addition of urine be performed four times.
Fewer washes will result in unacceptable
carryover of interfering substances. Likewise, it
is essential that at least four elutions be
performed with acetic acid to ensure adequate
recovery of PBG.
 982
Procainamide and N-Acetyl Procainamide

Procainamide and N-Acetylprocainamide

Matthew T. Olson and William Clarke
Name: Procainamide N-acetylprocainamide
Clinical Vaughan Williams class Ia antiarrhythmics with multiple indications
significance: Refer to Chapter 14, Therapeutic Drug Monitoring, in the 5th Edition of
Clinical Chemistry: Theory, Analysis, Correlation.
Molecular C13H21N3O C15H22N3O2
formula:
Molecular 235.54 D 277.37 D
weight:
Merck Index: 7553 7845
Chemical Amines Amines
class:
Structure:

i polarization immunoassay (FPIA), enzyme-multiplied

Principles of Analysis and Current Usage
Procainamide (PA) is a Vaughan Williams class Ia
antiarrhythmic drug that clinicians prescribe acutely for
pharmacological conversion of atrial and ventricular
arrhythmias and for chronic suppression of these
arrhythmias and others when they are refractory to more
common treatment modalities [1,2]. Hepatic arylamine N-
acetyltransferase 2 (NAT2) acetylates PA to yield an
equivalently therapeutic active metabolite, N-
acetylprocainamide (NAPA), which is at least equally
responsible for the adverse cardiac effects associated with
PA therapy [3,4]. The production of NAPA by acetylation
on NAT2 occurs at a genetically determined rate that
varies widely among patients [5, 6], so NAPA
concentrations cannot be predicted from those of PA
without genetic information about the NAT2
polymorphisms in the patient [7]. For these reasons,
therapeutic drug monitoring (TDM) of PA necessitates
monitoring of NAPA.
According to the 2007 College of American Pathologists
(CAP) survey [8], participating laboratories analyze PA
and NAPA concentrations with either fluorescence
i
Procainamide and N-Acetyl Procainamide
Previous and current authors of this method:
First edition: Not done
Methods edition: John E. Sherwin
Second edition: Not updated
Third edition: Not updated
Fourth edition: John E. Sherwin, Curt Grote
Fifth edition: Matthew T. Olson, William Clarke
immunoassay technique (EMIT), particle-enhanced
turbidimetric inhibition immunoassay (PETINIA), or
cloned-enzyme donor immunoassay (CEDIA). The
frequency of these assays in use is given in Table 1. This
chapter will focus on these four methods. Readers
interested in the historical use of colorimetry of a
derivative [9], gas chromatography [10], or high-
performance liquid chromatography [11-13] may consult
this chapter in the previous edition. Those interested in the
developmental use of tandem mass spectrometry for TDM
of antiarrhythmics may find recent investigations a useful
starting point [14, 15].
Methods involving FPIA [16] are based on the fact that a
stationary fluorophore fluoresces in the same plane of light
that excites it. If the fluorophore moves after excitation
as the result of rotational relaxationthe emitted light will
not be polarized, because the electronic field has been
changed to new planes. Small molecules have faster
rotational relaxation than large molecules, so fluorophores
that are bound to antibodies preserve the plane of polarized
excitation light more than those that are not bound to
antibodies. Figure 1 provides a schematic for FPIA. In
FPIA for PA and NAPA [17-19], a known amount of
fluorescein-tagged drug and anti-drug antibody is added to
the cuvette containing an aliquot of patient serum or
plasma. Tagged and untagged drug compete to bind the
antibody. When more drug is present, less tagged drug
binds the antibody, so a smaller proportion of the
fluorescence will be polarized. The instrument irradiates
the cuvette with polarized excitation light, and the intensity
of fluoresced light on the planes parallel, Iv, and
 983
Procainamide and N-Acetyl Procainamide
orthogonal, Ih, to the excitational plane is detected. These
values are converted into a value for polarization, P, by
equation 1(1):
P = (Iv Ih)/(Iv + Ih)
Drug concentration and P are inversely proportional at a
slope defined by the experimental conditions. Linear
interpolation of P onto a concentration-versus-P graph
yields the drug concentration. Drug concentration and
polarization are inversely related, as in Figure 2A.
Instruments based on PETINIA [20, 21] make
turbidimetric measurements of light scattering in the
cuvette. When drug-coated microparticles bind anti-drug
antibody, agglutination occurs, and more light is scattered.
Drug from the patient competes with the particle-bound
drug for antigen-binding sites and so prevents
agglutination in a concentration-dependent fashion.
Because agglutinated particles scatter light, the light
scattering is inversely related to the amount of drug
present. The inverse plot is shown in Figure 2B, and the
method is shown in Figure 3. PETINIA offers the
advantage of a higher dynamic range than other
immunoassays in exchange for more susceptibility to
interference [22].
Both EMIT [23] and CEDIA [24] involve
spectrophotometric monitoring of enzymatic reactions by
drug-labeled enzymes which are disabled by binding to
anti-drug antibody. Higher concentrations of drug bind
more antibody, so the enzymatic reaction rate increases in
a linear fashion. The rate of reaction is linearly related to
functional enzyme. The two methods differ on the basis of
how the enzyme is labeled with drug and how antibody
binding affects enzyme kinetics. As shown in Figure 1A,
EMIT methods use whole enzymes, such as glucose-6-
phosphate dehydrogenase (G6PD), which have the drug of
interest covalently attached. Free anti-drug antibodies bind
the drug on the enzyme and inactivate the enzyme by steric
hindrance. At higher drug concentrations in the blood,
more patient drug binds the free anti-drug antibodies, less
G6PD is inactivated, so more glucose-6-phosphate is
converted to glucose-6-phosphogluconolactone. This
reaction can be followed via the NADP+/NADPH
transition, which results in a change in absorbance at 340
nm. Linear interpolation into a positively sloped graph of
drug concentration versus spectrophotometric absorbance
yields the drug concentration [25,26]. In contrast to EMIT
methods, which use whole enzymes, CEDIA methods,
depicted in Figure 1B, use recombinant fragments of an
enzyme, usually -galactosidase. The method involves two
kinds of fragments: large enzyme acceptors (EA) and
small drug-labeled enzyme donors (ED). The ED consists
of an essential peptide fragment to make the EA
functional. Anti-drug antibody binding of the drug moiety
on an ED prevents EA-ED association, formation of active
enzyme, and the degradation of a galactopyranoside which
is absorbent at 420 and 570 nm [24]. Whereas both CEDIA
and EMIT require anti-drug antibody, CEDIA has the
advantage in that the only other drug-specific entity it
requires is a very small drug-labeled ED rather than whole
enzyme. The direct relationship between enzyme rate and
antigen is demonstrated in Figure 2A, EMIT is shown in
Figure 4, and CEDIA is shown in Figure 5.
Reference and Preferred Methods
Traditionally, TDM of PA and NAPA has been performed
by GC and HPLC, but the immunoassays described above
have largely replaced these methods as preferred methods
because of their speed and the fact that they obviate the
extraction sample preparation required in chromatographic
methods. There is no formally defined reference method
for PA and NAPA; however, the HPLC method functions
as a de facto reference method because it is not subject to
the immunochemical interferences inherent in the modern
methods [27]. Among the immunoassays, FPIA is the most
widely used method among respondents to the 2007 CAP
survey [8]. Several studies have evaluated intermethod
variability [19] of the immunoassays and compared
immunoassay to HPLC for this analyte [17, 18, 26]. These
results have shown good agreement. All the methods meet
the acceptable performance standards outlined by the
Clinical Laboratory Improvement Amendments of 1988
(CLIA-88).
Specimen
Because procainamide and NAPA clear the body rapidly
under normal conditions, the trough concentration is most
clinically useful. Blood should be drawn 1 hour or less
before the next scheduled dose. Serum or plasma can be
drawn, and either EDTA or oxalate are acceptable
anticoagulants. The sample can be kept at 2C to 8C for
up to 24 hours and at 20C for up to 2 weeks [28].
Heating the sample to 60C immediately prior to analysis
(in protocols that desire virus denaturation) is shown not to
be associated with a decrease in drug concentrations [29].
Excessive free heme, bilirubin, and lipids all interfere with
CEDIA, EMIT, and PETINIA. As discussed elsewhere,
NAPA must accompany PA measurement, and the
concentrations should be reported separately, not as a sum
[28, 30].
Procainamide and N-Acetylprocainamide Therapeutic
Range
Procainamide [28]
Therapeutic: 4.0-8.0 mg/L
Toxic: >10 mg/L
N-Acetylprocainamide [28]
Therapeutic: 10-20 mg/L
Toxic: >40 mg/L
Interpretation
Procainamide causes more clinical side effects than can be
discussed in detail here. Effectiveness [31] and toxicity
[32] are the central reasons for TDM of class Ia
antiarrhythmics. Procainamide typically achieves
therapeutic effect at the defined ranges, but patients with
 984
Procainamide and N-Acetyl Procainamide
extensive chronic ischemia require higher concentrations
of drug [31]. However, PA and NAPA commonly cause
QTc prolongation, ventricular extrasystoles, and
tachycardia. These side effects result from the mechanism
of therapeutic action: inhibition of fast sodium channels in
the myocardium and His-Purkinje conduction system. In
addition to TDM, patient monitoring also involves regular
electrocardiogram monitoring and assessment of renal
function by laboratory analysis of blood urea nitrogen and
plasma creatinine. Regular electrocardiograms are
essential in monitoring, because QTc prolongation cannot
be predicted from drug concentrations alone in many cases
[3,4] and can lead to the fatal torsades-de-pointes
arrhythmia.
The half-lives of PA and NAPA are 3.5 0.8 and 8.1 1.3
hours, respectively [28, 33, 34]. Organic cation
transporters eliminate both PA and NAPA by active
secretion in the proximal tubule of the nephron. Because
the same mechanism eliminates many other drugs, such as
fluoroquinolones [35] and central acetylcholinesterase
inhibitors [36], co-administration of these drugs can result
in potentially dangerous fluctuations of drug levels and
warrants TDM, especially since PA and NAPA already
compete for the same transporters and mutually prolong
the drug effects [3,34]. The organic anion transporters do
not appear to be involved to a clinically relevant extent
[37]. Concentrations of PA and NAPA are also followed in
patients with chronic renal disease, in cardiogenic shock,
and in edematous states [38-42]. Hemodialysis removes
NAPA but not PA [30,43], so it introduces an additional
need for TDM. Finally, PA ranks among the top drug
choices for treating supraventricular arrhythmias in
neonates, and standard of care includes intensive TDM of
PA and NAPA [44], because the clinical situation involves
rapidly changing conditions and immature renal function.
The relationship between drug levels and the occurrence
and severity of most common non-urgent systemic side
effects associated with PA and NAPA has not been fully
explored. However, contemporary interest in the drug-
induced lupus often associated with PA therapy has lead to
several investigations [45-47]. Chronic PA use is shown to
cause dose-dependent T-cell disturbances [45], leading
occasionally to drug-induced lupus [46]; this is a clinical
diagnosis supported by positive anti-chromatin antibody
tests [47]. There is no evidence that drug monitoring plays
any role in predicting or diagnosing these adverse effects.
Procainamide and N-Acetylprocainamide Performance
Goals
All the modern immunoassays show less than an 8%
coefficient of variation (CV) for PA. The performance
criteria outlined by CLIA-88 require no more than 25%
difference from the peer-group mean. These standards
apply to the minimal therapeutic concentration of 4.0
mg/L. The same performance criteria and observed CV
apply for NAPA, except that the clinical decision
concentration is 10.0 mg/L. Currently available assays
meet these necessary criteria.
References
1 Koch-Weser I, Klein S. Procainamide dosage
schedules, plasma concentrations, and clinical
effects. JAMA. 1971;215:1454-1460.
2 Atkinson AJ, Ruo T, Piergies A. Comparison of
the pharmacokinetic and pharmacodynamic
properties of procainamide and N-
acetylprocainamide. Angiology. 1988;39:655-657.
3 Funck-Bretano C, Light R, Lineberry M, Wright
G, Roden D, Woosley R. Pharmacokinetic and
pharmacodynamic interaction of N-acetyl
procainamide and procainamide in humans. J
Cardiovasc Pharmacol. 1989;14:364-373.
4 Miller J, Zipes D. Therapy for cardiac
arrhythmias. In: Zipes D, Libby P, Bonow R,
Braunwald E, eds. A Textbook of Cardiovascular
Medicine. 7th ed. Phladelphia: Saunders; 2005.
5 Okumura K, Kita T, Chikazawa S, Komada F,
Iwakawa S, Tanigawara Y. Genotyping of N-
acetylation polymorphism and correlation with
procainamide metabolism. Clin Pharmacol Ther.
1997;61:509-517.
6 Deguchi T, Mashimo M, Suzuki T. Correlation
between acetylator phenotypes and genotypes of
polymorphic arylamine N-acetyltransferase in
human liver. J Biol Chem. 1990;265:12757-
12760.
7 Grant D, Blum M, Beer M, Meyer U.
Monomorphic and polymorphic human arylamine
N-acetyltransferases: a comparison of liver
isoenzymes and expressed products of two gloned
Genes. Mol Pharmacol. 1990;39:184-191.
8 Z-B Therapeutic Drug Monitoring General
Survey: College of American Pathologists (CAP);
2007.
9 Sitar D, Graham D, Rango R, Dusfresne L,
Ogilvie R. Modified colorimetric method for
procainamide in plasma. Clin Chem.
1976;22:379-80.
10 Atkinson AJ, Parker M, Strong J. Rapid gas
chromatographic measurement of plasma
procainamide concentration. Clin Chem.
1972;18:643-647.
11 Kessler K, Ho-Tung, Steele B, Silver J, Pickoff
A, Narayanan S et al. Simultaneous quantitation
of quinidine, procainamide, and N-
acetylprocainamide in serum by gas-liquid
chromatography with a nitrogen-phosphorous
selective detector. Clin Chem. 1982;28:1187-
1190.
12 Stearns F. Determination of procainamide and N-
acetylprocainamide by high-performance liquid
chromatography. Clin Chem. 1981;27:2064-2067.
13 Dutcher J, Strong J. Determination of plasma and
N-acetylprocainamide concentration by high-
pressure liquid chromatography. Clin Chem.
1977;23:1318-1320.
14 Li S, Liu G, Jia J, Liu Y, Pan C, Yu C et al.
Simultaneous determination of ten antiarrhythmic
 985
Procainamide and N-Acetyl Procainamide
drugs and a metabolite in human plasma by liquid
chromatography tandem mass spectrometry. J
Chromatogr B Analyt Technol Biomed Life Sci.
2006;Nov17:174-181.
15 Dhananjeyan M, Bykowski C, Trendel J, Sarver J,
Ando H, Erhardt P. Simultaneous determination
of procaine and para-aminobenzoic acid by LC-
MS/MS method. J Chromatogr B Analyt Technol
Biomed Life Sci. 2007;847(2):224-230.
16 Gutierrez M, Gomez-Hens A, Perez-Bendito D.
Immunoassay methods based on fluorescence
polarization. Talanta. 1989;36:1187-1201.
17 Haver V, Audino N, Burris S, Nelson M. Four
fluorescence polarization immunoassays for
therapeutic drug monitoring evaluation. Clin
Chem. 1989;35:138-140.
18 Laurino J, McKay J, Fischberg E, Schwenzer K,
Kaufman R. Comparison of three immunoassays
for the determination of N-acetylprocainamide:
determination of false elevations by a polyclonal
antibody-based method. Ther Drug Monit.
1994;16:312-315.
19 Tisdale J, Padhi I, Ware J, Svensson C.
Comparison of FPIA with LC for quantification
of PA and NAPA in urine. Ther Drug Monit.
1996;18:693-697.
20 Cambiaso C, Riccomi H, Masson P, Heremans J.
Automated nephelometric immunoassay II: its
application to the determination of haptens. J
Immunol Methods. 1974;5:293-302.
21 Grange J, Roch A, Quash G. Nephelometric assay
of antigens and antibodies with latex particles. J
Immunol Methods. 1977;18:365-375.
22 Ritchie R. The foundations of immunochemistry.
In: Wild D, ed. The Immunoassay Handbook.
Oxford: Elsevier; 2005.
23 Rubenstein K, Schneider R, Ullman E.
Homogenous enzyme immunoassay: a new
immunochemical technique. Biochem Biophys
Res Commun. 1972;47:846-851.
24 Henderson D, Friedman S, Harris J, Manning W,
Zoccoli M. CEDIA, a new homogenous
immunoassay system. Clin Chem. 1986;32:1637-
1641.
25 Henry P, Dhruv R. More sensitive EMIT for PA
and NAPA in plasma, serum and urine. Clin
Chem. 1988;34:957-960.
26 Walberg C, Wan S. Clinical evaluation of the
EMIT PA and NAPA assay. Ther Drug Monit.
1979;1:47-56.
27 Mehta A. The how and why of therapeutic drug
monitoring. Anal Proc. 1995;32:347-351.
28 Valdes R, Jortani S, Gheorghiade M. Standards of
laboratory practice: cardiac drug monitoring. Clin
Chem. 1998;44:1096-1109.
29 Dasgupta A, Bard D. Effect of heating sera under
conditions necessary for deactivation of human
immunodeficiency virus on commonly monitored
therapeutic drugs. Ther Drug Monit.
1994;16:612-615.
30 Campbell T, Williams K. Therapeutic drug
monitoring: antiarrhythmic drugs. Br J Clin
Pharmacol. 1998;46(4):307-319.
31 Myerburg R, Kessler K, Kiem I, Pefkaros K,
Conde C, Cooper D et al. Relationship between
plasma levels of procainamide, suppression of
premature ventricular complexes and prevention
of recurrent ventricular tachycardia. Circulation.
1981;64:280-290.
32 Takada M, Goto T, Kotake T, Saito M, Kawato
N, Nakai M et al. Appropriate dosing of
antiarrhythmic drugs in Japan requires therapeutic
drug monitoring. J Clin Pharm Ther. 2005;30:5-
12.
33 Jamali F, Alballa R, Mehvar R, Lemko C. Longer
plasma half-life for procainamide, utilizing a very
sensitive high-performance liquid
chromatography assay. Ther Drug Monit.
1988;10:91-96.
34 Roden D, Beele S, Higgins S, Wilkinson G,
Smith R, Oates J et al. Antiarrhythmic efficacy,
pharmacokinetics, and safety of N-
acetylprocainamide in human subjects:
comparison with procainamide. Am J Cardiol.
1980;46:463-468.
35 Bauer L, Black D, Lill J, Garrison J, Raisys V,
Hooton T. Levofloxacin and ciprofloxacin
decrease procainamide and N-acetylprocainamide
renal clearances. Antimicrob Agents Chemother.
2005;49:1649-1651.
36 Cummings J, Frank J, Cherry D, Kohatsu N,
Kemp B, Hewett L et al. Guidelines for managing
Alzheimers disease: part II, treatment. Am Fam
Physician. 2002;65:2525-2534.
37 Lam Y, Boyd R, Chin S, Chang D, Giacomini K.
Effect of probenecid on the pharmacokinetics and
pharmacodynamics of procainamide. J Clin
Pharmacol. 1991;31:429-432.
38 Jurgens G, Graudal N, Kampmann J. Therapeutic
drug monitoring of antiarrhythmic drugs. Clin
Pharmacokinet. 2003;42:647-663.
39 Dresser M, Leabman M, Giacomini K.
Transporters involved in the elimination of drugs
in the kidney: organic anion transporters and
organic cation transporters. J Pharm Sci.
2001;90(4):397-421.
40 Lee W, Kim R. Transporters and renal drug
elimination. Ann Rev Pharmacol Toxicol.
2004;44:137-166.
41 Mizuno N, Niwa T, Yoshihisa Y, Sugiyama Y.
Impact of drug transporter studies on drug
discovery and development. Pharmacol Rev.
2003;55:425-461.
42 Raebel M, Carroll N, Andrade S, Chester E,
Lafata J, Feldstein A et al. Monitoring drugs with
a narrow therapeutic range in ambulatory care.
Am J Manag Care. 2006;12:268-274.
 986
Procainamide and N-Acetyl Procainamide

43 Braden G, Fitzgibbons J, Germain M, Ledewitz 45 Richardson B. DNA methylation and autoimmune

H. Hemoperfusion for treatment for N-
acetylprocainamide intoxication. Ann Intern Med.
1986;105:64-65.
44 Moffett B, Cannon B, Friedman R, Kertesz N.
Therapeutic levels of intravenous procainamide in
neonates: a retrospective assessment.
Pharmacotherapy. 2006;26:1687-1693.
disease. Clin Immunol. 2003;109:72-79.
46 Rubin R. Drug-induced lupus. Toxicology.
2005;209:135-147.
47 Schett G, Rubin R, Steiner G, Hiesberger H,
Muller S, Smolen J. Comparative analysis of
antichromatin antibody specificity in lupus
erythematosus cell-positive and cell-negative
sera. Arthritis Rheum. 2000;43:420-428.
987
Procainamide and N-Acetyl Procainamide

Table 1: The methods participants in the 2007 CAP survey use for the analysis of PA and NAPA are given with a
breakdown according to popularity in terms of raw numbers and percentages. These data are adapted from citation
[8].

Method N labs % labs

FPIA 278 70.6
EMIT 54 13.7
PETINIA 41 10.4
CEDIA 21 5.3
Total 394
CEDIA, Cloned-enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; FPIA,
fluorescence polarization immunoassay; PETINIA, particle-enhanced turbidimetric inhibition immunoassay.

Figure 1: Schematic for FPIA. When no drug is present, as in the top reaction, the polarity is conserved because the
fluorescein-labeled drug is bound to antibody so moves little. More drug prevents the labeled drug from binding, and
the polarity of the sample decreases.

Fluoroscein labeled Drug

+
YY Polarity conserved

YY Anti-drug Antibody Polarized Light

+
YY Polarity dispersed

Patient Drug
988
Procainamide and N-Acetyl Procainamide

Figure 2: Sample plots demonstrating the relationship between antigen concentration and observed parameters in
competitive (A) and non-competitive (B) relationships. In methods such as EMIT and CEDIA, the observed
parameter is the reaction rate of the enzyme, which correlates directly with antigen concentration, as in (B). In
methods such FPIA, which measures polarization, and PETINIA, which measures scattered light, more antigen
decreases the magnitude of these measurements, leading to an inverse relationship, as in (A).

(A) (B)
Observed parameter

Observed parameter

Antigen concentration Antigen concentration

Figure 3: PETINIA. In the presence of drug, anti-drug antibody binds drug rather than the particle-bound drug.
Therefore, less agglutination occurs, and less light scatters.

Y
Drug-coated
Latex Beads
Y Y
Light scatters
orthogonal to

+ incident

Incident Light

YYY Anti-drug
Source

+ antibody

Patient drug
YYY Light passes
parallel to
incident
989
Procainamide and N-Acetyl Procainamide

Figure 4: EMIT. Drug-labeled G6PD binds antibody in the absence of patient drug. The binding inactivates the
enzyme, so no reaction occurs. In the presence of patient drug, which occupies the antibody binding sites, the free
G6PD is able to convert G6P at a higher rate.

No reaction

+
Drug labeled
G6PD
YY
YY
Anti-drug antibody substrate
+

YY
Patient drug product

Figure 5: CEDIA. The top scenario involves the absence of patient blood. The enzyme acceptors are inactive in the
absence of enzyme donors, so no reaction takes place. At higher levels of patient drug, the antibody binding sites are
occupied with patient drug, and the reaction rate increases.

No reaction

Enzyme
acceptors

+
+ Enzyme donors
YY
Y+ Y
substrate

Anti-drug antibody
product

Patient drug

YY
990
Procainamide and N-Acetyl Procainamide

Table 2: Methods for Procainamide and N-Acetylprocainamide Analysis

Method 1: Fluorescence polarization immunoassay (FPIA)
Principle of analysis: Patient drug displaces drug-labeled fluorophores from fluorescent
plane polarity, conserving antibody complexes.
Comments: Most popular method in use for PA and NAPA TDM
Method 2: Enzyme-multiplied immunoassay technique (EMIT)
Principle of analysis: Patient drug displaces drug-labeled whole G6PD from a disabling
complexation with anti-drug antibody.
Comments: Commonly used
Method 3: Cloned-enzyme donor immunoassay (CEDIA)
Principle of analysis: Patient drug frees a drug-labeled recombinant peptide which
associates with incomplete -galactosidase to form an active enzyme.
Comments: Only requires a small drug-labeled peptide. Reaction rate monitored by
conversion of a galactopyranoside.
Method 4: Particle-enhanced turbidimetric inhibition assay (PETINIA)
Principle of analysis: Patient drug dissipates the light-scattering agglutination of drug-
labeled micro- or nanoparticles with anti-drug antibody.
Comments: Commonly used
991
Progesterone

Progesterone
John Galligan
Name: Progesterone
Clinical significance: Refer to Chapter 48, General Endocrinology, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular formula: C21H30O2
Molecular mass: 314.45 D
Merck Index: 57-83-0, (C A Registry Number)
Chemical class: Steroid

