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2nd Edition

HARRISONS
TM

HEMATOLOGY
and Oncology
Derived from Harrisons Principles of Internal Medicine, 18th Edition

Editors
Dan L. Longo, md Anthony S. Fauci, md
Professor of Medicine, Harvard Medical Chief, Laboratory of Immunoregulation;
School; Senior Physician, Brigham and Womens Hospital; Director, National Institute of Allergy and Infectious Diseases,
Deputy Editor, New England Journal of Medicine, Boston, National Institutes of Health, Bethesda, Maryland
Massachusetts

Dennis L. Kasper, md Stephen L. Hauser, md


William Ellery Channing Professor of Medicine, Robert A. Fishman Distinguished Professor and Chairman,
Professor of Microbiology and Molecular Genetics, Department of Neurology, University of California,
Harvard Medical School; Director, Channing Laboratory, San Francisco, San Francisco, California
Department of Medicine, Brigham and Womens Hospital,
Boston, Massachusetts

J. Larry Jameson, md, phd Joseph Loscalzo, md, phd


Robert G. Dunlop Professor of Medicine; Dean, Hersey Professor of the Theory and Practice of Medicine,
University of Pennsylvania School of Medicine; Harvard Medical School; Chairman, Department of Medicine;
Executive Vice-President of the University of Physician-in-Chief, Brigham and Womens Hospital,
Pennsylvania for the Health System, Philadelphia, Pennsylvania Boston, Massachusetts
2nd Edition

HARRISONS
TM

HEMATOLOGY
and Oncology

Dan L. Longo, md
Professor of Medicine, Harvard Medical School; Senior Physician, Brigham and
Womens Hospital; Deputy Editor, New England Journal of Medicine,
Boston, Massachusetts

ERRNVPHGLFRVRUJ
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Contents

Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 11 Aplastic Anemia, Myelodysplasia,


and Related Bone Marrow
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Failure Syndromes 127
Neal S. Young
Section I
12 Transfusion Biology and Therapy 142
The Cellular Basis of Jeffery S. Dzieczkowski, Kenneth C. Anderson
Hematopoiesis
1 Hematopoietic Stem Cells2 Section IV
David T. Scadden, Dan L. Longo Myeloproliferative Disorders
13 Polycythemia Vera and Other Myeloproliferative
Section II Diseases 152
Cardinal Manifestations of Jerry L. Spivak
Hematologic Disease
2 Anemia and Polycythemia 10 Section V
John W. Adamson, Dan L. Longo Hematologic Malignancies
3 Bleeding and Thrombosis 22 14 Acute and Chronic Myeloid Leukemia 164
Barbara Konkle Meir Wetzler, Guido Marcucci, Clara D. Bloomfield

4 Enlargement of Lymph Nodes and Spleen  32 15 Malignancies of Lymphoid Cells 182


Patrick H. Henry, Dan L. Longo Dan L. Longo

5 Disorders of Granulocytes and Monocytes  41 16 Less Common Hematologic Malignancies 205


Steven M. Holland, John I. Gallin Dan L. Longo

6 Atlas of Hematology and Analysis of Peripheral 17 Plasma Cell Disorders  214


Blood Smears  57 Nikhil C. Munshi, Dan L. Longo, Kenneth C. Anderson
Dan L. Longo
18 Amyloidosis  226
David C. Seldin, Martha Skinner
Section III
Anemias Section VI
7 Iron Deficiency and Other Hypoproliferative Disorders of Hemostasis
Anemias 70
19 Disorders of Platelets and Vessel Wall. . . . . . . . 236
John W. Adamson
Barbara Konkle
8 Disorders of Hemoglobin 80
20 Coagulation Disorders. . . . . . . . . . . . . . . . . . . 247
Edward J. Benz, Jr.
Valder R. Arruda, Katherine A. High
9 Megaloblastic Anemias 94
21 Arterial and Venous Thrombosis  260
A. Victor Hoffbrand
Jane E. Freeman, Joseph Loscalzo
10 Hemolytic Anemias and Anemia
22 Pulmonary Thromboembolism 267
Due to Acute Blood Loss 108
Samuel Z. Goldhaber
Lucio Luzzatto

v
vi Contents

23 Antiplatelet, Anticoagulant, and Fibrinolytic 38 Gastrointestinal Tract Cancer 501


Drugs 277 Robert J. Mayer
Jeffrey I. Weitz
39 Tumors of the Liver and Biliary Tree 518
Section VII Brian I. Carr
Biology of Cancer 40 Pancreatic Cancer 530
Irene Chong, David Cunningham
24 Cancer Genetics  300
Pat J. Morin, Jeffrey M. Trent, Francis S. Collins, 41 Bladder and Renal Cell Carcinomas 536
Bert Vogelstein Howard I. Scher, Robert J. Motzer
25 Cancer Cell Biology and Angiogenesis 312 42 Benign and Malignant Diseases
Dan L. Longo of the Prostate 544
Howard I. Scher
Section VIII
43 Testicular Cancer  558
Principles of Cancer Prevention Robert J. Motzer, George J. Bosl
and Treatment
44 Gynecologic Malignancies 565
26 Approach to the Patient with Cancer 334 Michael V. Seiden
Dan L. Longo
45 Soft Tissue and Bone Sarcomas
27 Prevention and Early Detection of Cancer  346 and Bone Metastases  574
Jennifer M. Croswell, Otis W. Brawley, Shreyaskumar R. Patel, Robert S. Benjamin
Barnett S. Kramer
46 Primary and Metastatic Tumors of the
28 Principles of Cancer Treatment  358 Nervous System  581
Edward A. Sausville, Dan L. Longo Lisa M. DeAngelis, Patrick Y. Wen
29 Infections in Patients with Cancer  389 47 Carcinoma of Unknown Primary 597
Robert Finberg Gauri R. Varadhachary, James L. Abbruzzese
30 Hematopoietic Cell Transplantation 404
Frederick R. Appelbaum Section X
31 Neoplasia During Pregnancy  414 Endocrine Neoplasia
Dan L. Longo
48 Thyroid Cancer  604
32 Palliative and End-of-Life Care  419 J. Larry Jameson, Anthony P. Weetman
Ezekiel J. Emanuel
49 Endocrine Tumors of the Gastrointestinal
Tract and Pancreas 612
Section IX Robert T. Jensen
Neoplastic Disorders
50 Multiple Endocrine Neoplasia  634
33 Cancer of the Skin 444 Camilo Jimenez Vasquez, Robert F. Gagel
Walter J. Urba, Carl V. Washington,
51 Pheochromocytoma and Adrenocortical
Hari Nadiminti
Carcinoma  643
34 Head and Neck Cancer 457 Hartmut P.H. Neumann, Wiebke Arlt,
Everett E. Vokes Dan L. Longo
35 Neoplasms of the Lung  462
Leora Horn, William Pao, David H. Johnson Section XI
36 Thymoma 485 Remote Effects of Cancer
Dan L. Longo 52 Paraneoplastic Syndromes: Endocrinologic and
37 Breast Cancer 488 Hematologic 656
Marc E. Lippman J. Larry Jameson, Dan L. Longo
Contents vii

53 Paraneoplastic Neurologic Syndromes 665 Appendix


Josep Dalmau, Myrna R. Rosenfeld Laboratory Values of Clinical Importance 699
Alexander Kratz, Michael A. Pesce, Robert C. Basner,
Andrew J. Einstein
Section XII
Oncologic Emergencies and Late Review and Self-Assessment 725
Effects Complications Charles Wiener,Cynthia D. Brown,
Anna R. Hemnes
54 Oncologic Emergencies 674
Rasim Gucalp, Janice Dutcher
Index 791
55 Late Consequences of Cancer
and Its Treatment  690
Carl E. Freter, Dan L. Longo
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CONTRIBUTORS

Numbers in brackets refer to the chapter(s) written or co-written by the contributor.


James L. Abbruzzese, MD Cynthia D. Brown, MD
Professor and Chair, Department of GI Medical Oncology; M.G. and Assistant Professor of Medicine, Division of Pulmonary and
Lillie Johnson Chair for Cancer Treatment and Research, University Critical Care Medicine, University of Virginia, Charlottesville,
of Texas, MD Anderson Cancer Center, Houston, Texas [47] Virginia [Review and Self-Assessment]

John W. Adamson, MD Brian I. Carr, MD, PhD, FRCP


Clinical Professor of Medicine, Department of Hematology/Oncology, Professor of Oncology and Hepatology, IRCCS De Bellis Medical
University of California, San Diego, San Diego, California [2, 7] Research Institute, Castellana Grotte, Italy [39]

Kenneth C. Anderson, MD Irene Chong, MRCP, FRCR


Kraft Family Professor of Medicine, Harvard Medical School; Chief, Clinical Research Fellow, Royal Marsden NHS Foundation Trust,
Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer London and Sutton, United Kingdom [40]
Institute, Boston, Massachusetts [12, 17]
Francis S. Collins, MD, PhD
Frederick R. Appelbaum, MD Director, National Institutes of Health, Bethesda, Maryland [24]
Director, Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, Washington [30] Jennifer M. Croswell, MD, MPH
Acting Director, Office of Medical Applications of Research,
Wiebke Arlt, MD, DSc, FRCP, FMedSci National Institutes of Health, Bethesda, Maryland [27]
Professor of Medicine, Centre for Endocrinology, Diabetes and
Metabolism, School of Clinical and Experimental Medicine, David Cunningham, MD, FRCP
University of Birmingham; Consultant Endocrinologist, University Professor of Cancer Medicine, Royal Marsden NHS Foundation
Hospital Birmingham, Birmingham, United Kingdom [51] Trust, London and Sutton, United Kingdom [40]

Valder R. Arruda, MD, PhD Josep Dalmau, MD, PhD


Associate Professor of Pediatrics, University of Pennsylvania School ICREA Research Professor, Institute for Biomedical Investiga-
of Medicine; Division of Hematology, The Childrens Hospital of tions, August Pi i Sunyer (IDIBAPS)/Hospital Clinic, Department
Philadelphia, Philadelphia, Pennsylvania [20] of Neurology, University of Barcelona, Barcelona, Spain; Adjunct
Professor of Neurology University of Pennsylvania, Philadelphia,
Robert C. Basner, MD Pennsylvania [53]
Professor of Clinical Medicine, Division of Pulmonary, Allergy, and
Lisa M. DeAngelis, MD
Critical Care Medicine, Columbia University College of Physicians
Professor of Neurology, Weill Cornell Medical College; Chair,
and Surgeons, New York, New York [Appendix]
Department of Neurology, Memorial Sloan-Kettering Cancer
Robert S. Benjamin, MD Center, New York, New York [46]
P.H. and Fay E. Robinson Distinguished Professor and Chair,
Janice Dutcher, MD
Department of Sarcoma Medical Oncology, University of Texas
Department of Oncology, New York Medical College, Montefiore,
MD Anderson Cancer Center, Houston, Texas [45]
Bronx, New York [54]
Edward J. Benz, Jr., MD
Jeffery S. Dzieczkowski, MD
Richard and Susan Smith Professor of Medicine, Professor of
Physician, St. Alphonsus Regional Medical Center; Medical
Pediatrics, Professor of Genetics, Harvard Medical School; President
Director, Coagulation Clinic, Saint Alphonsus Medical Group,
and CEO, Dana-Farber Cancer Institute; Director, Dana-Farber/
International Medicine and Travel Medicine, Boise, Idaho [12]
Harvard Cancer Center (DF/HCC), Boston, Massachusetts [8]
Andrew J. Einstein, MD, PhD
Clara D. Bloomfield, MD
Assistant Professor of Clinical Medicine, Columbia University
Distinguished University Professor; William G. Pace, III Professor
College of Physicians and Surgeons; Department of Medicine,
of Cancer Research; Cancer Scholar and Senior Advisor, The Ohio
Division of Cardiology, Department of Radiology, Columbia
State University Comprehensive Cancer Center; Arthur G. James
University Medical Center and New York-Presbyterian Hospital,
Cancer Hospital and Richard J. Solove Research Institute,
New York, New York [Appendix]
Columbus, Ohio [14]
Ezekiel J. Emanuel, MD, PhD
George J. Bosl, MD Vice Provost for Global Initiatives and Chair, Department of
Professor of Medicine, Weill Cornell Medical College; Chair, Medical Ethics and Health Policy, University of Pennsylvania,
Department of Medicine; Patrick M. Byrne Chair in Clinical Philadelphia, Pennsylvania [32]
Oncology, Memorial Sloan-Kettering Cancer Center, New York,
New York [43] Robert Finberg, MD
Chair, Department of Medicine, University of Massachusetts
Otis W. Brawley, MD Medical School, Worcester, Massachusetts [29]
Chief Medical Officer, American Cancer Society Professor of
Hematology, Oncology, Medicine, and Epidemiology, Emory
University, Atlanta, Georgia [27]
ix
x Contributors

Jane E. Freedman, MD David H. Johnson, MD, FACP


Professor, Department of Medicine, University of Massachusetts Donald W. Seldin Distinguished Chair in Internal Medicine;
Medical School, Worcester, Massachusetts [21] Professor and Chairman, Department of Internal Medicine,
University of Texas Southwestern Medical School, Dallas, Texas [35]
Carl E. Freter, MD, PhD
Professor, Department of Internal Medicine, Division of Barbara Konkle, MD
Hematology/Medical Oncology, University of Missouri; Ellis Professor of Medicine, Hematology, University of Washington;
Fischel Cancer Center, Columbia, Missouri [55] Director, Translational Research, Puget Sound Blood Center,
Seattle, Washington [3, 19]
Robert F. Gagel, MD
Professor of Medicine and Head, Division of Internal Medicine, Barnett S. Kramer, MD, MPH
University of Texas MD Anderson Cancer Center, Houston, Director, Division of Cancer Prevention,
Texas [50] National Cancer Institute, Bethesda, Maryland [27]
John I. Gallin, MD Alexander Kratz, MD, PhD, MPH
Director, Clinical Center, National Institutes of Health, Bethesda, Associate Professor of Pathology and Cell Biology, Columbia
Maryland [5] University College of Physicians and Surgeons; Director, Core
Laboratory, Columbia University Medical Center, New York,
Samuel Z. Goldhaber, MD New York [Appendix]
Professor of Medicine, Harvard Medical School; Director, Venous
Thromboembolism Research Group, Cardiovascular Division, Marc E. Lippman, MD, MACP
Brigham and Womens Hospital, Boston, Massachusetts [22] Kathleen and Stanley Glaser Professor; Chairman, Department of
Medicine, Deputy Director, Sylvester Comprehensive Cancer Center,
Rasim Gucalp, MD University of Miami Miller School of Medicine, Miami, Florida [37]
Professor of Clinical Medicine, Albert Einstein College of Medicine;
Associate Chairman for Educational Programs, Department of On- Dan L. Longo, MD
cology; Director, Hematology/Oncology Fellowship, Montefiore Professor of Medicine, Harvard Medical School; Senior Physician,
Medical Center, Bronx, New York [54] Brigham and Womens Hospital; Deputy Editor, New England
Journal of Medicine, Boston, Massachusetts [1, 2, 4, 6, 15, 16, 17,
Anna R. Hemnes, MD 25, 26, 28, 31, 36, 51, 52, 55]
Assistant Professor, Division of Allergy, Pulmonary, and
Critical Care Medicine, Vanderbilt University Medical Center, Joseph Loscalzo, MD, PhD
Nashville, Tennessee [Review and Self-Assessment] Hersey Professor of the Theory and Practice of Medicine, Harvard
Medical School; Chairman, Department of Medicine; Physician-in-
Patrick H. Henry, MD Chief, Brigham and Womens Hospital, Boston, Massachusetts [21]
Clinical Adjunct Professor of Medicine, University of Iowa, Iowa
City, Iowa [4] Lucio Luzzatto, MD, FRCP, FRCPath
Professor of Haematology, University of Genova, Scientific Director
Katherine A. High, MD Istituto Toscano Tumori, Italy [10]
Investigator, Howard Hughes Medical Institute; William H. Bennett
Professor of Pediatrics, University of Pennsylvania School of Medi- Guido Marcucci, MD
cine; Director, Center for Cellular and Molecular Therapeutics, Professor of Medicine; John B. and Jane T. McCoy Chair in Cancer
Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania [20] Research; Associate Director of Translational Research, Com-
prehensive Cancer Center, The Ohio State University College of
A. Victor Hoffbrand, DM Medicine, Columbus, Ohio [14]
Professor Emeritus of Haematology, University College, London;
Robert J. Mayer, MD
Honorary Consultant Haematologist, Royal Free Hospital, London,
Stephen B. Kay Family Professor of Medicine, Harvard Medical
United Kingdom [9]
School, Boston, Massachusetts [38]
Steven M. Holland, MD Pat J. Morin, PhD
Chief, Laboratory of Clinical Infectious Diseases, National Institute Senior Investigator, Laboratory of Molecular Biology and
of Allergy and Infectious Diseases, National Institutes of Health, Immunology, National Institute on Aging, National Institutes of
Bethesda, Maryland [5] Health, Baltimore, Maryland [24]
Leora Horn, MD, MSc Robert J. Motzer, MD
Division of Hematology and Medical Oncology, Vanderbilt Professor of Medicine, Weill Cornell Medical College;
University School of Medicine, Nashville, Tennessee [35] Attending Physician, Genitourinary Oncology Service, Memorial
J. Larry Jameson, MD, PhD Sloan-Kettering Cancer Center, New York, New York [41, 43]
Robert G. Dunlop Professor of Medicine; Dean, University of Nikhil C. Munshi, MD
Pennsylvania School of Medicine; Executive Vice President of the Associate Professor of Medicine, Harvard Medical School; Associate
University of Pennsylvania for the Health System, Philadelphia, Director, Jerome Lipper Multiple Myeloma Center, Dana Farber
Pennsylvania [48, 52] Cancer Institute, Boston, Massachusetts [17]
Robert T. Jensen, MD Hari Nadiminti, MD
Digestive Diseases Branch, National Institute of Diabetes; Clinical Instructor, Department of Dermatology, Emory University
Digestive and Kidney Diseases, National Institutes of Health, School of Medicine, Atlanta, Georgia [33]
Bethesda, Maryland [49]
Contributors xi

Hartmut P. H. Neumann, MD Jeffrey M. Trent, PhD, FACMG


Head, Section Preventative Medicine, Department of Nephrology President and Research Director, Translational Genomics Research
and General Medicine, Albert-Ludwigs-University of Freiburg, Institute, Phoenix, Arizona; Van Andel Research Institute, Grand
Germany [51] Rapids, Michigan [24]
William Pao, MD, PhD Walter J. Urba, MD, PhD
Associate Professor of Medicine, Cancer Biology, and Director of Cancer Research, Robert W. Franz Cancer Research
Pathology, Division of Hematology and Medical Oncology, Center, Providence Portland Medical Center, Portland, Oregon [33]
Vanderbilt University School of Medicine, Nashville,
Tennessee [35] Gauri R. Varadhachary, MD
Associate Professor, Department of Gastrointestinal Medical
Shreyaskumar R. Patel, MD Oncology, University of Texas MD Anderson Cancer Center,
Center Medical Director, Sarcoma Center; Professor of Medicine; Houston, Texas [47]
Deputy Chairman, Department of Sarcoma Medical Oncology, MD
Anderson Cancer Center, Houston, Texas [45] Camilo Jimenez Vasquez, MD
Assistant Professor, Department of Endocrine Neoplasia and
Michael A. Pesce, PhD Hormonal Disorders, Division of Internal Medicine, University of
Professor Emeritus of Pathology and Cell Biology, Columbia Texas MD Anderson Cancer Center, Houston, Texas [50]
University College of Physicians and Surgeons; Columbia
University Medical Center, New York, New York [Appendix] Bert Vogelstein, MD
Professor of Oncology and Pathology; Investigator, Howard Hughes
Myrna R. Rosenfeld, MD, PhD Medical Institute; Sidney Kimmel Comprehensive Cancer Center; Johns
Professor of Neurology and Chief, Division of Neuro-oncology, Hopkins University School of Medicine, Baltimore, Maryland [24]
University of Pennsylvania, Philadelphia, Pennsylvania [53]
Everett E. Vokes, MD
Edward A. Sausville, MD, PhD John E. Ultmann Professor and Chairman, Department of
Professor, Department of Medicine, University of Maryland School Medicine; Physician-in-Chief, University of Chicago Medical
of Medicine; Deputy Director and Associate Director for Clinical Center, Chicago, Illinois [34]
Research, University of Maryland Marlene and Stewart Greene-
baum Cancer Center, Baltimore, Maryland [28] Carl V. Washington, MD
Associate Professor of Dermatology, Winship Cancer Center, Emory
David T. Scadden, MD University School of Medicine, Atlanta, Georgia [33]
Gerald and Darlene Jordan Professor of Medicine, Harvard Stem
Cell Institute, Harvard Medical School; Department of Stem Cell Anthony P. Weetman, MD
and Regenerative Biology, Massachusetts General Hospital, Boston, University of Sheffield School of Medicine, Sheffield,
Massachusetts [1] United Kingdom [48]

Howard I. Scher, MD Jeffrey I. Weitz, MD, FRCP(C), FACP


Professor of Medicine, Weill Cornell Medical College; D. Wayne Professor of Medicine and Biochemistry; Executive Director,
Calloway Chair in Urologic Oncology; Chief, Genitourinary Thrombosis and Atherosclerosis Research Institute; HSFO/J. F.
Oncology Service, Department of Medicine, Memorial Mustard Chair in Cardiovascular Research, Canada Research Chair
Sloan-Kettering Cancer Center, New York, New York [41, 42] (Tier 1) in Thrombosis, McMaster University, Hamilton, Ontario,
Canada [23]
Michael V. Seiden, MD, PhD
Professor of Medicine; President and CEO, Fox Chase Cancer Patrick Y. Wen, MD
Center, Philadelphia, Pennsylvania [44] Professor of Neurology, Harvard Medical School; Dana-Farber
Cancer Institute, Boston, Massachusetts [46]
David C. Seldin, MD, PhD
Chief, Section of Hematology-Oncology, Department of Meir Wetzler, MD, FACP
Medicine; Director, Amyloid Treatment and Research Program, Professor of Medicine, Roswell Park Cancer Institute, Buffalo,
Boston University School of Medicine; Boston Medical Center, New York [14]
Boston, Massachusetts [18]
Charles M. Wiener, MD
Martha Skinner, MD Dean/CEO Perdana University Graduate School of Medicine,
Professor, Department of Medicine, Boston University School of Selangor, Malaysia; Professor of Medicine and Physiology,
Medicine, Boston, Massachusetts [18] Johns Hopkins University School of Medicine, Baltimore,
Maryland [Review and Self-Assessment]
Jerry L. Spivak, MD
Professor of Medicine and Oncology, Hematology Division, Neal S. Young, MD
Johns Hopkins University School of Medicine, Baltimore, Chief, Hematology Branch, National Heart, Lung and Blood
Maryland [13] Institute, National Institutes of Health, Bethesda, Maryland [11]
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PREFACE