Structure:

i before GC analysis employing flame ionization detection

Principles of Analysis and Current Usage
[8].
Progesterone is synthesized in the ovary, the adrenal
cortex, and the placenta from the biosynthetic precursor The application of a competitive protein-binding (CPB)
technique [9] (Table 1, Method 5) led to the
5-pregnenolone. The major excretory metabolite
development of a series of methods that employed
present in urine is pregnanediol.
various extraction solvents, different sources of binding
The original methods for assessing progesterone activity
proteins, different tritiated steroids, and a variety of
were bioassays. The increase in mouse uterine weight
techniques for separating the free and protein-bound
was correlated with progesterone content in the infusate
steroid fractions. Some of these methods purified the
(Table 1, Method 1) [1]. Subsequently, quantitative
steroid before CPB analysis. The radioactive steroid
chemical methods were described (Table 1, Method 2).
used was either tritiated corticosterone [9-15] or
These included the 2,4-dinitrophenylhydrazine
progesterone [16-18]. The corticosteroid-binding protein
derivative method [2] and methods employing
used in these competitive binding methods was obtained
spectrophotometric measurements at 240 nm [3], sulfuric
from various sources. Solvents used to extract the
acid chromogen [4], fluorescence [5,6], and
progesterone were petroleum ether [9-11,17,18], ethyl
thiosemicarbazide derivative [7].
acetate [13], hexane [14] and diethyl ether [15,16]. In
some publications, chromatographic purification of the
With the advent of isotopically-labeled steroids and
plasma extract before the protein-binding assay was
derivatization compounds, the technique of double-
reported to be essential for the removal of interfering
isotope derivatization was applied to the measurement of
steroids. Chromatographic systems employing thin-layer
plasma progesterone levels (Table 1, Method 3).
silica gel [9,11,16], paper [13], or Celite columns [15]
have been reported. Separation of the free and protein-
Gas chromatography (GC) has been used to improve the
bound fractions was performed with either Florisol
accuracy and rapidity of progesterone analysis (Table 1,
(activated magnesium silicate 60-200 mesh) [9-15,17] or
Method 4), with a series of solvent extractions and paper
Sephadex [16,18] as the absorbing medium.
chromatography being used to purify the plasma extract
The use of radioimmunoassay for the determination of
plasma progesterone concentration was first reported by
i
Progesterone Abraham et al. [19] and Furuyama and Nugent [20]
Previous and current authors of this method: (Table 1, Method 6). These reports were followed by a
First edition: Not done series of publications that reported the use of different
Methods edition: James A. Demitriou extraction solvents, chromatographic purification
Second edition: Not updated systems, radioactive steroids, antibodies, and methods
Third edition: Not updated for separating the free and antibody-bound fractions [21-
Fourth edition: James A. Demitriou 31].
Fifth edition: John Galligan
992
Progesterone
Non-isotopic immunoassays are now available, and they
have become the predominant method as various forms
of these assays have been adapted for use on automated
immunoassay analyzers [32-34]. Such non-isotopic
assays measure progesterone directly and do not require
its extraction from the sample. Reagents containing 8-
anilino-1-naphthalene sulfonic acid (ANS), salicylate,
danazol, dexamethasone, cortisol, or low pH have been
used to displace progesterone from endogenous binding
proteins. A range of chemiluminescent, fluorescent,
luminescent, and photometric substrates have been
utilized to monitor the activity of the assay. Separation
of the free and antibody-bound progesterone is achieved
by incorporating antibody-coated microparticles,
polystyrene beads, or paramagnetic particles.
Immunoassays for progesterone incorporate antibodies
generated against progesterone conjugated to protein,
such as bovine serum albumin through links at positions
C-3, C-4, C-6, C-7, C-11, or C-20. All antibodies so
generated have the potential to cross-react with other C-
21 corticosteroids present in the sample.
Reference and Preferred Methods
Isotope dilution gas chromatography/mass spectrometry
is the accepted reference method used to determine
target values for progesterone. Thienpont et al. [35] have
reported on five years experience in two laboratories,
determining reference-method values in serum-based
materials. For both laboratories, the mean overall
coefficient of variation (CV) was < 2%, the method bias
was < 1%, and the maximum total analytical error in all
cases was < 3%. More recently, developments of highly
sensitive liquid chromatography/tandem mass
spectrometry (LC/MS/MS) instrumentation have
resulted in precise, accurate, and sensitive methods for
the determination of low-concentration analytes,
including progesterone. As a result, a candidate
reference measurement procedure for the estimation of
progesterone has now been evaluated [36].
In the majority of clinical laboratories, automated non-
isotopic immunoassays are used to measure
progesterone. However, a recently published report [37]
which focused on the performance of six automated
immunoassay analyzers documented significant bias in
some methods and considerable variation in results for
the same sample assayed in different laboratories. This
report highlights the need for assays to be stringently
validated to ensure they are fit-for-purpose.
Specimen
Serum or plasma collected with heparin or EDTA as the
anticoagulant is the preferred specimen; however, the
specimen requirements for particular immunoassay
systems should be verified. Saliva has also been
suggested as a possible fluid [38]. A progressive
decrease in progesterone has been observed in specimens
collected into gel-barrier tubes when compared with
samples collected into tubes without gel [39]. No
significant differences have been noted between samples
collected in glass or plastic tubes [40]. Samples are
stable for 7 days at 4C or for up to 3 months at 20C.
Interferences
The presence of hemoglobin, lipemia, and icterus have
no significant effect on the described assays, but this
should be confirmed for automated immunoassay
systems.
Heterophilic antibodies present in some patient samples
can react with reagent immunoglobulins and interfere
with immunoassays. Patients who have been regularly
exposed to animals or have received immunotherapy or
diagnostic procedures utilizing immunoglobulins may
elicit such antibodies as human anti-mouse antibodies
(HAMA). Other heterophilic antibodies such as anti-
sheep, anti-rabbit or anti-goat may also be present in
patient samples. Although assay reagents now include
blocking agents to minimize the possibility of
interference, interaction between patients sera
containing heterophilic antibodies and assay reagents
still occurs [41], the possible outcome being anomalous
results and subsequent inappropriate treatment.
Progesterone Reference Intervals
Males, adult <0.4 ng/mL (<1.3 nmol/L)
Females, adults
Follicular 0.1 to 1.5 ng/mL (0.3 to 4.8 nmol/L)
Luteal 2.5 to 28 ng/mL (7.9 to 89 nmol/L)
Midluteal 5.7 to 28 ng/mL (18 to 89 nmol/L)
Postmenopausal
0.1 to 0.3 ng/mL (0.3 to 0.9 nmol/L)
The concentration of progesterone varies with time
before and after ovulation. During pregnancy, values
may increase by 10 to 100 times over those seen in
nonpregnant individuals. Marked increases are observed
after the 20th week of pregnancy [42].
Given the significant bias reported [37] for some
immunoassay analyzers, the use of laboratory-validated,
method-specific reference ranges would seem
mandatory.
Interpretation
Under the influence of follicle-stimulating and
luteinizing hormones, progesterone secretion by the
ovary starts to rise during the ovulatory phase of the
menstrual cycle and increases to a maximum 6 to 8 days
after ovulation. The corpus luteum formed after
ovulation is the major source of progesterone secretion
during the luteal phase of the menstrual cycle. If
fertilization of the ovum does not occur, the corpus
luteum atrophies, the progesterone concentration falls,
and menstruation occurs. However, with fertilization of
the ovum, the corpus luteum is stimulated by the
increasing levels of chorionic gonadotropin from the
fetoplacental unit, which continues to secrete the
hormone. Progesterone concentrations continue to rise
throughout pregnancy. During pregnancy, progesterone
is synthesized in the placenta from maternal cholesterol
as the substrate.
The major use of progesterone assays is the detection of
ovulation. Midluteal progesterone concentrations of
greater than 10 ng/mL of plasma indicate luteinization of
993
Progesterone
the follicle. However, there is evidence of corpus luteum
formation without ovulation (the luteinized, unruptured
follicle syndrome) in some infertile women [43]. In this
case, it is recommended that three or more samples be
obtained so that they will allow bracketing of the
midluteal peak (such as days 19, 21, and 33). It must be
stressed that the interpretation is dependent on accurate
determination of the start of the last menstrual period
[38]. Luteal-phase insufficiency or short luteal phase has
also been characterized in cases of infertility. Multiple
progesterone assays during the luteal phase are required
to identify this syndrome, which is characterized by low
progesterone concentrations throughout the cycle [44].
The measurement of progesterone in early pregnancy, as
an indicator of potential threatened abortion, has not
been found to be of clinical use and is not justified.
However, the measurement of progesterone (and hCG)
concentration may be a useful adjunct to
ultrasonography in the investigation of patients
presenting to emergency departments with first-trimester
bleeding or pain [45]. It was also reported that in such
circumstances, patients with progesterone concentration
5 ng/mL will almost certainly have a nonviable
pregnancy. In stable patients with a progesterone
concentration > 22 ng/mL, there is a strong likelihood of
a viable intrauterine pregnancy.
Estimation of progesterone concentration has been used
to determine the probability of pregnancy in women
undergoing ovarian stimulation with GNRH and
gonadotropins for in-vitro fertilization. However, a
recent systematic review and meta-analysis does not
support this association [46].
The level of progesterone in saliva samples has also been
shown to provide a noninvasive method for determining
profiles of menstrual cycles [47].
Progesterone Performance Goals
Data was obtained from the College of American
Pathologists (CAP) 2007 Y-A and Y-B Ligand (Special)
Participant Summary. The performance of 17
immunoassay analyzers (~1136 participants) in
measuring progesterone over the range ~1.6 to 31.5
ng/mL was documented. Of the 17 analyzer groups, 1
group had 5/6 samples with CVs > 10%, and 3 other
groups had 3/6 samples with > 10% CV. The evaluation
criteria for progesterone in this Survey is peer-group
target value 3 SD. Under this criteria, 16/17 analyzer
groups had acceptable performance.
When reviewing the all results medians, 1 analyzer
groups medians differed by > 15% in 6/6 samples,
another group differed in 4 samples, and a further 3
analyzer groups had 3 samples > 15% from the all
results medians [48].
References
1 Hooker CW, Forbes TR. A bioassay for minute
amounts of progesterone. Endocrinology
1947;41:158-169.
2 Zander J, Simmer H. Die chemische
Bestimmung von Progesterone in organischen
Substraten. Klin Wochenschr 1954;32:529-540.
3 Short RV. The chemical estimation of
progesterone in peripheral blood. J Endocrinol
1958;16:415-425.
4 Oertel GW, Weiss SP, Eik-Nes KB.
Determination of progesterone in human blood
plasma. J Clin Endocrinol Metab 1959;19:213-
218.
5 Short RV, Levett I. The fluorometric
determination of progesterone in human plasma
during pregnancy and the menstrual cycle. J
Endocrinol 1962;25:239-244.
6 Heap RB. A fluorescence assay for
progesterone. J Endocrinol 1964;30:293-305.
7 Sommerville IF, Pickett MT, Collins WP,
Denyer DC. A modified method for the
quantitative determination of progesterone in
human plasma. Acta Endocrinol 1963;43:101-
109.
8 Yannone MG, McComas DB, Goldfien A. The
assay of plasma progesterone. J Gas
Chromatogr 1964;2:30-33.
9 Neill JD, Johansson EDB, Datta JK, Knobil E.
Relationship between plasma levels and
luteinizing hormone and progesterone during
the menstrual cycle. J Clin Endocrinol Metab
1967;27:1167-1173.
10 Johansson EDB. Progesterone levels in
peripheral plasma during the luteal phase of the
normal menstrual cycle measured by a rapid
competitive binding technique. Acta Endocrinol
1969;61:592-606.
11 DeSouza MLA, Williamson HO, Moody LO,
Diczfalusy E. Further assessment of the
reliability of progesterone assays by
competitive protein binding. Acta Endocrinol
1970;64:(suppl 147)171-183.
12 Lurie AO, Patterson RJ. Progesterone in non-
pregnancy: an assay for the clinical chemistry
laboratory. Clin Chem 1970;16:856-860.
13 Martin BT, Cooke BA, Black WP. Evaluation
of a rapid method for the measurement of
plasma progesterone by competitive protein
binding. J Endocrinol 1970;46:369-377.
14 Demetriou JA, Austin FG. A rapid competitive
protein binding assay for plasma progesterone.
Clin Chim Acta 1971;33:21-32.
15 Stone S, Nakamura RM, Mishell DR Jr,
Thorneycroft IH. A modified technique for the
assay of progesterone in blood using Celite
column chromatography. Steroids 1971;17:411-
422.
16 Yoshimi T, Lipsett MB. The measurement of
plasma progesterone. Steroids 1968;11:527-
540.
17 Horth CE, Palmer RF. Measurement of
progesterone in human plasma. Clin Endocrinol
1972;1:199-207.
18 Pichon MF, Milgrom E. Competitive protein
binding assay of progesterone without
chromatography. Steroids 1973;21:335-346.
994
Progesterone
19 Abraham GE, Swerdloff R, Tulchinsky D,
Odell WD. Radioimmunoassay of progesterone.
J Clin Endocrinol Metab 1971;32:619-624.
20 Furuyama S, Nugent CA. A radioimmunoassay
for progesterone. Steroids 1971;17:663-674.
21 Morgan CA, Cooke ID. A comparison of the
competitive protein-binding assay and
radioimmunoassay for plasma progesterone
during the normal menstrual cycle. J Endocrinol
1972;54:445-456.
22 Kutas M, Chung A, Bartos D, Castro A. A
simple progesterone radioimmunoassay without
column chromatography. Steroids 1972;20:697-
716.
23 Youssefnejian E, Florensa E, Collins WP,
Sommerville IF. Radioimmunoassay of plasma
progesterone. J Steroid Biochem 1972;3:893-
901.
24 Thorneycroft IH, Stone SC. Radioimmunoassay
of serum progesterone in women receiving oral
contraceptive steroids. Contraception
1972;5:129-146.
25 DeVilla GO, Roberts K, Wiest WG, Mikhail G,
Flickinger G. A specific radioimmunoassay of
plasma progesterone. J Clin Endocrinol Metab
1972;35:458-460.
26 Cameron EDH, Scarisbrick JJ.
Radioimmunoassay of progesterone. Clin Chem
1973;19:1403-1408.
27 Calabresi E, Pazzagli M, Fiorelli G, Forti G,
Serio M. Determination of progesterone in
human plasma by radioimmunoassay. J Nucl
Biol Med 1974;18:30-37.
28 Tea NT, Castainer M, Roger M, Scholler R.
Simultaneous radioimmunoassay of plasma
progesterone and 17-hydroxyprogesterone
normal values in children, in men and women
throughout the menstrual cycle and in early
pregnancy. J Steroid Biochem 1975;6:1509-
1516.
29 Aso T, Guerrero R, Cekan A, Diczfalusy E. A
rapid 5-hour radioimmunoassay of progesterone
and oestradiol in human plasma. Clin
Endocrinol 1975;4:173-182.
30 Lebel M, Grosse JH. A rapid and precise
method for measurement of physiological
variations of human plasma progesterone. J
Steroid Biochem 1978;9:989-993.
31 Scarisbrick JJ, Cameron EHD.
Radioimmunoassay of progesterone:
comparison of [1,2,6,7-3H]-progesterone and
progesterone-[125I]-iodohistamine
radioligands. J Steroid Biochem 1975;6:51-56.
32 Product Insert. Architect System. Abbott
Laboratories 2004;69-6610/R5:2.
33 Product Insert. Access Immunoassay System.
Beckman Coulter 2005;33550:1.
34 Product Insert. Advia Centaur. Siemens
Medical Solutions Diagnostics 2004;118696:2.
35 Thienpont LM, Van Nieuwenhove B, Stockl D,
Reinauer H, De Leenheer A. Determination of
reference method values by isotope dilution gas
chromatography/mass spectrometry: a five-year
experience of two European reference
laboratories. Eur J Clin Chem Clin Biochem
1996;34:853-860.
36 Tai SS-C, Xu B, Welch MJ. Development and
evaluation of a candidate reference
measurement procedure for the determination of
progesterone in human serum using isotope
dilution liquid chromatography/tandem mass
spectrometry. Anal Chem 2006;78:6628-6633.
37 Coucke W, Devleeschouwer N, Libeer J-C,
Schiettecatte J, Martin M, Smitz J. Accuracy
and reproducibility of automated estradiol-17
and progesterone assays using native serum
samples: results obtained in the Belgian
external assessment scheme. Human
Reproduction 2007;22:3204-3209.
38 Wood P, Groom G, Moore A, Ratcliffe W,
Selby C. Progesterone assays: guidelines for the
provision of a clinical biochemistry service.
Ann Clin Biochem 1985;22:1-24.
39 Hilborn S, Krahn J. Effect of time of exposure
of serum to gel-barrier tubes on results for
progesterone and some other endocrine tests.
Clin Chem 1987;33:203-204.
40 Reinartz JJ, Ramey ML, Fowler MC. Plastic vs
glass SST evaluated serum-separator blood-
drawing tubes for endocrinologic assays. Clin
Chem 1993;39:2535-2536.
41 Kricka LJ. Interferences in immunoassay: still a
threat. Clin Chem 2000;46;1037-1038.
42 Edelstam G, Karlsson C, Westgren M, Lowbeer
C, Swahn M-L. Human chorionic gonadotropin
(hCG) during third-trimester pregnancy. Scand
J Clin Lab Invest 2007;67:519-522.
43 Abdulla U, Diver MJ, Hipkin LJ, Davis JC.
Plasma progesterone as an index of ovulation.
Br J Obstet Gynaecol 1983;90:543-548.
44 Hinney B, Henze C, Kuhn W, Wutke W. The
corpus luteum insufficiency: a multifactorial
disease. J Clin Endocrinol Metab 1996;81:565-
570.
45 Murray H, Baakdah H, Bardell T, Tulandi T.
Diagnosis and treatment of ectopic pregnancy.
CMAJ 2005;173;905-912.
46 Venetis CA, Kolibianakis EM, Papanikolaou E,
Bontis J, Devroey P, Tarlatzis BC. Is
progesterone elevation on the day of human
chorionic gonadotrophin administration
associated with the probability of pregnancy in
in-vitro fertilization? A systematic review and
meta-analysis. Human Reproduction Update
2007;13:343-355.
47 Gandara BK, Leresche L, Mancl L. Patterns of
salivary estradiol and progesterone across the
menstrual cycle. Ann N Y Acad Sci
2007;1098:446-450.
48 College of American Pathologists. Participant
summary surveys 2007: Y-A, Y-B, ligand
(special). Washington, DC: CAP; 2007.
995
Progesterone

Table 1: Methods for Measurement of Progesterone

Method 1: Biological; bioassay
Principle of analysis: Increase in mouse uterine weight is measured.
Comments: Historical interest
Method 2: Chemical; spectrometric or fluorometric measurement
Principle of analysis: Conversion of steroid to chromogen or fluorogen; absorbance or fluorescence is measured.
Comments: Many interferences, historical interest
Method 3: Isotopic; double-isotope derivatization
Principle of analysis: Isotope ratio of added steroid and radiolabeled endogenous steroid is determined.
Comments: Labor intensive; large quantity of isotope required, historical interest
Method 4: Vapor-phase chromatography; gas-liquid chromatography
Principle of analysis: Chromatographic separation and detection by flame ionization, electron capture, or
nitrogen sensors.
Comments: Labor intensive; limited in number of specimens that can be tested, historical interest
Method 5: Protein binding; competitive protein binding
Principle of analysis: Ratio of free to bound radioactive steroid determined after binding by steroid-specific
binding globulin.
Comments: Lacks specificity, but relatively large numbers of specimens can be processed
Method 6: Immunoassay
a. Radioisotopic/steroid-specific antibody
b. Enzyme/fluorescence/steroid-specific antibody
Principle of analysis:
a. Ratio of free to bound radioactive steroid determined after binding by steroid-specific antibody
b. Bound chemiluminescent, enzyme or fluorescent-labeled ligand determined after binding by steroid-
specific antibody
Comments:
a. Specific, simple, and relatively large numbers of specimens can be processed
b. Simple, readily automated, rapid, capable of processing greater numbers than other assays with no
radioactive waste generated
Method 7: Isotope dilution gas chromatography/mass spectrometry
Principle of analysis: Internal standard, ionization, sorting and separation of ions according to mass and charge,
detection and quantification
Comments: Complex instrumentation, high sensitivity, high specificity, medium throughput, reference method,
requires skilled analyst
Method 8: Liquid chromatography/tandem mass spectrometry
Principle of analysis: HPLC, ionization, sorting and separation of ions according to mass and charge, further
separation of compound fragments, detection, and quantification
Comments: Complex instrumentation, high sensitivity, high specificity, medium throughput, proposed reference
method, requires skilled analyst
996
Prolactin
Prolactin
Sheila Dawling
Name: Prolactin
Clinical interpretation: Refer to Chapter 48, General Endocrinology, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular mass: 22,000 D
Conversion factor: ng/mL 21.2 mIU/L [WHO 3rd IS 84/500]
Merck Index: 7683
Chemical class: Protein
i enzymatic activity of the eluate was assayed by use of a
Principles of Analysis and Current Usage
Human prolactin is a single-chain polypeptide hormone
of 198 amino acid residues with a molecular mass of
approximately 22,000 D [1]. It is secreted by lactotrophs
of the anterior pituitary gland under the control of
hypothalamic factors. The high structural homology of
prolactin with growth hormone (16%), their similar
cross-reactivity in bioassays, and the 20- to 100-fold
enrichment of growth hormone over prolactin in the
pituitary gland hampered early attempts at separation,
and it was only in 1970 that prolactin was definitively
identified as a discrete hormone [2]. Prolactin is assayed
as an aid in the clinical diagnosis and management of
amenorrhea, galactorrhea, and hypothalamic-pituitary
disorders. Magnetic resonance imaging (MRI) and other
scanning techniques play an important complementary
role. Assay methods have evolved from bioassays,
through radio-immunoassays and immuno-radiometric
assays, to modern non-isotopic immunometric assays
with an assortment of detection methods (see Table 1).
Bioassays for prolactin (Table 1, Method 1) are now
only used in research settings, particularly to
characterize variant molecular weight prolactins [3,4].
Early radio-iodine immunoassays were improved by the
use of purified human prolactin (Table 2, Method 2) [5].
Both bioactivity and immuno-reactivity results
correlated well in normal persons and from patients with
hyperprolactinemia under both basal and stimulated
conditions [6]. The manufacture of a monoclonal
prolactin antibody in the early 1970s and its
incorporation into immuno-radiometric kits using
radioisotope-labeled antibody as the tracer marked the
first clinically useful prolactin assays (Table 1, Method
3). The first of these assays was a competitive binding
enzyme assay [7] in which serum was incubated with
rabbit anti-prolactin antibody and -galactosidase-
labeled prolactin. After retention of the antibody-bound
prolactin on a column of anti-rabbit IgG-sepharose, the
i
Prolactin
Previous and current authors of this method:
First edition: Not done
Methods edition: C. Dennis Ashby
Second edition: Not updated
Third edition: Not updated
Fourth edition: C. Dennis Ashby
Fifth edition: Sheila Dawling
fluorogenic substrate, 4-methylumbelliferyl--
galactoside. Although 48-hour incubation was required
to optimize the sensitivity to 2.5 ng/mL, the results
correlated well with a radioimmunoassay.
The non-isotopic immunoassays available today are
rapid, sensitive, reliable, and easily automated. The most
common methods in use today are immunometric assays
employing two antibodies raised towards different
epitopes of the prolactin molecule (Table 1, Method 4).
One antibody, designated the capture antibody, is
usually immobilized in proprietary fashion to the
reaction vessel wall, to particle(s). The second
(detection) antibody forms a sandwich with the captured
prolactin and carries a label which is then used to
determine the extent of the binding (Figure 1a). Labels
can be enzymes, fluorophores, or chemiluminescent
probes, and to a large extent the nature of the detection
label determines the sensitivity of the method. All assays
are highly specific for prolactin with respect to growth
hormone and pituitary and reproductive hormones and
are described individually in the Reference and Preferred
Methods heading.
Prolactin circulates in human plasma in various forms.
Studies using gel-filtration chromatography [8,9] have
identified three distinct forms: approximately 60% to
90% is a 23kDa prolactin monomer; 15% to 30% is a 40
to 60 kDa big prolactin; and 0% to 10% is a >100 kDa
big, big prolactin, now commonly referred to as
macroprolactin. This normal distribution pattern is
preserved in the majority of patients with elevated
prolactin irrespective of the physiological/pathological
cause. The three forms react differently: in radio-
receptor assays, 77% of the receptor activity was found
with monomer prolactin; only 12% and 11%,
respectively, were found with big and macroprolactin. In
contrast, monomer prolactin represents 58% of the total
immunoreactivity, big prolactin 18%, and
macroprolactin 24% [10].
Macroprolactin
Little is known of the clinical significance of big
prolactin, and it has not received much attention to date.
Macroprolactin, however, has been studied extensively.
It is formed in the circulation and has a longer half-life
than monomeric prolactin [11]. Although active in vitro,
its lack of activity in vivo can be explained, since its
997
Prolactin
large size impedes transfer out of the capillaries to target
receptors [12,13]. However, macroprolactin is not a
homogeneous entity. The molecular mass of the
common fraction is in the range 150 to 170 kDa and is
frequently [14] though not always [15] associated with
IgG. There is evidence that it behaves as an anti-
prolactin autoantibody [16] and is commonly found in
patients with idiopathic hyperprolactinemia [17]. Recent
thought is that circulating acidic prolactin isoforms,
found only in these patients, may act as the antigenic
stimulus for continued macroprolactin production [18].
Lower (120 kDa) and higher (200 to 699 kDa) mass
forms, together with some heavily glycosylated forms
and those containing IgA and IgM, are less commonly
described [19,20]. The overall prevalence of
macroprolactinemia in the general population is thought
to be about 1% to 2% (0.5% in healthy people [21], and
in patients who have macroprolactinemia, the amount
present correlates with increases or decreases in
monomeric prolactin over time [22-25].
The reactivity of macroprolactin in immunoassays
causes problems when interpreting an increased serum
prolactin concentration. It is therefore of extreme
importance that laboratories recognize this potential
source of error and take steps to minimize the reporting
of misleading results. Identification of macroprolactin,
which has minimal biological activity but can be the
cause of high prolactin concentrations in serum by
immunoassay, can help resolve the diagnostic confusion
and avoid expensive investigations and inappropriate
treatment. The additional expense incurred by the
laboratory is offset many-fold by these avoidable costs.
Before an analysis for macroprolactin is undertaken, the
specimen must first be shown to have an increased total
prolactin concentration. A review of clinical studies [26]
reveals a cut-off of approximately 40 ng/mL (800
mIU/L), most commonly used to initiate a
macroprolactin assay, but this should be adapted to the
population under study.
One common approach has been to use two different
immunoassaysone with a broad spectrum of reactivity
(DELFIA or Elecsys) and the other with a low cross-
reactivity (BC Access or Bayer Centaur)and to
highlight discrepant results as possibly due to the
presence of macroprolactin. However, the case study by
Ismail shows the fallacy of this approach and highlights
the need for a more rigorous scientific base [27]. A
sounder approach is to re-run the prolactin analysis using
the same immunological method after removal of any
macroprolactin present. These methods are referred to as
macroprolactin assays and have recently been compared
and reviewed by Kavanagh et al. [28] (see Table 2 and
Appendix). Two general strategies are available for the
analysis of macroprolactin. The first is to separate the
prolactin fractions by gel-filtration chromatography
(GFC) and determine the immuno-reactivity of each
fraction. The second and more readily available and
cost-effective strategy is to remove any macroprolactin
present and then determine the residual
immunoreactivity due to monomeric prolactin. Both
ultrafiltration and precipitation (with polyethylene glycol
(PEG), Protein A, Protein G or anti-human IgG) have
been used.
GFC is the oldest method (Table 2, Method 1) and is
considered the gold standard in that it allows
simultaneous quantitation of all three molecular forms of
prolactin. It is, however, time consuming and expensive,
adding significantly to the cost of the analysis, and
requires equipment unlikely to be available in non-
specialist laboratories. Precipitation with PEG (Table 2,
Method 2) is relatively nonspecific but easy and
inexpensive to perform. PEG is readily obtained and
highly stable. PEG polymers sequester water from the
specimen into their matrix, causing macromolecules to
exceed their solubility and precipitate. The main
drawback is that some immunometric methods are
affected by the presence of the PEG in the supernate.
Precipitation of macroprolactinimmunoglobulin G
complexes has been described using a number of
relatively inexpensive reagents (Table 2, Method 3).
Ultrafiltration (Table 2, Method 4) employs a standard
membrane filter (available from Amicon in a variety of
pore sizes) with a molecular weight cut-off of around
100 kDa. These devices are freely porous to 45kDa and
completely retain above 100 kDa, with variable
penetrance in between. The clear filtrate is analyzed for
prolactin content by immunoassay. Since there are no
additives to interfere with immunoassays, it can be used
where precipitating techniques are not able to be applied.
Comparative studies show good agreement between
ultrafiltration, PEG precipitation, and GFC, most notably
when macroprolactin is the predominant species. The
additional sample processing and dilution steps for
macroprolactin elimination necessarily introduce
imprecision into the method, with assay coefficient of
variation (CV) ranging from 3% to16%, depending on
the methodology and choice of specimen [29].
Reference and Preferred Methods
There is no designated reference method for prolactin,
but the overwhelming preference is for an automated
immunoassay for clinical applications. These offer the
advantage of simplicity and make use of reagents that
have been optimized, validated, and quality controlled
by the manufacturer. Distribution of analytical methods
for prolactin among the 1500 laboratories returning
results for immunometric assays to the College of
American Pathologists (CAP) in 2006-7 is shown in
Table 3. Although 1500 laboratories participated in the
spot challenges, only one third as many took part in the
calibration and linearity verification surveys.
Comparative data are shown for the 312 laboratories
participating in the 2006 United Kingdom National
External Quality Assurance Scheme (UKNEQAS).
Performance in these surveys is discussed later in the
section under Prolactin Performance Goals.
For an objective comparison of available prolactin
methodologies, optimal characteristics and performance
criteria should be defined. Suggested criteria are
presented here. Immunoassay reagents (i.e., immunogen,
label, and calibration standards) should be of human
origin. The procedure should be calibrated against a
reference standard with known international unit
998
Prolactin
potency, such as WHO 84/500. An analytical
measurement range from 0.5 to 200 ng/mL is desirable,
preferably with an option for automatic dilution of
samples with high concentrations of prolactin. The
challenge is to extend the measurement range without
sacrificing precision at low concentrations. The ability of
the procedure to allow accurate and precise quantitation
of levels in the 20 to 40 ng/mL range is crucial, because
this range represents the discrimination between normal
and elevated results. The antibodies should be tested for
cross-reactivity and be specific enough not to show
responses at less than 0.1% at pathological
concentrations with growth hormone (GH), luteinizing
hormone (LH), follicle-stimulating hormone (FSH),
human placental lactogen (PL), and thyroid stimulating
hormone (TSH). Cross-reactivity with human chorionic
gonadotrophin (HCG) should be less than 0.01%. Apart
from growth hormone, human prolactin has no
significant homology with other polypeptide hormones,
and thus no other interferences should be expected.
The features of the most commonly used immunometric
prolactin assays methods are compared in detail in Table
4, and in Figures 2 through 4. The Wallac DELFIA
method is also included as this method, although not
approved for clinical use in the United States, is used
extensively in research because it shows almost equal
cross-reactivity with all molecular forms of prolactin.
All methods should be tested for the presence of a hook
effect. The hook effect occurs in immunometric assays
when the excess antigen in the patient sample saturates
the binding sites on both the capture and detection
antibodies, thus preventing the formation of the
sandwich and the immobilization of the detection
antibody. This is most likely to happen when both
capture and detection antibodies are incubated with the
patient specimen in a single step (see Table 4). In a two-
step assay, patient specimen is reacted with the capture
antibody prior to the addition of the capture antibody (in
some assays excess unbound prolactin is washed from
the reaction system prior to the addition of the detection
antibody). In this latter case, a maximal (though
underestimated) response is obtained, whereas in the
former case, a falsely low or even subnormal result is
obtained [30,31]. With the prevalence of MRI and other
imaging techniques, the diagnosis of a pituitary adenoma
is unlikely to missed, although it may be classified
incorrectly as a nonfunctioning pituitary adenoma rather
than a prolactinoma and be treated surgically rather than
with drugs [32]. In one series of patients, nonfunctioning
adenomas produced more pituitary damage (visual
impairment, deficiency of ACTH, FSH, LH, and TSH)
than did prolactinomas secreting sufficient hormone for
a hook effect [33] and will therefore carry a high degree
of clinical suspicion. The hook effect can be
demonstrated by serial dilution of the specimen either in
isotonic saline, zero-prolactin calibrator, or system
diluent, as specified in the manufacturers instructions.
Example results are shown in Table 5.
Interference by heterophile antibodies should be
suspected when serial specimens from the same patient
or repeat analysis of the same sample produce markedly
different results, or when recovery on dilution is not
linear. Interference from heterophile antibodies is
discussed in the section on Interferences below.
While some analytical problems encountered in prolactin
assays are shared with many other hormones that are
measured by immunometric assays, in the case of
prolactin, it is differences in cross-reactivity with
macroprolactin that are responsible for most of the
observed variation and inconsistencies between different
methods[34]. Methods are classed as being highly
reacting (DELFIA, Elecsys I), low reacting (Beckman
Access, Bayer Centaur), and intermediate reacting
(Immulite, Vitros ECi). None of the technical inserts for
the methods listed contain specific cross-reactivity with
macroprolactin, although the Roche insert does describe
a procedure for its precipitation with PEG.
For the identification and measurement of
macroprolactin, the specimen is first analyzed by
immunoassay to determine the total prolactin present. If
hyperprolactinemia is demonstrated, yet symptoms are
not supportive, or if there is over-recovery on dilution, a
treatment step is performed and the prolactin analysis
repeated on the treated products. Two sample treatments
are described in more detail in the appendix. Gel-
filtration chromatography (GFC) is the gold standard but
is labor intensive and expensive, whereas PEG
precipitation is simpler and far less expensive.
Macroprolactin assays have recently been compared and
reviewed by Kavanagh et al. [28], and a more thorough
account of the significance of this entity can be found in
the appendix.
Specimen
Serum is the sample of choice for prolactin, but EDTA
and heparin plasma may be acceptable, depending on the
methodology used (not for methods with active metal
ions). Gel separators do not affect results. However,
there may be instances where a specific formulation of
tube additives can affect one or more methods adversely
(see comments in Interferences Section below).
No special handling procedures are necessary, though
multiple freeze/thaw cycles should be avoided. Serum
can be stored up to 2 weeks at 2C to 8C and for longer
periods at or below 10C [35].
Physical stress has been reported to both increase and
decrease serum prolactin concentrations, whereas
emotional stress may elevate blood levels of prolactin
[36]. Patients should be under resting basal conditions
before sampling [37]. Diurnal variation in prolactin has
been described with mean concentrations greater
between 04:00 and 08:00 hours, and between 20:00 and
22:00 hours [38,39].
Interferences
The presence of hemolysis (below 0.5 g/dL
hemoglobin), hyperbilirubinemia (below 20 mg/dL), or
lipemia (below 1000 mg/dL triglycerides) generally do
not interfere with the reported immunometric assay
999
Prolactin
methods in Table 4. Cross-reactivity with other
reproductive and pituitary hormones is negligible and
was addressed earlier.
For the assays that have biotin-streptavidin components,
specimens from patients receiving high-dose biotin
supplements (>5mg/day) may be problematic. It is
recommended that a minimum of 8 hours elapse between
biotin ingestion and specimen collection for prolactin
analysis. Isolated cases of high titer antibodies against
streptavidin are also mentioned in the literature, but
supporting data are difficult to locate. The presence of
antibodies to the cross-linker used for attachment of the
ruthenium ion in the Roche assay (Method 4f) has been
documented in a few patients with the thyroid assays but
is equally applicable to all assays with this structure
[40].
Interference by heterophile antibodies should be
suspected when serial specimens from the same patient,
or repeat analysis of the same sample, produce markedly
different results or when recovery on dilution is not
linear. Heterophile antibodies are polyclonal
autoantibodies, either IgG or IgM, which bind across a
broad spectrum of different animal antibodies used in kit
manufacture, probably through the Fc region.
Heterophile antibodies thus form sandwiches between
the capture and detection antibodies, and so a positive
signal is produced even in the absence of prolactin
(Figure 1b). In rare cases (10%) the heterophile antibody
binds to just one of the reagent antibodies and prevents
formation of the sandwich, giving a false-negative
signal. Assays employing enzyme labels are rather more
prone to this type of interference because of the degree
of modification to the antibody during the conjugation
process. Human anti-mouse antibodies (HAMA) are one
specific example that binds mouse immunoglobulins.
Heterophile antibodies are more likely to be seen in
people who are exposed to animals, such as farm
workers, veterinarians, laboratory technicians, or those
exposed repeatedly to blood products or medications
based on blood products. Many manufacturers formulate
their reagents to minimize this possibility of interference
from HAMA, but such interferences have been the
subject of several recent reviews [41,42]. Analysis by an
alternative method is usually sufficient to align the
measured result with the clinical picture, but in some
cases it may be necessary to prove that these antibodies
exist. In this case, a broad spectrum passive precipitant
such as PEG can be used, but it is preferable to use an
active blocking agent such as The Scantibodies
Heterophilic Blocking Reagent Tubes (HBRT). The
reagent is a proprietary mix of lyophilized mouse anti-
human IgM with high affinity for human anti-animal
antibodies commonly used in reagent manufacture
(mouse, goat, sheep, and rabbit) and to rheumatoid
factor. Each tube contains sufficient reagent to inactivate
500 L of patient sera and is incubated for 1 hour at
room temperature. In comparison to PEG, the specimen
is not diluted, and the immunoassay signal is only
minimally affected [43,44]. Although FDA-approved for
this purpose, the value obtained from a sample treated
with HBR should not be used as a reportable result.
The most recent type of interference discovered was
from a blood collection tube additive, the organosilicone
surfactant Silwet L-720, present in certain lots of BD
Vacutainer SST tubes. The presence of this compound
was responsible for desorbing capture antibodies from
the solid phase polystyrene beads used in the Immulite
methodology [45]. Although the problem was first noted
with the total T3 assay, resulting in falsely increased
results, it is common to the methodology, and other
competitive binding assays were similarly affected. In
contrast, immunometric assays were negatively affected,
although to a lesser degree [45]. Such findings have led
to heightened awareness of potential interference in
immunoassays and have called for decanting of
calibrators and controls into the same tube type as used
for patient specimens [46].
Prolactin Reference Interval
A variety of physiological, pharmacological, and
pathological conditions can affect the circulating
concentrations of prolactin, and these conditions should
be considered when results are being interpreted. Greatly
increased concentrations of serum prolactin are present
during the first 6 weeks or so of life but rapidly decrease
thereafter to approximately 5 ng/mL, where it remains
throughout most of childhood. For both males and
females, prolactin concentrations increase progressively
from prepuberty to puberty and to adulthood levels of up
to approximately 20 ng/mL for males and 25 ng/mL for
females [47,48]. Although adult female prolactin levels
are usually higher than those in adult males, there is a
considerable overlap. Levels of prolactin are increased
over baseline in females who are taking oral
contraceptives and up to 10-fold during the later stages
of pregnancy, declining to basal levels by some 90 days
postpartum. Breast feeding stimulates prolactin secretion
and slows the decline postpartum. In postmenopausal
women, serum concentrations of prolactin decrease to
below those seen in cycling females [37].
Interpretation
A variety of pharmacological agents are known to affect
the circulating concentration of prolactin.
Hyperprolactinemia can result from dopamine receptor
antagonists, such as phenothiazines, butyrophenone
antipsychotics, risperidone, verapamil, and
metoclopramide, and from dopamine-depleting agents,
such as methyldopa and reserpine. Tricyclic
antidepressants, cocaine, opiates, and estrogens can also
increase prolactin concentrations, but these elevations
are usually modest (rarely exceeding 150 ng/mL) [49].
Administration of dopamine substitutes or receptor
agonists such as l-dopa, apomorphine, and ergot
derivatives suppress prolactin secretion [37].
Psychological stress is a common cause of modest
increases in serum prolactin concentration, but the most
common pathological cause of an elevated serum
prolactin is a pituitary adenoma. These are usually small,
slow growing, and benign. Prolactinomas are classified
by their size, and the serum prolactin concentration
parallels tumor size quite closely. Microprolactinomas
1000
Prolactin
(<10 mm in diameter) are typically associated with
prolactin concentrations of 40 to 100 ng/mL.
Macroprolactinomas (>10 mm diameter) are typically
associated with concentrations > 250 ng/mL, and which
do occasionally exceed 1000 ng/mL. It is important to
distinguish between a macroprolactinoma and the
compound macroprolactin; the two are quite distinct
entities. The discrepancy between a large pituitary
adenoma and only a mildly elevated prolactin (<100
ng/mL) should be a signal for further investigation for
either compression of the pituitary stalk by a non-
prolactin-secreting adenoma or to a heavily secreting
macroprolactinoma producing a hook effect on
immunoassay. This distinction is especially important,
since drug treatment is particularly effective for
prolactinomas, reducing both size and prolactin
secretion, whereas a non-secreting adenoma probably
requires surgical intervention. Bromocriptine and
cabergoline are the primary drug therapies for
prolactinomas [50]. A combined diagnostic and
treatment pathway is provided by Schlechte [32]. (Figure
5)
Prolactin is occasionally produced ectopically by oat cell
carcinomas of the lung and renal carcinomas. Circulating
concentrations of prolactin increase progressively with
the severity of chronic renal failure and as expected are
unaffected by hemodialysis [51]. Prolactin concentration
can be moderately increased with herpes zoster
infection, especially when prominent in dermatomes T4-
T5, in polycystic ovary syndrome, and with excessive
exercise or stress. Hyperprolactinemia is also observed
in patients with primary hypothyroidism, probably due
to the stimulation of pituitary lactotrophs by elevated
TSH and/or TRH concentrations. For this reason,
workup for hyperprolactinemia should include
assessment of thyroid status [32].
The most common symptoms of hyperprolactinemia in
premenopausal women are amenorrhea and infertility.
Galactorrhea occurs in about 80% of these patients, and
some have menstrual irregularities. The majority of
prolactinomas are small at diagnosis in women, and
headaches and neurological deficits are rare. By contrast,
prolactinomas in men typically tend to be large at
diagnosis and nay cause cranial nerve dysfunction,
visual-field loss, and panhypopituitarism, with
associated impotence, infertility, and loss of libido [32].
Prolactin Performance Goals
Acceptable performance criteria (CLIA-88) for
measurement of serum prolactin requires that
laboratories be accurate to within 3 SD of the peer
group mean. Precision data from the CAP 2006-7
Participant Summary Reports are shown in Table 3 for
1500 laboratories returning results for immunometric
assays to CAP in 2006-7. Only a third as many
participated in the calibration and linearity verification
surveys, and the spread of these data shows the bias
between methods. Comparative data are shown for the
312 laboratories participating in the 2006 United
Kingdom National External Quality Assurance Scheme
(UKNEQAS), showing imprecision and bias. Desirable
specifications for analytical imprecision derived from
studies of biological variation [52] indicate a desired
assay imprecision of 3.5%. Most assays have similar
imprecision values (% coefficient of variation) of 5% to
8%, although there is much larger variation across the
entire group (typically 20%). This is most likely
attributed to the presence of macroprolactin. UKNEQAS
specifically targets macroprolactin in its survey sets.
Over the last 12 years, they have circulated some 10
challenges containing macroprolactins and recently
introduced an interpretative challenge. This circulation
shows 83% of labs in the scheme screen routinely for
macroprolactin above 700 mIU/L (34 ng/mL) by PEG
precipitation [25].
Recent literature stresses the importance of adequately
identifying the presence of macroprolactin and
appreciating its significance. In one retrospective study
of patients previously diagnosed with prolactinoma,
macroprolactin has been proven to account entirely for
the hyperprolactinemia in 42% of patients, a third of
whom presented with signs and symptoms of
hyperprolactinemia [53]. This degree of misdiagnosis is
no longer acceptable. To this end, the onus has been
placed on kit manufacturers to characterize their
antibodies with respect to cross-reactivity with
macroprolactin, with a view to reducing it [54]. In
addition, the Pituitary Society and other leading groups
of endocrinologists are calling for automatic reflexing of
a PEG precipitation test to exclude the presence of
macroprolactin in all specimens with moderate
hyperprolactinemia (>35 ng/mL). Drafting of
appropriate guidelines are underway [55-57].
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62 Sustarsic LW, Fahie-Wilson M, Vanrieken L,
Walker K, Del Rosario I, El Shami AS. Matrix
effect of PEG precipitation on detection of
macroprolactin in IMMULITE and IMMULITE
1003
Prolactin

2000 prolactin assays. Clin Chem 2002; 48
(S6): A62.
63 Schiettecatte J, DeSchepper J, Velkeniers B,
Smitz J, van Steirteghem A. Rapid detection of
macroprolactin in the form of prolactin-
immunoglobulin G complexes by
immunoprecipitation with anti-human IgG-
agarose. Clin Chem Lab Med 2001; 39: 1244-8.
64 Bjorck L, Kronvall G. Purification and some
properties of streptococcal protein G, a novel
IgG binding reagent. J Immunol 1984; 133:
969-74.
65 Lindmark R, Thoren-Tolling K, Sjoquist J.
Binding of immunoglobulins to protein A and
immunoglobulin levels in mammalian sera. J
Immunol Methods 1983; 62: 1-13.
Appendix Data at
www.acb.org.uk/annclinbiochem/Webwise/Elli
sAppendix1.pdf

Table 1: Methods of Analysis for Prolactin

Method 1: Bioassay
Principle of analysis: Cell replication stimulated by prolactin
Comments: Historical; useful for establishing bioreactivity; insensitive for clinical samples
Method 2: Radioimmunoassay
Principle of analysis: Competitive binding assay; isotope-labeled prolactin competes with endogenous hormone
for limited amount of antibody.
Comments: Rare and mainly historical; applicable to most laboratory samples; lacks specificity
Method 3: Immuno-radiometric assay
Principle of analysis: Antibody linked to solid phase reacts with prolactin in test sample. Second prolactin
antibody radioactively labeled reacts in proportion to amount of prolactin bound to solid phase.
Comments: Rare; more specific than method 1; radioactivity a disadvantage
Method 4: Non-isotopic immunometric assays
Principle of analysis: Prolactin forms sandwich complex between a capture antibody attached to a solid phase
support and second labeled antibody. Label may be an enzyme, fluorophore, or chemiluminescent tag.
Comments: Most common methods in use; easily automated; good sensitivity and specificity; two-step assays are
less prone to hook effect. Take care to exclude macroprolactin.
(See table 4 for comparison of most commonly used commercially available methods.)

Table 2: Methods of Analysis for Macroprolactin

Method 1:
Principle of analysis: Gel-filtration chromatography (GFC)
Comments: Simultaneous quantitation of PRL in each molecular fraction identified; reference method;
expensive, time consuming; not readily available
Method 2:
Principle of analysis: PEG precipitation based on molecular size
Comments: Readily available and inexpensive; nonspecific for Ig class; most common method in use; correlates
well with GFC; may be incompatible with some immunoassays (Abbott Axsym)
Method 3:
Principle of analysis: Specific precipitation of immunoglobulin G and IgG-macroprolactins using anti-hIgG-
agarose, Protein A-agarose, Protein G-agarose
Comments: Easy to use and inexpensive; fractions containing other Ig classes are not precipitated.
Method 4:
Principle of analysis: Ultrafiltration by centrifugation
Comments: Readily available; inexpensive and easily performed; macroprolactins may dissociate; dilution of
specimen is not required; compatible with all immunoassays
1004
Prolactin

Table 3: Methods Used for Prolactin Measurement and Their Performance as Reported by CAP
and UKNEQAS During 2006-7

CAP 2006 - 7 2006

Average of 7 spot Linearity / Calibration UKNEQAS
challenges Verification
Method % of Labs mean ng/mL cv % low ng/mL high ng/mL % of labs cv % % Bias

4a + 4b 12.1 24.5 5.7 1.9 160.9 17.0 5.7 15.0

4c 29.5 19.0 5.9 1.8 132.8 28.2 5.5 -10.0
4d 22.7 19.6 5.6 1.7 129.6 8.3 7.0 3.0
4e 16.3 19.8 5.7 1.8 123.5 16.3 7.5 -15.0
4f 10.5 25.0 6.0 2.0 145.6 24.4 3.5 10.0
4g 7.2 19.7 5.3 6.0 124.7 <1 --- 10.0
Others 1.7 5
All Results 100.0 21.0 13.4 100.0 20.0

a Abbott Axsym
b Abbott Architect
c Bayer Centaur
d Beckman-Coulter Access / DxI
e Diagnostic Products Immulite Series
f Roche Elecsys Systems
g Vitros ECi

NEQAS data kindly supplied by Andy Ellis, UK NEQAS for Peptide Hormones, Edinburgh.

Table 4: Comparison of the Most Commonly Used Immunometric Assays for Prolactin
ULOQ ng/mL
Ab Hook Effect
Captue Ab*/ Detection Ab* / Conjugation Separation LLOQ ULOQ Extended by Calibration Type /
Method Addition Detection System Absent to:
solid phase Conjugate System method ng/mL ng/mL Automatic Frequency
Steps ng/mL
Dilution
mc mouse / mc rabbit / particles trap 4-Methylumbelliferyl-phosphate Wet
4a --- 1 0.6 200 no 10,000
microparticles ALP in fiber Fluorescent product Full curve 28 day

mc mouse / 2 with Acridinium (N-sulfonyl) carboxamide Electronic for lot#

mc rabbit /
4b paramagnetic --- interim magnet Chemiluminescence with 0.6 200 2,000 10,000 2 wet ajustors each
Acridinium ester
particles wash Peroxide and NaOH trigger kit and 28 day
mc mouse / Acridinium ester Electronic for lot#
pc goat /
4c paramagnetic Biotin - Streptavidin 2 magnet Chemiluminescence with 0.3 200 1,000 30,000 2 wet ajustors each
Acridinium ester
particles Acid and NaOH trigger kit and 28 day
mc mouse /
pc goat / pc goat anti-mouse Chemiluminescence with Lumi-Phos Wet
4d paramagnetic 1 magnet 0.25 200 no not stated
ALP IgG 530 substrate Full curve 28 day
particles
Electronic for lot#
mc mouse / mc goat / centrifugal Adamantyl-1,2-dioxetane phosphate
4e --- 1 0.16 150 3,000 20,500 2 wet ajustors each
plastic bead ALP wash substrate Fluorescent product
kit and 28 day
mc mouse / mc mouse / Electrochemiluminescence with Ru Electronic for lot#
4f paramagnetic Ruthenium Biotin - Streptavidin 2 magnet Electron amplification with 0.05 470 4,700 12,690 2 wet ajustors each
particles complex Tripropylamine kit and 28 day
Luminol derivative substrate
mc sheep / mc mouse / Peracid salt peroxide generator Wet
4g Biotin - Streptavidin 1 wash 1.4 329 no 20,680
well wall HRP Electron amplification with 3- Full curve 28 day
Chloro,4-hydroxy acetanilide
mc mouse / Acidic chelator dissociates Eu3+ Wet
mc mouse /
4h microtiter plate Biotin - Streptavidin 1 wash Micellar enhancer 0.04 250 no 2,500 Full curve each
Europium
well wall Time resolved fluorimetry at 613 nm assay

a Abbott Axsym ALP Alkaline phosphatase

b Abbott Architect HRP Horseradish Peroxidase
c Bayer Centaur * mc = monoclonal; pc = polyclonal
d Beckman-Coulter Access / DxI
e Diagnostic Products Immulite Series
f Roche Elecsys Systems
g Vitros ECi
h Wallac DELFIA
1005
Prolactin

Table 5: Demonstration of Hook Effect with Prolactin Assay

Sample Measured Final "Correct" 1600

Dilution Prolactin Result Response 1400

Measured PRL ng/mL

ng/mL ng/mL ng/mL 1200
Neat 12 12 392,000
1000
x2 15 30 196,000
800
x10 36 360 39,200
x50 133 6,650 7,840 600

x250 1504 376,000 1,568 400

x500 784 392,000 784 200
0
0 200 400 600
Dilution Made

Table 6: Incidence of Hyperprolactinemia Explained by Macroprolactin by Immunoassay

Prevalence
Immunoassay Method
%
Method used:
Data abstracted from Fahie- Abbott Architect 16 - 17
Wilson et al. [23] Bayer Centaur 5
Bayer Immuno-1 26
Beckman-Coulter Access / DxI <1
Diagnostic Products Immulite Series 10
Roche Elecsys Systems I 16 - 17
Roche Elecsys Systems II 5
Tosoh AIA 24
Wallac DELFIA 15

Figure 1:

1a: Typical sandwich immunoassay format with antibodies directed to different epitopes of the prolactin molecule. The
capture antibody may be attached to a surface such as a well wall or bead. The second antibody is labeled with a marker
that facilitates quantitation of the amount of sandwich formation, hence determination of prolactin concentration in the
specimen.
1b: Interference from heterophile antibody. The capture and detection antibodies are linked together by a nonspecific
antibody, producing a signal indistinguishable from that produced by prolactin.
1006
Prolactin

Figure 2

Prolactin in
patient sample Wash ALP

Y
off
YY

Y ALP
Substrate

YY
Detection Ab ALP

Y
Prolactin Capture Ab
coated Microparticles
Capture on Filter
Fluorescent
Product

CH3
CH3
Alkaline
O + PO4
Phosphatase
OH P O O O
O O
OH

4-Methylumbelliferyl phosphate 4-Methylumbelliferone

Microparticle-enhanced immunoassay (MEIA) see Table 4, Method 4a. Sample, microparticles (capture molecules) and
enzyme-labeled (ALP) detection antibodies are transferred to the reaction cell. During the incubation, analytes bind to both
antibodies, creating an immune complex with the microparticles. An aliquot is transferred to the matrix cell, where the
immune complex binds irreversibly to the glass fiber matrix. After washing the matrix to remove unbound materials, an
alkaline phosphatase substrate, 4-methylumbelliferyl phosphate (MUP), is added to the matrix. The bound ALP-conjugate
catalyzes the hydrolysis of 4-methylumbelliferyl phosphate (MUP) to the fluorescent product 4-methylumbelliferyl (MU).
The MEIA optics measures the rate at which MU is generated on the glass fiber matrix.
Figure 3

Biotinylated Capture Ab- Sandwich

Capture Ab Acridinium label Formation
YY

Incubate
Y
Prolactin in
patient sample
Streptavidin
coated magnetic
Y
micro-particles
 1007
Prolactin

Magnetic
Capture

Pre-trigger Y
Y Addition Y
RLU
Y Trigger
Addition
Measurement

Y Y
Two-step chemiluminescent immunometric assay Abbott Chemiflex Technology (Table 4, Method 4b).
In the first step, patient sample and paramagnetic microparticles coated with capture antibody are incubated. After washing
to remove unbound components (and reduce possibility of a hook effect from excess prolactin), detection antibody labeled
with an acridinium ester is added and incubated. Pre-trigger (peroxide) solution releases the complexed prolactin-labeled
detection antibody from the microparticles, which are drawn towards the magnet. Trigger solution (sodium hydroxide)
optimizes the pH for the release of chemiluminescence, which is detected by the liminometer and quantified in relative
light units (RLU). The acridinium ester used in this assay has been specially formulated with a sulfopropyl group to
improve its aqueous solubility and a sulfonamide-leaving group to improve its stability. Method 4c (Bayer Centaur)
follows a similar pathway.

Figure 4
Ru(tpy)2 2+
Ruthenium bis(2,2:6,2-
Biotinylated Capture Wash
Y

terpyridine)
Capture Ab Ab-Ru label off
YY

Y Photons

e- TPA
H+

Y Ru2+
base state
3+
RuTPA
TPA+
TPA
Y
Y

Prolactin in Electron donor

e-
patient sample Magnetic (Tripropylamine, TPA)
Capture + Platinum Electrode + ensures that the reaction
Streptavidin
Magnet continues to cycle,
coated magnetic
multiplying the signal
micro-particles

Two-step electrochemiluminescent immunometric assay Roche Diagnostics (Table 4, Method 4f).

In the first step, patient sample is incubated with biotinylated capture antibody. Next, streptavidin-coated paramagnetic
microparticles and detection antibody labeled with a ruthenium compound are incubated. A sandwich complex is formed,
which becomes bound to the magnetic particles by the interaction of streptavidin and biotin. After washing to remove
unbound components, the magnetic particles are attracted out of solution by a magnet onto the electrode. Application of a
voltage to the electrode then induces chemiluminescent emission, which is measured by the photomultiplier. The addition
of an electron donor (tripropylamine) ensures that the reaction continues to cycle, thus improving the sensitivity.
1008
Prolactin

Figure 5

A combined diagnostic and treatment pathway for prolactinoma.

Reproduced from Schlechte JA [32]: Prolactinoma, N Engl J Med 2003; 349:2035-41, with permission pending.

Figure 6

An 18-laboratory survey of 10 samples known to contain macroprolactin (defined as hyperprolactinemia that corrected on
treatment with PEG). Results are shown for nine different commercially-available immunometric assays.

Reproduced from Smith TP, Suliman AM, Fahie-Wilson MN, McKenna TJ [58]: Gross variability in the detection of
prolactin in sera containing big big prolactin (macroprolactin) by commercial immunoassays, J Clin Endocrinol Metab
87:5410-5, 2002, with permission pending.
1009
Prolactin

Figure 7

Calibration curve (o) for gel-filtration chromatography and the prolactin () curve of a serum containing a preponderance
of macroprolactin (bbPRL) and some monomeric prolactin (mPRL). Molecular weight markers are indicated as follows: A,
dextran blue; B, thyroglobulin; C, gamma globulin; D, ovalbumin; E, myoglobin; F, cyanocobalamin (vitamin B12).
Reproduced from Olukoga AO, Kane JW [59]. Macroprolactinemia: validation and application of the polyethylene glycol
precipitation test and clinical characterization of the condition, Clin Endocrinol 51:119-126, 1999, with permission.