Harrisons Principles of Internal Medicine has a long and dis- medicine subspecialties. There are now invasive and non-
tinguished tradition in the field of hematology. Maxwell invasive cardiologists, gastroenterologists who do and others
Wintrobe, whose work actually established hematol- who do not use endoscopes, and organ-focused subspecial-
ogy as a distinct subspecialty of medicine, was a found- ists (diabetologists, thyroidologists) instead of organ system
ing editor of the book and participated in the first seven focused subspecialists (endocrinologists). At a time when
editions, taking over for Tinsley Harrison as editor-in- the body of knowledge that must be mastered is increasing
chief on the sixth and seventh editions. Wintrobe, born dramatically, the duration of training has not been increased
in 1901, began his study of blood in earnest in 1927 as to accommodate the additional learning that is necessary
an assistant in medicine at Tulane University in New to become highly skilled. Extraordinary attention has been
Orleans. He continued his studies at Johns Hopkins from focused on the hours that trainees work. Apparently, the
1930 to 1943 and moved to the University of Utah in administrators are more concerned about undocumented
1943, where he remained until his death in 1986. He adverse effects of every third night call on trainees than they
invented a variety of the measures that are routinely are about the well-documented adverse effects on patients
used to characterize red blood cell abnormalities, includ- of frequent handoffs of patient responsibility to multiple
ing the hematocrit, the red cell indices, and erythrocyte caregivers.
sedimentation rate, and defined the normal and abnormal Despite the sub-sub-subspecialization that is pervasive
values for these parameters, among many other impor- in modern medicine, students, trainees, general inter-
tant contributions in a 50-year career. nists, family medicine physicians, physicians assistants,
Oncology began as a subspecialty much later. It came nurse practitioners, and specialists in nonmedicine spe-
to life as a specific subdivision within hematology. A sub- cialties still require access to information in hematology
set of hematologists with a special interest in hematologic and oncology that can assist them in meeting the needs
malignancies began working with chemotherapeutic agents of their patients. Given the paucity of single sources of
to treat leukemia and lymphoma in the mid-1950s and integrated information on hematology and oncology, the
early 1960s. As new agents were developed and the prin- editors of Harrisons Principles of Internal Medicine decided
ciples of clinical trial research were developed, the body of to pull together the chapters in the mother book related
knowledge of oncology began to become larger and mainly to hematology and oncology and bind them together in
independent from hematology. Informed by the laboratory a subspecialty themed book called Harrisons Hematology
study of cancer biology and an expansion in focus beyond and Oncology. The first edition of this book appeared
hematologic neoplasms to tumors of all organ systems, in 2010 and was based on the 17th edition of Harrisons
oncology developed as a separable discipline from hematol- Principles of Internal Medicine. Encouraged by the response
ogy. This separation was also fueled by the expansion of the to that book, we have embarked upon a second edition
body of knowledge about clotting and its disorders, which based on 18th edition of Harrisons Principles of Internal
became a larger part of hematology. Medicine.
In most academic medical centers, hematology and The book contains 55 chapters organized into 12
oncology remain connected. However, conceptual dis- sections: (I) The Cellular Basis of Hematopoiesis, (II)
tinctions between hematology and oncology have been Cardinal Manifestations of Hematologic Diseases, (III)
made. Differences are reinforced by separate fellowship Anemias, (IV) Myeloproliferative Disorders, (V) Hema-
training programs (although many joint training pro- tologic Malignancies, (VI) Disorders of Hemostasis, (VII)
grams remain), separate board certification examina- Biology of Cancer, (VIII) Principles of Cancer Preven-
tions, separate professional organizations, and separate tion and Treatment, (IX) Neoplastic Disorders, (X)
textbooks describing separate bodies of knowledge. In Endocrine Neoplasia, (XI) Remote Effects of Cancer,
some academic medical centers, oncology is not merely a and (XII) Oncologic Emergencies and Late Effects Com-
separate subspecialty division in a Department of Medi- plications.
cine but is an entirely distinct department in the medi- The chapters have been written by physicians who have
cal school with the same standing as the Department of made seminal contributions to the body of knowledge in
Medicine. Economic forces are also at work to separate their areas of expertise. The information is authoritative and
hematology and oncology. as current as we can make it, given the time requirements of
Perhaps I am only reflecting the biases of an old dog, producing books. Each chapter contains the relevant infor-
but I am unenthusiastic about the increasing fractionation of mation on the genetics, cell biology, pathophysiology,

xiii
xiv Preface

and treatment of specific disease entities. In addition, many areas of medicine, the body of knowledge relevant
separate chapters on hematopoiesis, cancer cell biology, to the practice of hematology and oncology is expand-
and cancer prevention reflect the rapidly growing body ing rapidly. New discoveries with clinical impact are
of knowledge in these areas that are the underpinning being made at an astounding rate; nearly constant effort is
of our current concepts of diseases in hematology and required to try to keep pace. It is our hope that this book
oncology. In addition to the factual information pre- is helpful to you in the struggle to master the daunt-
sented in the chapters, a section of test questions and ing volume of new findings relevant to the care of your
answers is provided to reinforce important principles. A patients.
narrative explanation of what is wrong with the wrong We are extremely grateful to Kim Davis and James
answers should be of further value in the preparation of Shanahan at McGraw-Hill for their invaluable assistance
the reader for board examinations. in the preparation of this book.
The bringing together of hematology and oncology
in a single text is unusual and we hope it is useful. Like Dan L. Longo, MD
NOTICE
Medicine is an ever-changing science. As new research and clinical experi-
ence broaden our knowledge, changes in treatment and drug therapy are
required. The authors and the publisher of this work have checked with
sources believed to be reliable in their efforts to provide information that is
complete and generally in accord with the standards accepted at the time of
publication. However, in view of the possibility of human error or changes
in medical sciences, neither the authors nor the publisher nor any other party
who has been involved in the preparation or publication of this work war-
rants that the information contained herein is in every respect accurate or
complete, and they disclaim all responsibility for any errors or omissions or
for the results obtained from use of the information contained in this work.
Readers are encouraged to confirm the information contained herein with
other sources. For example and in particular, readers are advised to check the
product information sheet included in the package of each drug they plan to
administer to be certain that the information contained in this work is accu-
rate and that changes have not been made in the recommended dose or in
the contraindications for administration. This recommendation is of particular
importance in connection with new or infrequently used drugs.

Review and self-assessment questions and answers were taken from Wiener CM,
Brown CD, Hemnes AR (eds). Harrisons Self-Assessment and Board Review, 18th ed.
New York, McGraw-Hill, 2012, ISBN 978-0-07-177195-5.

The global icons call greater attention to key epidemiologic and clinical differences in the practice of medicine
throughout the world.

The genetic icons identify a clinical issue with an explicit genetic relationship.
This page intentionally left blank
SECTION I

The Cellular Basis


ofHematopoiesis
Chapter 1

Hematopoietic Stem Cells

David T. Scadden n Dan L. Longo

All of the cell types in the peripheral blood and some to generate, maintain, and repair tissues. They function
cells in every tissue of the body are derived from hema- successfully if they can replace a wide variety of shorter-
topoietic (hemo: blood; poiesis: creation) stem cells. If lived mature cells over prolonged periods. The process
the hematopoietic stem cell is damaged and can no lon- of self-renewal (discussed later) assures that a stem cell
ger function (e.g., due to a nuclear accident), a person population can be sustained over time. Without self-
would survive 24 weeks in the absence of extraordi- renewal, the stem cell pool would become exhausted,
nary support measures. With the clinical use of hema- and tissue maintenance would not be possible. The pro-
topoietic stem cells, tens of thousands of lives are saved cess of differentiation leads to production of the effec-
each year (Chap. 30). Stem cells produce tens of billions tors of tissue function: mature cells. Without proper
of blood cells daily from a stem cell pool that is esti- differentiation, the integrity of tissue function would be
mated to be only in the hundreds of thousands. How compromised, and organ failure would ensue.
stem cells do this, how they persist for many decades In the blood, mature cells have variable average life
despite the production demands, and how they may spans, ranging from 7 h for mature neutrophils to a few
be better used in clinical care are important issues in months for red blood cells to many years for memory
medicine. lymphocytes. However, the stem cell pool is the cen-
The study of blood cell production has become a tral, durable source of all blood and immune cells,
paradigm for how other tissues may be organized and maintaining a capacity to produce a broad range of cells
regulated. Basic research in hematopoiesis that includes from a single cell source yet keeping itself vigorous
defining stepwise molecular changes accompanying over decades of life. As an individual stem cell divides,
functional changes in maturing cells, aggregating cells it has the capacity to accomplish one of three division
into functional subgroups, and demonstrating hemato- outcomes: two stem cells, two cells destined for differ-
poietic stem cell regulation by a specialized microenvi- entiation, or one stem cell and one differentiating cell.
ronment are concepts worked out in hematology, but The former two outcomes are the result of symmet-
they offer models for other tissues. Moreover, these ric cell division, whereas the latter indicates a different
concepts may not be restricted to normal tissue func- outcome for the two daughter cellsan event termed
tion but extend to malignancy. Stem cells are rare cells asymmetric cell division. The relative balance for these
among a heterogeneous population of cell types, and types of outcomes may change during development
their behavior is assessed mainly in experimental animal and under particular kinds of demands on the stem cell
models involving reconstitution of hematopoiesis. Thus, pool.
much of what we know about stem cells is imprecise
and based on inferences from genetically manipulated
animals. Developmental Biology of
Hematopoietic Stem Cells
During development, blood cells are produced at differ-
Cardinal Functions of ent sites. Initially, the yolk sac provides oxygen-carrying
Hematopoietic Stem Cells red blood cells, and then the placenta and several sites of
intraembryonic blood cell production become involved.
All stem cell types have two cardinal functions: self- These intraembryonic sites engage in sequential order,
renewal and differentiation (Fig. 1-1). Stem cells exist moving from the genital ridge at a site where the aorta,
2
Stem cell stem cells (through CD162 and CD44) engage the 3
lectins P- and E-selectin on the endothelial surface to
slow the movement of the cells to a rolling phenotype.

CHAPTER 1
Stem cell integrins are then activated and accomplish
Self-renewal Differentiation firm adhesion between the stem cell and vessel wall,
with a particularly important role for stem cell VCAM-1
engaging endothelial VLA-4. The chemokine CXCL12
(SDF1) interacting with stem cell CXCR4 recep-
tors also appears to be important in the process of stem
Stem cell
cells getting from the circulation to where they engraft

Hematopoietic Stem Cells


in the bone marrow. This is particularly true in the
developmental move from fetal liver to bone marrow;
Differentiated cells however, the role for this molecule in adults appears to
Figure 1-1
be more related to retention of stem cells in the bone
Signature characteristics of the stem cell. Stem cells
marrow rather the process of getting them there. Inter-
have two essential features: the capacity to differentiate rupting that retention process through specific molecular
into a variety of mature cell types and the capacity for self- blockers of the CXCR4/CXCL12 interaction, cleavage
renewal. Intrinsic factors associated with self-renewal include of CXCL12, or downregulation of the receptor can
expression of Bmi-1, Gfi-1, PTEN, STAT5, Tel/Atv6, p21, all result in the release of stem cells into the circula-
p18, MCL-1, Mel-18, RAE28, and HoxB4. Extrinsic signals tion. This process is an increasingly important aspect
for self-renewal include Notch, Wnt, SHH, and Tie2/Ang-1. of recovering stem cells for therapeutic use as it has
Based mainly on murine studies, hematopoietic stem cells permitted the harvesting process to be done by leu-
express the following cell surface molecules: CD34, Thy-1 kapheresis rather than bone marrow punctures in the
(CD90), c-Kit receptor (CD117), CD133, CD164, and c-Mpl operating room. Refining our knowledge of how stem
(CD110, also known as the thrombopoietin receptor). cells get into and out of the bone marrow may improve
our ability to obtain stem cells and make them more
efficient at finding their way to the specific sites for
blood cell production, the so-called stem cell niche.
gonadal tissue, and mesonephros are emerging to the fetal
liver and then, in the second trimester, to the bone mar-
row and spleen. As the location of stem cells changes,
the cells they produce also change. The yolk sac pro- Hematopoietic Stem Cell
vides red cells expressing embryonic hemoglobins while Microenvironment
intraembryonic sites of hematopoiesis generate red cells, The concept of a specialized microenvironment, or
platelets, and the cells of innate immunity. The produc- stem cell niche, was first proposed to explain why cells
tion of the cells of adaptive immunity occurs when the derived from the bone marrow of one animal could be
bone marrow is colonized and the thymus forms. Stem used in transplantation and again be found in the bone
cell proliferation remains high, even in the bone mar- marrow of the recipient. This niche is more than just
row, until shortly after birth, when it appears to dramati- a housing site for stem cells, however. It is an ana-
cally decline. The cells in the bone marrow are thought tomic location where regulatory signals are provided
to arrive by the bloodborne transit of cells from the fetal that allow the stem cells to thrive, to expand if needed,
liver after calcification of the long bones has begun. The and to provide varying amounts of descendant daughter
presence of stem cells in the circulation is not unique to cells. In addition, unregulated growth of stem cells may
a time window in development. Rather, hematopoi- be problematic based on their undifferentiated state and
etic stem cells appear to circulate throughout life. The self-renewal capacity. Thus, the niche must also regulate
time that cells spend freely circulating appears to be brief the number of stem cells produced. In this manner, the
(measured in minutes in the mouse), but the cells that do niche has the dual function of serving as a site of nur-
circulate are functional and can be used for transplanta- ture but imposing limits for stem cells: in effect, acting
tion. The number of stem cells that circulate can be as both a nutritive and constraining home.
increased in a number of ways to facilitate harvest and The niche for blood stem cells changes with each of
transfer to the same or a different host. the sites of blood production during development, but
for most of human life, it is located in the bone mar-
row. Within the bone marrow, at least two niche sites
Mobility of Hematopoietic StemCells have been proposed: on trabecular bone surfaces and in
Cells entering and exiting the bone marrow do so the perivascular space. Stem cells may be found in both
through a series of molecular interactions. Circulating places by histologic analysis, and functional regulation
4 has been shown at the highly vascular bone surface. little or different effects on progenitor cells. Hematopoi-
Specifically, bone-forming mesenchymal cells, osteo- etic stem cells have governing mechanisms that are dis-
blastic cells, participate in hematopoietic stem cell func- tinct from the cells they generate.
tion, affecting their location, proliferation, and number.
SECTION I

The basis for this interaction is through a number of


Hematopoietic Stem Cell
molecules mediating location, such as the chemokine
Differentiation
CXCL12 (SDF1), through proliferation signals medi-
ated by angiopoietin 1, and signaling to modulate self- Hematopoietic stem cells sit at the base of a branch-
renewal or survival by factors such as Notch ligands, kit ing hierarchy of cells, culminating in the many mature
ligand, and Wnts. Other bone components, such as the cell types that comprise the blood and immune system
The Cellular Basis ofHematopoiesis

extracellular matrix glycoprotein, osteopontin, and the (Fig. 1-2). The maturation steps leading to terminally
high ionic calcium found at trabecular surfaces, con- differentiated and functional blood cells take place both
tribute to the unique microenvironment, or stem cell as a consequence of intrinsic changes in gene expres-
niche, on trabecular bone. This physiology has practi- sion and niche- and cytokine-directed changes in the
cal applications. First, medications altering niche com- cells. Our knowledge of the details remains incomplete.
ponents may have an effect on stem cell function. This As stem cells mature to progenitors, precursors, and,
has now been shown for a number of compounds, and finally, mature effector cells, they undergo a series of
some are being clinically tested. Second, it is now pos- functional changes. These include the obvious acquisi-
sible to assess whether the niche participates in disease tion of functions defining mature blood cells, such as
states and to examine whether targeting the niche with phagocytic capacity or hemoglobin synthesis. They also
medications may alter the outcome of certain diseases. include the progressive loss of plasticity (i.e., the ability
to become other cell types). For example, the myeloid
Excess Capacity of Hematopoietic progenitor can make all cells in the myeloid series but
Stem Cells none in the lymphoid series. As common myeloid pro-
genitors mature, they become precursors for either
In the absence of disease, one never runs out of hema- monocytes and granulocytes or erythrocytes and mega-
topoietic stem cells. Indeed, serial transplantation studies karyocytes, but not both. Some amount of reversibil-
in mice suggest that sufficient stem cells are present to ity of this process may exist early in the differentiation
reconstitute several animals in succession, with each ani- cascade, but that is lost beyond a distinct stage. As cells
mal having normal blood cell production. The fact that differentiate, they may also lose proliferative capac-
allogeneic stem cell transplant recipients also never run ity (Fig. 1-3). Mature granulocytes are incapable of
out of blood cells in their life span, which can extend for proliferation and only increase in number by increased
decades, argues that even the limiting numbers of stem production from precursors. Lymphoid cells retain the
cells provided to them are sufficient. How stem cells capacity to proliferate but have linked their prolifera-
respond to different conditions to increase or decrease tion to the recognition of particular proteins or peptides
their mature cell production remains poorly understood. by specific antigen receptors on their surface. In most
Clearly, negative feedback mechanisms affect the level tissues, the proliferative cell population is a more imma-
of production of most of the cells, leading to the nor- ture progenitor population. In general, cells within the
mal tightly regulated blood cell counts. However, many highly proliferative progenitor cell compartment are
of the regulatory mechanisms that govern production of also relatively short-lived, making their way through the
more mature progenitor cells do not apply or apply dif- differentiation process in a defined molecular program
ferently to stem cells. Similarly, most of the molecules involving the sequential activation of particular sets of
shown to be able to change the size of the stem cell pool genes. For any particular cell type, the differentiation
have little effect on more mature blood cells. For exam- program is difficult to speed up. The time it takes for
ple, the growth factor erythropoietin, which stimulates hematopoietic progenitors to become mature cells is
red blood cell production from more mature precursor 1014 days in humans, evident clinically by the inter-
cells, has no effect on stem cells. Similarly, granulocyte val between cytotoxic chemotherapy and blood count
colony-stimulating factor drives the rapid proliferation recovery in patients.
of granulocyte precursors but has little or no effect on
the cell cycling of stem cells. Rather, it changes the loca-
Self-Renewal
tion of stem cells by indirect means, altering molecules
such as CXCL12 that tether stem cells to their niche. The hematopoietic stem cell must balance its three
Molecules shown to be important for altering the pro- potential fates: apoptosis, self-renewal, and differentia-
liferation, self renewal or survival of stem cells, such as tion. The proliferation of cells is generally not associ-
cyclin-dependent kinase inhibitors, transcription factors ated with the ability to undergo a self-renewing division
such as Bmi-1, or microRNAs such as miR125a, have except among memory T and B cells and among stem
Stem Cells Progenitor Cells Lineage Committed
Precursors
Mature Cells 5
Aiolos,
LEF1, E2A, PAX-5, AML-1

CHAPTER 1
Common EBF, PAX-5 B Cell
Lymphoid IL-4 T Cell
Progenitor B Cell
Progenitor IKAROS,
IL-7 Progenitor E2A, NOTCH1, NOTCH,CBF1
NOTCH1 GATA3 T Cell
IL-2
IL-7 IL-7
NOTCH1
T/NK Cell Id2, Ets-1
IL-7 NK Cell
Progenitor IL-15

Hematopoietic Stem Cells


IKAROS NK Cell
PU1 Progenitor
Plasmacytoid
IL-7 FLT-3 Ligand Dendritic Cell
Hematopoietic
stem cell
cMyb
RelB, ICSBP, ld2 Monocytoid
Multipotent Dendritic Cell
FLT-3 Ligand
Progenitor
Egn1, Myb
Monocyte
Hox, Pbx1, M-CSF
Granulocyte Monocyte
SCL, GATA2,
NOTCH Monocyte Progenitor
Progenitor Granulocyte
SCF
C/EBP
TPO
G-CSF
Basophil
GM-CSF IL-3, SCF
Granulocyte Mast Cell
GATA1, FOG Progenitor C/EBP
Common NF-E2, SCL
Myeloid Rbtn2
IL-5 Eosinophil
Progenitor Erythrocyte
IL-3, SCF Progenitor
TPO GATA1
RBCs
EPO EPO
Megakaryocyte Megakaryocyte
Erythroid Progenitor Fli-1
Progenitor TPO AML-1 Platelets
TPO

Figure 1-2
Hierarchy of hematopoietic differentiation. Stem cells are and immune system. Progress through the pathways is medi-
multipotent cells that are the source of all descendant cells ated by alterations in gene expression. The regulation of the
and have the capacity to provide either long-term (measured differentiation by soluble factors and cellcell communica-
in years) or short-term (measured in months) cell produc- tions within the bone marrow niche are still being defined.
tion. Progenitor cells have a more limited spectrum of cells The transcription factors that characterize particular cell tran-
they can produce and are generally a short-lived, highly pro- sitions are illustrated on the arrows; the soluble factors that
liferative population also known as transient amplifying cells. contribute to the differentiation process are in blue. EPO,
Precursor cells are cells committed to a single blood cell lin- erythropoietin; SCF, stem cell factor; TPO, thrombopoietin.
eage but with a continued ability to proliferate; they do not M-CSF is macrophage-colony-stimulating factor; GM-CSF is
have all the features of a fully mature cell. Mature cells are the granulocyte-macrophage-colony stimulating factor; G-CSF is
terminally differentiated product of the differentiation process granulocyte-colony-stimulating factor.
and are the effector cells of specific activities of the blood

cells. Self-renewal capacity gives way to differentiation hematopoietic system, stem cells are generally cyto-
as the only option after cell division when cells leave kine-resistant, remaining dormant even when cytokines
the stem cell compartment until they have the oppor- drive bone marrow progenitors to proliferation rates
tunity to become memory lymphocytes. In addition to measured in hours. Stem cells, in contrast, are thought
this self-renewing capacity, stem cells have an additional to divide at far longer intervals measured in months
feature characterizing their proliferation machinery. to years, for the most quiescent cells. This quiescence
Stem cells in many mature adult tissues may be hetero- is difficult to overcome in vitro, limiting the ability to
geneous with some being deeply quiescent, serving as effectively expand human hematopoietic stem cells. The
a deep reserve, while others are more proliferative and process may be controlled by particularly high levels of
replenish the short-lived progenitor population. In the cyclin-dependent kinase inhibitors that restrict entry of
6 <40kg. This limitation restricts what would otherwise
Stem Progenitor Precursor Mature be an extremely promising source of stem cells. Two
features of cord blood stem cells are particularly impor-
tant. (1) They are derived from a diversity of individuals
SECTION I

Differentiation state
that far exceeds the adult donor pool and therefore can
More Less
overcome the majority of immunologic cross-matching
obstacles. (2) Cord blood stem cells have a large num-
Self-renewal ability
ber of T cells associated with them, but (paradoxically)
they appear to be associated with a lower incidence of
graft-versus-host disease when compared with simi-
The Cellular Basis ofHematopoiesis

Proliferation activity larly mismatched stem cells from other sources. If stem
cell expansion by self-renewal could be achieved, the
Lymphoid
exception number of cells available might be sufficient for use in
(memory B larger adults. An alternative approach to this problem is
and T cells)
to improve the efficiency of engraftment of donor stem
cells. Graft engineering is exploring methods of adding
Figure 1-3 cell components that may enhance engraftment. Fur-
Relative function of cells in the hematopoietic hierarchy. thermore, at least some data suggest that depletion of
The boxes represent distinct functional features of cells in the host natural killer (NK) cells may lower the number of
myeloid (upper box) versus lymphoid (lower box) lineages. stem cells necessary to reconstitute hematopoiesis.
Some limited understanding of self-renewal exists
and, intriguingly, implicates gene products that are
stem cells into cell cycle, blocking the G1S transition. associated with the chromatin state, a high-order orga-
Exogenous signals from the niche also appear to enforce nization of chromosomal DNA that influences tran-
quiescence, including the activation of the tyrosine scription. These include members of the polycomb
kinase receptor Tie2 on stem cells by angiopoietin 1 on family, a group of zinc fingercontaining transcrip-
osteoblasts. tional regulators that interact with the chromatin struc-
The regulation of stem cell proliferation also appears ture, contributing to the accessibility of groups of genes
to change with age. In mice, the cyclin-dependent for transcription. One member, Bmi-1, is important in
kinase inhibitor p16INK4a accumulates in stem cells enabling hematopoietic stem cell self-renewal through
in older animals and is associated with a change in five modification of cell cycle regulators such as the cyclin-
different stem cell functions, including cell cycling. dependent kinase inhibitors. In the absence of Bmi-1 or
Lowering expression of p16INK4a in older animals of the transcriptional regulator, Gfi-1, hematopoietic
improves stem cell cycling and the capacity to recon- stem cells decline in number and function. In contrast,
stitute hematopoiesis in adoptive hosts, making them dysregulation of Bmi-1 has been associated with leuke-
similar to younger animals. Mature cell numbers are mia; it may promote leukemic stem cell self-renewal
unaffected. Therefore, molecular events governing when it is overexpressed. Other transcription regulators
the specific functions of stem cells are being gradually have also been associated with self-renewal, particularly
made clear and offer the potential of new approaches homeobox, or hox, genes. These transcription fac-
to changing stem cell function for therapy. One criti- tors are named for their ability to govern large numbers
cal stem cell function that remains poorly defined is the of genes, including those determining body patterning
molecular regulation of self-renewal. in invertebrates. HoxB4 is capable of inducing exten-
For medicine, self-renewal is perhaps the most sive self-renewal of stem cells through its DNA-binding
important function of stem cells because it is critical in motif. Other members of the hox family of genes have
regulating the number of stem cells. Stem cell num- been noted to affect normal stem cells, but they are
ber is a key limiting parameter for both autologous also associated with leukemia. External signals that may
and allogeneic stem cell transplantation. Were we to influence the relative self-renewal versus differentiation
have the ability to use fewer stem cells or expand lim- outcomes of stem cell cycling include the Notch ligands
ited numbers of stem cells ex vivo, it might be pos- and specific Wnt ligands. Intracellular signal transduc-
sible to reduce the morbidity and expense of stem ing intermediates are also implicated in regulating self-
cell harvests and enable use of other stem cell sources. renewal but, interestingly, are not usually associated
Specifically, umbilical cord blood is a rich source of with the pathways activated by Notch or Wnt receptors.
stem cells. However, the volume of cord blood units They include PTEN, an inhibitor of the AKT pathway,
is extremely small and, therefore, the total number of and STAT5, both of which are usually downstream of
hematopoietic stem cells that can be obtained is gener- activated growth factor receptors and necessary for nor-
ally only sufficient to transplant an individual weighing mal stem cell functions including, self-renewal, at least
in mouse models. The connections between these mol- may be more readily converted to a malignant pheno- 7
ecules remain to be defined, and their role in physio- type. This hypothesis has been tested experimentally
logic regulation of stem cell self-renewal is still poorly in the hematopoietic system. Taking advantage of the

CHAPTER 1
understood. cell-surface markers that distinguish hematopoietic cells
of varying maturity, stem cells, progenitors, precursors,
and mature cells can be isolated. Powerful transforming
gene constructs were placed in these cells, and it was
Cancer is similar to an organ
found that the cell with the greatest potential to pro-
with self-renewing capacity
duce a malignancy was dependent on the transforming
gene. In some cases it was the stem cell, but in others,

Hematopoietic Stem Cells


The relationship of stem cells to cancer is an important
evolving dimension of adult stem cell biology. Cancer the progenitor cell functioned to initiate and per-petuate
may share principles of organization with normal the cancer. This shows that cells can acquire stem cell-
tissues. Cancer might have the same hierarchical orga- like properties in malignancy.
nization of cells with a base of stem-like cells capable
of the signature stem-cell features, self-renewal, and
differentiation. These stem-like cells might be the basis What Else Can Hematopoietic
for perpetuation of the tumor and represent a slowly Stem Cells Do?
dividing, rare population with distinct regulatory
mechanisms, including a relationship with a specialized Some experimental data have suggested that hematopoi-
microenvironment. A subpopulation of self-renewing etic stem cells or other cells mobilized into the circu-
cells has been defined for some, but not all, cancers. lation by the same factors that mobilize hematopoietic
A more sophisticated understanding of the stem-cell stem cells are capable of playing a role in healing the
organization of cancers may lead to improved strate- vascular and tissue damage associated with stroke and
gies for developing new therapies for the many com- myocardial infarction. These data are controversial, and
mon and difficult-to-treat types of malignancies that the applicability of a stem-cell approach to nonhemato-
have been relatively refractory to interventions aimed poietic conditions remains experimental. However, the
at dividing cells. application of the evolving knowledge of hematopoietic
Does the concept of cancer stem cells provide insight stem cell biology may lead to wide-ranging clinical uses.
into the cellular origin of cancer? The fact that some The stem cell, therefore, represents a true dual-edged
cells within a cancer have stem celllike properties sword. It has tremendous healing capacity and is essen-
does not necessarily mean that the cancer arose in the tial for life. Uncontrolled, it can threaten the life it
stem cell itself. Rather, more mature cells could have maintains. Understanding how stem cells function, the
acquired the self-renewal characteristics of stem cells. signals that modify their behavior, and the tissue niches
Any single genetic event is unlikely to be sufficient to that modulate stem cell responses to injury and disease
enable full transformation of a normal cell to a frankly are critical for more effectively developing stem cell
malignant one. Rather, cancer is a multistep process, based medicine. That aspect of medicine will include
and for the multiple steps to accumulate, the cell of the use of the stem cells and the use of drugs to tar-
origin must be able to persist for prolonged periods. It get stem cells to enhance repair of damaged tissues. It
must also be able to generate large numbers of daugh- will also include the careful balance of interventions to
ter cells. The normal stem cell has these properties and, control stem cells where they may be dysfunctional or
by virtue of its having intrinsic self-renewal capability, malignant.
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SECTION II

Cardinal
Manifestations of
Hematologic Disease
chAPteR 2

ANEMIA AND POLYCYTHEMIA

John W. adamson dan l. longo

The mature red cell is 8 m in diameter, anucleate,


hemAtoPoieSiS AnD the
discoid in shape, and extremely pliable in order to tra-
PhySioloGic BASiS oF
verse the microcirculation successfully; its membrane
ReD cell PRoDUction integrity is maintained by the intracellular generation
Hematopoiesis is the process by which the formed ele- of ATP. Normal red cell production results in the daily
ments of blood are produced. The process is regulated replacement of 0.81% of all circulating red cells in the
through a series of steps beginning with the hemato- body, since the average red cell lives 100120 days. The
poietic stem cell. Stem cells are capable of producing organ responsible for red cell production is called the
red cells, all classes of granulocytes, monocytes, plate- erythron. The erythron is a dynamic organ made up of
lets, and the cells of the immune system. The precise a rapidly proliferating pool of marrow erythroid pre-
molecular mechanismeither intrinsic to the stem cursor cells and a large mass of mature circulating red
cell itself or through the action of extrinsic factors blood cells. The size of the red cell mass reflects the
by which the stem cell becomes committed to a balance of red cell production and destruction. The
given lineage is not fully defined. However, experi- physiologic basis of red cell production and destruction
ments in mice suggest that erythroid cells come from provides an understanding of the mechanisms that can
a common erythroid/megakaryocyte progenitor that lead to anemia.
does not develop in the absence of expression of the The physiologic regulator of red cell production, the
GATA-1 and FOG-1 (friend of GATA-1) transcrip- glycoprotein hormone EPO, is produced and released by
tion factors (Chap. 1). Following lineage commit- peritubular capillary lining cells within the kidney. These
ment, hematopoietic progenitor and precursor cells cells are highly specialized epithelial-like cells. A small
come increasingly under the regulatory influence of amount of EPO is produced by hepatocytes. The fun-
growth factors and hormones. For red cell produc- damental stimulus for EPO production is the availabil-
tion, erythropoietin (EPO) is the regulatory hormone. ity of O2 for tissue metabolic needs. Key to EPO gene
EPO is required for the maintenance of committed regulation is hypoxia-inducible factor (HIF)-1. In the
erythroid progenitor cells that, in the absence of the presence of O2, HIF-1 is hydroxylated at a key proline,
hormone, undergo programmed cell death (apoptosis). allowing HIF-1 to be ubiquitinylated and degraded
The regulated process of red cell production is erythro- via the proteasome pathway. If O2 becomes limiting,
poiesis, and its key elements are illustrated in Fig. 2-1. this critical hydroxylation step does not occur, allow-
In the bone marrow, the first morphologically rec- ing HIF-1 to partner with other proteins, translocate
ognizable erythroid precursor is the pronormoblast. to the nucleus, and upregulate the EPO gene, among
This cell can undergo four to five cell divisions, which others.
result in the production of 1632 mature red cells. Impaired O2 delivery to the kidney can result from
With increased EPO production, or the administration a decreased red cell mass (anemia), impaired O2 load-
of EPO as a drug, early progenitor cell numbers are ing of the hemoglobin molecule or a high O2 affin-
amplified and, in turn, give rise to increased numbers of ity mutant hemoglobin (hypoxemia), or, rarely, impaired
erythrocytes. The regulation of EPO production itself is blood flow to the kidney (renal artery stenosis). EPO
linked to tissue oxygenation. governs the day-to-day production of red cells, and
In mammals, O2 is transported to tissues bound to ambient levels of the hormone can be measured in
the hemoglobin contained within circulating red cells. the plasma by sensitive immunoassaysthe normal
10
functioning erythroid marrow, and an adequate supply 11
Iron folate B12 of substrates for hemoglobin synthesis. A defect in any
Erythroid
marrow Red cell mass
of these key components can lead to anemia. Generally,
Red cell anemia is recognized in the laboratory when a patients
Erythropoietin
destruction hemoglobin level or hematocrit is reduced below an
Plasma expected value (the normal range). The likelihood and
volume
Hb concentration
severity of anemia are defined based on the deviation
Kidney of the patients hemoglobin/hematocrit from values
tissue expected for age- and sex-matched normal subjects.

CHAPTER 2
O2 consumption PO2 Heart The hemoglobin concentration in adults has a Gauss-
ian distribution. The mean hematocrit value for adult
Lungs males is 47% ( SD 7) and that for adult females is 42%
Vessels
( 5). Any single hematocrit or hemoglobin value car-
Atmospheric O2 levels
ries with it a likelihood of associated anemia. Thus, a
Figure 2-1 hematocrit of 39% in an adult male or <35% in an
The physiologic regulation of red cell production by tis- adult female has only about a 25% chance of being

Anemia and Polycythemia


sue oxygen tension. Hb, hemoglobin. normal. Suspected low hemoglobin or hematocrit
values are more easily interpreted if previous values
for the same patient are known for comparison. The
level being 1025 U/L. When the hemoglobin con- World Health Organization (WHO) defines anemia as
centration falls below 100120 g/L (1012 g/dL), a hemoglobin level <130 g/L (13 g/dL) in men and
plasma EPO levels increase in proportion to the sever- <120 g/L (12 g/dL) in women.
ity of the anemia (Fig. 2-2). In circulation, EPO has The critical elements of erythropoiesisEPO pro-
a half-clearance time of 69 h. EPO acts by binding duction, iron availability, the proliferative capacity of
to specific receptors on the surface of marrow ery- the bone marrow, and effective maturation of red cell
throid precursors, inducing them to proliferate and precursorsare used for the initial classification of
to mature. With EPO stimulation, red cell production anemia (discussed later).
can increase four- to fivefold within a 1- to 2-week
period, but only in the presence of adequate nutrients,
especially iron. The functional capacity of the erythron,
therefore, requires normal renal production of EPO, a Anemia
Clinical Presentation of Anemia
104 Signs and symptoms
Serum erythropoietin (mU/mL)

Anemia is most often recognized by abnormal screen-


ing laboratory tests. Patients less commonly present
103
with advanced anemia and its attendant signs and symp-
toms. Acute anemia is due to blood loss or hemolysis.
If blood loss is mild, enhanced O2 delivery is achieved
102
through changes in the O2hemoglobin dissociation
Normal 926 mU/mL curve mediated by a decreased pH or increased CO2
101
(Bohr effect). With acute blood loss, hypovolemia domi-
nates the clinical picture, and the hematocrit and hemo-
3 6 9 12 15 globin levels do not reflect the volume of blood lost.
Hemoglobin (g/dL) Signs of vascular instability appear with acute losses of
Figure 2-2 1015% of the total blood volume. In such patients, the
Erythropoietin (EPO) levels in response to anemia. When issue is not anemia but hypotension and decreased organ
the hemoglobin level falls to 120 g/L (12 g/dL), plasma EPO perfusion. When >30% of the blood volume is lost
levels increase logarithmically. In the presence of chronic suddenly, patients are unable to compensate with the
kidney disease or chronic inflammation, EPO levels are usual mechanisms of vascular contraction and changes
typically lower than expected for the degree of anemia. As in regional blood flow. The patient prefers to remain
individuals age, the level of EPO needed to sustain normal supine and will show postural hypotension and tachy-
hemoglobin levels appears to increase. (From RS Hillman cardia. If the volume of blood lost is >40% (i.e., >2 L
et al: Hematology in Clinical Practice, 5th ed. New York, in the average-sized adult), signs of hypovolemic shock
McGraw-Hill, 2010.) including confusion, dyspnea, diaphoresis, hypotension,
12 and tachycardia appear (Chap. 10). Such patients have commonly in those of Middle Eastern or African origin,
significant deficits in vital organ perfusion and require including African Americans who have a high frequency
immediate volume replacement. of G6PD deficiency. Other information that may be use-
With acute hemolysis, the signs and symptoms ful includes exposure to certain toxic agents or drugs
depend on the mechanism that leads to red cell destruc- and symptoms related to other disorders commonly
tion. Intravascular hemolysis with release of free hemo- associated with anemia. These include symptoms and
globin may be associated with acute back pain, free signs such as bleeding, fatigue, malaise, fever, weight
hemoglobin in the plasma and urine, and renal failure. loss, night sweats, and other systemic symptoms. Clues
Symptoms associated with more chronic or progres- to the mechanisms of anemia may be provided on
sive anemia depend on the age of the patient and the
SECTION II

physical examination by findings of infection, blood in


adequacy of blood supply to critical organs. Symptoms the stool, lymphadenopathy, splenomegaly, or pete-
associated with moderate anemia include fatigue, loss chiae. Splenomegaly and lymphadenopathy suggest an
of stamina, breathlessness, and tachycardia (particu- underlying lymphoproliferative disease, and petechiae
larly with physical exertion). However, because of the suggest platelet dysfunction. Past laboratory measure-
intrinsic compensatory mechanisms that govern the ments are helpful to determine a time of onset.
O2hemoglobin dissociation curve, the gradual onset
Cardinal Manifestations of Hematologic Disease

In the anemic patient, physical examination may dem-


of anemiaparticularly in young patientsmay not onstrate a forceful heartbeat, strong peripheral pulses,
be associated with signs or symptoms until the anemia and a systolic flow murmur. The skin and mucous mem-
is severe (hemoglobin <7080 g/L [78 g/dL]). When branes may be pale if the hemoglobin is <80100 g/L
anemia develops over a period of days or weeks, the (810 g/dL). This part of the physical examination should
total blood volume is normal to slightly increased, and focus on areas where vessels are close to the surface
changes in cardiac output and regional blood flow help such as the mucous membranes, nail beds, and palmar
compensate for the overall loss in O2-carrying capac- creases. If the palmar creases are lighter in color than the
ity. Changes in the position of the O2hemoglobin dis- surrounding skin when the hand is hyperextended, the
sociation curve account for some of the compensatory hemoglobin level is usually <80 g/L (8 g/dL).
response to anemia. With chronic anemia, intracellular
levels of 2,3-bisphosphoglycerate rise, shifting the dis- Laboratory Evaluation Table 2-1 lists the
sociation curve to the right and facilitating O2 unload- tests used in the initial workup of anemia. A rou-
ing. This compensatory mechanism can only maintain tine complete blood count (CBC) is required as part
normal tissue O2 delivery in the face of a 2030 g/L of the evaluation and includes the hemoglobin,
(23 g/dL) deficit in hemoglobin concentration. hematocrit, and red cell indices: the mean cell vol-
Finally, further protection of O2 delivery to vital organs ume (MCV) in femtoliters, mean cell hemoglobin
is achieved by the shunting of blood away from organs (MCH) in picograms per cell, and mean concentra-
that are relatively rich in blood supply, particularly the tion of hemoglobin per volume of red cells (MCHC)
kidney, gut, and skin. in grams per liter (non-SI: grams per deciliter). The
Certain disorders are commonly associated with ane- red cell indices are calculated as shown in Table 2-2,
mia. Chronic inflammatory states (e.g., infection, rheu- and the normal variations in the hemoglobin and hema-
matoid arthritis, cancer) are associated with mild to tocrit with age are shown in Table 2-3. A number of
moderate anemia, whereas lymphoproliferative disorders, physiologic factors affect the CBC, including age, sex,
such as chronic lymphocytic leukemia and certain other pregnancy, smoking, and altitude. High-normal hemo-
B-cell neoplasms, may be associated with autoimmune globin values may be seen in men and women who live
hemolysis. at high altitude or smoke heavily. Hemoglobin elevations
from smoking reflect normal compensation due to the
displacement of O2 by CO in hemoglobin binding. Other
Approach to the
Patient Anemia important information is provided by the reticulocyte
count and measurements of iron supply, including serum
The evaluation of the patient with anemia requires a iron, total iron-binding capacity (TIBC; an indirect measure
careful history and physical examination. Nutritional of the transferrin level), and serum ferritin. Marked altera-
history related to drugs or alcohol intake and family his- tions in the red cell indices usually reflect disorders of
tory of anemia should always be assessed. Certain geo- maturation or iron deficiency. A careful evaluation of the
graphic backgrounds and ethnic origins are associated peripheral blood smear is important, and clinical laborato-
with an increased likelihood of an inherited disorder ries often provide a description of both the red and white
of the hemoglobin molecule or intermediary metabo- cells, a white cell differential count, and the platelet count.
lism. Glucose-6-phosphate dehydrogenase (G6PD) defi- In patients with severe anemia and abnormalities in red
ciency and certain hemoglobinopathies are seen more blood cell morphology and/or low reticulocyte counts, a
Table 2-1 Table 2-3 13
Laboratory Tests in Anemia Diagnosis Changes in Normal Hemoglobin/Hematocrit
I.Complete blood count II. Iron supply studies Values With Age and Pregnancy
(CBC) A.Serum iron Age/Sex Hemoglobin g/dL Hematocrit %
A. Red blood cell count B.Total iron-binding
1. Hemoglobin capacity At birth 17 52
2. Hematocrit C.Serum ferritin Childhood 12 36
3. Reticulocyte count III. Marrow examination
Adolescence 13 40
B.Red blood cell indices A.Aspirate
1.Mean cell volume 1.M/E ratioa Adult man 16 (2) 47 (6)

CHAPTER 2
(MCV) 2.Cell morphology Adult woman 13 (2) 40 (6)
2.Mean cell hemoglobin 3.Iron stain (menstruating)
(MCH) B.Biopsy
3.Mean cell hemoglobin 1.Cellularity Adult woman 14 (2) 42 (6)
concentration (MCHC) 2.Morphology (postmenopausal)
4.Red cell distribution During pregnancy 12 (2) 37 (6)
width (RDW)

Anemia and Polycythemia


C.White blood cell count Source: From RS Hillman et al: Hematology in Clinical Practice,
1.Cell differential 5th ed. New York, McGraw-Hill, 2010.
2.Nuclear segmenta-
tion of neutrophils
D.Platelet count xperienced laboratory technician will be able to iden-
e
E.Cell morphology
tify minor populations of large or small cells or hypo-
1.Cell size
2.Hemoglobin content
chromic cells before the red cell indices change.
3.Anisocytosis Peripheral Blood Smear The peripheral blood
4.Poikilocytosis smear provides important information about defects in
5.Polychromasia
red cell production (Chap. 6). As a complement to the
a
red cell indices, the blood smear also reveals variations
M/E ratio, ratio of myeloid to erythroid precursors.
in cell size (anisocytosis) and shape (poikilocytosis). The
degree of anisocytosis usually correlates with increases
in the RDW or the range of cell sizes. Poikilocytosis sug-
bone marrow aspirate or biopsy can assist in the diagno-
gests a defect in the maturation of red cell precursors
sis. Other tests of value in the diagnosis of specific ane-
in the bone marrow or fragmentation of circulating red
mias are discussed in chapters on specific disease states.
cells. The blood smear may also reveal polychromasia
The components of the CBC also help in the classi-
red cells that are slightly larger than normal and grayish
fication of anemia. Microcytosis is reflected by a lower
blue in color on the Wright-Giemsa stain. These cells are
than normal MCV (<80), whereas high values (>100)
reticulocytes that have been prematurely released from
reflect macrocytosis. The MCH and MCHC reflect defects
the bone marrow, and their color represents residual
in hemoglobin synthesis (hypochromia). Automated
amounts of ribosomal RNA. These cells appear in circu-
cell counters describe the red cell volume distribu-
lation in response to EPO stimulation or to architectural
tion width (RDW). The MCV (representing the peak of
damage of the bone marrow (fibrosis, infiltration of
the distribution curve) is insensitive to the appearance
the marrow by malignant cells, etc.) that results in their
of small populations of macrocytes or microcytes. An
disordered release from the marrow. The appearance
of nucleated red cells, Howell-Jolly bodies, target cells,
Table 2-2 sickle cells, and others may provide clues to specific dis-
Red Blood Cell Indices orders (Figs. 2-3 to 2-11).

Index Normal Value Reticulocyte Count An accurate reticulocyte count


is key to the initial classification of anemia. Normally,
Mean cell volume (MCV) = (hematocrit 90 8 fL
reticulocytes are red cells that have been recently released
10)/(red cell count 106)
from the bone marrow. They are identified by staining
Mean cell hemoglobin (MCH) = 30 3 pg with a supravital dye that precipitates the ribosomal RNA
(hemoglobin 10)/(red cell count
(Fig. 2-12). These precipitates appear as blue or black
106)
punctate spots. This residual RNA is metabolized over
Mean cell hemoglobin concentration = 33 2%
the first 2436 h of the reticulocytes life span in circula-
(hemoglobin 10)/hematocrit, or
MCH/MCV
tion. Normally, the reticulocyte count ranges from 1 to
2% and reflects the daily replacement of 0.81.0% of the
14
SECTION II

Figure 2-3 Figure 2-6


Normal blood smear (Wright stain). High-power field show- Howell-Jolly bodies. In the absence of a functional spleen,
ing normal red cells, a neutrophil, and a few platelets. (From nuclear remnants are not culled from the red cells and
Cardinal Manifestations of Hematologic Disease

RS Hillman et al: Hematology in Clinical Practice, 5th ed. remain as small homogeneously staining blue inclusions on
New York, McGraw-Hill, 2010.) Wright stain. (From RS Hillman et al: Hematology in Clinical
Practice, 5th ed. New York, McGraw-Hill, 2010.)

Figure 2-4
Severe iron-deficiency anemia. Microcytic and hypochro- Figure 2-7
mic red cells smaller than the nucleus of a lymphocyte asso- Red cell changes in myelofibrosis. The left panel shows a
ciated with marked variation in size (anisocytosis) and shape teardrop-shaped cell. The right panel shows a nucleated red
(poikilocytosis). (From RS Hillman et al: Hematology in Clini- cell. These forms are seen in myelofibrosis.
cal Practice, 5th ed. New York, McGraw-Hill, 2010.)