Appendix
Identification & Measurement of Macroprolactin
Different commercially-available assays have varied
responses to macroprolactins. This can be explained in
part by the orientation of the prolactin peptide within the
macromolecule relative to the epitopes to which the
antibodies were raised, and to a lesser extent by the
steric hindrance offered by the detection label. It was
initially assumed that the relative responses of
commercial assays would be stable for the majority of
specimens tested. Thus those labs using assays that react
only weakly might be tempted to assume that an elevated
prolactin is unlikely to be caused by macroprolactin.
However, the pattern does vary with some samples
[25,34], and hyperprolactinemia due to macroprolactin
can be observed in all assays tested to date. Figure 6
shows results of an 18-laboratory survey of ten samples
known to contain macroprolactin (defined as
hyperprolactinemia that corrected on treatment with
PEG) [58]. Results are shown for nine different
commercially-available immunometric assays, and there
is a 2.3- to 7.8-fold variance in results obtained for
individual specimens (since these data were generated,
Roche has reformulated the antibodies and reduced their
cross-reactivity with macroprolactin fourfold). The
results suggest that depending on the assay used,
macroprolactin may account for up to 25% of reported
hyperprolactinemias. Table 6 shows the estimated
prevalence of hyperprolactinemia due to macroprolactin
in samples with elevated total serum prolactin by
individual assay method [23]. A meta-analysis of
reported incidence in clinical studies shows good
agreement [26].
Two general strategies are available for the analysis of
macroprolactin. The first is to separate the prolactin
fractions by gel-filtration chromatography (GFC) and
determine the immuno-reactivity of each fraction. The
second and more readily-available and cost-effective
strategy is to remove any macroprolactin present and
then determine the residual immuno-reactivity due to
monomeric prolactin. Ultrafiltration, and precipitation
with polyethylene glycol (PEG), Protein A, Protein G, or
anti-human IgG have all been used for this purpose.
Both methods are described in more detail.
Gel-Filtration Chromatography (GFC) (see Table 2,
Method 1)
This is considered the gold standard because it allows
simultaneous quantitation of all three molecular forms of
prolactin. It is, however, time consuming and expensive,
adding almost $300 to the cost of the analysis, and
requires equipment unlikely to be available in non-
specialist laboratories. Many systems haven been used,
of which the description below is one example [59].
Principle
GFC separates molecules based on their size or
molecular weight. The gel (stationary phase) is
packed into a column equilibrated with elution
buffer. The column is calibrated with molecular
size markers either prior to or concurrently with
the mixture to be separated, which is applied to
the top of the column. Crevices or pores within
the gel particles can accommodate molecules
up to a certain size (the cut-off), while larger
molecules (above the cut-off) are excluded from
the pores and thus pass through the column
1010
Prolactin
quickly in the void volume. Molecules of
smaller size penetrate the pores and retain on
the column; over time they dissociate from the
pores and elute from the column in order of
decreasing molecular mass. The range of pore
sizes in the gel preparation determines the
speed and resolution of the analysis. The
column filtrate is collected for fixed time
intervals by use of an automatic fraction
collector. A UV detector signal provides
monitoring of the separation of the molecular
weight protein markers. Alternate fractions are
then subjected to prolactin analysis by
immunometric assay, and this is usually a
widely cross-reacting method such as the
DELFIA assay.
Materials required
Peristaltic pump
Automated fraction collector
UV spectrophotometer set at 280 nm
Sephacryl S-300 HR
Isotonic buffer
Tris Buffer, 10 mmol/L, pH 7.4,
containing 140 mmol/L sodium
chloride, 1.25 mol/L calcium chloride,
0.5 mmol/L magnesium chloride and
0.2% sodium azide
Molecular weight markers, for example:
Dextran blue (MW 2.5 106)
Bovine thyroglobulin (MW 6.7 105)
Bovine gamma globulin (MW 1.58 105)
Chicken ovalbumin (MW 4.4 104)
Horse myoglobin (MW 1.7 104)
Cyanocobalamin (MW 1350)
Prolactin standard 9000 mIU/L
Immunometric prolactin assay system
Method
1. A 1.6 70 cm column is packed to a bed height
of 55 to 60 cm and equilibrated at 4C with
isotonic buffer.
2. The column is calibrated with the molecular
weight markers.
3. 1 mL of filtered serum is applied to the column
and run at a flow rate of 30 mL/h 110 1.0 mL.
4. Fractions collected over the next 3.7 hr.
Protein in each fraction is measured by UV
spectrophotometry at 280 nm to determine the location
of the molecular weight markers. Within the molecular
weight areas of interest, alternate fractions are analyzed
for prolactin by immunoassay
Results
The amount of prolactin determined in each
fraction analyzed is plotted against the fraction
number.
Separation and location of the molecular weight
markers on this system is shown in Figure 7,
with the DELFIA prolactin immunoassay
concentrations overlaid in a patient with
macroprolactinemia.
Calculation
1. The area under the total prolactin
curve is determined by integration.
2. The regions of interest are identified
against the molecular size markers,
monomeric (23 kDa), big (40 to 60
kDa) and macro (>100 kDa) prolactin
fractions.
3. The area of the macroprolactin peak is
calculated as a percentage of the total
prolactin activity in the specimen.
Interpretation
Samples are considered macroprolactin positive
when the area under the prolactin curve 30%
of the total area of prolactin elution.
The normal pattern of activity is 60% to 90%
prolactin monomer; 15% to 30% big prolactin;
and 0% to 10% macroprolactin
Comment
Although considered the gold standard, there
are four main disadvantages:
In sera with a low affinity complex, there is the
potential for dissociation of the prolactin from
the antibody complexes during the lengthy GF
run. There is little evidence that this often
happens in practice. There is inherent
cumulative imprecision (and cost) when
measuring prolactin in 30 to 40 different
fractions. There may be disproportionate loss of
prolactin from some fractions by absorption
onto the gel during the run, which will skew the
percentage distribution pattern for prolactin. It
is labor intensive and costly because multiple
immunoassays are required, which limits its
application to research institutions. Although
longer columns give better molecular size
separations, they take longer and produce more
fractions. When using the S-300 gel, the big
prolactin peak may be shouldering on the
macroprolactin, or if only small in size may be
completely incorporated into it. It is, however,
useful for investigating specimens with unusual
macroprolactins. S-100 gel gives better
separation within the middle masses and can be
used to expand the area between macro and
monomeric prolactin, but macroprolactin elutes
close to the void volume[28]. S-200 gel may be
a good compromise for routine use.
PEG Precipitation Method
(Table 2, Method 2) Precipitation with PEG is easy and
inexpensive to perform, requiring only about 10 minutes
preparation time and adding about $11 to the cost of the
test. PEG is readily obtained and highly stable. It is,
however, relatively nonspecific.
Principle
The specimen is first analyzed by immunoassay
to determine the total prolactin present. If
hyperprolactinemia is demonstrated, a PEG
1011
Prolactin
precipitation is performed and the supernate
reanalyzed using the same immunoassay
method. Patient specimen is mixed with an
equal volume of 25% PEG solution, vortex
mixed, and incubated for several minutes. PEG
polymers sequester water from the specimen
into their matrix, causing macromolecules to
exceed their solubility and precipitate, leaving
smaller molecules in solution. The size of the
PEG will determine the molecular weight cut-
off. After centrifugation, the supernate is
analyzed by immunoassay.
Materials Required
Polyethylene glycol (PEG) 6000 or 8000
available from most standard chemical
manufacturers
Balance
DI or distilled water
Magnetic stir plate
Vortex or rotating shaker
Small centrifuge
General laboratory supplies
Working Reagent Preparation
1. 250 g/L (25% w/v) polyethylene glycol solution
in water
2. Add 25 g PEG to approx 60 mL deionized
water at room temperature. Stir till dissolved on
a magnetic plate. QS to 100 mL with deionized
water.
3. Working reagent can be stored at room
temperature for 7 days.
Method
Combine sufficient patient serum with an equal volume
of working-strength PEG solution in a small test tube
(typically 150 L).
Vortex mix for 30 seconds, or mix on a rotator for 10
min.
Centrifuge for 5 min between 1500 g and 10,000 g
within 5 to 30 min of preparation.
Analyze the supernate by the same immunoassay method
used with the native specimen.
Calculations
The specimen has been diluted by a factor of 2 during
the preparation, and this must be accounted for by
multiplication of the instrument result by the dilution
factor.
Approximately 14 % (range 0% to 40%) of monomeric
prolactin is co-precipitated with PEG [60]. The best
practice is to work up a calibrator and QC material,
processed similarly, to determine the assay recovery.
These should be factored into the calculation for the
specimen result.
Example Calculation:
Monomeric PRL = 2(PEG Patient Instrument Reading)
Set point value for calibrator or control
Divided by
2(PEG Control or Calibrator Instrument Reading)
Macro PRL = Original untreated PRL result
monomeric PRL treated result
Interpretation
The difference between the two results indicates the
contribution of macroprolactin to the original prolactin
determination. If the corrected prolactin result falls
within the reference range for the patient, they are
described as having macroprolactinemia. In the absence
of correction for dilution and recovery with controls or
calibrators, a drop of > 60% is considered definitive for
macroprolactinemia, while a drop of < 40% indicates
presence of prolactin monomer. A grey zone exists
between 40% and 60%.
Comment
Imprecision is 3% to 10%, and because of the small
dilution made, it is applicable to specimens with only
moderate hyperprolactinemia [59,61]. Although there is
under-recovery of monomeric prolactin (only
approximately 75% is recovered), when this is corrected
by concomitant analysis of calibrators or patient pools, it
correlates well (r = 0.80) with GFC and is much less
expensive and more easily performed. The main
drawback is that some immunometric methods are
affected by the presence of the PEG in the supernate.
These include Abbott Axsym and DPC Immulite [62].
However, this does not preclude use of PEG with these
assays, particularly if the purpose is to identify the
presence of macroprolactin rather than accurately
determine the monomeric prolactin concentration.
Other Methods
Precipitation of macroprolactin-immunoglobulin G
complexes has been described using a number of
reagents (Table 2, Method 3). They are all relatively
inexpensive, adding an additional $25 to the cost of the
assay. The first assay used goat anti-human IgG-agarose
with a binding capacity of 1,5 mg hIgG/mL of resin.
This assay had a 2-hour incubation and a dilution factor
of 20-fold, and therefore was not sufficiently sensitive to
detect mild macroprolactinemia [63]. A later
modification uses an IgG-agarose resin with a binding
capacity of 20 mg/mL of resin. This protein G
polypeptide binds to the Fc region of all subclasses of
the human IgG molecule. Protein G-agarose is available
in ready-to-use suspension (Roche) [64]. 150 L patient
serum is mixed and incubated with 300 L Protein G-
agarose suspension, giving a threefold dilution. After
centrifugation for 2 min at 9500 g at 20C the supernate
is analyzed by immunoassay. The imprecision is < 2%,
and results agree well with PEG precipitation when
macroprolactin is the predominant form. Two sera which
normalized on PEG precipitation but did not normalize
with protein G-agarose were shown to react with anti-
IgA- and anti-IgMsepharose, demonstrating the
specificity of this method and the conclusive presence of
IgA- and IgM-associated macroprolactins [20].
Protein A is another polypeptide that binds the Fc region
of human immunoglobulin molecules, especially hIgG1,
hIgG2, and hIgG4, but only a fraction of IgG3 [65]. 300
L of a Protein A-sepharose suspension (Protein A CL-
4B in 20 mmol/L borate buffer) is added to 150 L
patient serum. After incubation for 15 min at room
temperature, the sample is centrifuged at 2000 g for 5
1012
Prolactin
min at 20C. Prolactin is determined in the supernate by
immunoassay. This assay uses a shorter incubation time
and produces only a threefold dilution of sample so is
more applicable when there is only mild
hyperprolactinemia. Total imprecision is better than
12%. The assay appears to specifically target IgG
complexes, while other forms of macroprolactins are not
removed [60].
Ultrafiltration (Table 2, Method 4) employs a standard
membrane filter (available from Amicon in a variety of
pore sizes) with a molecular weight cut-off of around
100 kDa. These devices are freely porous to 45kDa and
completely retain above 100 kDa, with variable
penetrance in between. Specimen (typically 0.5 to 1 mL)
is loaded into a reservoir above the filter, and a small
tube is attached to the base of the device to collect the
filtrate. Some studies use a dilution of 25 L specimen in
500 L PBS. The entire apparatus is loaded into a fixed-
angle rotor centrifuge and spun at 1000 g for 30 to 45
minutes. Spin times of up to 5 hours have been used at
450 g, but it is likely that the filter clogs on prolonged
contact and results become more variable. The clear
filtrate is analyzed for prolactin content by
immunoassay. Unlike some other methods, the specimen
is not diluted so is ideal for patients with only moderate
hyperprolactinemia. Additionally, since there are no
additives to interfere with immunoassays, it can be used
where precipitating techniques are not able to be applied.
Comparative studies show good agreement with PEG
precipitation and GFC, most notably when
macroprolactin is the predominant species. Some
comparative studies demonstrate over-recovery of
monomeric prolactin, which is most likely caused by
dissociation of macroprolactin during centrifugation or
because of dilution in PBS, or because high dilution
factor (20) used in some studies introduces
imprecision. Values for imprecision range from a CV of
3% to 9% to 16%, depending on the methodology and
choice of specimen [29]. Additional cost are
approximately $16.
1013
Prostate-Specific Antigen (PSA)
Prostate-Specific Antigen (PSA)
Hassan M.E. Azzazy
Name: Serine protease, PSA
Clinical significance: Refer to Chapter 53, Neoplasia, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.
Molecular mass: 28.43 kDa
Chemical class: Glycoprotein
Subunit: Monomer (single chain)
Isoelectric pH: Range from 6.8 to 7.5
Coding gene location and size: Chromosome 19, 6 kb
i many of the available methods are difficult to compare
Principles of Analysis and Current Usage
Human prostate-specific antigen (PSA) is produced
exclusively by the epithelial cells lining the prostatic
acini and ducts of prostatic tissue. PSA is a single-chain
glycoprotein that contains 237 amino acids and N-linked
oligosaccharide [1]. Functionally, PSA is a kallikrein-
like serine protease with chymotrypsin- and trypsin-like
enzymatic activity [2]. Because of its high specificity for
prostate tissue, PSA is the preferred serum marker for
prostate cancer [3,4]. PSA is present in seminal fluid in
high concentrations and cleaves the high-molecular-mass
seminal vesicle protein, semenogelin, the major
structural protein of the seminal coagulum (to increase
sperm motility). Semenogelin is formed at ejaculation
and appears to be the physiological substrate for PSA
[4].
In serum, PSA can be joined to other proteins
(complexed PSA [cPSA]), or it can exist as free PSA
(fPSA). Several assays are available that measure free,
complexed, and/or total PSA. Assays for free or cPSA
are used when the total PSA result is marginally raised,
but not when total PSA result is highly elevated. Free
PSA measurements can be used to improve the
specificity of PSA for prostate cancer, especially when
total PSA values are between 4 and 10 ng/mL (gray
zone). Free PSA is expressed as a ratio of total PSA
[%fPSA = fPSA/total PSA 100]. Men with prostate
cancer may have a smaller proportion of fPSA and more
cPSA when compared with men with benign prostatic
hyperplasia (BPH) or prostatitis.
Methods for PSA Analysis
Immunoassays for PSA are widely available and
typically use non-isotopic labels such as enzymes,
fluorescent labels, or chemiluminescence. Unfortunately,
i
Prostate-Specific Antigen (PSA)
Previous and current authors of this method:
First edition: Not done
Methods edition: Not done
Second edition: Not done
Third edition: William R. Jackson, John A. Lott
Fourth edition: William R. Jackson, John A. Lott
Fifth edition: Hassan M.E. Azzazy
because their results are dependent on the standard that
is used, the specificity of the antibodies, and the purity
of the antigen employed in the generation of antibodies.
PSA occurs in serum in different molecular forms.
Approximately 70% is bound to protease inhibitors,
primarily 1-antichymotrypsin and 2-macroglobulin,
and a small amount is complexed with 1-antitrypsin and
protein C. Free PSA, defined as the portion of PSA not
bound to serum proteins, accounts for ~30% of total
PSA [5,6]. Antibodies can react with different epitopes,
and the different molecular forms of PSA can influence
antibody-binding characteristics.
Some manufacturers advocate the use of the free PSA
molecule as the standard, whereas others recommend the
PSAanti-chymotrypsin complex. The former is better
characterized, but the latter is the form that predominates
in plasma. Clearly, some effort is needed by the
manufacturers of the kits to promote intermethod
agreement [5].
Early methods used to measure PSA included isoelectric
focusing, immunodiffusion, immunoelectrophoresis, and
rocket immunoelectrophoresis [7]. Subsequent tests for
PSA incorporated standard enzyme-linked
immunosorbent assays and solid-phase immunometric
assays with polystyrene beads.
Zundel et al. [8] developed an EIA for PSA that uses
microtiter wells coated with monoclonal antibody to
PSA. After the specimen is added to the wells, a second
monoclonal antibody to PSA, labeled with horseradish
peroxidase, is added along with the substrate
tetramethylbenzene.
A solid-phase, two-site, immunoradiometric assay
(IRMA) that uses two murine monoclonal antibodies for
different epitopes on the PSA molecule has been
described [9]. A high-dose hook effect has been
reported for this assay, with artificially low measured
PSA values found with serum concentrations above 5000
ng/mL [10]. An EIA version of the IRMA PSA assay has
also been developed. The principle is the same as the
IRMA; however, the label is alkaline phosphatase rather
than 125I, and the substrate is p-nitrophenyl phosphate.
1014
Prostate-Specific Antigen (PSA)
Another FIA for PSA is available that uses monoclonal
antibodies to PSA coated on magnetic beads. The
resulting complex binds with an anti-PSAalkaline
phosphatase conjugate; 4-MUP is the substrate.
PSA can be measured using a microparticle-capture EIA
system. PSA binds to inert microparticles that hold
monoclonal antibody to PSA. A second antibody to
PSA, labeled with ALP, binds to the PSA on the
microparticles. A 4-MUP substrate is used.
A chemiluminescent immunoassay is also available for
PSA. Polyclonal antibodies to PSA labeled with
acridinium ester are used, plus a second antibody to
PSA. The labeled PSAanti-PSA complex is separated
magnetically, then chemiluminescence is generated and
measured with a luminometer. A summary of the
analytical principles of the commercially available PSA
assays is shown in Table 1.
Circulating levels of cPSA can be determined either
directly by cPSA specific assays [11] or indirectly by
subtracting fPSA from total PSA using assays
standardized against one another [12]. An assay that
measures cPSA has been developed. The assay measures
PSA complexes with 1-antichymotrypsin and other
minor PSA complexes. It employs an antibody to block
and inactivate fPSA in the sample [13]. Other cPSA
assays are available on automated platforms and in
ELISA format.
Long-term monitoring may represent a challenge
because of between-method variations caused by
calibration using different standard and differences in
method design. Additionally, different commercial PSA
immunoassays employ monoclonal-monoclonal or
monoclonal-polyclonal antibody combinations which
may differ in their recognition of the free and complexed
PSA forms. Two international standards representing the
main immunoreactive PSA components in serum, free
PSA and PSA 90:10 (ratio of bound-to-free PSA), are
now available for calibration of PSA immunoassays.[14]
Although these reference materials may help to
minimize between-method differences, studies designed
to characterize the molecular epitope structure and
specificities of diagnostically relevant antibodies specific
for PSA and its derivatives could improve comparability
of PSA immunoassay results [15].
Although several commercial assays for PSA and its
derivatives in serum are available, physicians making
clinical decisions should be alerted to the potential
standardization bias among PSA assays. For example,
AxSYM total and free PSA are standardized using the
World Health Organization (WHO) reference material
and the Stanford 90:10 reference material, respectively.
IMMULITE total and free PSA assays are referenced
against the WHO National Institute for Biological
Standards and Control First International Standard
(NIBSC 1st IS) 96/670 and WHO NIBSC 1st IS 96/688,
respectively [16]. Measurements for PSA and its
variations obtained by commercial assays are not
necessarily interchangeable and may lead to different
clinical outcomes. Sotelo et al. (2007) have compared
the free and total PSA values obtained using two
commercial PSA assays. Using a free/total PSA ratio of
20% as the threshold for biopsy, the free/total PSA
ratio was found to be discordant between the assays,
leading to a discrepancy in biopsy recommendations and
cancer detection rates [16].
Reference and Preferred Method
Currently, there is no reference method for PSA. Several
sensitive PSA immunoassays are commercially available
and generate acceptable results.
Specimen
PSA is stable in serum for up to 24 hours at 2C to 8C.
For longer and extended storage periods, serum should
be stored frozen at 20C and at 70C, respectively.
For complexed PSA (cPSA), serum is stable for up to 48
hours at 2C to 8C and at 70C for extended storage.
For PSA measurements, in particular for free PSA, blood
should be centrifuged within 3 hours of collection to
isolate serum or plasma. Free PSA is more susceptible to
decay than cPSA; decay is slower in plasma than in
serum [17].
The serum half-life of PSA is 2.2 to 3.2 days [18]. Small
but significant increases in PSA may occur following
prostatic manipulation, with greater increases occurring
in men with enlarged prostates [19]. In addition,
diagnostic intervention such as transrectal ultrasound or
biopsy can also result in increased PSA concentrations.
PSA is occasionally found in urine in men and is an ideal
forensic marker in rape cases. It is a component of
seminal plasma, even in vasectomized males. PSA is
detectable in semen stains for as long as 1 year and is
stable in the vagina for approximately 2 days.
Matsuzawa et al. [20] described a latex particle
agglutination test for PSA that is based on the use of an
antibody to PSA.
It is preferred to collect blood prior to prostate biopsy or
cystoscopy. Although DRE does not elevate PSA serum
levels, it is prudent to wait several days prior to blood
collection for PSA [21]. Blood drawing for PSA should
be delayed for several weeks following prostate biopsy,
surgery, or resolution of prostatitis [22].
Interferences
Serum PSA values may be decreased by 50% in men
using finasteride (Proscar) for over 6 months or after
surgical or medical castration. Total and free PSA serum
levels may increase upon transurethral resection of the
prostate (TURP), needle biopsy, ejaculation, or exercise;
cPSA levels are less affected. Antiandrogens, LHRH
agonists and antagonists and finasteride decrease cPSA
levels. Digital rectal exam (DRE) may not cause
clinically significant increases in PSA [23]. The
presence of heterophilic antibodies in serum may
interfere with results generated using immunoassays
employing murine antibodies.
1015
Prostate-Specific Antigen (PSA)
Reference Intervals
Reference intervals for PSA depend on the assay used.
Reference intervals for PSA in males are listed as 0 to 4
ng/mL when measured using immunoassays. Age-
specific reference intervals recommended using
chemiluminescent immunoassay are as follows: 40 to 49
years, 2.7 ng/mL; 50 to 59 years, 3.7 ng/mL; 60 to
69 years, 5.1 ng/mL; and 70 to 79 years; 7.0 ng/mL.
PSA is found in women in low concentrations, with
values typically less than 0.15 ng/mL [24].
The standard cutoff for biopsy consideration, PSA value
of 4.0 ng/mL, is equivalent to a cPSA concentration of
3.2 ng/mL. The lower threshold of 2.5 ng/mL is
equivalent to cPSA value of 2.2 ng/mL [23,25].
Interpretation
An ideal tumor marker is specific for a tissue, tumor, or
both. It is released from the tumor into the blood or
urine, has a short half-life, and indicates the presence of
tumor before clinical symptoms; also, its concentrations
fluctuate in proportion to the tumor mass. Although a
perfect tumor marker has not yet been discovered, PSA
exemplifies many qualities of an ideal tumor marker.
PSA is useful for staging, monitoring, and screening for
prostate cancer, as well as detecting recurrent disease.
Unfortunately, PSA is specific to prostate tissue not to
prostate cancer. It is also found in abnormal
concentrations in normal and benign changes of the
prostate such as benign prostatic hypertrophy (BPH)
[26]. Osterling et al. [27] reported that not only cancer
causes an elevation in the serum PSA; it can be elevated
from digital rectal examinations, cystoscopic
examinations, and prostatic biopsies and trauma. The
prevalence of foci of malignant cells is an incredible
40% in men over age 50. Because the incidence of
clinical prostate cancer in the United States was 165,000
cases in 1993, there is clearly a great deal of latent
disease. Although the normal range of PSA is
conventionally considered between 0.0 and 4.0 ng/mL,
there is no cutoff PSA value at which prostate cancer is
not present, but rather there is a continuum of prostate
cancer risk at all values of PSA [28].
The usefulness of PSA as an early detector of prostate
cancer by itself is questionable, owing to the overlap in
PSA values seen in patients with BPH and in those with
organ-confined prostate cancer (see 27a). PSA values
combined with digital rectal examination findings and/or
transrectal ultrasonography may lead to early detection
of prostate cancers. The sensitivity and specificity of a
screening test for prostatic cancer is markedly increased
when these test combinations are used.
PSA diffuses into the systemic circulation in increased
amounts only after significant changes have occurred in
the architecture of the prostate gland. Approximately
38% to 48% of patients with organ-confined prostate
cancer have PSA values in the reference interval [27].
Only 30% to 40% of men who present with moderate
PSA elevations (4 to 10 ng/mL) actually have prostate
cancer confirmed by biopsy. The specificity of the PSA
test for cancer rises to 40% to 50% or higher when
serum levels exceed 10 ng/mL [29].
PSA-level increases with age and day-to-day
intraindividual variation have been reported. According
to the American Society for Therapeutic Radiation,
recurrence after radiation therapy is defined as three
consecutive increases in PSA above nadir (<0.5 ng/mL)
[30].
Serum PSA concentrations correlate directly with the
various clinical stages of prostate cancer, but they cannot
be relied upon to determine the clinical stage of the
disease. PSA concentrations also correlate with the
weight of prostatic tissue resected during a transurethral
resection of the prostate and with the volume estimated
by transrectal ultrasonography. The ratio of PSA
concentration to the volume of prostatic tissue is useful
in differentiating patients with benign and malignant
diseases of the prostate [31,32]. Increasing PSA values
are associated with advancing pathological stages of
prostate cancer and correlate positively with increasing
tumor volume [33]. PSA concentrations are helpful in
determining the necessity of radionuclide bone scan in
newly diagnosed, untreated prostate cancer.
PSA values should decrease to undetectable
concentrations following radical prostatectomy. Since
the serum half-life of PSA is relatively long (3.2 days),
assessment of PSA measurements should be delayed
until 2 or 3 weeks after surgery. Positive PSA values of
0.2 to 0.6 ng/mL after radical prostatectomy are
indicative of residual disease. PSA is also useful in
monitoring prostatic cancer patients who have
undergone radiotherapy and anti-androgen therapy
[33,34].
The use of PSA as a screening test for prostate cancer is
controversial. Although it demonstrates good sensitivity,
PSA lacks clinical specificity. Also, the sensitivities and
specificities of serum PSA assays change when different
cutoff points are used. Armbruster [35] illustrated a
hypothetical use of PSA for screening the general male
population for prostate cancer.
Measurement of PSA cannot be the sole factor used for
the diagnosis of disease but must be used together with
clinical findings. PSA must always be used in
conjunction with a direct rectal examination (DRE) for
the initial screening of patients for prostate cancer. The
American Cancer Society recommends that the PSA test
and DRE should be offered annually to men of age 50
years with life expectancy of 10 years or more. Men at
higher risk, of sub-Saharan African descent, and men
with a first-degree relative diagnosed with prostate
cancer at < 65 years should begin testing at age 45 years
[29].
The fPSA and cPSA tests are frequently ordered when
total PSA values are between 4 and 10 ng/mL. Tests
from the same manufacturer should be used when
measuring total and fPSA values for the same patient.
The relative proportions of fPSA and cPSA vary
1016
Prostate-Specific Antigen (PSA)
between men with prostate cancer and those with other
benign prostatic diseases. Compared with men with
prostatic cancer, patients with prostate hypertrophy or
prostatitis have a higher proportion of free PSA and less
cPSA. Assays for cPSA are approved by the U.S. Food
and Drug Administration (FDA) for detection of prostate
cancer in men > 50 years in conjunction with DRE, as
well as for monitoring therapy in prostate cancer
patients.
PSA kinetics provides a dynamic picture of prostate
cancer activity, and PSA doubling time (PSADT) has
been proposed as a determinant of the presence and
pattern of disease recurrence after definitive therapy
[36]. Although definitive thresholds are yet to be
established, patients with a shorter PSADT should be
treated aggressively, and those with a longer PSADT
may represent a slow-growing process for which delayed
or local therapy may be more appropriate. Table 2
presents variations of PSA measurements and their
proposed clinical applications.
Performance Goals
Survey data from the 2007 College of American
Pathologists participant summary report show
imprecision values (% coefficient of variation [CV]) for
total prostate-specific antigen procedures ranging from
3.7% to 9.7% at a mean concentration of 10 ng/mL.
Imprecision values for free PSA assays range from 3.2%
to 7.0% at a mean concentration of 2 ng/mL; imprecision
values for complexed PSA assays range from 4.5% to
5.1% at a mean concentration of 6.5 ng/mL; and those
for PSA ratio range from 4.3% to 8.7% at a mean ratio
of 0.2 [37].
Acceptable Clinical Laboratory Improvement
Amendments performance criteria (CLIA 88) for
measurement of total, complexed, and free prostate-
specific antigen require that laboratories be accurate to
within the greater of 3 SD or 0.2 ng/mL of the peer-
group mean. PSA ratio is currently not graded. Within-
subject biological variation has been determined to be
approximately 14% [38]. Desirable specifications for
analytical imprecision derived from studies of biological
variation indicate an assay imprecision of no greater than
7.0% [39].
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7 Kuriyama M, Wang MC, Papsidero LD, Killian
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10 Ooi DS, Escares EA. High-dose hook effect
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11 Brawer MK, Meyer GE, Letran JL, Bankson
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13 Parsons JK, Brawer MK, Cheli CD, Partin AW,
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14 Rafferty B, Rigsby P, Rose M, Stamey T,
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15 Berger P, Sturgeon C, Bidart JM, Paus E, Gerth
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oriented standardization of pregnancy and
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16 Sotelo RJ, Mora KE, Prez LH, Novoa J,
Carmona O, De Andrade R et al. Assay
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17 Piironen T, Pettersson K, Suonp M, Stenman
UH, Oesterling JE, Lvgren T, Lilja H. In vitro
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18 Pontes JE, Jabalameli P, Montie J, Foemmel R,
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21 Lilja H, Haese A, Bjrk T, Friedrich MG,
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glandular kallikrein 2 in clinically localized
prostate cancer before and after radical
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22 Oberpenning F, Schmid HP, Fuchs-Surdel W,
Hertle L, Semjonow A. The impact of
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Biol Markers 2002;17:154-60.
23 Price CP, Allard J, Davies G, Dawnay A, Duffy
MJ, France M et al. Pre- and post-analytical
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24 Mayo Clinic Test Catalog, 2007.
25 Polascik TJ, Oesterling JE, Partin AW.
Prostate-specific antigen: a decade of discovery:
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26 Duffy MJ. New cancer markers. Ann Clin
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27 Oesterling JE, Chan DW, Epstein JI, Kimball
AW Jr, Bruzek DJ, Rock RC et al. Prostate-
specific antigen in the preoperative and
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27a.
http://www.cancer.gov/cancertopics/factsheet/d
etection/PSA
Schrder FH, Hugosson J, Roobol MJ, et al.
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randomized European study. New England
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Andriole G, Crawford E, Grubb R, et al. Seven
year mortality results and related findings from
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component of the PLCO randomized cancer
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March 18, 2009.
28 Thompson IM, Ankerst DP, Chi C, Lucia MS,
Goodman PJ, Crowley JJ et al. Operating
characteristics of prostate-specific antigen in
men with an initial PSA level of 3.0 ng/mL or
lower. JAMA 2005;294:66-70.
29 Smith RA, Cokkinides V, Eyre HJ. Cancer
screening in the United States, 2007: a review
of current guidelines, practices, and prospects.
CA Cancer J Clin 2007;57:90-104.
30 Freedland SJ, Sutter ME, Dorey F, Aronson
WJ. Defining the ideal cutpoint for determining
PSA recurrence after radical prostatectomy.
Prostate-specific antigen. Urology 2003;61:365-
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31 Vesey SG, Goble M, Ferro MA, Stower MJ,
Hammonds JC, Smith PJ. Quantification of
prostatic cancer metastatic disease using
prostate-specific antigen. Urology 1990;35:483-
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32 Veneziano S, Pavlica P, Querz R, Nanni G,
Lalanne MG, Vecchi F. Correlation between
prostate-specific antigen and prostate volume,
evaluated by transrectal ultrasonography:
usefulness in diagnosis of prostate cancer. Eur
Urol 1990;18:112-6.
33 Stamey TA, Kabalin JN, McNeal JE, Johnstone
IM, Freiha F, Redwine EA, Yang N. Prostate-
specific antigen in the diagnosis and treatment
of adenocarcinoma of the prostate. II. Radical
prostatectomy treated patients. J Urol
1989;141:1076-83.
34 Stamey TA, Kabalin JN, Ferrari M, Yang N.
Prostate-specific antigen and the diagnosis and
treatment of adenocarcinoma of the prostate.
IV. Anti-androgen treated patients. J Urol
1989;141:1088-90.
35 Armbruster DA. Prostate-specific antigen:
biochemistry, analytical methods, and clinical
application. Clin Chem 1993;39:181-95.
36 Ramrez ML, Nelson EC, Devere White RW,
Lara PN Jr, Evans CP. Current applications for
prostate-specific antigen doubling time. Eur
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37 College of American Pathologists. 2007
Participant Survey. Northfield, IL: CAP; 2007.
38 Panteghini M, Pagani F, Bonora R.
Preanalytical and biological variability of
prostatic acid phosphatase and prostate-specific
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pathology. Eur J Clin Chem Clin Biochem 43 Brawer MK, Cheli CD, Neaman IE, Goldblatt J,
1992;30:135-9. Smith C, Schwartz MK et al. Complexed
39 Rics C, Alvarez V, Cava F, Garca-Lario JV, prostate-specific antigen provides significant
Hernndez A, Jimnez CV et al. Current enhancement of specificity compared with total
databases on biologic variation: pros, cons and prostate-specific antigen for detecting prostate
progress. Scand J Clin Lab Invest 1999;59:491- cancer. J Urol 2000;163:1476-80.
500. 44 Benson MC, Whang IS, Pantuck A, Ring K,
40 Loeb S, Catalona WJ. Prostate-specific antigen Kaplan SA, Olsson CA, Cooner WH. Prostate-
in clinical practice. Cancer Lett 2007;249:30-9. specific antigen density: a means of
41 Catalona WJ, Smith DS, Ratliff TL, Dodds KM, distinguishing benign prostatic hypertrophy and
Coplen DE, Yuan JJ et al. Measurement of prostate cancer. J Urol 1992;147:815-6.
prostate-specific antigen in serum as a 45 DAmico AV, Renshaw AA, Sussman B, Chen
screening test for prostate cancer. N Engl J Med MH. Pretreatment PSA velocity and risk of
1991;324:1156-61. death from prostate cancer following external
42 Uzzo RG, Pinover WH, Horwitz EM, Parlanti beam radiation therapy. JAMA 2005;294:440-7.
A, Mazzoni S, Raysor S, et al. Free prostate- 46 Oudard S, Banu E, Scotte F, Banu A, Medioni
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Table 1
Table 1: Analytical Principles of Typical Commercially Available PSA Assays
Method 1:
Principle: RIA (polyclonal Abs)
Signal: 125I radiation
Formula:
Ab + PSA + PSA - 125I Ab - PSA + Ab - PSA - 125I + PSA + PSA - 125I
Separation step
Count bound radiation
Method 2:
Principle: IRMA (monoclonal Abs)
Signal: 125I radiation
Formula:
Plastic bead- Ab + PSA + Ab - 125I
Plastic bead- Ab - PSA - Ab - 125I + PSA + Ab - 125I
+ PSA - Ab - 125I
Separation step
Count bound radiation
Method 3:
Principle: EIA (monoclonal Abs)
Signal: PNP absorbance
Formula:
Plastic bead- Ab + PSA + Ab - ALP
Plastic bead- Ab - PSA - Ab - ALP + PSA +
Ab-ALP + PSA - Ab-ALP
1. Wash step
2. Add PNPP substrate
3. Quench
4. Measure PNP product absorbance at 405 and 450 nm