Figure 2-8
Figure 2-5 Target cells. Target cells have a bulls-eye appearance and
Macrocytosis. Red cells are larger than a small lymphocyte are seen in thalassemia and in liver disease. (From RS Hillman
and well hemoglobinized. Often macrocytes are oval shaped et al: Hematology in Clinical Practice, 5th ed. New York,
(macro-ovalocytes). McGraw-Hill, 2010.)
15

CHAPTER 2
Figure 2-9
Red cell fragmentation. Red cells may become fragmented Figure 2-12
in the presence of foreign bodies in the circulation, such as Reticulocytes. Methylene blue stain demonstrates residual

Anemia and Polycythemia


mechanical heart valves, or in the setting of thermal injury. RNA in newly made red cells. (From RS Hillman et al: Hematol-
(From RS Hillman et al: Hematology in Clinical Practice, 5th ogy in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.)
ed. New York, McGraw-Hill, 2010.)
c irculating red cell population. A corrected reticulocyte
count provides a reliable measure of red cell production.
In the initial classification of anemia, the patients
reticulocyte count is compared with the expected retic-
ulocyte response. In general, if the EPO and erythroid
marrow responses to moderate anemia (hemoglobin
<100 g/L [10 g/dL]) are intact, the red cell production rate
increases to two to three times normal within 10 days fol-
lowing the onset of anemia. In the face of established ane-
mia, a reticulocyte response less than two to three times
normal indicates an inadequate marrow response.
In order to use the reticulocyte count to estimate
marrow response, two corrections are necessary. The
Figure 2-10
first correction adjusts the reticulocyte count based on
Uremia. The red cells in uremia may acquire numerous regu- the reduced number of circulating red cells. With ane-
larly spaced, small, spiny projections. Such cells, called burr mia, the percentage of reticulocytes may be increased
cells or echinocytes, are readily distinguishable from irregu- while the absolute number is unchanged. To correct for
larly spiculated acanthocytes shown in Fig. 2-11. this effect, the reticulocyte percentage is multiplied by
the ratio of the patients hemoglobin or hematocrit to
the expected hemoglobin or hematocrit for the age
and gender of the patient (Table 2-4). This provides an
estimate of the reticulocyte count corrected for anemia.
In order to convert the corrected reticulocyte count to
an index of marrow production, a further correction is
required, depending on whether some of the reticulo-
cytes in circulation have been released from the marrow
prematurely. For this second correction, the peripheral
blood smear is examined to see if there are polychro-
matophilic macrocytes present.
These cells, representing prematurely released reticu-
locytes, are referred to as shift cells, and the relation-
ship between the degree of shift and the necessary shift
Figure 2-11 correction factor is shown in Fig. 2-13. The correction is
Spur cells. Spur cells are recognized as distorted red cells necessary because these prematurely released cells sur-
containing several irregularly distributed thornlike projec- vive as reticulocytes in circulation for >1 day, thereby
tions. Cells with this morphologic abnormality are also called providing a falsely high estimate of daily red cell pro-
acanthocytes. (From RS Hillman et al: Hematology in Clinical duction. If polychromasia is increased, the reticulocyte
Practice, 5th ed. New York, McGraw-Hill, 2010.)
16 Table 2-4 Table 2-5
Calculation of Reticulocyte Production Normal Marrow Response to Anemia
Index
RETICULOCYTES
Correction #1 for Anemia: Production (INCLUDING Marrow
Hematocrit Index CORRECTIONS) M/E Ratio
This correction produces the corrected reticulocyte count
In a person whose reticulocyte count is 9%, hemoglobin 45 1 1 3:1
7.5g/dL, hematocrit 23%, the absolute reticulocyte count = 35 2.03.0 4.8%/3.8/2.5 2:11:1
9 (7.5/15) [or (23/45)]= 4.5%
25 3.05.0 14%/8/4.0 1:11:2
Correction #2 for Longer Life of Prematurely Released
15 3.05.0 30%/10/4.0 1:11:2
SECTION II

Reticulocytes in the Blood:


This correction produces the reticulocyte production index
In a person whose reticulocyte count is 9%, hemoglobin
7.5gm/dL, hematocrit 23%, the reticulocyte production
index
(7.5/15)(hemoglobin correction) index, and it provides an estimate of marrow production
=9 = 2.25 relative to normal.
2(maturation timecorrection)
Cardinal Manifestations of Hematologic Disease

Premature release of reticulocytes is normally due to


increased EPO stimulation. However, if the integrity of
the bone marrow release process is lost through tumor
infiltration, fibrosis, or other disorders, the appearance
count, already corrected for anemia, should be divided of nucleated red cells or polychromatophilic macro-
again by 2 to account for the prolonged reticulocyte cytes should still invoke the second reticulocyte correc-
maturation time. The second correction factor varies tion. The shift correction should always be applied to a
from 1 to 3 depending on the severity of anemia. In patient with anemia and a very high reticulocyte count
general, a correction of 2 is commonly used. An appro- to provide a true index of effective red cell production.
priate correction is shown in Table 2-4. If polychro- Patients with severe chronic hemolytic anemia may
matophilic cells are not seen on the blood smear, the increase red cell production as much as six- to sevenfold.
second correction is not required. The now doubly cor- This measure alone, therefore, confirms the fact that the
rected reticulocyte count is the reticulocyte production patient has an appropriate EPO response, a normally
functioning bone marrow, and sufficient iron available to
meet the demands for new red cell formation. Table 2-5
demonstrates the normal marrow response to anemia.
Marrow Peripheral
normoblasts and blood If the reticulocyte production index is <2 in the face of
reticulocytes reticulocytes
Hematocrit (%) (days) (days)
established anemia, a defect in erythroid marrow prolif-
eration or maturation must be present.
45 3.5 1.0
Tests of Iron Supply and Storage The labo-
35 3.0 1.5
ratory measurements that reflect the availability of iron
25 2.5 2.0
for hemoglobin synthesis include the serum iron, the
TIBC, and the percent transferrin saturation. The per-
15 1.5 2.5 cent transferrin saturation is derived by dividing the
serum iron level ( 100) by the TIBC. The normal serum
SHIFT iron ranges from 9 to 27 mol/L (50150 g/dL), while
correction factor the normal TIBC is 5464 mol/L (300360 g/dL); the
Figure 2-13 normal transferrin saturation ranges from 25 to 50%. A
Correction of the reticulocyte count. In order to use the diurnal variation in the serum iron leads to a variation
reticulocyte count as an indicator of effective red cell pro- in the percent transferrin saturation. The serum ferritin
duction, the reticulocyte percentage must be corrected is used to evaluate total body iron stores. Adult males
based on the level of anemia and the circulating life span of have serum ferritin levels that average 100 g/L, corre-
the reticulocytes. Erythroid cells take 4.5 days to mature. At sponding to iron stores of 1 g. Adult females have lower
a normal hemoglobin, reticulocytes are released to the circu- serum ferritin levels averaging 30 g/L, reflecting lower
lation with 1 day left as reticulocytes. However, with differ- iron stores (300 mg). A serum ferritin level of 1015
ent levels of anemia, reticulocytes (and even earlier erythroid g/L represents depletion of body iron stores. However,
cells) may be released from the marrow prematurely. Most ferritin is also an acute-phase reactant and, in the pres-
patients come to clinical attention with hematocrits in the ence of acute or chronic inflammation, may rise several-
mid-20s, and thus a correction factor of 2 is commonly used fold above baseline levels. As a rule, a serum ferritin >200
because the observed reticulocytes will live for 2 days in the g/L means there is at least some iron in tissue stores.
circulation before losing their RNA.
17

CHAPTER 2
Figure 2-14 Figure 2-16
Normal bone marrow. This is a low-power view of a sec- Myeloid hyperplasia. This marrow shows an increase in

Anemia and Polycythemia


tion of a normal bone marrow biopsy stained with hematoxy- the fraction of cells in the myeloid or granulocytic lineage as
lin and eosin (H&E). Note that the nucleated cellular elements might be seen in a normal marrow responding to infection.
account for 4050% and the fat (clear areas) accounts for The myeloid/erythroid ratio is >3:1. (From RS Hillman et al:
5060% of the area. (From RS Hillman et al: Hematology in Hematology in Clinical Practice, 5th ed. New York, McGraw-
Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) Hill, 2010.)

Bone Marrow Examination A bone marrow an M/E ratio of 2 or 3:1. In contrast, patients with hemo-
aspirate and smear or a needle biopsy can be useful in the lytic disease and a production index >3 will have an M/E
evaluation of some patients with anemia. In patients with ratio of at least 1:1. Maturation disorders are identified
hypoproliferative anemia and normal iron status, a bone from the discrepancy between the M/E ratio and the
marrow biopsy is indicated. Marrow examination can reticulocyte production index discussed later). Either
diagnose primary marrow disorders such as myelofibro- the marrow smear or biopsy can be stained for the
sis, a red cell maturation defect, or an infiltrative disease presence of iron stores or iron in developing red cells.
(Figs. 2-14 to 2-16). The increase or decrease of one cell The storage iron is in the form of ferritin or hemosiderin.
lineage (myeloid vs. erythroid) compared with another On carefully prepared bone marrow smears, small ferri-
is obtained by a differential count of nucleated cells in a tin granules can normally be seen under oil immersion
bone marrow smear (the myeloid/erythroid [M/E] ratio). in 2040% of developing erythroblasts. Such cells are
A patient with a hypoproliferative anemia (see below) called sideroblasts.
and a reticulocyte production index <2 will demonstrate Other Laboratory Measurements
Additional laboratory tests may be of value in con-
firming specific diagnoses. For details of these tests
and how they are applied in individual disorders, see
Chaps. 7 to 11.

Definition and Classification of


Anemia
Initial classification of anemia
The functional classification of anemia has three major
categories. These are (1) marrow production defects
(hypoproliferation), (2) red cell maturation defects (inef-
fective erythropoiesis), and (3) decreased red cell survival
(blood loss or hemolysis). The classification is shown in
Figure 2-15
Fig. 2-17. A hypoproliferative anemia is typically seen
Erythroid hyperplasia. This marrow shows an increase in
with a low reticulocyte production index together with
the fraction of cells in the erythroid lineage as might be seen
little or no change in red cell morphology (a normo-
when a normal marrow compensates for acute blood loss or
cytic, normochromic anemia) (Chap. 7). Maturation
hemolysis. The myeloid/erythroid ratio is about 1:1. (From RS
Hillman et al: Hematology in Clinical Practice, 5th ed. New
disorders typically have a slight to moderately elevated
York, McGraw-Hill, 2010.)
reticulocyte production index that is accompanied by
18 ALGORITHM OF THE PHYSIOLOGIC CLASSIFICATION OF ANEMIA Hypoproliferative anemias
Anemia At least 75% of all cases of anemia are hypoproliferative
in nature. A hypoproliferative anemia reflects absolute
or relative marrow failure in which the erythroid mar-
CBC, reticulocyte
count row has not proliferated appropriately for the degree of
anemia. The majority of hypoproliferative anemias are
due to mild to moderate iron deficiency or inflamma-
Index <2.5 Index 2.5
tion. A hypoproliferative anemia can result from marrow
damage, iron deficiency, or inadequate EPO stimulation.
SECTION II

Red cell Hemolysis/ The last may reflect impaired renal function, suppression
morphology hemorrhage
of EPO production by inflammatory cytokines such as
Blood loss interleukin 1, or reduced tissue needs for O2 from meta-
Normocytic Micro- or
normochromic macrocytic Intravascular bolic disease such as hypothyroidism. Only occasionally
hemolysis
is the marrow unable to produce red cells at a normal
Metabolic defect rate, and this is most prevalent in patients with renal fail-
Cardinal Manifestations of Hematologic Disease

Hypoproliferative Maturation disorder


Membrane ure. With diabetes mellitus or myeloma, the EPO defi-
abnormality
Marrow damage Cytoplasmic defects ciency may be more marked than would be predicted
Infiltration/fibrosis
Aplasia
Iron deficiency Hemoglobinopathy by the degree of renal insufficiency. In general, hypop-
Thalassemia
Iron deficiency Sideroblastic Immune destruction roliferative anemias are characterized by normocytic,
anemia normochromic red cells, although microcytic, hypo-
Stimulation Fragmentation
Inflammation Nuclear defects hemolysis chromic cells may be observed with mild iron defi-
Metabolic defect Folate deficiency
Renal disease Vitamin B 12 deficiency ciency or long-standing chronic inflammatory disease.
Drug toxicity The key laboratory tests in distinguishing between the
Refractory anemia
various forms of hypoproliferative anemia include the
serum iron and iron-binding capacity, evaluation of
Figure 2-17 renal and thyroid function, a marrow biopsy or aspi-
The physiologic classification of anemia. CBC, complete rate to detect marrow damage or infiltrative disease,
blood count.
and serum ferritin to assess iron stores. An iron stain
of the marrow will determine the pattern of iron dis-
either macrocytic (Chap. 9) or microcytic (Chaps. 7 tribution. Patients with the anemia of acute or chronic
and 8) red cell indices. Increased red blood cell destruc- inflammation show a distinctive pattern of serum iron
tion secondary to hemolysis results in an increase in the (low), TIBC (normal or low), percent transferrin satura-
reticulocyte production index to at least three times tion (low), and serum ferritin (normal or high). These
normal (Chap. 10), provided sufficient iron is avail- changes in iron values are brought about by hepcidin,
able. Hemorrhagic anemia does not typically result in the iron regulatory hormone that is increased in inflam-
production indices of more than 2.02.5 times normal mation (Chap. 7). A distinct pattern of results is noted
because of the limitations placed on expansion of the in mild to moderate iron deficiency (low serum iron,
erythroid marrow by iron availability. high TIBC, low percent transferrin saturation, low
In the first branch point of the classification of ane- serum ferritin) (Chap. 7). Marrow damage by drugs,
mia, a reticulocyte production index >2.5 indicates that infiltrative disease such as leukemia and lymphoma, and
hemolysis is most likely. A reticulocyte production index marrow aplasia are diagnosed from the peripheral blood
<2 indicates either a hypoproliferative anemia or matu- and bone marrow morphology. With infiltrative disease
ration disorder. The latter two possibilities can often be or fibrosis, a marrow biopsy is required.
distinguished by the red cell indices, by examination of
the peripheral blood smear, or by a marrow examina-
tion. If the red cell indices are normal, the anemia is Maturation disorders
almost certainly hypoproliferative in nature. Matura- The presence of anemia with an inappropriately low
tion disorders are characterized by ineffective red cell reticulocyte production index, macro- or microcytosis
production and a low reticulocyte production index. on smear, and abnormal red cell indices suggests a mat-
Bizarre red cell shapesmacrocytes or hypochromic uration disorder. Maturation disorders are divided into
microcytesare seen on the peripheral blood smear. two categories: nuclear maturation defects, associated
With a hypoproliferative anemia, no erythroid hyperpla- with macrocytosis, and cytoplasmic maturation defects,
sia is noted in the marrow, whereas patients with inef- associated with microcytosis and hypochromia usually
fective red cell production have erythroid hyperplasia from defects in hemoglobin synthesis. The inappropri-
and an M/E ratio <1:1. ately low reticulocyte production index is a reflection
of the ineffective erythropoiesis that results from the are typically normocytic or slightly macrocytic, reflect- 19
destruction within the marrow of developing eryth- ing the increased number of reticulocytes. Acute blood
roblasts. Bone marrow examination shows erythroid loss is not associated with an increased reticulocyte pro-
hyperplasia. duction index because of the time required to increase
Nuclear maturation defects result from vitamin B12 EPO production and, subsequently, marrow prolifera-
or folic acid deficiency, drug damage, or myelodyspla- tion. Subacute blood loss may be associated with mod-
sia. Drugs that interfere with cellular DNA synthesis, est reticulocytosis. Anemia from chronic blood loss
such as methotrexate or alkylating agents, can produce a presents more often as iron deficiency than with the
nuclear maturation defect. Alcohol, alone, is also capa- picture of increased red cell production.
The evaluation of blood loss anemia is usually not

CHAPTER 2
ble of producing macrocytosis and a variable degree of
anemia, but this is usually associated with folic acid defi- difficult. Most problems arise when a patient pres-
ciency. Measurements of folic acid and vitamin B12 are ents with an increased red cell production index from
critical not only in identifying the specific vitamin defi- an episode of acute blood loss that went unrecognized.
ciency but also because they reflect different pathoge- The cause of the anemia and increased red cell produc-
netic mechanisms (Chap. 9). tion may not be obvious. The confirmation of a recov-
Cytoplasmic maturation defects result from severe ering state may require observations over a period of

Anemia and Polycythemia


iron deficiency or abnormalities in globin or heme syn- 23 weeks, during which the hemoglobin concentra-
thesis. Iron deficiency occupies an unusual position in tion will be seen to rise and the reticulocyte production
the classification of anemia. If the iron-deficiency ane- index fall (Chap. 10).
mia is mild to moderate, erythroid marrow proliferation Hemolytic disease, while dramatic, is among the least
is blunted, and the anemia is classified as hypoprolifera- common forms of anemia. The ability to sustain a high
tive. However, if the anemia is severe and prolonged, reticulocyte production index reflects the ability of the
the erythroid marrow will become hyperplastic despite erythroid marrow to compensate for hemolysis and, in
the inadequate iron supply, and the anemia will be clas- the case of extravascular hemolysis, the efficient recy-
sified as ineffective erythropoiesis with a cytoplasmic cling of iron from the destroyed red cells to support red
maturation defect. In either case, an inappropriately low cell production. With intravascular hemolysis, such as
reticulocyte production index, microcytosis, and a clas- paroxysmal nocturnal hemoglobinuria, the loss of iron
sic pattern of iron values make the diagnosis clear and may limit the marrow response. The level of response
easily distinguish iron deficiency from other cytoplas- depends on the severity of the anemia and the nature of
mic maturation defects such as the thalassemias. Defects the underlying disease process.
in heme synthesis, in contrast to globin synthesis, are Hemoglobinopathies, such as sickle cell disease
less common and may be acquired or inherited. and the thalassemias, present a mixed picture. The
Acquired abnormalities are usually associated with myelo- reticulocyte index may be high but is inappropriately
dysplasia, may lead to either a macro- or microcytic ane- low for the degree of marrow erythroid hyperplasia
mia, and are frequently associated with mitochondrial (Chap. 8).
iron loading. In these cases, iron is taken up by the Hemolytic anemias present in different ways. Some
mitochondria of the developing erythroid cell but not appear suddenly as an acute, self-limited episode of
incorporated into heme. The iron-encrusted mitochon- intravascular or extravascular hemolysis, a presentation
dria surround the nucleus of the erythroid cell, form- pattern often seen in patients with autoimmune hemo-
ing a ring. Based on the distinctive finding of so-called lysis or with inherited defects of the Embden-Meyerhof
ringed sideroblasts on the marrow iron stain, patients pathway or the glutathione reductase pathway. Patients
are diagnosed as having a sideroblastic anemiaalmost with inherited disorders of the hemoglobin molecule or
always reflecting myelodysplasia. Again, studies of iron red cell membrane generally have a lifelong clinical his-
parameters are helpful in the differential diagnosis of tory typical of the disease process. Those with chronic
these patients. hemolytic disease, such as hereditary spherocytosis, may
actually present not with anemia but with a complica-
tion stemming from the prolonged increase in red cell
Blood loss/hemolytic anemia destruction such as symptomatic bilirubin gallstones
In contrast to anemias associated with an inappropriately or splenomegaly. Patients with chronic hemolysis are
low reticulocyte production index, hemolysis is associ- also susceptible to aplastic crises if an infectious process
ated with red cell production indices 2.5 times nor- interrupts red cell production.
mal. The stimulated erythropoiesis is reflected in the The differential diagnosis of an acute or chronic
blood smear by the appearance of increased numbers hemolytic event requires the careful integration of
of polychromatophilic macrocytes. A marrow exami- family history, the pattern of clinical presentation,
nation is rarely indicated if the reticulocyte production andwhether the disease is congenital or acquired
index is increased appropriately. The red cell indices a careful examination of the peripheral blood smear.
20 Precise diagnosis may require more specialized labo- whereas polycythemia refers to any increase in red cells.
ratory tests, such as hemoglobin electrophoresis or a Often patients with polycythemia are detected through
screen for red cell enzymes. Acquired defects in red an incidental finding of elevated hemoglobin or hema-
cell survival are often immunologically mediated and tocrit levels. Concern that the hemoglobin level may
require a direct or indirect antiglobulin test or a cold be abnormally high is usually triggered at 170 g/L
agglutinin titer to detect the presence of hemolytic (17 g/dL) for men and 150 g/L (15 g/dL) for women.
antibodies or complement-mediated red cell destruc- Hematocrit levels >50% in men or >45% in women
tion (Chap. 10). may be abnormal. Hematocrits >60% in men and
>55% in women are almost invariably associated with
an increased red cell mass. Given that the machine that
SECTION II

Treatment Anemia quantitates red cell parameters actually measures hemo-


An overriding principle is to initiate treatment of mild globin concentrations and calculates hematocrits, hemo-
to moderate anemia only when a specific diagnosis is globin levels may be a better index.
made. Rarely, in the acute setting, anemia may be so Features of the clinical history that are useful in the
severe that red cell transfusions are required before a differential diagnosis include smoking history; current
living at high altitude; or a history of congenital heart
Cardinal Manifestations of Hematologic Disease

specific diagnosis is made. Whether the anemia is of


acute or gradual onset, the selection of the appropriate disease, sleep apnea, or chronic lung disease.
treatment is determined by the documented cause(s) Patients with polycythemia may be asymptomatic or
of the anemia. Often, the cause of the anemia is multi- experience symptoms related to the increased red cell
factorial. For example, a patient with severe rheuma- mass or the underlying disease process that leads to the
toid arthritis who has been taking anti-inflammatory increased red cell mass. The dominant symptoms from
drugs may have a hypoproliferative anemia associated an increased red cell mass are related to hyperviscosity
with chronic inflammation as well as chronic blood loss and thrombosis (both venous and arterial) because the
associated with intermittent gastrointestinal bleed- blood viscosity increases logarithmically at hematocrits
ing. In every circumstance, it is important to evalu- >55%. Manifestations range from digital ischemia to
ate the patients iron status fully before and during the Budd-Chiari syndrome with hepatic vein thrombosis.
treatment of any anemia. Transfusion is discussed in Abdominal vessel thromboses are particularly common.
Chap. 12; iron therapy is discussed in Chap. 7; treat- Neurologic symptoms such as vertigo, tinnitus, head-
ment of megaloblastic anemia is discussed in Chap. 9; ache, and visual disturbances may occur. Hypertension
treatment of other entities is discussed in their respec- is often present. Patients with polycythemia vera may have
tive chapters (sickle cell anemia, Chap. 8; hemolytic aquagenic pruritus and symptoms related to hepato-
anemias, Chap. 10; aplastic anemia and myelodysplasia, splenomegaly. Patients may have easy bruising, epi-
Chap. 11). staxis, or bleeding from the gastrointestinal tract. Peptic
Therapeutic options for the treatment of anemias ulcer disease is common. Patients with hypoxemia may
have expanded dramatically during the past 25 years. develop cyanosis on minimal exertion or have headache,
Blood component therapy is available and safe. Recom- impaired mental acuity, and fatigue.
binant EPO as an adjunct to anemia management has The physical examination usually reveals a ruddy
transformed the lives of patients with chronic renal fail- complexion. Splenomegaly favors polycythemia vera
ure on dialysis and reduced transfusion needs of anemic as the diagnosis (Chap. 13). The presence of cyanosis
cancer patients receiving chemotherapy. Eventually, or evidence of a right-to-left shunt suggests congenital
patients with inherited disorders of globin synthesis or heart disease presenting in the adult, particularly tetral-
mutations in the globin gene, such as sickle cell disease, ogy of Fallot or Eisenmengers syndrome. Increased
may benefit from the successful introduction of tar- blood viscosity raises pulmonary artery pressure; hypox-
geted genetic therapy. emia can lead to increased pulmonary vascular resistance.
Together, these factors can produce cor pulmonale.
Polycythemia can be spurious (related to a decrease in
plasma volume; Gaisbocks syndrome), primary, or sec-
Polycythemia ondary in origin. The secondary causes are all associated
with increases in EPO levels: either a physiologically
Polycythemia is defined as an increase in the hemoglo- adapted appropriate elevation based on tissue hypoxia
bin above normal. This increase may be real or only (lung disease, high altitude, CO poisoning, high-affin-
apparent because of a decrease in plasma volume (spu- ity hemoglobinopathy) or an abnormal overproduction
rious or relative polycythemia). The term erythrocyto- (renal cysts, renal artery stenosis, tumors with ectopic
sis may be used interchangeably with polycythemia, but EPO production). A rare familial form of polycythemia
some draw a distinction between them: erythrocyto- is associated with normal EPO levels but hyperrespon-
sis implies documentation of increased red cell mass, sive EPO receptors due to mutations.
Approach to the
blood radioactivity over a 2-h period. If the red cell
21
Patient Polycythemia
mass is normal (<36 mL/kg in men, <32 mL/kg in
As shown in Fig. 2-18, the first step is to document the women), the patient has spurious or relative polycy-
presence of an increased red cell mass using the prin- themia. If the red cell mass is increased (>36 mL/kg in
ciple of isotope dilution by administering 51Cr-labeled men, >32 mL/kg in women), serum EPO levels should
autologous red blood cells to the patient and sampling be measured. If EPO levels are low or unmeasurable,
the patient most likely has polycythemia vera. Tests
that support this diagnosis include an elevated white
blood cell count, increased absolute basophil count,

CHAPTER 2
AN APPROACH TO DIAGNOSING PATIENTS WITH POLYCYTHEMIA
and thrombocytosis. A mutation in JAK-2 (Val617Phe),
Increased hct or hgb
a key member of the cytokine intracellular signal-
normal
ing pathway, can be found in 7095% of patients with
Measure RBC mass Dx: Relative
erythrocytosis polycythemia vera.
elevated If serum EPO levels are elevated, one needs to distin-
Measure serum
low Confirm guish whether the elevation is a physiologic response

Anemia and Polycythemia


Dx: Polycythemia JAK-2
EPO levels vera mutation to hypoxia or is related to autonomous EPO produc-
elevated tion. Patients with low arterial O2 saturation (<92%)
low should be further evaluated for the presence of heart
Measure arterial Diagnostic evaluation for
O2 saturation heart or lung disease,
or lung disease if they are not living at high altitude.
e.g., COPD, high altitude, Patients with normal O2 saturation who are smokers
normal AV or intracardiac shunt
may have elevated EPO levels because of CO displace-
no
Smoker? Measure hemoglobin ment of O2. If carboxyhemoglobin (COHb) levels are
yes
O2 affinity high, the diagnosis is smokers polycythemia. Such
normal
increased
patients should be urged to stop smoking. Those who
Measure normal cannot stop smoking require phlebotomy to control
carboxyhemoglobin Dx: O2 affinity
levels hemoglobinopathy
their polycythemia. Patients with normal O2 saturation
who do not smoke either have an abnormal hemoglo-
elevated Search for tumor as source of EPO bin that does not deliver O2 to the tissues (evaluated
IVP/renal ultrasound (renal Ca or cyst)
Dx: Smokers CT of head (cerebellar hemangioma) by finding elevated O2hemoglobin affinity) or have
polycythemia CT of pelvis (uterine leiomyoma)
CT of abdomen (hepatoma)
a source of EPO production that is not responding to
the normal feedback inhibition. Further workup is dic-
tated by the differential diagnosis of EPO-producing
Figure 2-18 neoplasms. Hepatoma, uterine leiomyoma, and renal
An approach to the differential diagnosis of patients with cancer or cysts are all detectable with abdominopel-
an elevated hemoglobin (possible polycythemia). AV,
vic CT scans. Cerebellar hemangiomas may produce
atrioventricular; COPD, chronic obstructive pulmonary dis-
EPO, but they present with localizing neurologic
ease; CT, computed tomography; Dx, diagnosis; EPO, eryth-
signs and symptoms rather than polycythemia-related
ropoietin; hct, hematocrit; IVP, intravenous pyelogram; RBC,
symptoms.
red blood cell.
chAPter 3

BLEEDING AND THROMBOSIS

Barbara Konkle

The human hemostatic system provides a natural bal- platelets undergo the release reaction, during which
ance between procoagulant and anticoagulant forces. they secrete contents that further promote aggregation
The procoagulant forces include platelet adhesion and and inhibit the naturally anticoagulant endothelial cell
aggregation and fibrin clot formation; anticoagulant factors. During platelet aggregation (platelet-platelet
forces include the natural inhibitors of coagulation and interaction), additional platelets are recruited from the
fibrinolysis. Under normal circumstances, hemosta- circulation to the site of vascular injury, leading to the
sis is regulated to promote blood flow; however, it is formation of an occlusive platelet thrombus. The plate-
also prepared to clot blood rapidly to arrest blood flow let plug is anchored and stabilized by the developing
and prevent exsanguination. After bleeding is success- fibrin mesh.
fully halted, the system remodels the damaged vessel to The platelet glycoprotein (Gp) IIb/IIIa (IIb3) com-
restore normal blood flow. The major components of plex is the most abundant receptor on the platelet sur-
the hemostatic system, which function in concert, are face. Platelet activation converts the normally inactive
(1) platelets and other formed elements of blood, such Gp IIb/IIIa receptor into an active receptor, enabling
as monocytes and red cells; (2) plasma proteins (the binding to fibrinogen and VWF. Because the surface
coagulation and fibrinolytic factors and inhibitors); and of each platelet has about 50,000 Gp IIb/IIIa-binding
(3) the vessel wall. sites, numerous activated platelets recruited to the site of
vascular injury can rapidly form an occlusive aggregate
by means of a dense network of intercellular fibrino-
stePs oF normAL hemostAsis gen bridges. Since this receptor is the key mediator of
platelet aggregation, it has become an effective target for
PlATeleT Plug FormATion antiplatelet therapy.
On vascular injury, platelets adhere to the site of injury,
usually the denuded vascular intimal surface. Platelet
Fibrin CloT FormATion
adhesion is mediated primarily by von Willebrand fac-
tor (VWF), a large multimeric protein present in both Plasma coagulation proteins (clotting factors) normally cir-
plasma and the extracellular matrix of the subendothe- culate in plasma in their inactive forms. The sequence
lial vessel wall, which serves as the primary molecu- of coagulation protein reactions that culminate in the
lar glue, providing sufficient strength to withstand the formation of fibrin was originally described as a water-
high levels of shear stress that would tend to detach fall or a cascade. Two pathways of blood coagulation
them with the flow of blood. Platelet adhesion is also have been described in the past: the so-called extrinsic,
facilitated by direct binding to subendothelial collagen or tissue factor, pathway and the so-called intrinsic, or
through specific platelet membrane collagen receptors. contact activation, pathway. We now know that coag-
Platelet adhesion results in subsequent platelet acti- ulation is normally initiated through tissue factor (TF)
vation and aggregation. This process is enhanced and exposure and activation through the classic extrinsic path-
amplified by humoral mediators in plasma (e.g., epineph- way but with critically important amplification through
rine, thrombin); mediators released from activated plate- elements of the classic intrinsic pathway, as illustrated in
lets (e.g., adenosine diphosphate, serotonin); and vessel Fig. 3-1. These reactions take place on phospholipid
wall extracellular matrix constituents that come in contact surfaces, usually the activated platelet surface. Coagula-
with adherent platelets (e.g., collagen, VWF). Activated tion testing in the laboratory can reflect other influences
22
Vessel IX
23
injury
IX
TF VIIa
VIIIa
IXa XIa
X

TFPI
X
XI

CHAPTER 3
Va
Xa II
(Prothrombin)
Thrombin (IIa)

Fibrinogen Fibrin

Bleeding and Thrombosis


Figure 3-1
Coagulation is initiated by tissue factor (TF) exposure, formed, tissue factor pathway inhibitor (TFPI) inhibits the TF/
which, with factor (F)VIIa, activates FIX and FX, which in FVIIa pathway, making coagulation dependent on the ampli-
turn, with FVIII and FV as cofactors, respectively, results in fication loop through FIX/FVIII. Coagulation requires calcium
thrombin formation and subsequent conversion of fibrino- (not shown) and takes place on phospholipid surfaces, usu-
gen to fibrin. Thrombin activates FXI, FVIII, and FV, amplify- ally the activated platelet membrane.
ing the coagulation signal. Once the TF/FVIIa/FXa complex is

due to the artificial nature of the in vitro systems used


(discussed later). A D E D
The immediate trigger for coagulation is vascular
damage that exposes blood to TF that is constitutively Thrombin Fibrin assembly

expressed on the surfaces of subendothelial cellular com-


ponents of the vessel wall, such as smooth muscle cells D E D D E D D E D
B
and fibroblasts. TF is also present in circulating micropar-
D E D D E D D E D
ticles, presumably shed from cells including monocytes
and platelets. TF binds the serine protease factor VIIa; the Factor XIIIa
Fibrin
cross-linking
complex activates factor X to factor Xa. Alternatively,
the complex can indirectly activate factor X by initially
D E D D E D D E D
converting factor IX to factor IXa, which then activates C
factor X. The participation of factor XI in hemostasis is D E D D E D D E D
not dependent on its activation by factor XIIa but rather
on its positive feedback activation by thrombin. Thus,
factor XIa functions in the propagation and amplification, Plasmin Clot lysis

rather than in the initiation, of the coagulation cascade.


Factor Xa can be formed through the actions of
D D D E
either the tissue factor/factor VIIa complex or factor
IXa (with factor VIIIa as a cofactor) and converts pro-
Figure 3-2
thrombin to thrombin, the pivotal protease of the coag- Fibrin formation and dissolution. (A). Fibrinogen is a tri-
ulation system. The essential cofactor for this reaction nodular structure consisting of 2 D domains and 1 E domain.
is factor Va. Like the homologous factor VIIIa, factor Va Thrombin activation results in an ordered lateral assembly of
is produced by thrombin-induced limited proteolysis protofibrils (B) with noncovalent associations. FXIIIa cross-
of factor V. Thrombin is a multifunctional enzyme that links the D domains on adjacent molecules (C). Fibrin and
converts soluble plasma fibrinogen to an insoluble fibrin fibrinogen (not shown) lysis by plasmin occurs at discrete
matrix. Fibrin polymerization involves an orderly pro- sites and results in intermediary fibrin(ogen) degradation
cess of intermolecular associations (Fig. 3-2). Throm- products (not shown). D-Dimers are the product of complete
bin also activates factor XIII (fibrin-stabilizing factor) lysis of fibrin (D), maintaining the cross-linked D domains.
24 to factor XIIIa, which covalently cross-links and thereby XII
stabilizes the fibrin clot.
TF
The assembly of the clotting factors on activated cell XI
membrane surfaces greatly accelerates their reaction VII VIIa XIIa
IX
rates and also serves to localize blood clotting to sites
of vascular injury. The critical cell membrane compo- VIIa/TF XIa
TFPI
nents, acidic phospholipids, are not normally exposed
on resting cell membrane surfaces. However, when IXa
platelets, monocytes, and endothelial cells are activated VIIIa
by vascular injury or inflammatory stimuli, the proco-
SECTION II

agulant head groups of the membrane anionic phos-


pholipids become translocated to the surfaces of these X Xa AT

cells or released as part of microparticles, making them PC


Va
available to support and promote the plasma coagulation PS
reactions.
PT Th
Cardinal Manifestations of Hematologic Disease

Antithrombotic Mechanisms Fibrinogen Fibrin FDP

Several physiologic antithrombotic mechanisms act in Plasmin


concert to prevent clotting under normal circumstances.
PA
These mechanisms operate to preserve blood fluid- Plasminogen
ity and to limit blood clotting to specific focal sites of
vascular injury. Endothelial cells have many antithrom- Figure 3-3
botic effects. They produce prostacyclin, nitric oxide, Sites of action of the four major physiologic antithrom-
and ectoADPase/CD39, which act to inhibit platelet botic pathways: antithrombin (AT); protein C/S (PC/PS);
binding, secretion, and aggregation. Endothelial cells tissue factor pathway inhibitor (TFPI); and the fibrinolytic sys-
produce anticoagulant factors including heparan pro- tem, consisting of plasminogen, plasminogen activator (PA),
teoglycans, antithrombin, TF pathway inhibitor, and and plasmin. FDP, fibrin(ogen) degradation products. PT, pro-
thrombomodulin. They also activate fibrinolytic mech- thrombin; Th, thrombin; (Modified from BA Konkle, AI Schafer,
anisms through the production of tissue plasminogen in DP Zipes et al [eds]: Braunwalds Heart Disease, 7th ed.
activator 1, urokinase, plasminogen activator inhibitor, Philadelphia, Saunders, 2005.)
and annexin-2. The sites of action of the major physi-
ologic antithrombotic pathways are shown in Fig. 3-3.
Antithrombin (or antithrombin III) is the major
plasma protease inhibitor of thrombin and the other thrombinthrombomodulin complex, thereby enhancing
clotting factors in coagulation. Antithrombin neutral- its activation efficiency. Activated protein C acts as an
izes thrombin and other activated coagulation fac- anticoagulant by cleaving and inactivating activated fac-
tors by forming a complex between the active site of tors V and VIII. This reaction is accelerated by a cofac-
the enzyme and the reactive center of antithrombin. tor, protein S, which, like protein C, is a glycoprotein
The rate of formation of these inactivating complexes that undergoes vitamin Kdependent posttranslational
increases by a factor of several thousand in the presence modification. Quantitative or qualitative deficiencies
of heparin. Antithrombin inactivation of thrombin and of protein C or protein S, or resistance to the action
other activated clotting factors occurs physiologically on of activated protein C by a specific mutation at its tar-
vascular surfaces, where glycosoaminoglycans, including get cleavage site in factor Va (factor V Leiden), lead to
heparan sulfates, are present to catalyze these reactions. hypercoagulable states.
Inherited quantitative or qualitative deficiencies of anti- Tissue factor pathway inhibitor (TFPI) is a plasma
thrombin lead to a lifelong predisposition to venous protease inhibitor that regulates the TF-induced extrin-
thromboembolism. sic pathway of coagulation. TFPI inhibits the TF/FVIIa/
Protein C is a plasma glycoprotein that becomes FXa complex, essentially turning off the TF/FVIIa ini-
an anticoagulant when it is activated by thrombin. tiation of coagulation, which then becomes dependent
The thrombin-induced activation of protein C occurs on the amplification loop via FXI and FVIII activa-
physiologically on thrombomodulin, a transmembrane tion by thrombin. TFPI is bound to lipoprotein and can
proteoglycan-binding site for thrombin on endothe- also be released by heparin from endothelial cells, where
lial cell surfaces. The binding of protein C to its recep- it is bound to glycosoaminoglycans, and from plate-
tor on endothelial cells places it in proximity to the lets. The heparin-mediated release of TFPI may play a
role in the anticoagulant effects of unfractionated and further. This creates a highly efficient mechanism to 25
low-molecular-weight heparins. generate plasmin focally on the fibrin clot, which then
becomes plasmins substrate for digestion to fibrin deg-
The Fibrinolytic System radation products. Plasmin cleaves fibrin at distinct sites
of the fibrin molecule, leading to the generation of char-
Any thrombin that escapes the inhibitory effects of the acteristic fibrin fragments during the process of fibrino-
physiologic anticoagulant systems is available to con- lysis (Fig. 3-2). The sites of plasmin cleavage of fibrin
vert fibrinogen to fibrin. In response, the endogenous are the same as those in fibrinogen. However, when plas-
fibrinolytic system is then activated to dispose of intra- min acts on covalently cross-linked fibrin, D-dimers are
vascular fibrin and thereby maintain or reestablish the

CHAPTER 3
released; hence, D-dimers can be measured in plasma
patency of the circulation. Just as thrombin is the key as a relatively specific test of fibrin (rather than fibrino-
protease enzyme of the coagulation system, plasmin is gen) degradation. D-dimer assays can be used as sensi-
the major protease enzyme of the fibrinolytic system, tive markers of blood clot formation, and some have
acting to digest fibrin to fibrin degradation products. been validated for clinical use to exclude the diagnosis of
The general scheme of fibrinolysis and its control is deep-venous thrombosis (DVT) and pulmonary embo-
shown in Fig. 3-4. lism in selected populations.

Bleeding and Thrombosis


The plasminogen activators, tissue type plasmino- Physiologic regulation of fibrinolysis occurs primar-
gen activator (tPA) and the urokinase type plasminogen ily at three levels: (1) plasminogen activator inhibitors
activator cleave (uPA), cleave the Arg560-Val561 bond (PAIs), specifically PAI-1 and PAI-2, inhibit the physi-
of plasminogen to generate the active enzyme plasmin. ologic plasminogen activators; (2) the thrombin-activatable
The lysine-binding sites of plasmin (and plasminogen) fibrinolysis inhibitor (TAFI) limits fibrinolysis; and (3)
permit it to bind to fibrin, so that physiologic fibrino- 2-antiplasmin inhibits plasmin. PAI1 is the primary
lysis is fibrin specific. Both plasminogen (through its inhibitor of tPA and uPA in plasma. TAFI cleaves the
lysine-binding sites) and tPA possess specific affinity for N-terminal lysine residues of fibrin, which aid in local-
fibrin and thereby bind selectively to clots. The assembly ization of plasmin activity. 2-Antiplasmin is the main
of a ternary complex, consisting of fibrin, plasminogen, inhibitor of plasmin in human plasma, inactivating any
and tPA, promotes the localized interaction between nonfibrin clotassociated plasmin.
plasminogen and tPA and greatly accelerates the rate of
plasminogen activation to plasmin. Moreover, partial
degradation of fibrin by plasmin exposes new plasmin- APPROACH TO THE
PATIENT Bleeding and Thrombosis
ogen and tPA-binding sites in carboxy-terminus lysine
residues of fibrin fragments to enhance these reactions
Clinical Presentation Disorders of hemo-
stasis may be either inherited or acquired. A detailed
personal and family history is key in determining the
chronicity of symptoms and the likelihood of the dis-
order being inherited, and it provides clues to underly-
ing conditions that have contributed to the bleeding
or thrombotic state. In addition, the history can give
UPA Plasminogen clues as to the etiology by determining (1) the bleed-
tPA ing (mucosal and/or joint) or thrombosis (arterial and/
PAI or venous) site and (2) whether an underlying bleeding
Plasmin
Thrombin or clotting tendency was enhanced by another medical
condition or the introduction of medications or dietary
2PI-Plasmin supplements.

FDPs History of Bleeding A history of bleeding is the


most important predictor of bleeding risk. In evaluating
a patient for a bleeding disorder, a history of at-risk situa-
Figure 3-4 tions, including the response to past surgeries, should be
A schematic diagram of the fibrinolytic system. Tissue assessed. Does the patient have a history of spontane-
plasminogen activator (tPA) is released from endothelial cells, ous or trauma- or surgery-induced bleeding? Spontane-
binds the fibrin clot, and activates plasminogen to plasmin. ous hemarthroses are a hallmark of moderate and severe
Excess fibrin is degraded by plasmin to distinct degradation factor VIII and IX deficiency and, in rare circumstances,
products (FDPs). Any free plasmin is complexed with 2- of other clotting factor deficiencies. Mucosal bleeding
antiplasmin (2Pl). PAI, plasminogen activator inhibitor; UPA, symptoms are more suggestive of underlying platelet
urokinase type plasminogen activator.
26 disorders or von Willebrand disease (VWD), termed dis- Epistaxis is a common symptom, particularly in children
orders of primary hemostasis or platelet plug formation. and in dry climates, and may not reflect an underly-
Disorders affecting primary hemostasis are shown in ing bleeding disorder. However, it is the most common
Table 3-1. symptom in hereditary hemorrhagic telangiectasia
A bleeding score has been validated as a tool to pre- and in boys with VWD. Clues that epistaxis is a symp-
dict patients more likely to have Type 1 VWD. Studies tom of an underlying bleeding disorder include lack of
are under way to validate additional formats, including seasonal variation and bleeding that requires medical
ones that are easier to administer and improving per- evaluation or treatment, including cauterization. Bleed-
formance in pediatric populations. Bleeding symptoms ing with eruption of primary teeth is seen in children
SECTION II

that appear to be more common in patients with bleed- with more severe bleeding disorders, such as moder-
ing disorders include prolonged bleeding with surgery, ate and severe hemophilia. It is uncommon in children
dental procedures and extractions, and/or trauma; men- with mild bleeding disorders. Patients with disorders
orrhagia or postpartum hemorrhage; and large bruises of primary hemostasis (platelet adhesion) may have
(often described with lumps). increased bleeding after dental cleanings and other pro-
Easy bruising and menorrhagia are common com- cedures that involve gum manipulation.
Cardinal Manifestations of Hematologic Disease

plaints in patients with and without bleeding disorders. Menorrhagia is defined quantitatively as a loss of
Easy bruising can also be a sign of medical conditions >80 mL of blood per cycle, based on blood loss required
in which there is no identifiable coagulopathy; instead, to produce iron-deficiency anemia. A complaint of
the conditions are caused by an abnormality of blood heavy menses is subjective and has a poor correlation
vessels or their supporting tissues. In Ehlers-Danlos with excessive blood loss. Predictors of menorrhagia
syndrome, there may be posttraumatic bleeding and a include bleeding resulting in iron-deficiency anemia or
history of joint hyperextensibility. Cushings syndrome, a need for blood transfusion, passage of clots >1 inch
chronic steroid use, and aging result in changes in skin in diameter, and changing a pad or tampon more than
and subcutaneous tissue, and subcutaneous bleeding hourly. Menorrhagia is a common symptom in women
occurs in response to minor trauma. The latter has been with underlying bleeding disorders and is reported in
termed senile purpura. the majority of women with VWD and factor XI defi-
ciency and in symptomatic carriers of hemophilia A.
Women with underlying bleeding disorders are more
likely to have other bleeding symptoms, including
Table 3-1
bleeding after dental extractions, postoperative bleed-
ing, and postpartum bleeding, and are much more
Primary Hemostatic (Platelet Plug)
Disorders
likely to have menorrhagia beginning at menarche than
women with menorrhagia due to other causes.
Defects of Platelet Adhesion
Postpartum hemorrhage (PPH) is a common symp-
Von Willebrand disease tom in women with underlying bleeding disorders.
Bernard-Soulier syndrome (absence of dysfunction of In women with type 1 VWD and symptomatic carriers
GpIb-IX-V)
of hemophilia in whom levels of VWF and FVIII usu-
Defects of Platelet Aggregation ally normalize during pregnancy, PPH may be delayed.
Glanzmanns thrombasthenia (absence or dysfunction Women with a history of postpartum hemorrhage have
ofGpIIbIIIa) a high risk of recurrence with subsequent pregnancies.
Afibrinogenemia Rupture of ovarian cysts with intraabdominal hemor-
Defects of Platelet Secretion rhage has also been reported in women with underlying
Decreased cyclooxygenase activity bleeding disorders.
Drug-induced (aspirin, nonsteroidal anti-inflammatory Tonsillectomy is a major hemostatic challenge, as
agents, thienopyridines) intact hemostatic mechanisms are essential to prevent
Inherited excessive bleeding from the tonsillar bed. Bleeding may
Granule storage pool defects occur early after surgery or after approximately 7 days
Inherited
postoperatively, with loss of the eschar at the operative
Acquired
Nonspecific inherited secretory defects site. Similar delayed bleeding is seen after colonic polyp
Nonspecific drug effects resection. Gastrointestinal (GI) bleeding and hematu-
Uremia ria are usually due to underlying pathology, and proce-
Platelet coating (e.g., paraprotein, penicillin) dures to identify and treat the bleeding site should be
Defect of Platelet Coagulant Activity undertaken, even in patients with known bleeding dis-
Scotts syndrome
orders. VWD, particularly types 2 and 3, has been associ-
ated with angiodysplasia of the bowel and GI bleeding.
Table 3-2 27
Hemarthroses and spontaneous muscle hematomas
Herbal Supplements Associated With
are characteristic of moderate or severe congenital fac-
Increased Bleeding
tor VIII or IX deficiency. They can also be seen in moder-
ate and severe deficiencies of fibrinogen; prothrombin; Herbs With Potential Antiplatelet Activity
and of factors V, VII, and X. Spontaneous hemarthro- Ginkgo (Ginkgo biloba L.)
ses occur rarely in other bleeding disorders except for Garlic (Allium sativum)
severe VWD, with associated FVIII levels <5%. Muscle Bilberry (Vaccinium myrtillus)
Ginger (Gingiber officinale)
and soft tissue bleeds are also common in acquired FVIII
Dong quai (Angelica sinensis)
deficiency. Bleeding into a joint results in severe pain Feverfew (Tanacetum parthenium)

CHAPTER 3
and swelling, as well as loss of function, but is rarely Asian ginseng (Panax ginseng)
associated with discoloration from bruising around the American ginseng (Panax quinquefolius)
joint. Life-threatening sites of bleeding include bleeding Siberian ginseng/eleuthero (Eleutherococcus senticosus)
into the oropharynx, where bleeding can obstruct the Turmeric (Circuma longa)
airway, into the central nervous system, and into the ret- Meadowsweet (Filipendula ulmaria)
Willow (Salix spp.)
roperitoneum. Central nervous system bleeding is the