Method 4:
Principle: Sandwich ELISA (monoclonal/polyclonal Abs)
Formula:
Plate Ab + PSA + Ab HRP Plate Ab PSA Ab HRP + PSA + PSA Ab HRP + Ab HRP
1. Wash step
2. Add TMB (3, 3, 5, 5 tetramethyl-benzidine) substrate
3. Quench (sulfuric acid)
4. Measure color change absorbance at 450 nm
1019
Prostate-Specific Antigen (PSA)

Method 5:
Principle: MEIA (monoclonal Abs)
Signal: 4-MU fluorescence
Formula:
Microparticle- Ab + PSA + Ab - ALP
Microparticle- Ab - PSA - Ab-ALP + PSA +
Ab-ALP + PSA - Ab - ALP
1. Wash step
2. Add 4-MU substrate
3. Quench
4. Measure 4-MU product fluorescence at 448 nm

Method 6:
Principle: FIA (polyclonal Abs)
Signal: 4-MU fluorescence
Formula:
Glass-fiber paper tab Ab + PSA + Ab - ALP
Glass-fiber paper tabAb - PSA - Ab - ALP + PSA +
Ab - ALP + PSA - Ab - ALP
1. Wash step
2. Add 4-MU substrate
3. 360 nm excitation light
4. Measure 4-MU product fluorescence at 450 nm
Method 7:
Principle: CIA (monoclonal/polyclonal Abs)
Signal: Acridinium ester chemiluminescence
Formula:
Paramagnetic particle- Ab + PSA + Ab - AE
Paramagnetic particle- Ab - PSA - Ab - AE + PSA +
Ab-AE + PSA - Ab-AE
1. Wash/separation
2. Add H2O2
3. OH-
4. Measure chemiluminescence of acridinium ester at 400 nm

See text for further details. Some of these assays are available for experimental use only, pending FDA approval. Some
assays are available for routine use in Europe.
Ab, Antibody; CIA, chemiluminescence immunoassay.[35]

Table 2: Variations of PSA Measurements [40]

PSA Measurement Definition Application
Total PSA [41] Includes free and bound PSA in Most commonly reported value
serum FDA approved for detection of prostate cancer (upper
limit of normal 4.0 ng/mL)
Useful in management and monitoring recurrence after
treatment
fPSA [42] Unbound PSA Higher values are observed in benign conditions
Improves prostate cancer detection in high-risk
population of men with a normal total PSA and DRE
cPSA [43] PSA complexed with other Higher values are observed in malignancies
proteins such as 1-
antichymotrypsin
PSA density [44] Serum PSA divided by the Facilitates interpretation of PSA values by reference to
prostate volume (as estimated gland size
by ultrasound)
PSA velocity [45] Measurement of longitudinal May be used to predict presence and aggressiveness of
change in PSA over a specific prostate cancer prior to intervention
time interval
PSA doubling time [46] Measurement of time for PSA Dependent upon initial PSA measurement
levels to double Useful for detecting relapse after therapy
 1020
Pyruvic Acid

Pyruvic Acid
Steven C. Kazmierczak
Name: Pyruvic acid, pyruvate
Clinical significance: see Interpretation page 2, line 94, right column.
Molecular mass: 88.06 D
Merck Index: 7925
Chemical class: -Ketocarboxylic acid

Structure:
Refer to Chapter 29, Acid-Base Control and Acid-Base Disorders, in the 5th edition of Clinical
Chemistry: Theory, Analysis, Correlation.

Principles of Analysis and Current Usage

i Many reports describe the use of the enzymatic method
The earliest method for measuring blood pyruvate was
based on the fact that pyruvate is a ketone. Ketones react
with 2,4-dinitrophenylhydrazine (DNPH) to produce a
colored hydrazone (Table 1, Method 1).
This reaction was usually performed on a protein-free
filtrate after treatment of serum with a strong acid
(trichloroacetic acid [TCA], perchloric acid [PCA], or
metaphosphoric acid [MPA]). Since other ketones react
with DNPH, most methods were an attempt to extract the
acid hydrazones [1] with an organic solvent such as
ethyl acetate, xylene, toluene, or benzene. The extract was
then back extracted with an alkaline solution (10%
Na2CO3) to separate the acid hydrazones. After a wash
with the organic solvent, the aqueous solution was made
more alkaline with NaOH, resulting in a red color (Amax
about 430 nm). Attempts to purify the pyruvic acid
hydrazone used chromatography on alumina or silica-gel
columns and paper chromatography [2].
Current methods for the analysis of pyruvic acid employ a
reaction catalyzed by muscle lactate dehydrogenase (LD)
(Table 1, Method 2):
LD, pH 7.4
Pyruvate + NADH + H+ lactate + NAD+
using protein-free filtrates formed by acid precipitation of
serum proteins. At pH 7.4, the reaction strongly favors the
quantitative conversion of pyruvate to lactate. The reaction
is easily monitored by following the consumption of
NADH, either spectrophotometrically at 340 nm [3-5] or
fluorometrically [6,7]. This method is easily adapted to
many automated instruments that monitor the reaction by
measuring either absorbance [3] or fluorescence [6,7].
A biosensor has also been developed for the measurement
of pyruvate in whole blood or serum (Table 1, Method 3)
[8]. The pyruvate sensor is a hydrogen peroxide sensor
covered with a membrane containing immobilized
pyruvate oxidase and protected with a cellulose acetate
membrane. The reaction of pyruvate in the presence of
pyruvate oxidase is as follows:
pyruvate
Pyruvate + HPO42 + O2 + H2O
acetyl phosphate + CO2 + H2O2
The H2O2 is measured amperometrically and related to
pyruvate concentrations present in the sample. Several
cofactors, including thiamin pyrophosphate, phosphate,
and calcium chloride, are required for the reaction
catalyzed by pyruvate oxidase [8].

A rapid HPLC method has also been described that

measures branched-chain keto acids and pyruvate in blood
i
Pyruvic Acid [9]. The pyruvate and other keto acids are derivatized with
Previous and current authors of this method: o-phenylenediamine to give fluorescent derivatives which
First edition: Nancy Gau are then separated chromatographically on a reversed-
phase column using a binary gradient.
Methods edition: Nancy Gau
Second edition: Not updated A method for measuring pyruvic acid by capillary
Third edition: Steven C. Kazmierczak electrophoresis with amperometric detection has recently
Fourth edition: Nancy Gau been described [10]. In this technique, pyruvic acid reacts
Fifth edition: Steven C. Kazmierczak with NH2OH to form oxime as the final product. Pyruvic
 1021
Pyruvic Acid
acid is quantified by capillary electrophoretic separation,
followed by amperometric detection of remaining NH2OH.
Reference and Preferred Methods
There is no reference method for the measurement of
pyruvate.
The hydrazone method was a manual, laborious procedure
that lacked specificity. In the method of Friedemann and
Haugen [1], it was estimated that 70% to 96% of the color
extracted was produced by the pyruvate hydrazone [11-
13]. Other endogenous ketones interfered, including
acetoacetate, levulinic acid, and -ketoglutaric acid.
Chromatographic purification of the pyruvate hydrazone
only made the analysis more laborious, and the analysis
was further complicated by the presence of two hydrazone
isomers for each ketone.
The enzymatic method is the method of choice. The
reagents are reasonably stable, and the reaction is easily
automated. Recovery of added pyruvate is close to 100%
[3-5], and the precision of 2.5% (percent coefficient of
variation) at 0.08 mmol/L is acceptable [4]. The analysis
can be performed in an end-point or kinetic mode.
A major concern in all methods is the instability of pyruvic
acid at room temperature. To preserve the sample, many
workers recommend preparing a protein-free supernatant
of serum or plasma as rapidly as possible. Although PCA
and TCA are widely used, Marbach and Weil recommend
the use of metaphosphoric acid [4]. They found that
NADH was much more stable when MPA was used as the
precipitating agent.
Specimen
Blood should be drawn in an iodoacetate (gray-top)
phlebotomy tube to prevent metabolism of glucose to
pyruvate by blood cells. Pyruvate is very unstable in blood,
and proper technique is essential. The blood should be
drawn from a fasting patient. A tourniquet can be used
during phlebotomy, since blood stasis for up to 2 min does
not produce any change in pyruvate concentration [5].
Clenching and unclenching of a fist also does not affect
pyruvate levels. Mild exercise will increase the blood
pyruvate concentration.
The serum should be separated immediately at refrigerated
temperatures and kept at 4C to 8C until analysis.
Analysis should be performed as soon as possible. Delays
of 1 hour or more before deproteinization have been found
unacceptable [8]. Preservation of the sample by
preparation of a protein-free supernatant of serum or
plasma is recommended. The sample can be stored frozen,
but for long-term stability, it is best to treat the sample
with metaphosphoric acid (to a final concentration of 40
g/L) and separate the protein-free supernatant by
centrifugation. This supernatant is stable for 6 days at
room temperature, 8 days at refrigerated temperatures, and
for up to 42 days when frozen [4].
Interferences
Hemolysis can cause an increase in pyruvate. One recent
study found that samples with plasma hemoglobin
concentrations of 0.9 g/dL showed increased measured
pyruvate concentrations by approximately 30% [14].
Decreases in measured pyruvate have been described for
enzymatic methods where the reagent LD is contaminated
with pyruvate kinase [15].
Pyruvic Acid Reference Interval
Pyruvate concentrations in fasting venous and arterial
blood are < 0.10 mmol/L. Pyruvate measured in capillary
blood of 141 healthy newborns ranged from 0.01 to 0.14
mmol/L, with a mean of 0.03 mmol/L [16]. Measurement
of pyruvate, in addition to lactate, in cerebrospinal fluid is
important in the assessment of numerous primary and
acquired disorders affecting the central nervous system.
The upper 90th percentile limit for cerebrospinal fluid
pyruvate from birth to 15 years of age showed a linear
decrease from 0.148 to 0.139 mmol/L [17].
Interpretation
Pyruvate is a product of the Embden-Meyerhof pathway,
which oxidizes glucose to three-carbon compounds. When
peripheral cells are sufficiently oxygenated, most of the
pyruvic acid produced is further oxidized in mitochondria
to CO2 and water by the citric acid cycle. However, under
conditions of tissue anoxia, when the cellular PO2 is low
and the NAD/NADH ratio is low, pyruvate is reduced to
lactate. This raises the NAD/NADH ratio and allows
continued anaerobic metabolism of glucose by the
Embden-Meyerhof pathway. Thus the ratio of serum
lactate to pyruvate is a measure of the oxidative-reductive
state of the body. Mild exercise will cause an increase in
both lactate and pyruvate but will not substantially change
the ratio. The determination of the lactate/pyruvate ratio
has also been suggested as an aid to estimate the severity
of circulatory failure, since the ratio is considered to be a
marker of cytosolic redox status [18,19]. The higher the
ratio, the more severe the tissue anoxia is presumed to be.
Similarly, this ratio can be useful in allowing
determination of the extent of an alcohol-induced ketosis.
Measurement of lactate and pyruvate in cerebrospinal fluid
can help in the diagnosis of inborn errors of metabolism
that can affect the central nervous system, such as pyruvate
dehydrogenase deficiency [20] and respiratory chain
disorders [21].
Pyruvic Acid Performance Goals
Pyruvic acid is not currently regulated for proficiency
testing. Inter- and intraindividual biological variation
measured in healthy individuals over a 5-day period was
determined to be 13.0% and 15.2%, respectively [22].
Desirable specifications for analytical imprecision derived
from studies of biological variation indicate an assay
imprecision of no greater than 7.6% [23].
 1022
Pyruvic Acid
References
1 Friedmann TE, Haugen GE. Pyruvic acid. II. The
determination of keto acids in blood and urine. J
Biol Chem 1943;147:415-442.
2 Seligson, D, Shapiro B. Alpha-keto acids in blood
and urine studied by paper chromatography. Anal
Chem 1952;24:754-755.
3 Hansen JL, Freier EF. Direct assays of lactate,
pyruvate, beta-hydroxybutyrate and acetoacetate
with a centrifugal analyzer. Clin Chem
1978;24:475-479.
4 Marbach EP, Weil MH. Rapid enzymatic
measurement of blood lactate and pyruvate. Clin
Chem 1967;13:314-325
5 Segal S, Blair AE, Wyngaarden JB. An enzymatic
spectrophotometric method for the determination
of pyruvic acid in blood. J Lab Clin Med
1956;48:137-143.
6 Antonius A, Clark M, Pilkington JRE. A
semiautomated fluorometric method for the
enzymatic determination of pyruvate, lactate,
acetoacetate and beta-hydroxybutyrate levels in
plasma. J Lab Clin Med 1966;68:340-356.
7 Cramp DG. Automated enzymatic fluorometric
method for the determination of pyruvic and
lactic acids in blood. J Clin Pathol 1968;21:171-
174.
8 Chariot P, Tariney R, Ami-Said M, Herigault R,
Adnot S, Gherardi R. Optimal handling of blood
samples for routine measurement of lactate and
pyruvate. Arch Path Lab Med 1994;118:695-697.
9 Pailla K, Blonde-Cynober, F, Aussel C, DeBandt
JP, Cynober L. Branched-chain keto-acids and
pyruvate in blood: measurement by HPLC with
fluorometric detection and changes in older
subjects. Clin Chem 2000;46:848-853.
10 Lu X, Huang WH, Ai F, Wang ZL, Cheng JK.
Indirect determination of pyruvic acid by
capillary electrophoresis with amperometric
determination. J Chromatogr B Analyt Technol
Biomed Life Sci 2007;857(2):347-351.
11 McArdle B. The quantitative estimation of
pyruvic and (L-oxyglutaric acids by paper
chromatography in blood, urine and cerebrospinal
fluid. Biochem J 1957;66:144-148.
12 Kulonen E, Carpen E, Ruokolainen T. Experience
with paper chromatography in the study of -keto
acids. Scand J Clin Lab Invest 1952;4:189-191.
13 Drewes PA. Carbohydrate derivatives and
metabolites. In: Henry RJ, Cannon DC,
Winkelman JW, eds. Clinical Chemistry:
Principles and Technics. 2nd ed. Hagerstown,
MD: Harper & Row; 1974:1335-1340.
14 Chuang CK, Wang TJ, Yeung CY, Hsieh WS,
Lin DS, Ho SC et al. Interference and blood
sample preparation for a pyruvate enzymatic
assay. Clin Biochem 2006;39:74-77.
15 Henderson AR. A source of error in blood
pyruvate determination. J Clin Pathol
1971;24:475.
16 Nielsen J, Ytrebo LM, Borud O. Lactate and
pyruvate concentrations in capillary blood from
newborns. Acta Paediatr 1994;83:920-922.
17 Benoist JF, Alberti C, Leclercq S, Rigal O, Jean-
Louis R, Ogier de Baulny H et al. Cerebrospinal
fluid lactate and pyruvate concentrations and their
ratio in children: age-related reference intervals.
Clin Chem 2003;49:487-494.
18 Huckabee WE. Relationships of pyruvate and
lactate during anaerobic metabolism. III. Effect of
breathing low-oxygen gases. J Clin Invest
1958;37:264-271.
19 Medina JM, Tabernero A, Martin-Barrientos J.
Metabolic fuel utilization and pyruvate oxidation
during the postnatal period [review]. J inherit
Metab Dis 1996;19:432-442.
20 Matsuda J, Ito M, Naito E, Yokota I, Kuroda Y.
DNA diagnosis of pyruvate dehydrogenase
deficiency in female patients with congenital
lactic academia. J Inherit Metab Dis 1995;18:534-
546.
21 Jackson MJ, Schaefer JA, Johnson MA, Morris
AA, Turnbull DM, Bindoff LA. Presentation and
clinical investigation of mitochondrial respiratory
chain disease. A study of 51 patients. Brain
1995;118:339-357.
22 Panteghini M, Pagani F. Biological variation of
lactate and pyruvate in blood. Clin Chem
1993;39:908.
23 Ricos C, Alvarez V, Cava F, Garcia-Lario JV,
Hernndez A, Jimnez CV et al. Current database
on biologic variation: pros, cons and progress.
Scand J Clin Lab Invest 1999;59:491-500.
 1023
Pyruvic Acid

Table 1: Pyruvate Methods

Method 1: Dinitrophenylhydrazone
Principle of analysis: Pyruvate and other ketones react with 2,4-dinitrophenylhydrazine to form the hydrazone
derivative; acid hydrazones are extracted and alkalinized to produce a red color.
Comments: Plasma, serum; nonspecific, of historical interest
Method 2: Enzymatic
Principle of analysis: Pyruvate is quantitatively converted to lactate at pH 7.4 by lactate dehydrogenase; the reaction
is monitored at 340 nm by following the concomitant conversion of NADH to NAD+ or by fluorescence using
excitation at 360 nm and emission at 470 nm.

LD
Pyruvate + NADH + H lactate + NAD
Comments: Plasma, serum; highly specific, automated, preferred method
Method 3: Amperometric
Principle of analysis: Pyruvate reacts with pyruvate oxidase which has been immobilized on an artificial membrane.
H2O2 produced measured amperometrically.
Pyruvate + HPO42 + O2 + H2O acetyl phosphate + CO2 + H2O2
Pyruvate oxidase

4. Lactic dehydrogenase (LD). Muscle LD can be

Procedure: Enzymatic Analysis of Pyruvic Acid purchased from Boehringer Mannheim Diagnostics
(including reaction conditions for enzymatic measurement
of pyruvic acid) (Indianapolis, IN; No. 127230) as an ammonium sulfate
suspension. Prepare working reagent by diluting 50 mL of
Principle the stock suspension with 450 mL of distilled water. Use
Muscle lactate dehydrogenase is used to immediately.
catalyze the reaction between pyruvic acid and 5. Working reagent. Add 40 mL of diluted LD
solution and 400 mL of NADH solution to 10 mL with
NADH to form lactate and NAD+. The reaction working buffer. Stable 24 hr at refrigerated temperatures.
is monitored by measurement of the decreasing 6. Stock pyruvic acid, 25 mmol/L. Place 2.2 g of
absorbance at 340 nm. This procedure can be sodium pyruvate in a 1-L flask. Dissolve in 200 mL of 0.1
readily adapted to most automated analyzers. M HCl, and bring to volume with additional 0.1 M HCl.
Stable for 3 months at refrigerated temperatures.
7. Working pyruvic acid standard, 0.5 mmol/L.
Reagents Dilute 1 mL of stock pyruvic acid to 50 mL with distilled
1. Stock buffers
water in a volumetric flask. Prepare fresh daily.
0.1 M Na2HPO4. Dissolve 1.42 g of Na2HPO4
salt in 80 mL of distilled water in a 100-mL volumetric Assay
flask, and bring to volume with additional distilled water. Equipment: ABA-100 or any automated
Stable for 1 year at refrigerated temperatures. spectrophotometric analyzer capable of reading at 340 nm.
0.1 M KH2PO4. Dissolve 1.36 g of KH2PO4 in
80 mL of distilled water in a 100-mL volumetric flask, and
bring to volume with additional distilled water. Stable for Instrument Parameters
1 year at refrigerated temperatures. Temperature 37C
2. Working buffer, 0.1 mol/L phosphate buffer. Mode Kinetic
Mix 20 mL of 0.1 M KH2PO4 and 80 mL of Na2HPO4 Direction Down
buffers. Check that the pH is 7.4 0.05; adjust if necessary Time 15 sec
with 0.1 M HCI or NaOH. Stable for 2 months at Revolutions Infinity
refrigerated temperatures. Check pH periodically, and Filter 340/380 nm
examine for the presence of bacterial growth or a
precipitate. Syringe plate 1:11
3. NADH. Weigh 20 mg of pure NADH. Dissolve Decimal 0.000
in 1 mL of distilled water. Prepare fresh, and use within 1 Calibration factor 0.500
h. Zero setting 0.000
 1024
Pyruvic Acid

Calculation
1. Prime working reagent through syringe plate. Subtract the first reading of position 1 (blank)
2. Set above parameters, and arrange sample tray in from the first reading of other positions to get change of
following manner: absorbances (A); then:
a. H2O
b. 0.5 mmol/L pyruvate standard
c. 0.5 mmol/L pyruvate standard
d. Patient sample
e. Patient sample, diluted fourfold with
saline solution
3. Rotate to position 31, go to test mode, and set Notes
zero. Push calibrate button, and set scale to read 0.500 with 1. This method is linear up to 1 mmol/L.
scaling knob. Push start button. As the carousel rotates, it
2. High lactate (to 40 mmol/L), bilirubin (to 200
will begin printing the change of absorbance readings
mg/L), and gross hemolysis (2 g of
every 15 sec for the first sample. It will print a total of
hemoglobin/L) will not interfere with the
three readings for each sample and then rotate to the next
analysis.
sample.
3. Grossly lipemic sera will not affect the analysis.
4. After the third absorbance reading on the last
sample is completed, stop instrument. Carousel will not
stop automatically.