Bleeding and Thrombosis


major cause of bleeding-related deaths in patients with Coumarin-Containing Herbs
severe congenital factor deficiencies. Motherworth (Leonurus cardiaca)
Chamomile (Matricaria recutita, Chamaemelum mobile)
Prohemorrhagic Effects of Medications and Horse chestnut (Aesculus hippocastanum)
Dietary Supplements Aspirin and other nonsteroidal Red clover (Trifolium pratense)
anti-inflammatory drugs (NSAIDs) that inhibit cyclooxy- Fenugreek (Trigonella foenum-graecum)
genase 1 impair primary hemostasis and may exacer-
bate bleeding from another cause or even unmask a
previously occult mild bleeding disorder such as VWD.
All NSAIDs, however, can precipitate GI bleeding, which
may be more severe in patients with underlying bleed- of a patient with a bleeding tendency must therefore
ing disorders. The aspirin effect on platelet function include a thorough assessment for evidence of under-
as assessed by aggregometry can persist for up to 7 lying disease. Bruising or mucosal bleeding may be
days, although it has frequently returned to normal by the presenting complaint in liver disease, severe renal
3 days after the last dose. The effect of other NSAIDs impairment, hypothyroidism, paraproteinemias or amy-
is shorter, as the inhibitor effect is reversed when the loidosis, and conditions causing bone marrow failure.
drug is removed. Thienopyridines (clopidogrel and pra- All coagulation factors are synthesized in the liver, and
sugrel) inhibit ADP-mediated platelet aggregation and hepatic failure results in combined factor deficiencies.
like NSAIDs can precipitate or exacerbate bleeding This is often compounded by thrombocytopenia from
symptoms. splenomegaly due to portal hypertension. Coagulation
Many herbal supplements can impair hemostatic func- factors II, VII, IX, X and proteins C, S, and Z are depen-
tion (Table 3-2). Some are more convincingly associated dent on vitamin K for posttranslational modification.
with a bleeding risk than others. Fish oil or concentrated Although vitamin K is required in both procoagulant
omega 3 fatty acid supplements impair platelet function. and anticoagulant processes, the phenotype of vitamin
They alter platelet biochemistry to produce more PGI3, a K deficiency or the warfarin effect on coagulation is
more potent platelet inhibitor than prostacyclin (PGI2), bleeding.
and more thromboxane A3, a less potent platelet acti- The normal blood platelet count is 150,000450,000/
vator than thromboxane A2. In fact, diets naturally rich L. Thrombocytopenia results from decreased pro-
in omega 3 fatty acids can result in a prolonged bleed- duction, increased destruction, and/or sequestration.
ing time and abnormal platelet aggregation studies, but Although the bleeding risk varies somewhat by the rea-
the actual associated bleeding risk is unclear. Vitamin E son for the thrombocytopenia, bleeding rarely occurs
appears to inhibit protein kinase Cmediated platelet in isolated thrombocytopenia at counts <50,000/L
aggregation and nitric oxide production. In patients with and usually not until <10,00020,000/L. Coexisting
unexplained bruising or bleeding, it is prudent to review coagulopathies, as is seen in liver failure or dissemi-
any new medications or supplements and discontinue nated coagulation, infection, platelet-inhibitory drugs,
those that may be associated with bleeding. and underlying medical conditions, can all increase the
Underlying Systemic Diseases That Cause risk of bleeding in the thrombocytopenic patient. Most
or Exacerbate a Bleeding Tendency Acquired procedures can be performed in patients with a platelet
bleeding disorders are commonly secondary to, or asso- count of 50,000/L. The level needed for major surgery
ciated with, systemic disease. The clinical evaluation will depend on the type of surgery and the patients
28 underlying medical state, although a count of approxi- 200 per year among octogenarians. Family history is
mately 80,000/L is likely sufficient. helpful in determining if there is a genetic predisposi-
tion and how strong that predisposition appears to be.
History of Thrombosis The risk of throm-
A genetic thrombophilia that confers a relatively small
bosis, like that of bleeding, is influenced by both genetic
increased risk, such as being a heterozygote for the pro-
and environmental influences. The major risk factor for
thrombin G20210A or factor V Leiden mutation, may be a
arterial thrombosis is atherosclerosis, while for venous
minor determinant of risk in an elderly individual under-
thrombosis, the risk factors are immobility, surgery,
going a high-risk surgical procedure. As illustrated in
underlying medical conditions such as malignancy,
Fig. 3-5, a thrombotic event usually has more than one
medications such as hormonal therapy, obesity, and
SECTION II

contributing factor. Predisposing factors must be care-


genetic predispositions. Factors that increase risks for
fully assessed to determine the risk of recurrent thrombo-
venous and for both venous and arterial thromboses are
sis and, with consideration of the patients bleeding risk,
shown in Table 3-3.
determine the length of anticoagulation. Similar consid-
The most important point in a history related to
eration should be given in determining the need to test
venous thrombosis is determining whether the throm-
the patient and family members for thrombophilias.
botic event was idiopathic (meaning there was no
Cardinal Manifestations of Hematologic Disease

clear precipitating factor) or was a precipitated event. Laboratory Evaluation Careful history tak-
In patients without underlying malignancy, having an ing and clinical examination are essential components
idiopathic event is the strongest predictor of recur- in the assessment of bleeding and thrombotic risk. The
rence of venous thromboembolism. In patients who use of laboratory tests of coagulation complement, but
have a vague history of thrombosis, a history of being
treated with warfarin suggests a past DVT. Age is an
important risk factor for venous thrombosisthe risk
of DVT increasing per decade, with an approximate
incidence of 1/100,000 per year in early childhood to
Thrombotic risk

OCP use
Leg in cast
HRT use
DVT
Thrombosis

Table 3-3 Surgery

Risk Factors for Thrombosis


Venous Venous and Arterial

Inherited Inherited
Factor V Leiden Homocystinuria
Prothrombin G20210A Dysfibrinogenemia
Antithrombin deficiency Mixed (inherited and iden
Protein C deficiency acquired) or V Le
Fact
Protein S deficiency Hyperhomocysteinemia
Elevated FVIII Acquired
Acquired Malignancy Age
Age Antiphospholipid antibody
Previous thrombosis syndrome Figure 3-5
Immobilization Hormonal therapy Thrombotic risk over time. Shown schematically is an
Major surgery Polycythemia vera individuals thrombotic risk over time. An underlying fac-
Pregnancy and puerperium Essential thrombocythemia tor V Leiden mutation provides a theoretically constant
Hospitalization Paroxysmal nocturnal increased risk. The thrombotic risk increases with age and,
Obesity hemoglobinuria intermittently, with oral contraceptive (OCP) or hormone
Infection APC resistance, Thrombotic thrombocyto- replacement (HRT) use; other events may increase the risk
nongenetic penic purpura further. At some point the cumulative risk may increase to the
Smoking Heparin-induced thrombo- threshold for thrombosis and result in deep-venous throm-
Unknown* cytopenia
bosis (DVT). Note: The magnitude and duration of risk por-
Elevated factor II, IX, XI Disseminated intravascular
trayed in the figure is meant for example only and may not
Elevated TAFI levels coagulation
Low levels of TFPI precisely reflect the relative risk determined by clinical study.
(From BA Konkle, A Schafer, in DP Zipes et al [eds]: Braunwalds
*
Unknown whether risk is inherited or acquired. Heart Disease, 7th ed. Philadelphia, Saunders, 2005; modified
Abbreviations: APC, activated protein C; TAFI, thrombin-activatable with permission from FR Rosendaal: Venous thrombosis: A mul-
fibrinolysis inhibitor; TFPI, tissue factor pathway inhibitor. ticausal disease. Lancet 353:1167, 1999.)
c annot substitute for, clinical assessment. No test exists The INR was developed to assess anticoagulation due 29
that provides a global assessment of hemostasis. The to reduction of vitamin Kdependent coagulation fac-
bleeding time has been used to assess bleeding risk; tors; it is commonly used in the evaluation of patients
however, it does not predict bleeding risk with surgery with liver disease. While it does allow comparison
and it is not recommended for this indication. The PFA- among laboratories, reagent sensitivity as used to deter-
100, an instrument that measures platelet-dependent mine the ISI is not the same in liver disease as with war-
coagulation under flow conditions, is more sensitive and farin anticoagulation. In addition, progressive liver fail-
specific for platelet disorders and VWD than the bleed- ure is associated with variable changes in coagulation
ing time; however it is not sensitive enough to rule out factors; the degree of prolongation of either the PT or

CHAPTER 3
underlying mild bleeding disorders. Also, its utility in pre- the INR only roughly predicts the bleeding risk. Throm-
dicting bleeding risk has not been determined. bin generation has been shown to be normal in many
For routine preoperative and preprocedure testing, patients with mild to moderate liver dysfunction. As the
an abnormal prothrombin time (PT) may detect liver dis- PT only measures one aspect of hemostasis affected by
ease or vitamin K deficiency that had not been previously liver dysfunction, we likely overestimate the bleeding
appreciated. Studies have not confirmed the usefulness risk of a mildly elevated INR in this setting.

Bleeding and Thrombosis


of an activated partial thromboplastin time (aPTT) in pre- The aPTT assesses the intrinsic and common coagula-
operative evaluations in patients with a negative bleed- tion pathways, factors XI, IX, VIII, X, V, II; fibrinogen; and
ing history. The primary use of coagulation testing should also prekallikrein, high-molecular-weight kininogen, and
be to confirm the presence and type of bleeding disorder factor XII (Fig. 3-6). The aPTT reagent contains phospho-
in a patient with a suspicious clinical history. lipids derived from either animal or vegetable sources
Because of the nature of coagulation assays, proper that function as a platelet substitute in the coagula-
sample acquisition and handling is critical to obtaining tion pathways and includes an activator of the intrinsic
valid results. In patients with abnormal coagulation coagulation system, such as nonparticulate ellagic acid
assays who have no bleeding history, repeat stud- or the particulate activators kaolin, celite, or micronized
ies with attention to these factors frequently results silica.
in normal values. Most coagulation assays are per- The phospholipid composition of aPTT reagents
formed in sodium citrate anticoagulated plasma that varies, which influences the sensitivity of individual
is recalcified for the assay. Because the anticoagulant is reagents to clotting factor deficiencies and to inhibitors
in liquid solution and needs to be added to blood in such as heparin and lupus anticoagulants. Thus, aPTT
proportion to the plasma volume, incorrectly filled or
inadequately mixed blood collection tubes will give
erroneous results. Vacutainer tubes should be filled
to >90% of the recommended fill, which is usually aPTT
denoted by a line on the tube. An elevated hematocrit
(>55%) can result in a false value due to a decreased HMWK
PT
plasma to anticoagulant ratio.
PK
Screening Assays The most commonly used
FXII FVII
screening tests are the PT, aPTT, and platelet count.
The PT assesses the factors I (fibrinogen), II (prothrom- FXI
bin), V, VII, and X (Fig. 3-6). The PT measures the time FIX
for clot formation of the citrated plasma after recalcifi-
cation and addition of thromboplastin, a mixture of TF FVIII
FX
and phospholipids. The sensitivity of the assay varies by
the source of thromboplastin. The relationship between FV
defects in secondary hemostasis (fibrin formation) and
Prothrombin (FII)
coagulation test abnormalities is shown in Table 3-4.
To adjust for this variability, the overall sensitivity of Fibrinogen (FI)
different thromboplastins to reduction of the vitamin
Kdependent clotting factors II, VII, IX, and X in antico-
agulation patients is now expressed as the International
Sensitivity Index (ISI). An inverse relationship exists Figure 3-6
between ISI and thromboplastin sensitivity. The interna- Coagulation factor activity tested in the activated partial
tional normalized ratio (INR) is then determined based thromboplastin time (aPTT) in red and prothrombin time (PT)
on the formula: INR = (PTpatient/PTnormal mean)ISI. in green, or both. F, factor; HMWK, high-molecular-weight
kininogen; PK, prekallikrein.
30 Table 3-4
between a factor deficiency and an inhibitor. In this
Hemostatic Disorders and Coagulation Test
assay, normal plasma and patient plasma are mixed in
Abnormalities
a 1:1 ratio, and the aPTT or PT is determined immediately
Prolonged Activated Partial Thromboplastin Time and after incubation at 37C for varying times, typically
(aPTT)
30, 60, and/or 120 min. With isolated factor deficiencies,
No clinical bleeding factors XII, high-molecular-weight the aPTT will correct with mixing and stay corrected with
kininogen, prekallikrein incubation. With aPTT prolongation due to a lupus anti-
Variable, but usually mild, bleeding factor XI, mild
coagulant, the mixing and incubation will show no cor-
FVIII and FIX
Frequent, severe bleedingsevere deficiencies of FVIII rection. In acquired neutralizing factor antibodies, such
SECTION II

and FIX as an acquired factor VIII inhibitor, the initial assay may
Heparin or may not correct immediately after mixing but will pro-
Prolonged Prothrombin Time (PT) long or remain prolonged with incubation at 37C. Fail-
ure to correct with mixing can also be due to the pres-
Factor VII deficiency
Vitamin K deficiencyearly ence of other inhibitors or interfering substances such as
Warfarin anticoagulation heparin, fibrin split products, and paraproteins.
Cardinal Manifestations of Hematologic Disease

Prolonged aPTT and PT Specific Factor Assays Decisions to proceed


Factor II, V, X, or fibrinogen deficiency with specific clotting factor assays will be influenced
Vitamin K deficiencylate by the clinical situation and the results of coagulation
Direct thrombin inhibitors screening tests. Precise diagnosis and effective manage-
Prolonged Thrombin Time ment of inherited and acquired coagulation deficiencies
Heparin or heparin-like inhibitors necessitate quantitation of the relevant factors. When
Mild or no bleedingdysfibrinogenemia bleeding is severe, specific assays are often urgently
Frequent, severe bleedingafibrinogenemia required to guide appropriate therapy. Individual fac-
Prolonged PT and/or aPTT Not Correct with Mixing tor assays are usually performed as modifications of the
with Normal Plasma mixing study, where the patients plasma is mixed with
Bleedingspecific factor inhibitor plasma deficient in the factor being studied. This will
No symptoms, or clotting and/or pregnancy losslupus correct all factor deficiencies to >50%, thus making pro-
anticoagulant longation of clot formation due to a factor deficiency
Disseminated intravascular coagulation dependent on the factor missing from the added plasma.
Heparin or direct thrombin inhibitor
Testing for Antiphospholipid Antibodies Anti
Abnormal Clot Solubility
bodies to phospholipids (cardiolipin) or phospholipid-
Factor XIII deficiency binding proteins (2-microglobulin and others) are
Inhibitors or defective cross-linking
detected by enzyme-linked immunosorbent assay
Rapid Clot Lysis (ELISA). When these antibodies interfere with phos-
Deficiency of 2-antiplasmin or plasminogen activator pholipid-dependent coagulation tests, they are termed
inhibitor 1 lupus anticoagulants. The aPTT has variability sensitivity
Treatment with fibrinolytic therapy to lupus anticoagulants, depending in part on the aPTT
reagents used. An assay using a sensitive reagent has
been termed an LA-PTT. The dilute Russell viper venom
test (dRVVT) and the tissue thromboplastin inhibition
results will vary from one laboratory to another, and the (TTI) test are modifications of standard tests with the
normal range in the laboratory where the testing occurs phospholipid reagent decreased, thus increasing the sen-
should be used in the interpretation. Local laboratories sitivity to antibodies that interfere with the phospholipid
can relate their aPTT values to the therapeutic heparin component. The tests, however, are not specific for lupus
anticoagulation by correlating aPTT values with direct anticoagulants, as factor deficiencies or other inhibitors
measurements of heparin activity (anti-Xa or protamine will also result in prolongation. Documentation of a lupus
titration assays) in samples from heparinized patients, anticoagulant requires not only prolongation of a phos-
although correlation between these assays is often pholipid-dependent coagulation test but also lack of cor-
poor. The aPTT reagent will vary in sensitivity to individ- rection when mixed with normal plasma and correction
ual factor deficiencies and usually becomes prolonged with the addition of activated platelet membranes or cer-
with individual factor deficiencies of 3050%. tain phospholipids, e.g. hexagonal phase.

Mixing Studies Mixing studies are used to evaluate Other Coagulation Tests The thrombin time
a prolonged aPTT or, less commonly PT, to distinguish and the reptilase time measure fibrinogen conversion
to fibrin and are prolonged when the fibrinogen level of treatment, and testing performed at least 3 weeks 31
is low (usually <80100 mg/dL) or qualitatively abnor- later. Sensitive markers of coagulation activation, nota-
mal, as seen in inherited or acquired dysfibrinogen- bly the D-dimer assay and the thrombin generation test,
emias, or when fibrin/fibrinogen degradation products hold promise as predictors, when elevated, of recurrent
interfere. The thrombin time, but not the reptilase thrombosis when measured at least 1 month from dis-
time, is prolonged in the presence of heparin. Measure- continuation of warfarin.
ment of antifactor Xa plasma inhibitory activity is a
Measures of Platelet Function The bleeding
test frequently used to assess low-molecular-weight
time has been used to assess bleeding risk; however, it
heparin (LMWH) levels or as a direct measurement of

CHAPTER 3
has not been found to predict bleeding risk with sur-
unfractionated heparin (UFH) activity. Heparin in the
gery, and it is not recommended for use for this indica-
patient sample inhibits the enzymatic conversion of a
tion. The PFA-100 and similar instruments that measure
Xa-specific chromogenic substrate to colored product
platelet-dependent coagulation under flow conditions
by factor Xa. Standard curves are created using mul-
are generally more sensitive and specific for platelet
tiple concentrations of UFH and LMWH and are used
disorders and VWD than the bleeding time; however,
to calculate the concentration of anti-Xa activity in the
data are insufficient to support their use to predict

Bleeding and Thrombosis


patient plasma.
bleeding risk or monitor response to therapy. When
Laboratory Testing for Thrombophilia Lab- they are used in the evaluation of a patient with bleed-
oratory assays to detect thrombophilic states include ing symptoms, abnormal results, as with the bleeding
molecular diagnostics and immunologic and func- time, require specific testing, such as VWF assays and/or
tional assays. These assays vary in their sensitivity and platelet aggregation studies. Since all of these screen-
specificity for the condition being tested. Furthermore, ing assays may miss patients with mild bleeding disor-
acute thrombosis, acute illnesses, inflammatory condi- ders, further studies are needed to define their role in
tions, pregnancy, and medications affect levels of many hemostasis testing.
coagulation factors and their inhibitors. Antithrombin is For classic platelet aggregometry, various agonists
decreased by heparin and in the setting of acute throm- are added to the patients platelet-rich plasma, and
bosis. Protein C and S levels may be increased in the set- platelet aggregation is observed. Tests of platelet secre-
ting of acute thrombosis and are decreased by warfarin. tion in response to agonists can also be measured.
Antiphospholipid antibodies are frequently transiently These tests are affected by many factors, including
positive in acute illness. Testing for genetic thrombo- numerous medications, and the association between
philias should, in general, only be performed when minor defects in aggregation or secretion in these
there is a strong family history of thrombosis and results assays and bleeding risk is not clearly established.
would affect clinical decision making.
Because thrombophilia evaluations are usually per-
formed to assess the need to extend anticoagulation, Acknowledgment
testing should be performed in a steady state, remote
Robert I. Handin, MD, contributed this chapter in the 16th edi-
from the acute event. In most instances, warfarin anti-
tion of Harrisons Principles of Internal Medicine and some mate-
coagulation can be stopped after the initial 36 months
rial from that chapter has been retained here.
CHapTeR 4

ENLARGEMENT OF LYMPH NODES


AND SPLEEN

Patrick H. Henry Dan L. Longo

This chapter is intended to serve as a guide to the or metastatic adenocarcinoma). Of the patients with
evaluation of patients who present with enlargement of benign lymphadenopathy, 63% had a nonspecific or
the lymph nodes (lymphadenopathy) or the spleen (spleno- reactive etiology (no causative agent found), and the
megaly). Lymphadenopathy is a rather common clinical remainder had a specific cause demonstrated, most
finding in primary care settings, whereas palpable spleno- commonly infectious mononucleosis, toxoplasmosis,
megaly is less so. or tuberculosis. Thus, the vast majority of patients with
lymphadenopathy will have a nonspecific etiology
requiring few diagnostic tests.
lympHaDenopaTHy
CLInICaL assessment The physician will be
Lymphadenopathy may be an incidental finding in aided in the pursuit of an explanation for the lymphade-
patients being examined for various reasons, or it may nopathy by a careful medical history, physical examina-
be a presenting sign or symptom of the patients ill- tion, selected laboratory tests, and perhaps an excisional
ness. The physician must eventually decide whether lymph node biopsy.
the lymphadenopathy is a normal finding or one that The medical history should reveal the setting in which
requires further study, up to and including biopsy. Soft, lymphadenopathy is occurring. Symptoms such as sore
flat, submandibular nodes (<1 cm) are often palpable in throat, cough, fever, night sweats, fatigue, weight loss,
healthy children and young adults; healthy adults may or pain in the nodes should be sought. The patients
have palpable inguinal nodes of up to 2 cm, which are age, sex, occupation, exposure to pets, sexual behavior,
considered normal. Further evaluation of these nor- and use of drugs such as diphenylhydantoin are other
mal nodes is not warranted. In contrast, if the physician important historic points. For example, children and
believes the node(s) to be abnormal, then pursuit of a young adults usually have benign (i.e., nonmalignant)
more precise diagnosis is needed. disorders that account for the observed lymphadenop-
athy such as viral or bacterial upper respiratory infec-
tions; infectious mononucleosis; toxoplasmosis; and,
aPPROaCH TO THE
PATIENT Lymphadenopathy in some countries, tuberculosis. In contrast, after age
50 years, the incidence of malignant disorders increases
Lymphadenopathy may be a primary or secondary and that of benign disorders decreases.
manifestation of numerous disorders, as shown in The physical examination can provide useful clues
Table 4-1. Many of these disorders are infrequent such as the extent of lymphadenopathy (localized or
causes of lymphadenopathy. In primary care practice, generalized), size of nodes, texture, presence or absence
more than two-thirds of patients with lymphadenopa- of nodal tenderness, signs of inflammation over the
thy have nonspecific causes or upper respiratory ill- node, skin lesions, and splenomegaly. A thorough ear,
nesses (viral or bacterial) and <1% have a malignancy. nose, and throat (ENT) examination is indicated in adult
In one study, 84% of patients referred for evalua- patients with cervical adenopathy and a history of
tion of lymphadenopathy had a benign diagnosis. tobacco use. Localized or regional adenopathy implies
The remaining 16% had a malignancy (lymphoma involvement of a single anatomic area. Generalized

32
Table 4-1 33
adenopathy has been defined as involvement of three
Diseases Associated With Lymphadenopathy or more noncontiguous lymph node areas. Many of the
1. Infectious diseases causes of lymphadenopathy (Table 4-1) can produce
a. Viralinfectious mononucleosis syndromes (EBV, localized or generalized adenopathy, so this distinction
CMV), infectious hepatitis, herpes simplex, herpes- is of limited utility in the differential diagnosis. Never-
virus-6, varicella-zoster virus, rubella, measles, ade-
theless, generalized lymphadenopathy is frequently
novirus, HIV, epidemic keratoconjunctivitis, vaccinia,
herpesvirus-8 associated with nonmalignant disorders such as infec-
b. Bacterialstreptococci, staphylococci, cat-scratch tious mononucleosis (Epstein-Barr virus [EBV] or cyto-
disease, brucellosis, tularemia, plague, chancroid, megalovirus [CMV]), toxoplasmosis, AIDS, other viral

CHAPTER 4
melioidosis, glanders, tuberculosis, atypical myco- infections, systemic lupus erythematosus (SLE), and
bacterial infection, primary and secondary syphilis, mixed connective tissue disease. Acute and chronic lym-
diphtheria, leprosy phocytic leukemias and malignant lymphomas also pro-
c. Fungalhistoplasmosis, coccidioidomycosis, para-
duce generalized adenopathy in adults.
coccidioidomycosis
d. Chlamydiallymphogranuloma venereum, trachoma The site of localized or regional adenopathy may pro-
e. Parasitictoxoplasmosis, leishmaniasis, trypanoso- vide a useful clue about the cause. Occipital adenopathy

Enlargement of Lymph Nodes andSpleen


miasis, filariasis often reflects an infection of the scalp, and preauricular
f. Rickettsialscrub typhus, rickettsialpox, Q fever adenopathy accompanies conjunctival infections and
.Immunologic diseases
2 cat-scratch disease. The most frequent site of regional
a. Rheumatoid arthritis adenopathy is the neck, and most of the causes are
b. Juvenile rheumatoid arthritis benignupper respiratory infections, oral and den-
c. Mixed connective tissue disease tal lesions, infectious mononucleosis, or other viral ill-
d. Systemic lupus erythematosus nesses. The chief malignant causes include metastatic
e. Dermatomyositis
cancer from head and neck, breast, lung, and thyroid
f. Sjgrens syndrome
g. Serum sickness primaries. Enlargement of supraclavicular and scalene
h. Drug hypersensitivitydiphenylhydantoin, hydrala- nodes is always abnormal. Because these nodes drain
zine, allopurinol, primidone, gold, carbamazepine, etc. regions of the lung and retroperitoneal space, they
i. Angioimmunoblastic lymphadenopathy can reflect lymphomas, other cancers, or infectious
j. Primary biliary cirrhosis processes arising in these areas. Virchows node is an
k. Graft-vs.-host disease enlarged left supraclavicular node infiltrated with meta-
l. Silicone-associated
static cancer from a gastrointestinal primary. Metastases
m. Autoimmune lymphoproliferative syndrome
to supraclavicular nodes also occur from lung, breast,
3.Malignant diseases testis, or ovarian cancers. Tuberculosis, sarcoidosis, and
a. HematologicHodgkins disease, non-Hodgkins lym-
toxoplasmosis are nonneoplastic causes of supracla-
phomas, acute or chronic lymphocytic leukemia, hairy
cell leukemia, malignant histiocytosis, amyloidosis vicular adenopathy. Axillary adenopathy is usually due
b. Metastaticfrom numerous primary sites to injuries or localized infections of the ipsilateral upper
extremity. Malignant causes include melanoma or lym-
4.Lipid storage diseasesGauchers, Niemann-Pick,
Fabry, Tangier phoma and, in women, breast cancer. Inguinal lymph-
adenopathy is usually secondary to infections or trauma
5.Endocrine diseaseshyperthyroidism
of the lower extremities and may accompany sexually
6.Other disorders transmitted diseases such as lymphogranuloma vene-
a. Castlemans disease (giant lymph node hyperplasia)
reum, primary syphilis, genital herpes, or chancroid.
b. Sarcoidosis
c. Dermatopathic lymphadenitis
These nodes may also be involved by lymphomas and
d. Lymphomatoid granulomatosis metastatic cancer from primary lesions of the rectum,
e. Histiocytic necrotizing lymphadenitis (Kikuchis disease) genitalia, or lower extremities (melanoma).
f. Sinus histiocytosis with massive lymphadenopathy The size and texture of the lymph node(s) and the
(Rosai-Dorfman disease) presence of pain are useful parameters in evaluat-
g. Mucocutaneous lymph node syndrome (Kawasakis ing a patient with lymphadenopathy. Nodes <1.0 cm2
disease) in area (1.0 cm 1.0 cm or less) are almost always sec-
h. Histiocytosis X
ondary to benign, nonspecific reactive causes. In one
i. Familial Mediterranean fever
j. Severe hypertriglyceridemia retrospective analysis of younger patients (925 years)
k. Vascular transformation of sinuses who had a lymph node biopsy, a maximum diameter
l. Inflammatory pseudotumor of lymph node of >2 cm served as one discriminant for predicting
m. Congestive heart failure that the biopsy would reveal malignant or granuloma-
tous disease. Another study showed that a lymph node
Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus. size of 2.25 cm2 (1.5 cm 1.5 cm) was the best size
34 limit for distinguishing malignant or granulomatous the patients history and physical findings. One study
lymphadenopathy from other causes of lymphadenopa- from a family practice clinic evaluated 249 younger
thy. Patients with node(s) 1.0 cm2 should be observed patients with enlarged lymph nodes, not infected or
after excluding infectious mononucleosis and/or toxo- lymphadenitis. No laboratory studies were obtained in
plasmosis unless there are symptoms and signs of an 51%. When studies were performed, the most common
underlying systemic illness. were a complete blood count (CBC) (33%), throat cul-
The texture of lymph nodes may be described as ture (16%), chest x-ray (12%), or monospot test (10%).
soft, firm, rubbery, hard, discrete, matted, tender, mov- Only eight patients (3%) had a node biopsy, and half of
able, or fixed. Tenderness is found when the capsule is those were normal or reactive. The CBC can provide use-
SECTION II

stretched during rapid enlargement, usually secondary ful data for the diagnosis of acute or chronic leukemias,
to an inflammatory process. Some malignant diseases EBV or CMV mononucleosis, lymphoma with a leukemic
such as acute leukemia may produce rapid enlargement component, pyogenic infections, or immune cytopenias
and pain in the nodes. Nodes involved by lymphoma in illnesses such as SLE. Serologic studies may demon-
tend to be large, discrete, symmetric, rubbery, firm, strate antibodies specific to components of EBV, CMV,
mobile, and nontender. Nodes containing metastatic HIV, and other viruses; Toxoplasma gondii; Brucella; etc.
Cardinal Manifestations of Hematologic Disease

cancer are often hard, nontender, and nonmovable If SLE is suspected, antinuclear and anti-DNA antibody
because of fixation to surrounding tissues. The coexis- studies are warranted.
tence of splenomegaly in the patient with lymphade- The chest x-ray findings are usually negative, but the
nopathy implies a systemic illness such as infectious presence of a pulmonary infiltrate or mediastinal lymph-
mononucleosis, lymphoma, acute or chronic leukemia, adenopathy would suggest tuberculosis, histoplasmosis,
SLE, sarcoidosis, toxoplasmosis, cat-scratch disease, or sarcoidosis, lymphoma, primary lung cancer, or meta-
other less common hematologic disorders. The patients static cancerand demands further investigation.
story should provide helpful clues about the underlying A variety of imaging techniques (computed tomog-
systemic illness. raphy [CT], magnetic resonance imaging [MRI], ultraso-
Nonsuperficial presentations (thoracic or abdomi- nography color Doppler ultrasonography) have been
nal) of adenopathy are usually detected as the result of employed to differentiate benign from malignant lymph
a symptom-directed diagnostic workup. Thoracic ade- nodes, especially in patients with head and neck can-
nopathy may be detected by routine chest radiography cer. CT and MRI are comparably accurate (6590%) in
or during the workup for superficial adenopathy. It may the diagnosis of metastases to cervical lymph nodes.
also be found because the patient complains of a cough Ultrasonography has been used to determine the long
or wheezing from airway compression; hoarseness (L) axis, short (S) axis, and a ratio of long to short axis in
from recurrent laryngeal nerve involvement; dysphagia cervical nodes. An L/S ratio of <2.0 has a sensitivity and a
from esophageal compression; or swelling of the neck, specificity of 95% for distinguishing benign and malig-
face, or arms secondary to compression of the superior nant nodes in patients with head and neck cancer. This
vena cava or subclavian vein. The differential diagnosis ratio has greater specificity and sensitivity than palpation
of mediastinal and hilar adenopathy includes primary or measurement of either the long or the short axis alone.
lung disorders and systemic illnesses that characteris- The indications for lymph node biopsy are impre-
tically involve mediastinal or hilar nodes. In the young, cise, yet it is a valuable diagnostic tool. The decision
mediastinal adenopathy is associated with infectious to biopsy may be made early in a patients evaluation
mononucleosis and sarcoidosis. In endemic regions, or delayed for up to 2 weeks. Prompt biopsy should
histoplasmosis can cause unilateral paratracheal lymph occur if the patients history and physical findings sug-
node involvement that mimics lymphoma. Tuberculosis gest a malignancy; examples include a solitary, hard,
can also cause unilateral adenopathy. In older patients, nontender cervical node in an older patient who is a
the differential diagnosis includes primary lung cancer chronic user of tobacco; supraclavicular adenopathy;
(especially among smokers), lymphomas, metastatic and solitary or generalized adenopathy that is firm,
carcinoma (usually lung), tuberculosis, fungal infection, movable, and suggestive of lymphoma. If a primary
and sarcoidosis. head and neck cancer is suspected as the basis of a soli-
Enlarged intraabdominal or retroperitoneal nodes tary, hard cervical node, then a careful ENT examination
are usually malignant. Although tuberculosis may pres- should be performed. Any mucosal lesion that is suspi-
ent as mesenteric lymphadenitis, these masses usually cious for a primary neoplastic process should be biop-
contain lymphomas or, in young men, germ cell tumors. sied first. If no mucosal lesion is detected, an excisional
biopsy of the largest node should be performed. Fine-
Laboratory Investigation The laboratory needle aspiration should not be performed as the first
investigation of patients with lymphadenopathy must diagnostic procedure. Most diagnoses require more
be tailored to elucidate the etiology suspected from tissue than such aspiration can provide, and it often
delays a definitive diagnosis. Fine-needle aspiration Splenomegaly 35
should be reserved for thyroid nodules and for confir-
mation of relapse in patients whose primary diagnosis Structure and Function
is known. If the primary physician is uncertain about oftheSpleen
whether to proceed to biopsy, consultation with a The spleen is a reticuloendothelial organ that has its
hematologist or medical oncologist should be helpful. embryologic origin in the dorsal mesogastrium at
In primary care practices, <5% of lymphadenopathy about 5 weeks gestation. It arises in a series of hillocks,
patients will require a biopsy. That percentage will be migrates to its normal adult location in the left upper
considerably larger in referral practices, i.e., hematol- quadrant (LUQ), and is attached to the stomach via the

CHAPTER 4
ogy, oncology, or ENT. gastrolienal ligament and to the kidney via the lienore-
Two groups have reported algorithms that they nal ligament. When the hillocks fail to unify into a sin-
claim will identify more precisely those lymphadenopa- gle tissue mass, accessory spleens may develop in around
thy patients who should have a biopsy. Both reports 20% of persons. The function of the spleen has been
were retrospective analyses in referral practices. The elusive. Galen believed it was the source of black bile
first study involved patients 925 years of age who or melancholia, and the word hypochondria (literally,

Enlargement of Lymph Nodes andSpleen


had a node biopsy performed. Three variables were beneath the ribs) and the idiom to vent ones spleen
identified that predicted those young patients with attest to the beliefs that the spleen had an important
peripheral lymphadenopathy who should undergo influence on the psyche and emotions. In humans, its
biopsy; lymph node size >2 cm in diameter and abnor- normal physiologic roles seem to be the following:
mal chest x-ray had positive predictive values, whereas
recent ENT symptoms had negative predictive values. 1. Maintenance of quality control over erythrocytes in
The second study evaluated 220 lymphadenopathy the red pulp by removal of senescent and defective
patients in a hematology unit and identified five vari- red blood cells. The spleen accomplishes this function
ables (lymph node size, location [supraclavicular or through a unique organization of its parenchyma and
nonsupraclavicular], age [>40 years or <40 years], tex- vasculature (Fig. 4-1).
ture [nonhard or hard], and tenderness) that were used 2. Synthesis of antibodies in the white pulp.
in a mathematical model to identify patients requiring 3. Removal of antibody-coated bacteria and antibody-
a biopsy. Positive predictive value was found for age coated blood cells from the circulation.
>40 years, supraclavicular location, node size >2.25 cm2, An increase in these normal functions may result in
hard texture, and lack of pain or tenderness. Negative splenomegaly.
predictive value was evident for age <40 years, node The spleen is composed of red pulp and white pulp,
size <1.0 cm2, nonhard texture, and tender or pain- which are Malpighis terms for the red bloodfilled
ful nodes. Ninety-one percent of those who required sinuses and reticuloendothelial celllined cords and the
biopsy were correctly classified by this model. Because white lymphoid follicles arrayed within the red pulp
both of these studies were retrospective analyses and matrix. The spleen is in the portal circulation. The rea-
one was limited to young patients, it is not known how son for this is unknown but may relate to the fact that
useful these models would be if applied prospectively in lower blood pressure allows less rapid flow and mini-
a primary care setting. mizes damage to normal erythrocytes. Blood flows into
Most lymphadenopathy patients do not require a the spleen at a rate of about 150 mL/min through the
biopsy, and at least half require no laboratory studies. splenic artery, which ultimately ramifies into central
If the patients history and physical findings point to a arterioles. Some blood goes from the arterioles to cap-
benign cause for lymphadenopathy, careful follow-up illaries and then to splenic veins and out of the spleen,
at a 2- to 4-week interval can be employed. The patient but the majority of blood from central arterioles flows
should be instructed to return for reevaluation if there into the macrophage-lined sinuses and cords. The blood
is an increase in the size of the nodules. Antibiotics are entering the sinuses reenters the circulation through
not indicated for lymphadenopathy unless strong evi- the splenic venules, but the blood entering the cords
dence of a bacterial infection is present. Glucocorticoids is subjected to an inspection of sorts. To return to the
should not be used to treat lymphadenopathy because circulation, the blood cells in the cords must squeeze
their lympholytic effect obscures some diagnoses (lym- through slits in the cord lining to enter the sinuses that
phoma, leukemia, Castlemans disease), and they con- lead to the venules. Old and damaged erythrocytes are
tribute to delayed healing or activation of underlying less deformable and are retained in the cords, where
infections. An exception to this statement is the life- they are destroyed and their components recycled. Red
threatening pharyngeal obstruction by enlarged lym- cellinclusion bodies such as parasites, nuclear residua
phoid tissue in Waldeyers ring that is occasionally seen (Howell-Jolly bodies, or denatured hemoglobin (Heinz
in infectious mononucleosis. bodies) are pinched off in the process of passing through
36 Central artery and particulates from the blood, (2) the generation of
Primary follicle immune responses to certain pathogens, and (3) the
(B cell area) Secondary follicle with generation of cellular components of the blood under
germinal center
(B cell area)
circumstances in which the marrow is unable to meet
Lymphoid
T cell area the needs (i.e., extramedullary hematopoiesis). The latter
Marginal lymphoid
zone
adaptation is a recapitulation of the blood-forming func-
tion the spleen plays during gestation. In some animals,
Arterial capillaries the spleen also serves a role in the vascular adaptation to
Pulp sinus stress because it stores red blood cells (often hemocon-
Pulp sinus centrated to higher hematocrits than normal) under
SECTION II

Pulp cord
normal circumstances and contracts under the influ-
Pulp cord
ence of -adrenergic stimulation to provide the animal
with an autotransfusion and improved oxygen-carrying
Splenic venous system capacity. However, the normal human spleen does not
sequester or store red blood cells and does not contract
in response to sympathetic stimuli. The normal human
Cardinal Manifestations of Hematologic Disease

Arterioles
spleen contains approximately one-third of the total
Sinusoidal body platelets and a significant number of marginated
pores
neutrophils. These sequestered cells are available when
needed to respond to bleeding or infection.

aPPROACH TO THE
PATIENT Splenomegaly

Pulp Clinical Assessment The most common symp-


cord
Sinusoids toms produced by diseases involving the spleen are
Pulp sinuses
pain and a heavy sensation in the LUQ. Massive sple-
nomegaly may cause early satiety. Pain may result from
RE cells
acute swelling of the spleen with stretching of the cap-
sule, infarction, or inflammation of the capsule. For
To splenic
venous
many years, it was believed that splenic infarction was
system clinically silent, which, at times, is true. However, Soma
Weiss, in his classic 1942 report of the self-observa-
Figure 4-1
tions by a Harvard medical student on the clinical
Schematic spleen structure. The spleen comprises many
course of subacute bacterial endocarditis, documented
units of red and white pulp centered around small branches
that severe LUQ and pleuritic chest pain may accom-
of the splenic artery, called central arteries. White pulp is
pany thromboembolic occlusion of splenic blood flow.
lymphoid in nature and contains B cell follicles, a marginal
zone around the follicles, and T cellrich areas sheathing
Vascular occlusion, with infarction and pain, is com-
arterioles. The red pulp areas include pulp sinuses and pulp monly seen in children with sickle cell crises. Rupture
cords. The cords are dead ends. In order to regain access to of the spleen, from either trauma or infiltrative disease
the circulation, red blood cells must traverse tiny openings in that breaks the capsule, may result in intraperitoneal
the sinusoidal lining. Stiff, damaged, or old red cells cannot bleeding, shock, and death. The rupture itself may be
enter the sinuses. RE, reticuloendthelial. (Bottom portion of painless.
figure from RS Hillman, KA Ault: Hematology in Clinical Prac- A palpable spleen is the major physical sign produced
tice, 4th ed. New York, McGraw-Hill, 2005.) by diseases affecting the spleen and suggests enlarge-
ment of the organ. The normal spleen weighs <250 g,
decreases in size with age, normally lies entirely within
the slits, a process called pitting. The culling of dead the rib cage, has a maximum cephalocaudad diameter
and damaged cells and the pitting of cells with inclu- of 13 cm by ultrasonography or maximum length of
sions appear to occur without significant delay because 12 cm and/or width of 7 cm by radionuclide scan, and
the blood transit time through the spleen is only slightly is usually not palpable. However, a palpable spleen
slower than in other organs. was found in 3% of 2200 asymptomatic, male, fresh-
The spleen is also capable of assisting the host in man college students. Follow-up at 3 years revealed
adapting to its hostile environment. It has at least that 30% of those students still had a palpable spleen
three adaptive functions: (1) clearance of bacteria without any increase in disease prevalence. Ten-year
follow-up found no evidence for lymphoid malignan- margin inferiorly. The patient is supine with the left 37
cies. Furthermore, in some tropical countries (e.g., New arm slightly abducted. During normal breathing, this
Guinea), the incidence of splenomegaly may reach 60%. space is percussed from medial to lateral margins,
Thus, the presence of a palpable spleen does not always yielding a normal resonant sound. A dull percussion
equate with presence of disease. Even when disease is note suggests splenomegaly.
present, splenomegaly may not reflect the primary dis-
Studies comparing methods of percussion and palpation
ease but rather a reaction to it. For example, in patients
with a standard of ultrasonography or scintigraphy have
with Hodgkins disease, only two-thirds of the palpable
revealed sensitivity of 5671% for palpation and
spleens show involvement by the cancer.

CHAPTER 4
5982% for percussion. Reproducibility among examin-
Physical examination of the spleen uses primarily the
ers is better for palpation than percussion. Both tech-
techniques of palpation and percussion. Inspection may
niques are less reliable in obese patients and patients
reveal fullness in the LUQ that descends on inspiration,
who have just eaten. Thus, the physical examination
a finding associated with a massively enlarged spleen.
techniques of palpation and percussion are imprecise
Auscultation may reveal a venous hum or friction rub.
at best. It has been suggested that the examiner per-
Palpation can be accomplished by bimanual palpa-
form percussion first and, if results are positive, proceed

Enlargement of Lymph Nodes andSpleen


tion, ballotment, and palpation from above (Middle-
to palpation; if the spleen is palpable, then one can be
ton maneuver). For bimanual palpation, which is at
reasonably confident that splenomegaly exists. How-
least as reliable as the other techniques, the patient is
ever, not all LUQ masses are enlarged spleens; gastric
supine with flexed knees. The examiners left hand is
or colon tumors and pancreatic or renal cysts or tumors
placed on the lower rib cage and pulls the skin toward
can mimic splenomegaly.
the costal margin, allowing the fingertips of the right
The presence of an enlarged spleen can be more
hand to feel the tip of the spleen as it descends while
precisely determined, if necessary, by liverspleen
the patient inspires slowly, smoothly, and deeply. Pal-
radionuclide scan, CT, MRI, or ultrasonography. The
pation is begun with the right hand in the left lower
latter technique is the current procedure of choice for
quadrant with gradual movement toward the left costal
routine assessment of spleen size (normal = a maxi-
margin, thereby identifying the lower edge of a mas-
mum cephalocaudad diameter of 13 cm) because it
sively enlarged spleen. When the spleen tip is felt, the
has high sensitivity and specificity and is safe, nonin-
finding is recorded as centimeters below the left costal
vasive, quick, mobile, and less costly. Nuclear medicine
margin at some arbitrary point, e.g. 1015 cm, from the
scans are accurate, sensitive, and reliable but are costly,
midpoint of the umbilicus or the xiphisternal junction.
require greater time to generate data, and use immo-
This allows other examiners to compare findings or the
bile equipment. They have the advantage of demon-
initial examiner to determine changes in size over time.
strating accessory splenic tissue. CT and MRI provide
Bimanual palpation in the right lateral decubitus posi-
accurate determination of spleen size, but the equip-
tion adds nothing to the supine examination.
ment is immobile and the procedures are expensive.
Percussion for splenic dullness is accomplished with any
MRI appears to offer no advantage over CT. Changes in
of three techniques described by Nixon, Castell, or Barkun:
spleen structure such as mass lesions, infarcts, inhomo-
1. Nixons method: The patient is placed on the right side geneous infiltrates, and cysts are more readily assessed
so that the spleen lies above the colon and stomach. by CT, MRI, or ultrasonography. None of these tech-
Percussion begins at the lower level of pulmonary niques is very reliable in the detection of patchy infiltra-
resonance in the posterior axillary line and proceeds tion (e.g., Hodgkins disease).
diagonally along a perpendicular line toward the
lower midanterior costal margin. The upper border of Differential Diagnosis Many of the diseases
dullness is normally 68 cm above the costal margin. associated with splenomegaly are listed in Table 4-2.
Dullness >8 cm in an adult is presumed to indicate They are grouped according to the presumed basic
splenic enlargement. mechanisms responsible for organ enlargement:
2. Castells method: With the patient supine, percussion 1. Hyperplasia or hypertrophy related to a particular
in the lowest intercostal space in the anterior axillary splenic function such as reticuloendothelial hyperpla-
line (8th or 9th) produces a resonant note if the spleen sia (work hypertrophy) in diseases such as hereditary
is normal in size. This is true during expiration or full spherocytosis or thalassemia syndromes that require
inspiration. A dull percussion note on full inspiration removal of large numbers of defective red blood cells,
suggests splenomegaly. immune hyperplasia in response to systemic infec-
3. Percussion of Traubes semilunar space: The borders tion (infectious mononucleosis, subacute bacterial
of Traubes space are the sixth rib superiorly, the endocarditis) and or immunologic diseases (immune
left midaxillary line laterally, and the left costal thrombocytopenia, SLE, Feltys syndrome).
38 Table 4-2
Diseases Associated With Splenomegaly Grouped by Pathogenic Mechanism
Enlargement Due to Increased Demand for Splenic Function
Reticuloendothelial system hyperplasia (for removal Malaria
of defective erythrocytes) Leishmaniasis
Spherocytosis Trypanosomiasis
Early sickle cell anemia Ehrlichiosis
Ovalocytosis Disordered immunoregulation
Thalassemia major Rheumatoid arthritis (Feltys syndrome)
Hemoglobinopathies Systemic lupus erythematosus
SECTION II

Paroxysmal nocturnal hemoglobinuria Collagen vascular diseases


Pernicious anemia Serum sickness
Immune hyperplasia Immune hemolytic anemias
Response to infection (viral, bacterial, fungal, parasitic) Immune thrombocytopenias
Infectious mononucleosis Immune neutropenias
AIDS Drug reactions
Viral hepatitis Angioimmunoblastic lymphadenopathy
Cardinal Manifestations of Hematologic Disease

Cytomegalovirus Sarcoidosis
Subacute bacterial endocarditis Thyrotoxicosis (benign lymphoid hypertrophy)
Bacterial septicemia Interleukin-2 therapy
Congenital syphilis Extramedullary hematopoiesis
Splenic abscess Myelofibrosis
Tuberculosis Marrow damage by toxins, radiation, strontium
Histoplasmosis Marrow infiltration by tumors, leukemias, Gauchers disease
Enlargement Due to Abnormal Splenic or Portal Blood Flow
Cirrhosis Splenic artery aneurysm
Hepatic vein obstruction Hepatic schistosomiasis
Portal vein obstruction, intrahepatic or extrahepatic Congestive heart failure
Cavernous transformation of the portal vein Hepatic echinococcosis
Splenic vein obstruction Portal hypertension (any cause including the above): Bantis
disease
Infiltration of the Spleen
Intracellular or extracellular depositions Hodgkins disease
Amyloidosis Myeloproliferative syndromes (e.g., polycythemia vera, essential
Gauchers disease thrombocytosis)
Niemann-Pick disease Angiosarcomas
Tangier disease Metastatic tumors (melanoma is most common)
Hurlers syndrome and other mucopolysaccharidoses Eosinophilic granuloma
Hyperlipidemias Histiocytosis X
Benign and malignant cellular infiltrations Hamartomas
Leukemias (acute, chronic, lymphoid, myeloid, mono- Hemangiomas, fibromas, lymphangiomas
cytic) Splenic cysts
Lymphomas
Unknown Etiology
Idiopathic splenomegaly Iron-deficiency anemia
Berylliosis