Table 2: Reaction Conditions for Enzymatic Measurement of Pyruvic Acid

Temperature: 37C
pH: 7.4
Sample volume: 25 L
Fraction of sample volume: 0.0909
Final concentration of reagents:
Phosphate buffer: 90.9 mmol/L
NADH: 1.1 mmol/L
LD: varies with lot
Linearity: 0 to 1 mmol/L
1025
Renin
Renin
Greg Ward i
Name: Renin (PRA, plasma renin activity, direct renin concentration, DRC)
Clinical significance: Refer to Chapter 28, Physiology of Body Water and Electrolytes, in the
5th Edition of Clinical Chemistry: Theory, Analysis, Correlation.
Molecular mass: ~ 40,000 D
Chemical class: Glycoprotein, enzyme EC 3.4.23.15
Principles of Analysis and Current Usage
Renin (EC 3.4.23.15; formerly EC 3.4.4.15 and EC
3.4.99.19) is a 340-amino-acid aspartyl protease
glycoprotein (MW ~ 40000 daltons) which catalyses the
cleavage of angiotensin I from angiotensinogen [1,2].
The juxtaglomerular cells of the kidney synthesize the
inactive precursor prorenin that is cleaved into the active
proteolytic enzyme renin, which is stored and released in
response to stimuli from secretory granules.
Physiological factors important for renin release include
sodium depletion, hypotension, hypovolemia, and -
adrenergic stimulation [1,2,3].
Active renin released into the circulation cleaves the
decapeptide angiotensin I from its substrate serum
angiotensinogen, an 2-globulin synthesized in liver and
kidney, to angiotensin I, which is converted by
angiotensin-converting enzyme to the potent
vasoconstrictor, angiotensin II. Angiotensin II, together
with ACTH and potassium, are responsible for synthesis
and secretion of aldosterone. The renin-angiotensin-
aldosterone system is critical to both sodium
homeostasis and maintenance of blood volume and
blood pressure. This system is controlled by renin
activity, which is the rate-limiting step [1,2,3].
Prorenin is secreted constitutively from the kidney and
other organs in contrast to the tight regulation of active
renin release. Prorenin concentrations in normal serum
are approximately 10-fold greater than those of active
renin, and conversion to active renin appears not to
occur in plasma in vivo. In-vitro irreversible
cryoactivation of prorenin to active renin occurs in liquid
plasma at refrigerated temperatures below 25C,
increasing with decreasing temperature [1,4,5]. Plasma
active renin concentrations are very low, on the order of
approximately 0.2 to 0.8 pmol/L (8 to 32 ng/L or 12 to
48 mU/L, depending on the conversion factor for the
assay), which corresponds to a plasma renin activity of
approximately 1 to 4 ng/mL/hr.
Assays for renin have been classified as those which
measure enzyme activity (plasma renin activity [PRA])
and immunoassays for the renin molecule [4,5,6,7].
Enzyme activity assays have also been referred to as
i
Renin
Previous and current authors of this method:
New method
Fifth edition: Greg Ward
indirect or enzyme kinetic assays, whereas whole-
molecule assays have been referred to as direct renin
concentration (DRC) or active renin assays or total
renin assays for those assays which measure both active
renin and prorenin. Currently, prorenin assays or total
renin assays do not have routine clinical application and
will not be further discussed. In general, the terminology
PRA and DRC will be used, consistent with the 2008
Endocrine Society Clinical Practice Guideline [7].
Plasma Renin Activity (Table 1, Method 1)
Plasma renin activity is measured by the determination
of the amount of angiotensin I generated after incubation
of plasma at 37C, buffered to pH 6.0 for optimal
activity. The substrate angiotensinogen is not added, but
rather endogenous angiotensinogen normally present in
excess amounts is utilized. The amount of endogenous
angiotensinogen is not at the concentration required for
Vmax and is sometimes closer to the Km. Research
methods where addition of exogenous angiotensinogen
or synthetic substrates are used have been described and
are termed assays for plasma renin concentration (PRC).
The PRA is dependent on both the amount of active
renin and angiotensinogen for generation of angiotensin
I, and since this is the rate-limiting step, it is considered
to reflect the ability to generate angiotensin II in vivo
[1,2,5,6].
Angiotensin I generated is protected from degradation or
modification by the addition to reaction buffer of phenyl
methyl sulfonyl fluoride (PMSF) and EDTA, which
respectively inhibit the enzymes angiotensinase and
angiotensin-converting enzyme. The angiotensin I
generated during the incubation is quantitated by
competitive radioimmunoassay using125I-angiotensin I as
tracer and separation of antibody-bound tracer from free
achieved by PEG separation or antibody-coated tubes.
Although a blank tube is recommended in some
procedures, it has been demonstrated that a blank is not
necessary, provided the amount of angiotensin I
generated is 10 times greater than the blank signal. This
is achieved by extending the incubation times for
samples with low PRA. The standard incubation time for
the generation of angiotensin I is 90 to 180 min. Samples
with PRA < 1 ng/mL/hr should be incubated for 18
hours, which allows for lower activity to be accurately
1026
Renin
and precisely quantitated. The limit of detection for
incubation times of 90 min, 3 hours, and 18 hours is,
respectively, 0.65 ng/mL/hr, 0.33 ng/mL/hr, and 0.06
ng/mL/hr. The PMSF inhibition of angiotensinase cannot
be maintained during the 18-hour incubation unless pH
6.0 is maintained. Further renin is unstable at pH > 8.0,
and at pH 3.5, conversion of prorenin to renin occurs
[1,2,5].
Angiotensin I formation in PRA assays has also been
determined by EIA using -galactosidase-labeled
angiotensin I [8] or liquid chromatography/mass
spectrometry [9].
Direct Renin Concentration
All two-site immunometric (sandwich) assays for DRC
utilize capture antibodies which immuno-extract both
active renin and prorenin. Specificity is achieved by
using signal antibodies towards renin in the active (open)
conformation and short incubation times at 37C.
Two-site immunoradiometric assays (IRMA) with 125I-
labeled signal antibodies have used capture antibodies,
which are either coupled to magnetic beads [10] or
biotinylated and react with avidin-coated beads [11] to
create a solid phase for separation of the sandwich
complex from unreacted signal antibody (Table 1,
Method 2). Deinum and co-workers [12] demonstrated
that overestimation of renin in samples with low
concentrations was not due to cross-reactivity with
inactive prorenin, but rather the conformation of
prorenin had changed to that of the active form under the
conditions of incubation, resulting in formation of
additional measurable sandwich complexes. Thus these
assays are specific for both active renin and activated
prorenin. The conformational change and overestimation
was eliminated by reducing the incubation time from 24
to 6 hours and increasing the incubation temperature
from 22C to 37C. The functional sensitivity (interassay
coefficient of variation [CV] < 20%) for the latter assay
which eliminated prorenin interference was 4 mU/L.
Automation of DRC assays has been achieved with
immunoluminometric assays (ILMA) which utilize
chemiluminescence labels (Table 1, Method 3). In the
Nichols Advantage System, the signal antibody was
labeled with an acridinium ester and the capture
antibody biotinylated. Separation of the sandwich
complex was achieved by the use of paramagnetic
particles coated with streptavidin. The functional
sensitivity of this method was 2.65 mU/L [13]. In routine
practice, this system could not reliably measure low
renin levels [5]. The Nichols Advantage System is no
longer commercially available. The DiaSorin Liaison
automated analyzer has a DRC method (currently not
available in the United States) where the capture
antibody is coupled to paramagnetic particles, and the
signal antibody is labeled with amino-butyl-ethyl-
isoluminol. The manufacturers quoted functional
sensitivity is < 2 mU/L
(http://www.diasorin.com/upload/prodottiesistemi/Direct
%20Renin.pdf). Prorenin interference (conversion to
active conformation) was eliminated in both systems by
using an incubation time of 30 minutes at 37C.
Reference and Preferred Methods
There is no reference method for renin.
According to the 2007 General Ligand Survey by the
American College of Pathologists, more than 90% of 40
participating laboratories used PRA methods with
incubation at pH 6.0 and RIA of angiotensin I. The
remainder (<5 labs) measured DRC with no data
available.
Both DRC and PRA assays are suitable for measurement
of normal or low amounts of renin. In the case of low
renin levels, PRA with an 18-hour incubation is
preferred over DRC [5,7,14].
Specimen
Critical to the collection and preparation of samples for
renin analysis are procedures which prevent
cryoactivation of renin resulting in falsely elevated
levels. EDTA is an inhibitor of angiotensin-converting
enzyme and is the preferred anticoagulant for PRA and
can also be used for DRC [1,2,5]. In the case of DRC
assays, all anticoagulants tested were observed to be
suitable [13].
It is recommended that the sample be collected mid-
morning, after the patient has been ambulant for a
minimum of 2 hours but seated for 5 to 15 minutes prior
to collection [7]. The sample should be collected at room
temperature (not placed on ice, because the lower
temperature will cause cryoactivation). The sample
should be centrifuged at room temperature and the
plasma snap frozen.
Renin is stable in whole EDTA blood and plasma at
room temperature for 24 hours. Samples stored at 20C
are stable for up to 15 months [2,5].
To avoid cryoactivation, frozen samples should be
thawed rapidly prior to analysis. Fans and lukewarm
water have been variously used. Frozen samples should
not be thawed in a refrigerator, and frozen samples
which thaw during transportation should be rejected for
analysis [1,2,5].
Interferences
No analytical interferences in renin assays have been
reported. Heterophilic antibody interference is possible
for the two-site immunometric DRC assays, but this has
not yet been reported.
Many medications can cause plasma renin levels to
either decrease (-adrenergic blockers, clonidine, -
methyldopa, NSAIDS) or increase (potassium wasting or
sparing diuretics, ACE inhibitors, dihydropyridines,
angiotensin IItype I receptor blockers). This can result
in false-positive or false-negative aldosterone/renin
ratios, and recommendations are made in clinical
practice guidelines for medication use during
investigation of hypertension [1,2,7].
1027
Renin

pseudohypoaldosteronism [2,14]. PRA is best used for

Renin Reference Interval measurement of low renin levels; both PRA and DRC
As with all diagnostic tests, it is recommended that are suitable for normal and high renin levels.
reference intervals be determined by each laboratory to
conform to the characteristics of the population being In most clinical situations, renin levels are measured
tested. together with aldosterone and the aldosterone-to-renin
ratio (ARR). A recent assessment of current literature
Renin levels are highest in newborn children, with a demonstrated ARR to be superior to measurement of
rapid fall after 3 months, whereby they slowly decrease aldosterone or renin in isolation for patients with PA [7].
to adult levels. In adults, renin levels decrease with Renin levels are low in PA. The use of PRA and DRC in
aging, presumably due to decreased renal function PA is controversial [4, 5, 16-19]. In general, two-site
[2,15]. immunometric assays are unable to reliably measure low
renin levels [4, 5, 15, 16]. Early two-site IRMAs
Renin has a diurnal variation, with the highest level measured activated prorenin in addition to active renin
observed on awakening and values then progressively [4,5,12]. In normal patients, the prorenin concentration is
falling through the day. approximately 10 times the renin concentration, whereas
patients with PA have up to 100 times more prorenin
Renin levels are influenced by posture, with the change than active renin. Thus at low DRC, even small
in blood pressure on standing resulting in an increased conversion of prorenin can affect the accuracy and
renin level. clinical reliability of the assay. The problem of prorenin
activation was overcome by short incubation times at
Renin levels are lower in blacks than whites. 37C [12]. Initial studies on a two-site ICMA
demonstrated that this assay could measure DRC in
Dietary sodium intake influences the renin level, with patients with low PRA [5, 20-22]. In routine use, the
salt restriction or high-salt diets respectively stimulating two-site ICMA could not reliably measure low DRC.
or suppressing renin levels. This has been attributed to a variety of factors, including
insufficient range of low renin levels used in the
In general, morning reference intervals for PRA and standard curve, variation between reagent lots, false high
DRC are 1 to 4 ng/mL/hr and 8 to 35 mU/L, levels (this could be unproven heterophilic antibody
respectively. interference), and machine maintenance [5]. It has been
recommended that the best test for measurement of low
Reference intervals in use are shown below. renin levels is PRA, with an 18-hour incubation followed
by two-site immunometric assay [5, 14].
Plasma Renin Activity Reference Intervals
(ARUP, Renin levels are low in adrenal disease where there is
http://www.aruplab.com/guides/ug/tests/00 excess production of deoxycorticosterone (DOC),
70105.jsp) including: 11--hydroxlase deficiency; 17--hydroxylase
deficiency; primary glucocorticoid resistance; DOC
Adults, normal sodium diet: producing tumors; and sometimes ectopic ACTH
Supine: 0.2-1.6 ng/mL/hr syndrome [2, 14].
Upright: 0.5-4.0 ng/mL/hr
Newborn (1-7 days): 2.0-35.0 ng/mL/hr Renin levels are elevated in the following adrenal
Cord blood: 4.0-32.0 ng/mL/hr diseases: primary adrenal insufficiency; 21-hydroxylase
Children, normal sodium diet, supine: deficiency; aldosterone synthase deficiency; 3--
1-12 months: 2.4-37.0 ng/mL/hr hydroxysteroid dehydrogenase deficiency; side-chain
13 months-3 years: 1.7-11.2 ng/mL/hr cleavage enzyme deficiency; and steroid acute
4-5 years: 1.0-6.5 ng/mL/hr regulatory protein deficiency [2, 14].
6-10 years: 0.5-5.9 ng/mL/hr
11-15 years: 0.5-3.3 ng/mL/h Renin levels are normal in secondary adrenal
insufficiency (hypopituitarism).
Direct Renin Concentration Reference Intervals [2]
Adults: Renin levels can be either normal or elevated in renal
Supine: 3-21 ng/L (5-34 mU/L) artery stenosis (RAS) or reninoma [2, 14]. In RAS, the
Upright: 5-30 ng/L (8-48 mU/L) clinical sensitivity and specificity is improved by using
Children: captopril renography rather than captopril-stimulated
Mean DRC, neonatal period 73.1 ng/L PRA or peripheral PRA [14]. In renography, renin is
Mean DRC at 3 months 30.4 ng/L measured in the renal veins both before and after
administration of captopril, with a renal-vein ratio
Interpretation greater than 1.5 used to demonstrate lateralization of
Renin levels are used in the investigation of patients with renin excess. Reninoma is rare, and lateralized renin
secondary hypertension, most commonly primary secretion in the absence of RAS is suggestive; further
hyperaldosteronism (PA) and renal artery stenosis but radiological studies are required to search for a small
also reninoma, congenital adrenal hyperplasia, and kidney mass. Renin secretion in these patients varies
1028
Renin
over time, and a single renin measurement does not
exclude reninoma [14].
Renin Performance Goals
Survey data from the 2007 CAP participant summary
report show imprecision values (CV) of PRA to be
approximately 20% for specimens with mean activities
ranging from approximately 1 to 12 ng/mL/hr.
Acceptable Clinical Laboratory Improvement
Amendments performance criteria (CLIA 88) for
measurement of renin require that laboratories be
accurate to within 3 SD of the peer-group mean. The
ranges of renin in the CAP survey are higher than those
seen in PA and may not reflect the ability of laboratories
to measure low PRA.
Most renin measurements are used for screening of PA
where low renin levels are observed. Currently, only 18-
hour incubation PRA can consistently measure these low
renin levels. DRC assays using two-site ICMA have the
potential to measure these low concentrations but so far
have been inconsistent when used routinely.
References
1 Sealey JE. Plasma renin activity and plasma
prorenin assays. Clin Chem 1991;37:1811-
1819.
2 Cartledge S, Lawson N. Aldosterone and renin
measurements. Ann Clin Biochem
2000;37:262-278.
3 Lavoie JL, Sigmund CD. Minireview: overview
of the renin-angiotensin system, an endocrine
and paracrine system. Endocrinology
2003;144:2179-2183.
4 Sealey JE, Laragh JH. Renin and prorenin:
advances and declines in methodology. Clin
Chem 1996;42:993-994.
5 Sealey JE, Gordon RD, Mantero F. Plasma
renin and aldosterone measurements in low
renin hypertensive states. Trends Endocrinol
Metab 2005;16:86-91.
6 Schalekamp MADH, Derkx FHM, Deinum J,
Danser AHJ. Newly developed renin and
prorenin assays and the clinical evaluation of
renin inhibitors. J Hypertens 2008;26:928-937.
7 Funder JW, Carey RM, Fardella C, Gomez-
Sanchez CE, Mantero F, Stowasser M, Young
WF Jr, Montori VM. Case detection, diagnosis
and treatment of patients with primary
aldosteronism: an Endocrine Society Clinical
Practice Guideline. J Clin Endocrinol Metab
2008;93:3266-3281.
8 Sharpe S, Verkerk R, Sasmito E, Theauws M.
Enzyme immunoassay of angiotensin and
rennin. Clin Chem 1987;33:1774-1777.
9 Fredline VF, Kovacs EM, Taylor PJ, Johnson
AG. Measurement of plasma renin activity with
use of HPLC-electrospray-tandem mass
spectrometry. Clin Chem 1999;45:659-664.
10 Simon D, Hartmann DJ, Badouallie G, Caillot
G, Guyenne T-T, Corvol P et al. Two-site direct
immunoassay specific for active renin. Clin
Chem 1992;38:1959-1962.
11 Derkx FHM, de Bruin RJA, van Gool JMG, van
den Hoek MJ, Beerendonk CCM, Rosmalen F
et al. Clinical validation of renin monoclonal
antibody-based sandwich assays of renin and
prorenin, and use of renin inhibitor to enhance
prorenin immunoreactivity. Clin Chem
1996;42:1051-1063.
12 Deinum J, Derkx FHM, Schalekamp MADH.
Improved immunoradiometric assay for plasma
renin. Clin Chem 1999;45:847-854.
13 de Bruin RA, Bouhuizen A, Diederich S,
Perschel FH, Boomsma F, Deinum J.
Validation of a new automated renin assay. Clin
Chem 2004;50:2111-2116.
14 Rossi GP, Seccia TM, Pessina AC. Clinical use
of laboratory tests for the identification of
secondary forms of arterial hypertension. Crit
Rev Clin Lab Sci 2007;44:1-85.
15 Gordon RD. The challenge of more robust and
reproducible methodology in screening for
primary aldosteronism. J Hypertens
2004;22:251-255.
16 Sealey JE, Trenkwalder P, Gahnem F,
Catanzaro D, Laragh JH. Plasma renin
methodology: inadequate sensitivity and
accuracy of direct renin assay for clinical
applications compared with traditional
enzymatic plasma renin activity assay
[commentary]. J Hypertens 1995;13:27-30.
17 Morganti A, Pelizzola D, Mantero F, Gazzano
G, Opocher G, Piffanelli A.
Immunoradiometric versus enzymatic renin
assay: results of the Italian Multicenter
Comparative Study. Authors reply. J
Hypertens 1995;13:31.
18 Mnard J, Guyene T-T. Renin assays: a debate
for clinicians, not only for specialists
[commentary]. J Hypertens 1995;13:367-369.
19 Morganti A, Pelizzola D, Mantero F, Gazzano
G, Opocher G, Piffanelli A.
Immunoradiometric versus enzymatic renin
assay: results of the Italian Multicenter
Comparative Study. J Hypertens 1995;13:19-
26.
20 Hartman D, Sagnella GA, Chesters CA,
MacGregor GA. Direct renin assay and plasma
renin activity compared. Clin Chem
2004;50:2159-2161.
21 Perschel FH, Schemer R, Seiler L, Reincke M,
Deinum J, Maser-Gluth C et al. Rapid screening
test for primary hyperaldosteronism: ratio of
plasma aldosterone to renin concentration
determined by fully automated
chemiluminescence immunoassays. Clin Chem
2004;50:1650-1655.
22 Unger N, Schmidt IL, Pitt C, Walz MK, Philipp
T, Mann K, Petersenn S. Comparison of active
renin concentration and plasma renin activity
for the diagnosis of primary hyperaldosteronism
in patients with an adrenal mass. Eur J
Endocrinol 2004;150:517-523.
1029
Renin

Table 1: Methods of Analysis for Renin

Method 1: Plasma renin activity (PRA) radioimmunoassay (RIA) for angiotensin I
Principle of analysis: Competitive binding of angiotensin I with 125I-labeled angiotensin I for limited amount of
anti-angiotensin I
Marker: 125I
Signal: Radioactivity
Comments: Manual; 18-hour incubation required for low renin levels. Uses endogenous angiotensinogen as
substrate.
Method 2: Immunoradiometric assay (IRMA) for direct renin concentration (DRC)
Principle of analysis: Noncompetitive binding of active renin to excess 125I-labeled antibody
Marker: 125I
Signal: Radioactivity
Comments: Not commonly used now; manual
Method 3: Immunoluminometric assay (ILMA) for DRC
Principle of analysis: Noncompetitive binding of active renin to excess acridinium derivativelabeled antibody
Marker: Acridinium derivatives
Signal: Luminescence
Comments: Fully automated; functional sensitivity indicates that low renin can be measured, but not observed so
far in routine practice. Assay has 30-minute incubation at 37C to eliminate conversion of prorenin to activated
prorenin.
1030
Rheumatoid Factor
Rheumatoid Factor
Terry Pry i
Name: Rheumatoid factor
Clinical significance: Refer to Chapter 7, Immunological Reactions, and Chapter 8,
Immunochemical Techniques, in the 5th edition of Clinical Chemistry: Theory, Analysis, and
Correlation.
Principles of Analysis and Current Usage
Rheumatoid factors (RF) are immunoglobulins of any
isotype with antibody activity directed against antigenic
sites on the Fc region of human or animal
immunoglobulin G (IgG). After the discovery by Waaler
[1] that the sera from patients with rheumatoid arthritis
agglutinated sheep erythrocytes coated with rabbit anti-
sheep erythrocyte antibody, it was determined that the
serum factor responsible for the agglutination was a
high-molecular immunoglobulin of the IgM class. IgM-
RF is the main isotype identified by clinically available
diagnostic assays for RF detection. Assays for RF are the
most widely used serological tests as an aid for the
diagnosis of rheumatoid arthritis (RA).
Traditionally, RF was detected by a latex flocculation
test and the sensitized sheep-cell agglutination (Waaler-
Rose) test [1,2]. Conventional qualitative methods for
the measurement of RF-IgM utilize visual detection and
have depended upon the agglutination of particles (e.g.,
latex, erythrocytes) coated with human or animal IgG.
The latex-IgG agglutination technique of Singer and
Plotz has become the most popular method for detection
of RF [3]. Qualitative (positive/negative) and
semiquantitative (titer unit) assessment of RF are
provided. Inherent problems accompany these methods,
such as subjectivity, difficulty in standardization, and
imprecision [3,4].
Measurement of RF was greatly improved through
adaptation of automated quantitative serological tests
such as enzyme immunoassay (EIA), nephelometry, and
turbidimetry that utilize the advantage of objective
instrument measurement on a single sample dilution.
These tests are superior in that they are easy to use,
provide rapid results, have good precision, and have the
ability to measure RF in absolute terms.
The principle of commonly used methods for
demonstrating RF involves use of an indicator system
such as latex or erythrocytes, to which human IgG is
attached. The presence of RF is recognized by
aggregation, flocculation, or precipitation of various
indicator systems upon addition of specimen. Several
assay methods are available for RF determination and
are listed in Table 1.
i
Rheumatoid Factor
New method
Fifth edition: Terry Pry
Reference and Preferred Methods
There are no reference methods for RF measurement that
have been approved at this time. Preferred methods for
detecting RF are the following:
Latex Agglutination
The earliest tests, which are still widely used clinically,
rely on the agglutinating properties of the IgM class of
RF. IgG, usually human or rabbit, is bound to a
particulate carrier, and the presence of RF is then
detected by agglutination or flocculation of the
respective indicator system. Carrier particles frequently
used include latex and erythrocytes. In one application of
this method, the procedure is performed by mixing IgG-
coated latex particles with serum on a Teflon-coated
card (Table 1, Method 1). Latex particles function as
inert spacers in the antigen-antibody lattice, increasing
the size of the complexes.
The test is considered positive if agglutination of the
latex particles is observed following the mixing period.
Advantages of the latex agglutination procedure include
a rapid assay time and no need for specialized or
expensive equipment. Either qualitative or semi-
quantitative analysis is provided. The RF antibody
content of a serum involves doubling dilutions of the
serum and determination of an end point (the last
doubling dilution at which agglutination can be
visualized). The reciprocal of this dilution is known as
antibody titer. It is not possible to quantify RF by these
methods except by titer, which is recognized to be
inaccurate [4,5].
The latex agglutination test is sensitive, but it can result
in a fairly high number of false positives [6].
Nonspecific agglutination of latex particles by sera from
normal individuals is not uncommon [7].
Immunoturbidimetric
An immunoturbidimetric RF assay involves an antigen-
antibody reaction between RF in the sample and
denatured human IgG, which has been adsorbed to latex
particles (Table 1, Method 2) [4]. Immunoturbidimetric
methods measure the resulting agglutination as an
absorbance change, with the magnitude of the change
being proportional to the quantity of RF in the sample.
The actual concentration is then determined by
interpolation from a calibration curve prepared from
calibrators of known concentration.
1031
Rheumatoid Factor
Nephelometry
Similar to immunoturbidimetric methods, nephelometry
involves mixing RF and antibody under antibody-excess
conditions. The concentration of resulting antigen-
antibody complexes can be determined by light
dispersion (Table 1, Method 3) [7,8]. When a beam of
light is passed through tubes containing a fixed amount
of antibody and variable concentrations of RF, the
concentration of immune complexes formed in the tube
will determine the extent of light scattering. In contrast
to the immunoturbidimetric method, which measures
transmitted light in the reaction solution, nephelometry
measures the amount of light scattered at angles varying
from 0 to 90 degrees. Since antibody concentration
remains constant, the light scattered is proportional to
the concentration of RF in the mixture.
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) is used
for the qualitative, quantitative, or semi-quantitative
determination of IgM RF in human serum. Purified RF
antigen (human IgG) is attached to the wells of a
polystyrene microtiter plate (Table 1, Method 4) [9].
Diluted patient specimens, controls, and calibrators are
incubated at room temperature in the microwells. Any
RF-IgM antibody present binds to the immobilized
human IgG to form antigen-antibody complexes.
Unbound antibody is washed from the wells, and
enzyme-conjugated antihuman IgM is added. The
enzyme conjugate binds to the antigen-antibody
complex. Excess conjugate is washed away and a
specific substrate added. Bound enzyme conjugate
begins a hydrolytic reaction, causing color development.
After a specific time, the reaction is stopped.
The intensity of the generated color is proportional to the
amount of RF-specific IgM antibody bound to the wells.
The results are read on a spectrophotometer (ELISA
reader). The net absorbance is calculated by subtracting
the absorbance value for the specimen blank from the
value for the antigen-coated microwell. A calibration
standard that is assayed with each plate is then used to
calculate the RF-IgM activity in IU/mL from the net
absorbance value.
Specimen
Serum is the preferred specimen for RF testing.
Acceptable storage of specimen has been suggested for
20C to 25C (1 day), 2C to 8C (3 days), and 20C (1 IgM-RF accompanies secondary immune responses and
month) [10].
Interferences
All fluid-phase immunochemical assays may be
susceptible to nondetection of antigen excess and should
be carefully assessed. Pharmaceuticals may affect RF
concentration as follows: (1) interferon alfa-2a and
methotrexate may decrease serum RF levels; (2)
methyldopa, oral contraceptives, and oxyphenisatin may
increase serum RF levels; and (3) nonsteroidal
antiinflammatory drugs may decrease or have no
significant effect on serum RF levels [11]. Several
potential interferences have been described for
qualitative agglutination methods, including false
positives from bacterial contamination, lipemia, and
drying effect due to extended reaction times.
RF Reference Interval
Reference intervals are method dependent but are
typically < 30 IU/mL [12]. It is recommended that each
laboratory determine its own reference range based upon
its particular locale and population characteristics.
Results expressed in KU/L are equivalent to IU/mL. A
WHO International Standard Preparation of Rheumatoid
Arthritis Serum (NIBSC code: 64/2) is available to
achieve traceability of results.
Interpretation
Rheumatoid arthritis is an autoimmune disorder of
uncertain etiology, but there is evidence that an interplay
of genetic predisposition (HLA-DR shared-epitope
genes) and environmental factors (e.g., smoking) are
involved [13]. RA is characterized by symmetrical and
erosive synovitis that typically affects hands, wrists, and
feet initially and later may involve any joint lined by
synovial membrane. Chronic fluctuating disease course,
if left untreated, results in progressive joint destruction,
deformity, disability, and premature death. RA is the
most common autoimmune disease, affecting 1% to 2%
of the adult population, with twice as many women as
men [14]. The age of onset varies, peaking between 20
and 40 years for women, while often occurring at age 60
to 80 in men. Since structural joint damage is
irreversible, early recognition and treatment are currently
being emphasized, with the goal of halting the
progression of the disease.
Autoimmunity is one of the hallmarks of RA that
distinguishes this disease from other inflammatory and
degenerative joint diseases such as psoriatic arthritis or
osteoarthritis. The first autoimmune response described
in RA was RF. This observation formed the basis for a
pathogenetic concept that considers RA as one of the
systemic autoimmune diseases closely related to
systemic lupus erythematosus, scleroderma, or Sjgrens
syndrome.
Although RF has proven to be a useful diagnostic and
prognostic test, the etiology of RFs and the precise role
they play in the pathogenesis of RA is still unresolved.
IgM-RF synthesis can be induced by immune complexes
and polyclonal B-cell activators. Transient synthesis of
is part of the immunoregulatory process. RF is produced
during bacterial and viral infections, probably in
response to immune complexes containing microbial
antigens. Polyclonal stimulation induces low-affinity
polyreactive IgM-RF, which can also be found in healthy
individuals. There is evidence of a preclinical or
symptomatic phase of the disease.
The diagnosis of RA is made on the basis of history and
physical examination findings together with laboratory
testing and imaging of joints. The diagnosis may be
difficult in early stages of the disease. The
internationally accepted 1987 classification criteria of
1032
Rheumatoid Factor
the American College of Rheumatology, although
primarily developed as a basis for recruitment of patients
into clinical trials, are often also used as a diagnostic tool
for individual patients [15]. For the latter purpose,
especially in early diagnosis, performance is far from
optimal. These guidelines include a set of seven common
criteria that should be considered: (1) morning stiffness
> 1 hour, (2) arthritis of > 3 of 14 joints/joint groups, (3)
arthritis of hand joints, (4) symmetrical swelling of joint,
(5) rheumatoid nodules in specific locations, (6) positive
RF, and (7) radiographic changes suggestive of joint
erosion. The presence of four of seven criteria is
required to establish the presence of RA for study
purposes. In addition, the first four criteria must be
present for 6 weeks. The European League Against
Rheumatism (EULAR) recently recommended that the
following factors predicting persistent and erosive
disease should be measured: number of swollen and
tender joints, ESR or CRP, levels of RF and anti-
citrullinated protein (anti-CCP) antibodies, and
radiographic damage [16].
The presence of RF is still considered an important
feature of RA. RF can be found in 70% to 90% of RA
patients, though they are far from specific for this
disease. The autoantibodies most frequently found in
patients with RA are antibodies against IgG (IgM
rheumatoid factor) and antibodies against citrullinated
proteins (anti-CCP). Meta-analysis of clinical studies
involving anti-CCP and RF conclude that anti-CCP
antibodies are more specific than RF for diagnosing RA
and may better predict erosive disease [17,18].
As a diagnostic test, the use of RF is limited because a
positive RF can also be detected in other rheumatic
disorders, infections, and in apparently healthy
individuals (Table 2) [19]. The prevalence of RF
positivity in the normal population increases with age;
by age 70, up to 10% of the population may be positive.
Thus the presence of a positive serum RF is not
sufficient in itself to make a diagnosis of RA. In
addition, about 15% of patients meet diagnostic criteria
for RA and do not have a positive test for RF.
Importantly, combining the presence of RF with anti-
CCP can further increase the specificity for RA. The
combined use of RF and anti-CCP is currently
recommended [16]. A negative result in both assays
makes RA unlikely, although it does not exclude the
disease entirely. A positive result in both tests raises the
probability of RA to approximately 90% to 100%. The
prevalence of RA is markedly increased in individuals
with RF antibodies of more than one isotype, most
frequently a combination of IgM and IgA.
Rheumatoid Factor Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report summarize
both qualitative and quantitative assay methods. A
majority of laboratories reporting quantitative RF results
utilized the immunoturbidimetric method. Quantitative
RF test results were reported using turbidimetric
immunoassay methods (62% of laboratories; n = 526),
nephelometric methods (37% of laboratories; n = 315),
and enzyme immunoassay (1% of laboratories, n = 13).
Imprecision values (% coefficient of variation) for
quantitative measurement of RF were acceptable and
generally less than 10%. Criteria for measurement of RF
require that laboratories achieve 80% participant
consensus of qualitative results (negative or positive) or
within 3 SD of peer group mean for quantitative
results. Acceptable performance was achieved for
qualitative reporting, with > 98.5% of laboratories
reporting the consensus test result.
References
1 Whaler E. On the occurrence of a factor in
human serum activating the specific
agglutination of sheep blood corpuscles. Acta
Pathol Microbiol Scand. 1940;17:172-178.
2 Rose HM, Ragan C, Pearce E, Lipmann MO.
Differential agglutination of normal and
sensitized sheep erythrocytes by sera of patients
with rheumatoid arthritis. Proc Soc Exp Biol
Med. 1948;68:1-11.
3 Singer J, Plotz C. The latex fixation test.
Application to the serologic diagnosis of
rheumatoid arthritis. Am J Med. 1956;21:888-
92.
4 Melamies L, Ruutsalo H, Nissila H. Evaluation
of a quantitative immunoturbidimetric assay for
Rheumatoid factors. Clin Chem. 1986;32:1890-
1894.
5 Finley P, Hicks J, Williams J, Hinlicky J, Lichtl
D. Rate nephelometric measurement of
rheumatoid factor in serum. Clin Chem.
1979;25:1909-1914.
6 National Committee for Clinical Laboratory
Standards. User Comparison of Quantitative
Clinical Laboratory Methods Using Patient
Samples: Proposed Guideline. Vol 5. Wayne,
PA, 1985, NCCLS.
7 Ash K. Reference intervals (normal ranges): a
challenge to laboratories. Am J Med Tech.
1980;46:504-11.
8 Roberts-Thomson P, McEvoy R, Langhans T,
Bradley J. Routine quantification of rheumatoid
factor by rate nephelometry. Ann Rheum Dis.
1985;44:379-383.
9 Highton J, Hessian P, Small B, Palmer D.
Clinical advantages from measurement of IgM-
rheumatoid factor by enzyme immunoassay.
Rheumatology. 1986;25:20-25.
10 Guder WG, Narayanan S, Wisser H, Zawta B.
Samples: From the Patient to the Laboratory:
The Impact of Preanalytical Variables on the
Quality of Laboratory Results. Darmstadt,
Germany: Git Verlag; 1996.
11 Young DS. Effects of Drugs on Clinical
Laboratory Tests. 4th ed. Washington, DC:
AACC Press; 1995:3-527.
12 Tietz NW. Clinical Guide to Laboratory Tests.
3rd ed. Philadelphia: Saunders; 1995:544-5.
13 Klareskog L, Padyukov L, Lorentzen J,
Alfredsson L. Mechanisms of disease: genetic
susceptibility and environmental triggers in the
1033
Rheumatoid Factor
development of rheumatoid arthritis.
Rheumatology. 2006;2:425-433.
14 Lee DM, Weinblatt ME. Rheumatoid arthritis.
Lancet. 2001;358:903-11.
15 Arnett FC, Edworthy SM, Bloch DA, McShane
DJ, Fries JF, Cooper NS et al. The American
Rheumatism Association 1987 revised criteria
for the classification of rheumatoid arthritis.
Arthritis Rheum. 1988;31:315-24.
16 Combe B, Landewe R, Lukas C, Bolosiu H,
Breedveld F, Dougados M et al. EULAR
recommendations for the management of early
arthritis: report of a task force of the European
Standing Committee for International Clinical
Studies Including Therapeutics (ESCIST). Ann
Rheum Dis. 2007;66:34-45.
17 Nishimura K, Sugiyam D, Kogat Y, Tsuji G,
Nakazawa T, Kawano S et al. Meta-analysis:
diagnostic accuracy of anti-cyclic citrullinated
peptide antibody and rheumatoid factor of
rheumatoid arthritis. Ann Intern Med.
2007;146:797-808.
18 Avouac J, Gossec L, Dougados M. Diagnostic
and predictive value of anti-cyclic citrullinated
protein antibodies in rheumatoid arthritis: a
systematic literature review. Ann Rheum Dis.
2006;65:845-851.
19 Mierau R, Genth E. Diagnosis and prognosis of
early rheumatoid arthritis, with special
emphasis on laboratory analysis. Clin Chem
Lab Med. 2006;44:138-143.
1034
Rheumatoid Factor

Table 1: Methods for Rheumatoid Factor

Method 1: Latex agglutination

Principle: Latex particles coated with IgG (usually human or rabbit) are mixed with serum; presence of RF in
test serum results in agglutination of latex particles. Provides qualitative or semiquantitative results.
Usage: Frequently used
Comments: Visual detection method; large inter-observer variation when Ab titers are near assay sensitivity.
Method 2: Turbidimetry
Principle: Agglutination of latex particles coated with IgG antigen. Increasing levels of RF cause increasing
agglutination of latex particles, resulting in increased turbidity of the solution. The level of turbidity is measured
by absorbance spectrophotometry and is proportional to the RF level in the sample.
Usage: Frequently used (62% of laboratories reporting quantitative results)
Comments: Provides rapid (typically 10 minutes) quantitative as well as semi-quantitative or qualitative
results. Readily applied to automated general chemistry analyzers utilized to process routine chemistry tests on
patient specimens.
Method 3: Nephelometry
Principle: Agglutination of latex particles coated with IgG antigen. Increasing levels of RF cause increasing
agglutination of latex particles, resulting in increased turbidity of the solution. Measuring light reflected at various
angles using a nephelometer assesses turbidity.
Usage: Frequently used (37% of laboratories reporting quantitative results)
Comments: Provides quantitative as well as semi-quantitative or qualitative results. Requires specialized
instrumentation (nephelometer).
Method 4: Enzyme-Linked Immunosorbent Assay
Principle: ELISA technology in which human IgG attached to wells of a polystyrene micro-titer plate. Addition
of specimen containing RF binds to immobilized IgG, resulting in antigen-antibody complex. Subsequent addition
of enzyme-conjugated anti-human IgM binds the RF immune complex. The level of RF is proportional to the
level of enzyme activity.
Usage: Rarely used (<1% of laboratories reporting quantitative results)
Comments: Provides quantitative as well as semi-quantitative or qualitative results.

Table 2: Causes of Rheumatoid Factor Positivity

Age older than 60 years

Rheumatoid diseases
Rheumatoid arthritis
Sjgrens syndrome
Systemic lupus erythematosus

Viral infections
Hepatitis C
Hepatitis B
HIV infection
Parvovirus
Influenza

Bacterial infections
Endocarditis
Osteomyelitis
Tuberculosis

Chronic inflammatory conditions

Inflammatory bowel disease
Sarcoidosis
Pulmonary fibrosis
Primary biliary cirrhosis

Malignancy
1035
Rubella
Rubella
Terry Pry and Greg Maine i
Name: Rubella
Clinical significance: Refer to Chapter 7, Immunological Reactions, and Chapter 8,
Immunochemical Techniques, in the 5th edition of Clinical Chemistry: Theory, Analysis, and
Correlation.
Principles of Analysis and Current Usage
Rubella, also known as German measles, is a common
and normally benign viral disease that can occur in
unimmunized individuals. It has been largely eradicated
in countries with well-established vaccination programs,
but outbreaks still occur, and the disease is still
widespread in developing countries. It is a highly
contagious viral disease, usually transmitted from person
to person via the respiratory route. Following respiratory
transmission of rubella virus, replication of the virus is
thought to occur in the nasopharynx and regional lymph
nodes. Rubella spreads via the lymph nodes into the
blood, where it induces an immune response resulting in
lasting immunity. Viremia occurs 5 to 7 days after
exposure, with spread of the virus throughout the body
and the chance of transplacental infection of the fetus.
The virus can be detected in secretions from the
nasopharynx at approximately 4 days before to 4 days
following the onset of the typical rash associated with a
rubella infection.
Although relatively mild in most instances, rubella can
lead to serious complications for pregnant women,
especially during the first 4 months of gestation.
Intrauterine transmission of a primary rubella infection
can cause severe fetal damage, leading to miscarriage or
severe birth defects in the newborn. Up to 20% of infants
born to mothers infected during the first half of
pregnancy develop congenital rubella syndrome (CRS).
The most common congenital defects are cataracts, heart
disease, deafness, and mental retardation.
Clinical diagnosis of rubella infection is difficult; only
half of illnesses diagnosed clinically as acute rubella
infection are actually found to be caused by the rubella
virus. Since rubella-like illnesses can be induced by
other viruses that have no teratogenic potential,
laboratory differentiation of rubella from other rash-
causing infections (e.g., measles, parvovirus B19, human
herpes virus 6, enteroviruses in developed countries, and
various endemic arbovirus) is essential [1]. Accurate
laboratory diagnosis of past or recent rubella infection is
vital for counseling a pregnant patient, as well as for
i
Rubella
Previous and current authors of this method:
First edition: Not done
Methods edition: Not done
Second edition: Not done
Third edition: Not done
Fourth edition: Steven C. Kazmierczak
Fifth edition: Terry Pry, Greg Maine
epidemiological reasons. As such, in many countries,
determining a patients immune status to rubella IgG is
part of the routine antenatal screening panel.
The laboratory diagnosis of rubella infection became
possible following the isolation of the virus in cell
cultures in 1962 [2]. Rubella virus, a Togavirus, is an
enveloped virus with a single-stranded RNA genome.
The structural proteins consist of capsid and
glycoproteins E1 and E2. Since viremia is usually
detectable only in the last week before the onset of rash,
virus isolation techniques are not suited for routine
diagnosis because of the short period of viral excretion
into the blood stream.
The diagnostic laboratory tests developed for the
assessment of rubella infection are based on a number of
serological methods discussed below. Figure 1 shows the
typical relation between the virological and
immunological features of infection. The appearance of
IgM antibody to rubella virus is seen very early
following the onset of the illness. IgM typically reaches
peak concentrations within 7 to 10 days after infection
and then gradually disappears within 4 to 5 weeks. The
measurement of rubella IgM is primarily used for the
assessment of primary rubella infection in pregnant
women. In contrast to IgM, IgG antibody continues to
increase for several months following the onset of rash,
and elevated levels of serum antirubella IgG may persist
for decades. The avidity of rubella IgG antibody is low
following primary antigenic stimulus but matures rapidly
to high avidity within 2 to 3 months. Hence, the clinical
utility of the rubella IgG avidity assay is limited and
must be used in the context of other rubella serological
testing described below to distinguish between a primary
and non-primary rubella infection.
Neutralization Antibody Test
The isolation of the rubella virus gave rise to tests based
on the ability of viral-specific antibodies to neutralize
the infectivity of the virus (Table 1, Method 1).
Neutralization is used to identify both virus isolates and
immune response to the virus. Neutralizing antibodies,
1036
Rubella
which may be of the IgG or IgM class, appear in serum
approximately 1 to 2 days following the onset of rash
and reach peak titers usually within 3 to 4 weeks.
Rubella IgM antibody is rarely detectable for longer than
4 to 5 weeks following an acute illness. This test is
rarely used today because it is too time consuming to
perform and is a labor-intensive procedure.
Complement Fixation Test
Like the neutralization procedure, the complement
fixation test is rarely used today. This test is based upon
the fixation of complement added to antigen-antibody
complexes (Table 1, Method 2). Visualization of the
complement fixation reaction is mediated through the
use of sensitized erythrocytes. If a specimen contains
rubella-specific antibodies, complement will attach to
the antigen-antibody complexes and be unavailable for
lysis of the sensitized erythrocytes. Rubella-specific
complement-fixation antibodies generally appear in
serum within 2 to 3 weeks following infection and may
remain detectable for 2 to 5 years after the infection.
The complement fixation test is relatively insensitive and
cannot be used as a single test for the diagnosis of a
rubella infection. Test results are dependent upon the
quality of the antigen used in the procedure, particularly
the proportions of soluble and virion components [3].
Both the neutralization and complement fixation
procedures have been replaced by the hemagglutination
inhibition (HI) test (see below).
Hemagglutination Inhibition (HI) Test
The HI test was the first technique that was widely
available for the detection of antibodies to rubella. This
test is based on the ability of rubella virus to agglutinate
avian erythrocytes (Table 1, Method 3) [4]. Inhibition of
agglutination by the binding of specific antirubella
antibodies to the agglutinin is utilized in this test.
Following infection with rubella, hemagglutination-
inhibition antibodies increase very rapidly, with
maximum titers occurring approximately 8 days
following the onset of rash. The hemagglutination
inhibition test detects all classes of antibody and
therefore can be used for the selective detection of IgM-
class specific antibodies.
The HI test demonstrates excellent correlation with the
neutralization procedure but is simpler to perform and
more sensitive. In addition, the HI procedure is able to
detect all classes of antibody and does not require live
virus, instead utilizing antigen that can be stored
lyophilized. A titer of 1:8 is considered negative, and a
titer of > 1:32 suggests an earlier infection or successful
vaccination [5]. The test, however, does have drawbacks.
The presence of nonspecific inhibitors, particularly low-
density beta-lipoproteins and agglutinins, can interfere
with the assay, and these must be removed prior to
analysis. Kaolin, heparinmanganous chloride, dextran,
and red blood cells have been utilized for this purpose.
Passive Hemagglutination Test
The passive hemagglutination test is based on the
agglutination of erythrocytes that have been coated with
rubella virus antigen (Table 1, Method 4). This test is
most useful for detecting serological evidence of past
rubella infection (IgG antibodies) and is thus suited for
screening of immunity to rubella.
Hemolysis-in-Gel Test
The single radial hemolysis, or the hemolysis-in-gel test,
has been used as a simple and sensitive method for
rubella screening (Table 1, Method 5). The test is based
on the diffusion of antibodies through an agarose gel
plate containing erythrocytes coated with rubella
antigen. Complement is included within the gel or may
be layered on top of the gel. Patient serum is added to
wells punched into the gel. Complement-mediated lysis
of erythrocytes occurs if the serum diffusing through the
gel contains antibodies to rubella. Following an
overnight incubation, the diameter of the hemolytic
zones is measured. The area of the hemolyzed zone is
proportional to the concentration of antibody within the
sample.
Although this test is simple to perform and requires no
special equipment, the shelf life of reagents is limited to
a few weeks. Some sera have been found to give atypical
hemolysis zones. The presence of a nonhemolytic area in
the center of the zone, called a halo, has been attributed
to rheumatoid factor or some other factors [6]. In
addition, some acute-phase sera contain a blocking
factor which, when present in high concentration,
completely blocks hemolysis.
Enzyme Immunoassays
Rubella-IgG Assay: Most assay formats are
indirect, in which rubella antigen (peptide, recombinant,
or whole-virus antigen) is attached to the solid phase
(Table 1, Method 6). The sensitivity and specificity of
the assay have been shown to be affected by the source
of antigen. The typical test sample is maternal or fetal
blood. The limitation with this assay is that it cannot
distinguish between active infection and immunity.
Ideally, rubella IgG serology should be performed in
women prior to pregnancy to verify rubella immunity. If
the patient is nonimmune, the rubella vaccine should be
administered and the test repeated to verify the
development of humoral immunity to the rubella virus.
Rubella-IgM Assay: The development of
sensitive and specific enzyme-linked immunoassay
(ELISA) techniques for IgM, which is present in serum
for a limited period following infection, has been a
major advancement in the diagnosis of rubella in early
pregnancy (Table 1, Method 7). ELISA tests are simple
to perform, and many have been automated. Two assay
formats are described: (1) indirect ELISA and (2) IgM-
capture ELISA. In indirect ELISA, viral antigen is
bound to the solid phase, and a labeled antihuman IgM is
used as the conjugate. These assays now generally use a
pretreatment step that includes an absorbent reagent to
remove rubella IgG from the sample. In IgM-capture
ELISA, antihuman IgM antibodies are attached to the
solid phase, and a conjugate of rubella virus antigen and
labeled antirubella virus antibody is used for detection.
Although, IgM capture ELISA assays generally
1037
Rubella
demonstrate reduced non-specific assay interference,
interference is still observed and must be considered [7].
The increased sensitivity of recent rubella IgM assays
can lead to the detection of nonspecific IgM. Since the
incidence of rubella has diminished in countries with
high rubella vaccination coverage, the proportion of
nonspecific rubella IgM reactivity among all samples
with rubella IgM reactivity has increased. Nonspecific
false positives are possible due to (1) other IgM
antibodies which cross-react arising in patients infected
with Epstein-Barr virus, cytomegalovirus, and human
parvovirus B19, and (2) rheumatoid factor resulting from
patients with autoimmune diseases such as rheumatoid
arthritis and systemic lupus erythematous [5]. Therefore,
rheumatoid factor, parvovirus IgM, and heterophile tests
should be done if a false-positive result is suspected. In
addition, low levels of IgM may not be indicative of
primary infection when sensitive tests are used, because
IgM antibody has been shown to persist for much greater
than the typical 8 to 12 weeks [8]. A rubella IgM-
positive result alone does not accurately predict the risk
of fetal infection and should be followed by other rubella
serological tests described below prior to prenatal
diagnosis.
Rubella-IgG Avidity Assay: The rubella IgG avidity
assay measures the functional binding affinity (Avidity
Index) of the IgG class of antibody in response to
infection (Table 1, Method 8). Avidity is a measure of
the overall strength of binding of an antigen with many
antigenic determinants and multivalent antibodies,
whereas affinity refers to the strength of binding between
a single antigenic determinant and an individual
antibody combining site. The rubella IgG avidity test is
ordered when both IgG and IgM rubella antibodies are
detected in the mother. Following primary infection with
rubella virus, low-avidity IgG is produced first. As the
infection progresses, the affinity of the rubella IgG
increases with time, and the presence of high-avidity
rubella IgG can be used in conjunction with other
serological tests as a marker of past or recurrent
infection. The rubella-IgG avidity assay differentiates
between antibodies with high or low avidity to the
antigen and therefore helps to differentiate between
primary and non-primary infection. The assay measures
the functional binding affinity of the IgG class of
antibody in response to infection.
In avidity assays, the patients IgG is allowed to bind to
its antigen, followed by elution with or without a protein
denaturant, such as urea or diethylamine. The protein
denaturant dissociates weakly bound antibodies from
their specific antigens, but strongly bound antigens
remain intact. Thus the proportion of IgG remaining
antigen-bound indicates the relative amount of high-
affinity IgG. The avidity index (AI) is defined as the
ratio of (high avidity rubella IgG present)/(total rubella
IgG titer) 100. The cutoff point differentiates the low
avidity index and the high avidity index. Different values
of the low avidity index cutoff points have been
reported, ranging from < 30% to 55% [9-12]. The
patients serum rubella IgG levels should be above 29 IU
to provide a reliable AI result [13].
Latex Agglutination
The latex agglutination procedure for antibodies to
rubella is often used for the semiquantitative
determination of immune status. The procedure is
performed by mixing latex particles, previously coated
with rubella virus antigen, with untreated serum on a
Teflon-coated card (Table 1, Method 9). Once added, the
mixture is mechanically rotated for a short period, and
the results read macroscopically. The test is considered
positive if agglutination of the latex particles is observed
following the mixing period. Advantages of the latex
agglutination procedure include a rapid assay time, no
need for specialized or expensive equipment, and no
need for pretreatment of serum prior to analysis.
PCR
The use of reverse transcription followed by PCR
amplification has enabled the detection of rubella virus
RNA in tissue (Table 1, Method 10) [14]. This method is
typically used as a reflex to test for fetal infection
following detection of a maternal primary infection to
increase the reliability of the diagnosis [15]. Studies
show detection of rubella RNA by PCR has a sensitivity
of 83% to 100% and 100% specificity [16,17]. A rubella
IgM EIA recombinant assay uses a recombinant rubella
virus-like particle and is based upon the IgM capture
principle.
Reference and Preferred Methods
The hemagglutination inhibition test is generally
considered the gold-standard test against which other
rubella tests are measured. The use of ELISA has
become the predominant method of analysis for both
IgG and IgM antibody to rubella, because they are
easier, more rapid, and more sensitive than earlier
neutralization and HI tests. A number of compounds
have been employed as substrates, including those
whose products are measured spectrophotometrically, by
fluorescence, or by chemiluminescence. This technique
is the most common in use for the determination of
recent infection (IgM) and immune status (IgG).
Specimen
Serum or plasma for rubella serological testing should
ideally be collected at the onset of disease and 2 to 3
weeks later in order to determine acute and convalescent
titers. Serum can be stored at refrigerator temperatures
(4C to 8C) for 5 to 7 days and should be frozen if
testing is to be delayed. There is no significant
difference in antibody levels from blood collected by
fingerstick versus venipuncture [18]. Adaptation of
rubella IgM immunoassay using dried venous blood
spots has also been described [19]. Saliva also proved
useful as an alternative, less invasive sample [20].
Interferences
The potential for assay interference varies by assay
method. As described earlier, potential interferents
include rheumatoid factor, human anti-mouse antibodies,
1038
Rubella
and cross-reacting IgM antibodies (Epstein-Barr, CMV,
parvovirus B19).
Rubella Reference Interval
IgM antibodies in response to a primary rubella infection
can be detected in the patient by a sensitive EIA within 5
to 10 days after the onset of the rash; they disappear
rapidly, usually within 7 to 10 weeks. In some
individuals, low levels of IgM may last for an extended
time, up to 1 year. IgM antibodies in response to
vaccination can be detected in the vaccinee between 3
and 12 weeks post-vaccination.
Interpretation
Rubella was first discovered in the early 18th century
and described as a relatively mild, self-limiting disease
with few complications. However, following a rubella
epidemic in 1941, an increased incidence of neonatal
cataract cases associated with deafness, cardiac
anomalies, and microcephaly was observed [21]. From
these initial observations, the medical community
recognized the teratogenic effects of the rubella virus.
These and other defects (including mental retardation
and low birth weight) are now collectively known as the
congenital rubella syndrome (CRS) [8].
Immunization programs in most developed countries
have essentially eradicated rubella infections. However,
rubella may reemerge if vaccination programs cannot be
adequately sustained. In undeveloped nations, outbreaks
among individuals not vaccinated against the virus are
still common. Between immunization and natural
infections, > 85% of people carry antibodies to the
rubella virus, and > 90% of these will have lifelong
immunity to the disease [22]. Primary rubella virus
infection occurring after birth is typically a mild, self-
limiting disease characterized by a maculopapular rash,
pyrexia (fever), tiredness, and lymphadenopathy.
Maternal infection acquired within the first trimester of
pregnancy can be a serious threat to the developing
fetus. If a pregnant woman is infected with rubella
during the first trimester, there is an approximately 90%
chance that the fetus will be infected, and the majority of
these will have congenital defects. If the fetus is exposed
to rubella between the 12th and 20th weeks of gestation,
the risk of fetal infection declines to approximately 50%,
with about 30% of infants suffering complications.
Rubella infections that occur after 20 weeks of gestation
are not normally associated with any complications.
An algorithm for assessment of rubella infection in
pregnancy is shown in Figure 2. Screening pregnant
women for the presence of antirubella IgG antibodies
typically is best performed when a women plans to
become pregnant, or at the first prenatal visit to her
physician. If the antirubella IgG is detected, the woman
has immune status. If there are no detectable antibodies,
a nonpregnant woman is usually offered vaccination but
should avoid pregnancy for 3 months.
Protection and immunity is considered with antibody
levels of 10 to 15 international units (IU) of IgG per mL
[23]. Specimens taken very early in the acute phase of
infection may not contain any detectable amounts of
rubella IgG antibodies or may have antibody levels less
than 10 IU/mL. Moreover, antibody levels after
vaccination may drop under the threshold of 10 IU/mL.
Rubella Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report indicate that
approximately 67% of laboratories (n = 626) reporting
quantitative results used a chemiluminescence
immunoassay, and 24% used an enzyme immunoassay
to measure rubella antibody. Imprecision values (%
coefficient of variation) for quantitative measurement of
rubella antibody was generally less than 15%. Criteria
for measurement of rubella antibody require that
laboratories achieve 90% participant consensus of
qualitative results (negative or positive), and acceptable
performance was achieved across all assay methods.
References
1 Banatvala J, Best J, OShea S, Dudgeon J.
Persistence of rubella antibodies after
vaccination: detection after experimental
challenge. Rev Infect Dis. 1985;7(suppl.1):S86-
90.
2 Weller TH, Neva FA. Propagation in tissue
culture of cytopathic agents from patients with
rubella-like illness. Proc Soc Exp Biol Med.
1962;111:215-225.
3 Cradock-Watson JE. Laboratory diagnosis of
rubella: past, present and future. Epidemiol
Infect. 1991;107:1-15.
4 Stewart G, Parkman P, Hopps H, Douglas R,
Hamilton J, Meyer H. Rubella-virus
hemagglutination-inhibition tests. N Engl J
Med. 1967;276:554-7.
5 Mendelson E, Aboudy Y, Smetana Z,
Tepperberg M, Grossman Z. Laboratory
assessment and diagnosis of congenital viral
infections: Rubella, cytomegalovirus (CMV),
varicella-zoster virus (VZV), herpes simplex
virus (HSV), parvovirus B19 and human
immunodeficiency virus (HIV). Reprod
Toxicol. 2006;21:350-382.
6 Hedman K, Salonen EM, Keski-Oja J, and
Rih K. Single-serum radial hemolysis to
detect recent rubella virus infection. J Infect
Dis. 1986;154:1018-1023.
7 Tipples G, Hamkar R, Mohktari-Azid T, Gray
M, Ball J, Head C et al. Evaluation of rubella
IgM enzyme immunoassays. J Clin Virol.
2004;30:233-238.
8 Banatvala J, Brown D. Rubella. Lancet.
2004;363:1127-37.
9 Bottinger B, Jensen P. Maturation of rubella
IgG avidity over time after acute rubella
infection. Clin Diagn Virol. 1997;8:105-111.
10 Thomas H, Charlett A, Cubie H. Specific IgG1
avidity maturation after rubella vaccination; a
comparison with avidity maturation after
primary infection with wild rubella virus.
Serodiag Immunother Infect Dis. 1995;7:75-80.
1039
Rubella
11 Guttierrez J, Rodriguez M, Ory F, Piedrola G,
Maroto M. Reliability of low-avidity IgG and
of IgA in the diagnosis of primary infection by
rubella virus with adaptation of a commercial
test. J Clin Lab Anal. 1999;13:1-4.
12 Mubareka S, Richards H, Gray M, Tipples G.
Evaluation of commercial rubella
immunoglobin G avidity assays. J Clin
Microbiol. 2007;45:231-233.
13 Enders G, Knotek F, Pacher U. Comparison of
various serological methods and diagnostic kits
for the detection of acute, recent, and previous
rubella infection, vaccination, and congenital
infections. J Med Virol. 1985;16:219-232.
14 Grangeot-Keros L, Enders G. Evaluation of a
new enzyme immunoassay based on
recombinant rubella virus-like particles for
detection of immunoglobulin M antibodies to
rubella virus. J Clin Microbiol. 1997;35:398-
401.
15 Tang J, Aarons E, Hesketh L, Strobel S,
Schalasta G, Jauniauz E. Prenatal diagnosis of
congenital rubella infection in the second
trimester of pregnancy. Prenat Diagn. 2003;
23:509-512.
16 Mace M, Cointe D, Six C, Levy-Bruhl D,
Parent du Chatelet I, Ingrand D et al.
Diagnostic value of reverse transcription-PCR
of amniotic fluid for prenatal diagnosis of
congenital rubella infection in pregnant women
with confirmed primary rubella infection. J Clin
Microbiol. 2004;42:4818-20.
17 Revello M, Baldanti F, Sarasini A, Zavattoni
M, Torsellini M, Gerna G. Prenatal diagnosis of
rubella virus by direct detection and
semiquantitation of viral RNA in clinical
samples by reverse transcription PCR. J Clin
Microbiol. 1997;35:708-13.
18 Pinsky N, Loepfe T, Jacobson R, Vierkant R,
Poland G. Comparison of fingerstick versus
venipuncture for antibody testing of measles
and rubella. Scand J Infect Dis. 2003;35:107-
109.
19 Karapangiotidis T, Riddell M, Kelly H.
Detection of rubella immunoglobin M from
dried venous blood spots using a commercial
enzyme immunoassay. Diagn Microbiol Infect
Dis. 2005;53:107-111.
20 Vijaylakshmi P, Muthukkaruppan V,
Rajassundari A, Korukluoglu G, Nigatu W,
Warrener L et al. Evaluation of a commercial
rubella IgM assay for use on oral fluid samples
for diagnosis and surveillance of congenital
rubella syndrome and postnatal rubella. J Clin
Virol. 2006;17:265-268.
21 Gregg NM. Congenital cataract following
German measles in the mother. Trans
Opthalmol Soc Aust. 1941;3:35-46.
22 Llewellyn-Jones D. Obstetrics and Gynecology.
St Louis: Mosby; 1994:135.
23 Skendzel L. Rubella immunity: defining the
level of protective antibody. Am J Clin Pathol.
1996;106:170-74.
1040
Rubella

Table 1: Methods for Rubella

Method 1: Neutralization test

Principle: Antibodies neutralize the infectivity of virus
Usage: Infrequently used
Comments: Detects IgG and IgM antibodies; laborious; performed only in reference labs
Method 2: Complement fixation test
Principle: Presence of rubella antibodies results in attachment of complement to AgAb complexes; complement
not available for lysis of hemolysin-sensitized cells
Usage: Infrequently used
Comments: Detects IgG-class antibodies only
Method 3: Hemagglutination inhibition test
Principle: Based on inhibition of erythrocyte agglutination by binding of antirubella antibodies to an agglutinin
Usage: Infrequently used
Comments: Detects all classes of antibody; interference by nonspecific inhibitors
Method 4: Passive hemagglutination test
Principle: Agglutination of erythrocytes coated with rubella virus antigen
Usage: Infrequently used
Comments: Detects evidence of past infection; useful for screening of rubella immunity
Method 5: Hemolysis-in-gel test
Principle: Antibody diffusion through agarose gel containing erythrocytes coated with rubella antigen;
complement-mediated cell lysis occurs if sera contains antibodies to rubella
Usage: Infrequently used
Comments: Limited reagent shelf life; some interferences noted
Method 6: Rubella IgG
Principle: ELISA: Detection of virus-specific IgG Ab bound to a solid phase by a labeled secondary anti-IgG
Ab.
Usage: Marker of past infection
Comments: Limitation of result: not conclusive of latent or active infection. Comparisons between
commercial IgG assays correlate well with each other, however; no international standard yet exists. Fast and
sensitive commercialized assays available. Detection typically via chemiluminescent or fluorescent substrate
Method 7: Rubella IgM
Principle: ELISA: Detection of virus-specific IgM Ab bound to a solid phase by a labeled secondary anti-IgM
Ab. Detection typically via chemiluminescent or fluorescent substrate.
Usage: Marker of active or recent rubella infection
Comments: IgM antibodies may persist for months or years; false positives a limitation; fast and sensitive
commercialized assays available
Method 8: Rubella IgG Avidity
Principle: ELISA: Two rubella IgG ELISA tests are performed on the same sample. After primary antibody
incubation, the solid phase of each assay is washed with either buffer (control) or a chaotropic reagent (urea,
diethylamine) to remove low-avidity antibodies. After another wash, the conjugate is added, and the signal is
measured. The avidity index is the ratio of (rubella IgG titer with chaotropic wash step/rubella IgG titer with
buffer wash step) 100, that is, the ratio of rubella high-avidity IgG over the total rubella IgG titer.
Usage: Differentiates between primary infection (low avidity IgG) and recurrent or persistent infection (high
avidity IgG).
Comments: Avidity testing should be performed early in gestation (within the first trimester). Fully
automated high-throughput analytical systems available with integrated avidity assays.
Method 9: Latex agglutination
Principle: Latex particles coated with virus antigen mixed with serum; presence of rubella antibody in test serum
results in agglutination of latex particles
Usage: Frequently used
Comments: Visual detection method; large inter-observer variation when Ab titers are near assay sensitivity
Method 10: PCR
Principle: RNA extraction, reverse transcription, amplification, and detection of amplified sequences using
fluorescent probes or dye in a specialized analyzer.
Usage: Detection of viral RNA in clinical samples
Comments: Not prone to contamination. Interpretation of very low result questionable. Samples can be
frozen for subsequent analysis by other labs.
1041
Rubella

Figure 1:

IgG / IgG Avidity

1042
Rubella

Figure 2: Algorithm for assessment of rubella infection in pregnancy:

1043
Salicylates

Salicylates
Gus Koerbin and Julia M. Potter
Name: Salicylate, acetylsalicylic acid, aspirin
Clinical significance: Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.
Acetylsalicylic acid Salicylic acid
Molecular formula: C9H8O4 C7H6O3
Molecular mass: 180.15 D 138.12 D
Chemical class: Benzoic acid derivative Hydroxybenzoic acid
Structure:

Acetylsalicylic acid Salicylic acid

Principles of Analysis and Current Usage

i plasma by extraction into acidified ethylene dichloride,
Acetylsalicylic acid (aspirin) is absorbed from the
stomach and rapidly hydrolyzed to salicylic acid
(salicylate) and acetic acid in the blood. Aspirin which
passes into the small intestine is hydrolyzed prior to
absorption or within the mucosa. Salicylate is rapidly
absorbed into the blood. There are a variety of methods
for determination of salicylate in serum or plasma. The
most widely used methods in the clinical laboratory
employ either colorimetry or fluorescence polarization
immunoassay, but other methods include high-
performance liquid chromatography (HPLC), gas
chromatography (GC), ultraviolet spectroscopy, and
fluorescence spectrophotometry.
Historically, the earliest method for measurement of
salicylates is the colorimetric method, which measures
the absorbance of the complex formed between
salicylates and ferric ions (Table 1, Method 1). The first
widely accepted method using this technique was
described by Brodie et al. [1] (Table 1, Method 1a) in
1944. In this procedure, salicylate is separated from
i
Salicylates
Previous and current authors of this method:
First edition: Joseph Svirbely
Methods edition: Joseph Svirbely
Second edition: Joseph Svirbely
Third edition: Ross L.G. Norris, Donald Davis, Julia
M. Potter
Fourth edition: Not updated
Fifth edition: Gus Koerbin, Julia M. Potter
followed by back extraction into an aqueous phase as the
ferric complex, which is then measured at 540nm. In
1948, Keller published a modification of this method [2]
which eliminated the extraction steps (Table 1, Method
1b), and total salicylate was measured as the sum of
acetylsalicylate and its metabolites. In 1950, Trinder [3]
published the well-known protein precipitation method
using mercuric chloride and hydrochloric acid (Table 1,
Method 1c). Color is produced when salicylate is
complexed with ferric ion supplied as ferric nitrate.
A second type of colorimetric method involves reducing
salicylate with a mixture of phosphotungstic and
phosphomolybdic acids (Folin-Ciocalteu reagent) (Table
1, Method 2) [4]. Weichselbaum and Shapiro [5]
published a method in which serum was mixed with this
reagent, and the resulting blue color was read at 660 nm.
In 1950, Smith and Talbot [6] published a similar
method but shortened the color development step from
40 min to 3 min.
Ultraviolet spectrophotometry has also been employed
as an analytical technique to measure acetylsalicylate
plus salicylate (Table 1, Method 3). Routh et al. [7]
extracted the acetylsalicylate using the Brodie procedure
and quantified the drugs using differential
spectrophotometry by measuring the differences in
absorbance at pH 9 and pH 13.5.
Spectrofluorometry has also been used for the
measurement of salicylates (Table 1, Method 4). In
1948, Saltzman [8] published a spectrofluorometric
method in which the salicylate was separated from
1044
Salicylates
protein by precipitation with dilute tungstic acid. Strong
alkali was added to convert the salicylate into a more
fluorescent form. The ion was excited at 370 nm, and
fluorescent emission was monitored at 460 nm. Potter
and Guy [9] have published a micromethod for plasma
that allows determination of protein-bound salicylate,
free salicylate, acetylsalicylate, and total salicylate in
serum. Sephadex gel columns were used to separate the
protein-bound salicylate from the free forms. Salicylate
was measured by spectrofluorometry, with excitation at
305 nm and emission at 405 nm.
In another fluorometric assay, Lever and Powell [10] in
1973 described an enhancement and alteration of the
salicylate fluorescence by the addition of magnesium
acetate tetrahydrate and sodium barbitone to the serum.
Fluorescence is measured with excitation at 340 nm and
emission at 390 nm.
The fluorescence polarization immunoassay (FPIA)
(Table 1, Method 5) assay for salicylates is one of the
most frequently used methods. A change in the
polarization of fluorescent radiation emitted by the
fluorescein-labeled tracer is measured.
You and Bittikofer [11] reported an enzymatic assay that
has good precision at low salicylate levels (less than 10
mg/dL). The assay utilizes salicylate hydroxylase
derived from Pseudomonas cepacia (Table 1, Method 7).
Salicylate hydroxylase catalyzes the stoichiometric,
unidirectional conversion of salicylate and NAD(P)H to
catechol and NAD(P) in the presence of molecular
oxygen. The change in absorbance at 340 nm is
monitored and compared to a standard. A more specific
modification of this method, which is claimed to be
adaptable to most discrete analyzers, was reported by
Morris [12]. The improved specificity is gained by
monitoring the production of catechol rather than the
consumption of NADH. Several metabolites and analogs
of salicylate which interfere in the former assay because
they also consume NADH do not interfere in this
modification in that they do not produce catechol.
Gas chromatography has been used for measuring
salicylates, since acetylsalicylate and its metabolites can
be separated and quantitated independently (Table 1,
Method 6). GC procedures [13,14] incorporate a
sialylation step after extraction of the salicylates from
the biological sample. In the method of Thomas et al.
[13], plasma is extracted into ether along with p-toluic
acid (the internal standard). Following evaporation of the
ether, silylation is achieved by heating in the presence of
bis (trimethylsilyl) trifluoroacetamide (BSTFA).
Separation on the GC is achieved with a 5% OV-17
column run isothermally at a temperature of 150C, and
detection is with a flame ionization detector (FID).
Acetylsalicylate and salicylate are both quantitated
separately. Rance et al. [14] used a similar procedure,
but by using temperature programming, they were able
to measure salicylamide in addition to salicylate and
acetylsalicylate.
HPLC has been applied successfully to the quantitation
of salicylate (Table 1, Method 8). OKruk et al. [15]
have described a method which enables the simultaneous
determination of acetylsalicylic acid and its metabolites
on 0.2 mL of plasma or serum. Sample preparation is a
relatively simple protein precipitation step using
perchloric acid. Chromatography utilizes a reversed-
phase column with a mobile phase of methanol in
aqueous buffer. The eluent is monitored with a UV
detector at 235 nm. Limit of detection was quoted as
being 0.16 mg/dL, which is adequate for most clinical
laboratory applications. The HPLC method of Mays et
al. [16] has an improved limit of detection, which may
be an advantage in pharmacokinetic studies where low-
dose salicylates are used. However, the more complex
sample preparation requiring 1 mL of plasma offsets this
advantage for most clinical laboratory applications. The
method of Davis [17] utilizes a simple trichloroacetic
acid precipitation of proteins from 0.2 mL of plasma,
and reverse-phase chromatography and use of
fluorescence detection avoids potential theophylline
interference as reported by Miceli et al. [18].
Reference and Preferred Methods
There is no reference method for salicylate
measurement. The simplest method for measuring
salicylates on a routine basis is the spectrophotometric
method of Keller, which yields a measure of total
salicylate and its metabolites. The method of Brodie et
al. requires time-consuming extraction steps and
involves the use of a toxic solvent, ethylene dichloride.
Recovery with the Brodie method is less than 90%
because of the double extraction. Likewise, Trinders
method offers no advantages over Kellers method and
also makes use of toxic mercuric ion.
Ultraviolet spectrophotometry measures salicylate and
acetylsalicylate and has a sensitivity of 0.5 mg/dL.
However, because of its lack of specificity, it offers no
advantages over colorimetric procedures. The main
advantage of spectrofluorometric methods is that they
have better sensitivity than the colorimetric methods.
These methods do not measure acetylsalicylate directly,
since acetylsalicylate does not fluoresce. Instead they
derive the value indirectly by subtracting free salicylate
from the total salicylate measured after the sample is
hydrolyzed.
HPLC is the method of choice for separating and
quantitating acetylsalicylate and its major metabolites.
Its use is recommended in studies requiring
discrimination between these compounds. HPLC, in
common with GC, has good sensitivity and specificity,
but because no derivatization is required, it has the
advantage of more rapid and simple sample preparation.
Another benefit of both GC and HPLC is that large
numbers of samples can be processed simultaneously,
provided that the instrument is equipped with an
automatic injector. Because of economies of scale,
substantial cost benefits can be attained using automated
HPLC or GC where large numbers of samples are to be
assayed.
Spectrophotometric assays remain the overall methods
of choice because they are relatively free from
1045
Salicylates
interference, have adequate accuracy and precision, and
are inexpensive and readily available to all laboratories.
However, where equipment and expertise is available,
HPLC is to be preferred.
Clearly the final decision on the most appropriate
method needs to be tailored for the individual laboratory.
Factors to be considered can be broken down into
technical and nontechnical factors. Technical factors
include accuracy, specificity, sensitivity and precision.
These need to be considered within the anticipated range
of concentrations to be assayed. Nontechnical factors
include cost, numbers of samples to be processed, and
availability of expertise (i.e., if more technically
demanding assays such as HPLC are to be used).
Specimen
Either serum or plasma are suitable samples for analysis.
There is no known interference by commonly used
anticoagulants. Most methods can be readily adapted for
urine specimens.
Interferences
A major shortcoming of methods using the Folin-
Ciocalteu reagent is the false-positive color produced by
reaction with tyrosine, tryptophan, and uric acid. A
report by Duffens et al. [19] suggests that diflunisal, a
difluorophenyl analog of salicylic acid, interferes in both
the colorimetric and FPIA assays. They review other
reports, showing cross-reactivities of greater than 200%
with the FPIA assay and greater than 50% for the
Trinder method. As diflunisal fluoresces, it would be
expected to interfere in the fluorometric assays also.
Sarma et al. [20] have shown that HPLC is not subject to
diflunisal interference.
Salicylates Therapeutic Range
Analgesia Low dose 210 mg/dL (0.14 - 0.72mmol/L)
Anti-inflammatory High dose 530 mg/dL (0.36 2.16mmol/L)
Salicylism >20 mg/dL (>1.44mmol/L)
Marked toxicity >30 mg/dL (>2.16mmol/L)
Interpretation
Aspirin and nonacetylated salicylates (e.g., sodium
salicylate) are available in numerous pharmaceutical
formulations, many of which have been developed to
limit gastric irritation, microulceration, and the
gastrointestinal intolerance and blood loss which may
ensue. In general, the formulations (enteric-coated and
slow-release) are designed to encourage passage of the
drug from the stomach into the small intestine, from
where absorption then occurs. These changes alter the
pharmacokinetic profiles, particularly of the absorptive
phase. Nonionized aspirin is absorbed from the stomach
and then undergoes rapid hydrolysis within plasma
(Table 2). Hydrolysis is also rapid within the intestine
and its mucosa. The compound benorilate is an ester of
salicylic acid and acetaminophen (paracetamol) which
undergoes hydrolysis following absorption, releasing
both pharmacologically active moieties.
The pharmacokinetics of salicylate are dose dependent
(Table 2), and hepatic clearance is saturable. The major
metabolites are the glucuronide conjugate and salicyluric
and gentisic acids. Further, the drug is protein bound,
and both plasma protein binding and clearance alter over
the range of standard therapeutic doses. With saturation
kinetics and changing protein binding, accumulation of
salicylate is not uncommon, and the syndrome of
salicylism occurs (including tinnitus, nausea,
hyperventilation, and confusion). With increasing
concentrations and in overdose (either chronic or acute),
there will also be respiratory alkalosis, metabolic
acidosis, vomiting, hyperthermia, coma, or seizures,
among other signs and symptoms.
Although at usual therapeutic doses and urinary pH only
a small percentage of salicylate (<5%) is cleared by the
kidney, renal excretion can be used to advantage in the
treatment of overdose. Salicylate is a weak acid (pKa 3),
and if the pH of urine is made alkaline, the excretion of
salicylic acid can be increased significantly by
preventing the back-diffusion of ionized salicylate into
peritubular capillaries. In addition to alkalinization of
urine in this circumstance, gastric lavage should be
carried out and activated charcoal used. The further
therapy of forced diuresis is not recommended or
considered necessary, since the additional fluid load
carries the risk of pulmonary edema and further
complicates the already complex systemic acid-base and
disturbed electrolyte balances. In cases of severe
toxicity, such as that with signs of cerebral involvement
(seizure, coma, etc.) and/or hyperpyrexia, hemodialysis
should be considered. This allows redress of the acid-
base and electrolyte problems, as well as removal of
salicylate.
Salicylates Performance Goals
Survey data from the 2007 College of American
Pathologists Participant Summary Report shows
imprecision values (% coefficient of variation) for
measurement of salicylates range from 1.8% to 7.0% at
concentrations of approximately 60 mg/dL for assays
with > 20 participants. Acceptable performance criteria
(Clinical Laboratory Improvement Amendments 88) for
measurement of salicylate requires that laboratories be
accurate to within 3.0 SD or 10% of the peer-group
mean, whichever is greater.
References
1 Brodie BB, Udenfriend S, Coburn AF. The
determination of salicylic acid in plasma. J
Pharmacol 1944;80:114-7.
2 Keller W. A rapid method for the determination
of salicylates in serum or plasma. Am J Clin
Pathol 1947;17:415-7.
1046
Salicylates
3 Trinder P. Rapid determination of salicylate in
biological fluid. Biochem J 1954;57:301-3.
4 Folin O, Ciocalteu V. On tyrosine and
tryptophan determinations in proteins. J Biol 14
Chem 1927;73:627-50.
5 Weichselbaum TE, Shapiro I. A rapid and
simple method for the determination of salicylic
acid in small amounts in blood plasma. Am J
Clin Pathol 1945;9:42-4. 15
6 Smith MJ, Talbot JM. The estimation of plasma
salicylate levels. Br J Exp Pathol 1950;31:65-
69.
7 Routh JI, Shane NA, Arredondo EG, Paul WD.
Method for the determination of acetylsalicylic 16
acid in the blood. Clin Chem 1967;13:734-43.
8 Saltzmann . Fluorophotometric method for the
estimation of salicylate in blood. J Biol Chem
1948;174:399-404.
9 Potter GD, Guy J. A micromethod for the
analysis of plasma salicylate. Proc Soc Exp Biol 17
Med 1964;116:658-60.
10 Lever M, Powell JC. Simplified fluorimetric
determination of salicylate. Biochem Med 18
1973;7:203-7.
11 You K, Bittikofer JA. Quantification of
salicylate in serum by use of salicylate
hydroxylase. Clin Chem 1984;30:1549-51. 19
12 Morris HC, Overton PD, Ramsey JR, Campbell
RS, Hammond PM, Atkinson T, Price CP.
Development and validation of an automated,
enzyme-mediated colorimetric assay of 20
salicylate in serum. Clin Chem 1990;36:131-5.
13 Thomas BJ, Solomonraj G, Coldwell BB. The
estimation of acetylsalicylic acid and salicylate
in biological fluids by gas-liquid
chromatography. J Pharm Pharmacol
1973;25:201-4.
Rance ML, Jordan BI, Nichols JD. A
simultaneous determination of acetylsalicylic
acid, salicylic acid and salicylamide in plasma
by gas-liquid chromatography. J Pharm
Pharmacol 1975;27:425-9.
OKruk RJ, Adams MA, Philp RB. Rapid and
sensitive determination of acetylsalicylic acid
and its metabolites using reversed-phase high-
performance liquid chromatography. J
Chromatogr 1984;310:343-52.
Mays DC, Sharp DE, Beach CA, Kershaw RA,
Bianchine JR, Gerber N. Improved method for
the determination of aspirin and its metabolites
in biological fluids by high-performance liquid
chromatography: application to human and
animal studies. J Chromatogr 1984;311:301-9.
Davis D. Method of salicylate quantitation
utilizing trichloroacetic acid and reverse-phase
chromatography. Unpublished data.
Miceli JN, Aravind MK, Cohen SN, Done AK.
Simultaneous measurements of acetaminophen
and salicylate in plasma by liquid
chromatography. Clin Chem 1979;25:1002-4.
Duffens KR, Smilkstein MJ, Bessen HA,
Rumack BH. Falsely elevated salicylate levels
due to diflunisal overdose. J Emerg Med
1987;5:499-503.
Sarma L, Wong SH, Dellafera S. Diflunisal
significantly interferes with salicylate
measurements by FPIA-TDx and UV-VIS aca
methods. Clin Chem 1985;31:1922-3.
1047
Salicylates

Tables
Table 1: Salicylates Methods Summary Table
Method 1: Ferric nitrate complex; spectrophotometric
Principle of analysis: Salicylates + Fe3+ Ferric complex (Amax 540 nm)
a. Extracts salicylate into ethylene dichloride
b. Eliminates extraction step
c. Deproteinates with mercuric chloride
Comments: Most commonly used clinical salicylate procedure; sensitive to 20 mg/dL; linear to 100
mg/dL; only interference is salicylate metabolites
a. Method 1a uses toxic ethylene dichloride
b. Method 1b less complicated but specific enough for clinical needs
c. Method 1c uses toxic mercuric chloride
Method 2: Folin-Ciocalteu reagent reduction; spectrophotometric
Principle of analysis: Phosphotungstic acid + phosphomolybdic acid reduce salicylates to form blue
complex (Amax 660 nm)
Comments: Not commonly used because of interferences; tryptophan, tyrosine, and uric acid react
with reagent to give falsely high readings
Method 3: Spectrophotometry; differential ultraviolet spectrophotometric
Principle of analysis: Measures salicylates + acetylsalicylate by differences in absorbance at pH 9.0
and pH 13.5
Comments: Not commonly used; has advantage of measuring acetylsalicylate; requires narrow
bandpass ultraviolet spectrophotometer; sensitive to 0.5 mg/dL
Method 4: Fluorometry; spectrofluorometric
Principle of analysis: Protein can be removed by precipitation; alkali intensifies fluorescence of
salicylate
Comments: Not commonly used; has advantage of greater sensitivity; requires smaller sample than
spectrophotometry
Method 5: Fluorescence polarization immunoassay; polarization immunoassay
Principle of analysis: Drug in sample competes with fluorescent-labeled drug for antibody sites;
change in fluorescent radiation is monitored
Comments: Widely used; commercially available and convenient; fewer interferences than
colorimetric methods but diflunisal a problem
Method 6: Gas chromatography; chromatographic separation
Principle of analysis: Acetylsalicylate and major metabolites extracted with ether; derivation with
BSTFA (see text); analysis with gas chromatograph using temperature programming and OV-17
column monitored by flame ionization detector
Comments: Used in research; reference method; sensitive to 0.1 mg/dL; linear to 200 mg/dL; no
interfering compounds
Method 7: Enzymatic; spectrophotometric
Principle of analysis: Salicylate hydroxylate converts salicylate to catechol with the oxidation of
NADPH to NADP; reaction rate, monitored by measurement of decrease in A340, is proportional to
salicylate concentration
Comments: Increasing in use since commercially available; highly specific
Method 8: High-performance liquid chromatography; chromatographic separation
Principle of analysis: Plasma or serum cleaned up by a protein precipitation; salicylate and/or
metabolites separated on reversed-phase column and quantitated by UV or fluorescence detection
Comments: Research; reference method some clinical labs use; sensitive to 0.1 mg/dL; linear to > 500
mg/dL; no interfering compounds; simpler sample preparation of GC
1048
Salicylates

Table 2: Pharmacokinetic Parameters of Aspirin and Salicylates

Compound: Aspirin
Dose:
Volume of distribution (L/kg): 0.15
Protein binding (%): 80 to 90
Half-life: 10 to 15 min
Comments: Protein binding < salicylate
Compound: Salicylate
Dose: 600 mg bolus
Volume of distribution (L/kg): 0.15
Protein binding (%): 80 to 90
Half-life: 2 to 4 hr
Comments: Protein binding decreases as salicylate concentration increases; also altered in uremia; NB
hypoalbuminemia
Compound: Salicylate
Dose: 4 g/day
Volume of distribution (L/kg): 0.15
Protein binding (%): 80 to 90
Half-life: 20 hr

*Whether given as aspirin or salicylate or molar equivalent dose of benorilate.

Figures

Figure 1: Absorbance characteristics of colorimetric salicylate procedure.

1, Ferric nitrate reagent versus water; 2, acetylsalicylate standard in ferric nitrate reagent
versus water; 3, acetylsalicylate standard in ferric nitrate reagent versus ferric nitrate reagent
blank.
1049
Salicylates

Figure 2: Example of standard curve for ferric nitrate assay for salicylic acid.
Absorbance at 540 nm versus concentration of salicylate (g/mL).

Figure 3: Examples of HPLC chromatograms for salicylate assay.

Chromatogram A is of blank plasma, and chromatogram B is of the same plasma spiked with
250 mg/L salicylate. Compound I is the internal standard, and compound II is salicylate.
1050
Salicylates

Figure 4: Typical standard curve for the assay of salicylate by HPLC.

This demonstrates linearity up to at least 600 mg/L. Regression analysis produced the
following equation: y = 0.040x + 0.0040, with a correlation coefficient of 0.999.

Procedure: Spectrophotometric Method

Assay
Principle Equipment: 10 nm bandpass spectrophotometer.
The following spectrophotometric method is based 1. Label six test tubes: standard blank,
on salicylates complexing with ferric ion, which is control, control blank unknown(s), and unknown(s)
supplied by ferric nitrate in dilute nitric acid. The blank.
resulting complex is water soluble, absorbs at 540 2. To the standard, standard blank, control,
nm (Figure 1), and is read against a serum blank and unknown tubes, add 3.6 mL of ferric nitrate
pipetted into dilute nitric acid. solution.
3. To unknown and control blank tubes, add
Specimen 3.6 mL of dilute nitric acid.
4. Measure 0.3 mL of standard into standard
Reagents and Materials
tube. Mix.
1. Ferric nitrate solution (6.7 g/L, 24.8
5. Measure 0.3 mL of control into control
mmol/L). Dissolve 2.5 g of Fe(NO3)39H2O in
and control blank tubes. Mix.
1.0 mL of concentrated nitric acid, and dilute to 6. Measure 0.3 mL of unknown into
250 mL with distilled water. Filter through unknown and unknown blank tubes. Mix.
Whatman No. 1 filter paper into a glass bottle. 7. Read the standard, control, and unknown
Stable for 1 year at room temperature. against their respective blanks in the
2. Dilute nitric acid (62 mmol/L). Dilute spectrophotometer at a wavelength of 540 nm.
1.0 mL of concentrated nitric acid to 250 mL with
distilled water. This is stable for 2 years at room Calculation
temperature. [Absorbanceunknown/Absorbancestandard] x 10 mg/dL
3. Stock salicylate standard (1 g/L, 7.24 = salicylate (mg/dL)
mmol/L). Dissolve 100 mg of salicylic acid in 100
mL of water. This is stable for 1 year at 4C to 8C. Notes
4. Working salicylate standard (10 mg/dL, 1. Because of the presence of iron in the
0.724 mmol/L). In a 10-mL volumetric flask, unknown serum or plasma sample, a low
dilute 1.0 mL of stock standard to 10 mL with absorbance reading corresponding to
distilled water. This is stable for 3 months at 4C to approximately 2 mg/dL or less should not be
8C. considered significant.
5. Control. A serum sample containing 2. The colorimetric reaction is linear from 0
known amounts of salicylate. to 100 mg/dL (Salicylates: Figure 2).
1051
Salicylates
3. Analysis of urine. This procedure can be
used for a urine specimen after a heating step to
remove volatile interferences (especially ketones).
Place a test tube containing 5 mL of centrifuged
urine in a 100C heating block or boiling water for
15 min. After cooling, use as a specimen in the
ferric nitrate procedure. Phenothiazine,
homogentisic acid, and thiocyanates will interfere
and cause false-positive results. Salicylate cannot
be confirmed in urine in the presence of these
compounds.
Procedure: High-Performance Liquid
Chromatography
Principle
In the HPLC procedure, the native fluorescence of
salicylate is used for quantitation after preliminary
sample cleanup by protein precipitation and
separation on a reversed-phase column.
Specimen
Reagents and Materials
1. Deionized water. 18 MOhm quality or
better.
2. Glacial acetic acid. Analytical reagent
(AR) grade.
3. Acetonitrile. HPLC grade.
4. Mobile phase. Mix 700 mL deionized
water, 50 mL glacial acetic acid, and 250 mL
acetonitrile. Filter through a 0.22-mm filter prior to
use. May be recycled (with mixing) for up to 1
week or 200 injections.
5. Stock salicylate standard (1.5 g/L, 10.9
mmol/L). Prepare a stock standard solution
containing 1.500 g salicylic acid in 1 L by
weighing 1.74 g of sodium salicylate and
transferring to a 1-L volumetric flask. Use
deionized water to dissolve the sodium salicylate,
and dilute to the mark.
6. Serum salicylate standard (30 mg/dL,
2.2 mmol/L). Prepare a 30 mg/dL serum standard
by mixing 10 mL of stock standard with 50 mL of
drug-free serum. This can be stored in 0.5-mL
aliquots at 18C for at least 12 months.
7. o-Methoxybenzoic acid internal
standard (200 mg/L, 1.31 mmol/L). Dissolve 20
mg o-methoxybenzoic acid in 100 mL of 5%
trichloroacetic acid. AR-grade trichloroacetic acid
from the Ajax Company produces the least
chromatographic baseline noise. This solution is
stable at 4C for at
Procedure: To respective 1-mL centrifuge tubes,
add 200 L of serum or plasma. Add 200 L of
internal standard solution, and vortex. Centrifuge at
high speed for 5 sec in a Microfuge (Beckman
Instruments). Decant the supernatant into clean
tubes, and inject 50-L volumes onto the HPLC
column.
Equipment: An HPLC system equipped with a
fluorometric detector and a radial compression
module for the column (Millipore-Waters).
Column: Millipore Waters Radial-PAK cartridge
(8 NV C 18 4 (NOVA-PAK) part #86342). Flow
rate: 2.0 mL/min. Excitation wavelength: 300 nm.
Emission wavelength: 370 nm.
Calculation
The retention time for salicylate is approximately
3.3 min for the internal standard and 5.0 min for
salicylate. Sample chromatograms are presented as
Salicylates: Figure 3.
Compute peak height ratio of salicylate versus
internal standard for the standard and unknowns:
Peak height ratio = Peak height of compound
Peak height internal standard
Calculate a standard curve by using the ratio for the
single standard and assuming a straight line passing
through the origin. The concentrations of unknown
samples can then be determined by interpolation
from this line.
Notes
1. Both between- and within-day coefficients
of variation for this method were less than
1.2% when assessed using spiked plasma
at concentrations of 0.75, 15, and 30
mg/dL salicylate.
2. Limit of sensitivity is 0.1 mg/dL (0.007
mmol/L), with linearity up to at least 60
mg/dL (4.32 mmol/L). Linearity is
demonstrated in Salicylate: Figure 4.
3. There are no known interfering
substances.
4. It is essential that internal controls at both
the high and low ends of the reporting
range be included in each batch of
samples assayed when single-point
calibration is used as recommended above.