2. Passive congestion due to decreased blood flow from The differential diagnostic possibilities are much fewer
the spleen in conditions that produce portal hyperten- when the spleen is massively enlarged or palpable
sion (cirrhosis, Budd-Chiari syndrome, congestive heart more than 8 cm below the left costal margin or when its
failure). drained weight is 1000 g (Table 4-3). The vast major-
3. Infiltrative diseases of the spleen (lymphomas, ity of such patients will have non-Hodgkins lymphoma,
metastatic cancer, amyloidosis, Gauchers disease, chronic lymphocytic leukemia, hairy cell leukemia,
myeloproliferative disorders with extramedullary chronic myeloid leukemia, myelofibrosis with myeloid
hematopoiesis). metaplasia, or polycythemia vera.
Table 4-3 other diagnostic tests that suggest underlying disease. 39
Diseases Associated With Massive More often, splenectomy is performed for symptom
Splenomegaly* control in patients with massive splenomegaly, for
Chronic myeloid leukemia Gauchers disease disease control in patients with traumatic splenic rup-
Lymphomas Chronic lymphocytic leukemia ture, or for correction of cytopenias in patients with
Hairy cell leukemia Sarcoidosis hypersplenism or immune-mediated destruction of one
Myelofibrosis with Autoimmune hemolytic anemia or more cellular blood elements. Splenectomy is nec-
myeloid metaplasia essary for staging of patients with Hodgkins disease
Polycythemia vera Diffuse splenic hemangiomatosis
only in those with clinical stage I or II disease in whom

CHAPTER 4
*
radiation therapy alone is contemplated as the treat-
The spleen extends >8 cm below left costal margin and/or weighs
>1000 g.
ment. Noninvasive staging of the spleen in Hodgkins
disease is not a sufficiently reliable basis for treatment
decisions because one-third of normal-sized spleens
Laboratory Assessment The major labo- will be involved with Hodgkins disease and one-third
ratory abnormalities accompanying splenomegaly are of enlarged spleens will be tumor-free. The widespread
determined by the underlying systemic illness. Erythro- use of systemic therapy to test all stages of Hodgkins

Enlargement of Lymph Nodes andSpleen


cyte counts may be normal, decreased (thalassemia major disease has made staging laparotomy with splenectomy
syndromes, SLE, cirrhosis with portal hypertension), or unnecessary. Although splenectomy in chronic myeloid
increased (polycythemia vera). Granulocyte counts may leukemia (CML) does not affect the natural history of
be normal, decreased (Feltys syndrome, congestive sple- disease, removal of the massive spleen usually makes
nomegaly, leukemias), or increased (infections or inflam- patients significantly more comfortable and simplifies
matory disease, myeloproliferative disorders). Similarly, their management by significantly reducing transfusion
the platelet count may be normal, decreased when there requirements. The improvements in therapy of CML
is enhanced sequestration or destruction of platelets in have reduced the need for splenectomy for symptom
an enlarged spleen (congestive splenomegaly, Gauchers control. Splenectomy is an effective secondary or ter-
disease, immune thrombocytopenia), or increased in the tiary treatment for two chronic B cell leukemias, hairy
myeloproliferative disorders such as polycythemia vera. cell leukemia and prolymphocytic leukemia, and for
The CBC may reveal cytopenia of one or more the very rare splenic mantle cell or marginal zone lym-
blood cell types, which should suggest hypersplen- phoma. Splenectomy in these diseases may be associ-
ism. This condition is characterized by splenomegaly, ated with significant tumor regression in bone marrow
cytopenia(s), normal or hyperplastic bone marrow, and and other sites of disease. Similar regressions of systemic
a response to splenectomy. The latter characteristic is disease have been noted after splenic irradiation in some
less precise because reversal of cytopenia, particularly types of lymphoid tumors, especially chronic lympho-
granulocytopenia, is sometimes not sustained after cytic leukemia and prolymphocytic leukemia. This has
splenectomy. The cytopenias result from increased been termed the abscopal effect. Such systemic tumor
destruction of the cellular elements secondary to responses to local therapy directed at the spleen sug-
reduced flow of blood through enlarged and con- gest that some hormone or growth factor produced by
gested cords (congestive splenomegaly) or to immune- the spleen may affect tumor cell proliferation, but this
mediated mechanisms. In hypersplenism, various cell conjecture is not yet substantiated. A common thera-
types usually have normal morphology on the periph- peutic indication for splenectomy is traumatic or iat-
eral blood smear, although the red cells may be sphe- rogenic splenic rupture. In a fraction of patients with
rocytic due to loss of surface area during their longer splenic rupture, peritoneal seeding of splenic fragments
transit through the enlarged spleen. The increased can lead to splenosisthe presence of multiple rests of
marrow production of red cells should be reflected as spleen tissue not connected to the portal circulation. This
an increased reticulocyte production index, although ectopic spleen tissue may cause pain or gastrointestinal
the value may be less than expected due to increased obstruction, as in endometriosis. A large number of hema-
sequestration of reticulocytes in the spleen. tologic, immunologic, and congestive causes of spleno-
The need for additional laboratory studies is dictated megaly can lead to destruction of one or more cellular
by the differential diagnosis of the underlying illness of blood elements. In the majority of such cases, splenec-
which splenomegaly is a manifestation. tomy can correct the cytopenias, particularly anemia
and thrombocytopenia. In a large series of patients seen
in two tertiary care centers, the indication for splenec-
tomy was diagnostic in 10% of patients, therapeutic in
Splenectomy 44%, staging for Hodgkins disease in 20%, and inciden-
Splenectomy is infrequently performed for diagnostic tal to another procedure in 26%. Perhaps the only con-
purposes, especially in the absence of clinical illness or traindication to splenectomy is the presence of marrow
40 failure, in which the enlarged spleen is the only source group, and the recommendation discounts the pos-
of hematopoietic tissue. sibility that administration of the vaccine may actually
The absence of the spleen has minimal long-term lower the titer of specific pneumococcal antibodies. A
effects on the hematologic profile. In the immediate more effective pneumococcal conjugate vaccine that
postsplenectomy period, leukocytosis (25,000/L) involves T cells in the response is now available (Pre-
and thrombocytosis (1 106/L) may develop, but venar, 7-valent). The vaccine to Neisseria meningitidis
within 23 weeks, blood cell counts and survival of should also be given to patients in whom elective sple-
each cell lineage are usually normal. The chronic mani- nectomy is planned. Although efficacy data for H. influ-
festations of splenectomy are marked variation in size enzae type b vaccine are not available for older children
and shape of erythrocytes (anisocytosis, poikilocyto- or adults, it may be given to patients who have had a
SECTION II

sis) and the presence of Howell-Jolly bodies (nuclear splenectomy.


remnants), Heinz bodies (denatured hemoglobin), Splenectomized patients should be educated to con-
basophilic stippling, and an occasional nucleated eryth- sider any unexplained fever as a medical emergency.
rocyte in the peripheral blood. When such erythrocyte Prompt medical attention with evaluation and treat-
abnormalities appear in a patient whose spleen has not ment of suspected bacteremia may be life-saving. Rou-
been removed, one should suspect splenic infiltration tine chemoprophylaxis with oral penicillin can result
Cardinal Manifestations of Hematologic Disease

by tumor that has interfered with its normal culling and in the emergence of drug-resistant strains and is not
pitting function. recommended.
The most serious consequence of splenectomy is In addition to an increased susceptibility to bacterial
increased susceptibility to bacterial infections, particu- infections, splenectomized patients are also more suscep-
larly those with capsules such as Streptococcus pneumoniae, tible to the parasitic disease babesiosis. Splenectomized
Haemophilus influenzae, and some gram-negative enteric patient should avoid areas where the parasite Babesia is
organisms. Patients younger than age 20 years are par- endemic (e.g., Cape Cod, MA).
ticularly susceptible to overwhelming sepsis with S. Surgical removal of the spleen is an obvious cause of
pneumoniae, and the overall actuarial risk of sepsis in hyposplenism. Patients with sickle cell disease often suf-
patients who have had their spleens removed is about fer from autosplenectomy as a result of splenic destruc-
7% in 10 years. The case-fatality rate for pneumococ- tion by the numerous infarcts associated with sickle
cal sepsis in splenectomized patients is 5080%. About cell crises during childhood. Indeed, the presence of a
25% of patients without spleens will develop a serious palpable spleen in a patient with sickle cell disease after
infection at some time in their; oves. The frequency is age 5 years suggests a coexisting hemoglobinopathy,
highest within the first 3 years after splenectomy. About e.g., thalassemia or hemoglobin C. In addition, patients
15% of the infections are polymicrobial, and lung, skin, who receive splenic irradiation for a neoplastic or auto-
and blood are the most common sites. No increased immune disease are also functionally hyposplenic. The
risk of viral infection has been noted in patients who term hyposplenism is preferred to asplenism in referring to
have no spleen. The susceptibility to bacterial infections the physiologic consequences of splenectomy because
relates to the inability to remove opsonized bacteria asplenia is a rare, specific, and fatal congenital abnor-
from the bloodstream and a defect in making antibod- mality in which there is a failure of the left side of the
ies to T cellindependent antigens such as the polysac- coelomic cavity (which includes the splenic anlagen) to
charide components of bacterial capsules. Pneumococcal develop normally. Infants with asplenia have no spleens,
vaccine should be administered to all patients 2 weeks but that is the least of their problems. The right side of
before elective splenectomy. The Advisory Com- the developing embryo is duplicated on the left so there
mittee on Immunization Practices recommends that is liver where the spleen should be, there are two right
these patients receive repeat vaccination 5 years post- lungs, and the heart comprises two right atria and two
splenectomy. Efficacy has not been proven for this right ventricles.
chaPter 5

DISORDERS OF GRANULOCYTES AND


MONOCYTES

Steven M. Holland John I. Gallin

Leukocytes, the major cells comprising inflammatory and only in the bone marrow. The minimum number of
immune responses, include neutrophils, T and B lympho- stem cells necessary to support hematopoiesis is esti-
cytes, natural killer (NK) cells, monocytes, eosinophils, mated to be 400500 at any one time. Human blood
and basophils. These cells have specific functions, such as monocytes, tissue macrophages, and stromal cells pro-
antibody production by B lymphocytes or destruction of duce CSFs, hormones required for the growth of
bacteria by neutrophils, but in no single infectious disease monocytes and neutrophils in the bone marrow. The
is the exact role of the cell types completely established. hematopoietic system not only produces enough neu-
Thus, whereas neutrophils are classically thought to be trophils (1.3 1011 cells per 80-kg person per day)
critical to host defense against bacteria, they may also play to carry out physiologic functions but also has a large
important roles in defense against viral infections. reserve stored in the marrow, which can be mobilized
The blood delivers leukocytes to the various tis- in response to inflammation or infection. An increase in
sues from the bone marrow, where they are produced. the number of blood neutrophils is called neutrophilia,
Normal blood leukocyte counts are 4.310.8 109/L, and the presence of immature cells is termed a shift to
with neutrophils representing 4574% of the cells, the left. A decrease in the number of blood neutrophils is
bands 04%, lymphocytes 1645%, monocytes 410%, called neutropenia.
eosinophils 07%, and basophils 02%. Variation Neutrophils and monocytes evolve from pluripo-
among individuals and among different ethnic groups tent stem cells under the influence of cytokines and
can be substantial, with lower leukocyte numbers for CSFs (Fig. 5-2). The proliferation phase through the
certain African-American ethnic groups. The various metamyelocyte takes about 1 week, while the matura-
leukocytes are derived from a common stem cell in the tion phase from metamyelocyte to mature neutrophil
bone marrow. Three-fourths of the nucleated cells of takes another week. The myeloblast is the first recogniz-
bone marrow are committed to the production of leu- able precursor cell and is followed by the promyelocyte. The
kocytes. Leukocyte maturation in the marrow is under promyelocyte evolves when the classic lysosomal gran-
the regulatory control of a number of different factors, ules, called the primary, or azurophil, granules are pro-
known as colony-stimulating factors (CSFs) and inter- duced. The primary granules contain hydrolases, elastase,
leukins (ILs). Because an alteration in the number and myeloperoxidase, cathepsin G, cationic proteins,
type of leukocytes is often associated with disease pro- and bactericidal/permeability-increasing protein, which
cesses, total white blood cell (WBC) count (cells per is important for killing gram-negative bacteria. Azuro-
L) and differential counts are informative. This chap- phil granules also contain defensins, a family of cysteine-
ter focuses on neutrophils, monocytes, and eosinophils. rich polypeptides with broad antimicrobial activity
against bacteria, fungi, and certain enveloped viruses.
The promyelocyte divides to produce the myelocyte,
neutroPhils a cell responsible for the synthesis of the specic, or sec-
ondary, granules, which contain unique (specific) con-
MaturatioN
stituents such as lactoferrin, vitamin B12binding
Important events in neutrophil life are summarized in protein, membrane components of the reduced nico-
Fig. 5-1. In normal humans, neutrophils are produced tinamide-adenine dinucleotide phosphate (NADPH)
41
42 Microbial killing
tissue damage
Activation of other
BONE MARROW CIRCULATION limbs of host defense
Redness
Stem
C3a (Rubor) O2, H2O2, .OH,
cell
C5a Edema HOCl (bleach)
Histamine Vasodilation (Tumor)
Bradykinin Fluid leakage Pain
Serotonin (Dolor)
Warmth
SECTION II

(Calor) Figure 5-1


Schematic events in neutrophil
Chemokines, other Ingestion
PMN chemoattractants Bacteria production, recruitment, and
Diapedesis
or fungi inflammation. The four cardinal
G-CSF
Steroids Integrins signs of inflammation (rubor, tumor,
Endotoxin calor, dolor) are indicated, as are
Increased
Fever Cytokine secretion
endothelial the interactions of neutrophils with
Cardinal Manifestations of Hematologic Disease

stickiness IL-8, TNF-, IL-12


other cells and cytokines. G-CSF,
Vessel wall IL-1, TNF-
granulocyte colony-stimulating factor;
Endothelium Selectins Recruitment IL, interleukin; PMN, polymorpho-
Macrophages nuclear leukocyte; TNF-, tumor
Lymphocytes
necrosis factor .

Cell Stage Surface Markersa Characteristics

MYELOBLAST CD33, CD13, Prominent


CD15 nucleoli

PROMYELOCYTE CD33, CD13, Large cell


CD15 Primary granules
appear

MYELOCYTE CD33, CD13, Secondary


CD15, CD14, granules appear
CD11b

METAMYELOCYTE CD33, CD13, Kidney bean-


CD15, CD14, shaped nucleus
CD11b

BAND FORM CD33, CD13, Condensed, band-


CD15, CD14, shaped nucleus
CD11b CD10, Figure 5-2
CD16 Stages of neutrophil development
shown schematically. G-CSF (granu-
locyte colony-stimulating factor) and
NEUTROPHIL CD33, CD13, Condensed,
CD15, CD14, multilobed GM-CSF (granulocyte-macrophage col-
CD11b CD10, nucleus ony-stimulating factor) are critical to this
CD16 process. Identifying cellular character-
istics and specific cell-surface markers
aCD= Cluster determinant; Nucleolus; Primary granule; Secondary granule. are listed for each maturational stage.
43

CHAPTER 5
Figure 5-3
Neutrophil band with Dhle body. The neutrophil with a

Disorders of Granulocytes and Monocytes


sausage-shaped nucleus in the center of the field is a band
form. Dhle bodies are discrete, blue-staining nongranular
areas found in the periphery of the cytoplasm of the neu-
trophil in infections and other toxic states. They represent
aggregates of rough endoplasmic reticulum. Figure 5-4
Normal granulocyte. The normal granulocyte has a seg-
mented nucleus with heavy, clumped chromatin; fine neutro-
philic granules are dispersed throughout the cytoplasm.
oxidase required for hydrogen peroxide production,
histaminase, and receptors for certain chemoattrac-
tants and adherence-promoting factors (CR3) as well In severe acute bacterial infection, prominent neu-
as receptors for the basement membrane component, trophil cytoplasmic granules, called toxic granulations,
laminin. The secondary granules do not contain acid are occasionally seen. Toxic granulations are immature
hydrolases and therefore are not classic lysosomes. Pack- or abnormally staining azurophil granules. Cytoplasmic
aging of secondary granule contents during myelo- inclusions, also called Dhle bodies (Fig. 5-3), can be
poiesis is controlled by CCAAT/enhancer binding seen during infection and are fragments of ribosome-
protein-. Secondary granule contents are read- rich endoplasmic reticulum. Large neutrophil vacuoles
ily released extracellularly, and their mobilization is are often present in acute bacterial infection and prob-
important in modulating inflammation. During the ably represent pinocytosed (internalized) membrane.
final stages of maturation, no cell division occurs,
and the cell passes through the metamyelocyte stage
and then to the band neutrophil with a sausage-
shaped nucleus (Fig. 5-3). As the band cell matures,
the nucleus assumes a lobulated configuration. The
nucleus of neutrophils normally contains up to four seg-
ments (Fig. 5-4). Excessive segmentation (more than
five nuclear lobes) may be a manifestation of folate or
vitamin B12 deficiency or the congenital neutropenia
syndrome of warts, hypogammaglobulinemia, infec-
tions, and myelokathexis (WHIM) described later. The
Pelger-Het anomaly (Fig. 5-5), an infrequent domi-
nant benign inherited trait, results in neutrophils with
distinctive bilobed nuclei that must be distinguished
from band forms. Acquired bilobed nuclei, pseudo
Pelger-Het anomaly, can occur with acute infec-
tions or in myelodysplastic syndromes. The physiologic
role of the normal multilobed nucleus of neutrophils Figure 5-5
is unknown, but it may allow great deformation of Pelger-Het anomaly. In this benign disorder, the majority
neutrophils during migration into tissues at sites of of granulocytes are bilobed. The nucleus frequently has a
inflammation. spectacle-like, or pince-nez, configuration.
44 Basal
Circulating pool

Bone marrow Tissue

Marginated pool

Circulating pool
Infection
Bone marrow Tissue

Marginated pool
SECTION II

Circulating pool
Epinephrine
Bone marrow Tissue

Marginated pool

Figure 5-6 Circulating pool


Steroids (Acute)
Normal eosinophil and basophil. The eosinophil contains
Cardinal Manifestations of Hematologic Disease

large, bright orange granules and usually a bilobed nucleus. Bone marrow Tissue
The basophil contains large purple-black granules that fill the Marginated pool
cell and obscure the nucleus.
Leukocyte Adhesion Deficiency Circulating pool

Bone marrow Tissue


Neutrophils are heterogeneous in function. Mono-
Marginated pool
clonal antibodies have been developed that recognize
only a subset of mature neutrophils. The meaning of Figure 5-7
neutrophil heterogeneity is not known. Schematic neutrophil distribution and kinetics between the
The morphology of eosinophils and basophils is different anatomic and functional pools.
shown in Fig. 5-6.

neutrophils and endothelial cells, among others, that


Marrow Release and Circulating cause a low-affinity interaction, resulting in rolling of
Compartments the neutrophil along the endothelial surface. On neu-
Specific signals, including IL-1, tumor necrosis factor trophils, the molecule L-selectin (cluster determinant
(TNF-), the CSFs, complement fragments, and che- [CD] 62L) binds to glycosylated proteins on endothelial
mokines, mobilize leukocytes from the bone marrow cells (e.g., glycosylation-dependent cell adhesion mole-
and deliver them to the blood in an unstimulated state. cule [GlyCAM1] and CD34). Glycoproteins on neutro-
Under normal conditions, 90% of the neutrophil pool phils, most importantly sialyl-Lewisx (SLex, CD15s), are
is in the bone marrow, 23% in the circulation, and the targets for binding of selectins expressed on endothe-
remainder in the tissues (Fig. 5-7). lial cells (E-selectin [CD62E] and P-selectin [CD62P])
The circulating pool exists in two dynamic compart- and other leukocytes. In response to chemotactic
ments: one freely flowing and one marginated. The stimuli from injured tissues (e.g., complement product
freely flowing pool is about one-half the neutrophils in C5a, leukotriene B4, IL-8) or bacterial products (e.g.,
the basal state and is composed of those cells that are N-formylmethionylleucylphenylalanine [f-metleuphe]),
in the blood and not in contact with the endothelium. neutrophil adhesiveness increases, and the cells stick
Marginated leukocytes are those that are in close physi- to the endothelium through integrins. The integrins are
cal contact with the endothelium (Fig. 5-8). In the leukocyte glycoproteins that exist as complexes of a
pulmonary circulation, where an extensive capillary common CD18 chain with CD11a (LFA-1), CD11b
bed (1000 capillaries per alveolus) exists, margination (called Mac-1, CR3, or the C3bi receptor), and CD11c
occurs because the capillaries are about the same size as (called p150,95 or CR4). CD11a/CD18 and CD11b/
a mature neutrophil. Therefore, neutrophil fluidity and CD18 bind to specific endothelial receptors (intercellu-
deformability are necessary to make the transit through lar adhesion molecules [CAM] 1 and 2).
the pulmonary bed. Increased neutrophil rigidity and On cell stimulation, L-selectin is shed from neutro-
decreased deformability lead to augmented neutrophil phils, and E-selectin increases in the blood, presum-
trapping and margination in the lung. In contrast, in ably because it is shed from endothelial cells; receptors
the systemic postcapillary venules, margination is medi- for chemoattractants and opsonins are mobilized; and
ated by the interaction of specific cell-surface molecules the phagocytes orient toward the chemoattractant source
called selectins. Selectins are glycoproteins expressed on in the extravascular space, increase their motile activity
Pulmonary capillary bed 45

Alveolus
Chemotactic
factor

Free flowing

Rolling Systemic circulation postcapillary venules


Tight adhesion

CHAPTER 5
CD15s CD62L Diapedesis
CD18 CD11a,b
CD31

CD54

CD102
GlyCAM-1
CD34
CD62P
CD62E

Endothelium
Nucleus

Disorders of Granulocytes and Monocytes


Activation Chemoattractant

Figure 5-8
Neutrophil travel through the pulmonary capillaries is cells; CD62L (l-selectin) on neutrophils binds to CD34 and
dependent on neutrophil deformability. Neutrophil rigid- other molecules (e.g., GlyCAM-1) expressed on endothe-
ity (e.g., caused by C5a) enhances pulmonary trapping lium. Chemokines or other activation factors stimulate inte-
and response to pulmonary pathogens in a way that is not grin-mediated tight adhesion: CD11a/CD18 (LFA-1) and
so dependent on cell-surface receptors. Intraalveolar che- CD11b/CD18 (Mac-1, CR3) bind to CD54 (ICAM-1) and
motactic factors, such as those caused by certain bacte- CD102 (ICAM-2) on the endothelium. Diapedesis occurs
ria (e.g., Streptococcus pneumoniae), lead to diapedesis of between endothelial cells: CD31 (PECAM-1) expressed by
neutrophils from the pulmonary capillaries into the alveolar the emigrating neutrophil interacts with CD31 expressed at
space. Neutrophil interaction with the endothelium of the the endothelial cellcell junction. CD, cluster determinant;
systemic postcapillary venules is dependent on molecules GlyCAM, glycosylation-dependent cell adhesion molecule;
of attachment. The neutrophil rolls along the endothelium ICAM, intercellular adhesion molecule; PECAM, platelet/
using selectins: neutrophil CD15s (sialyl-Lewisx) binds to endothelial cell adhesion molecule.
CD62E (E-selectin) and CD62P (P-selectin) on endothelial

(chemokinesis), and migrate directionally (chemotaxis) release enzymes, such as collagenase and elastase, which
into tissues. The process of migration into tissues is may help establish abscess cavities. Neutrophils ingest
called diapedesis and involves the crawling of neutrophils pathogenic materials that have been opsonized by IgG
between postcapillary endothelial cells that open junc- and C3b. Fibronectin and the tetrapeptide tuftsin also
tions between adjacent cells to permit leukocyte passage. facilitate phagocytosis.
Diapedesis involves platelet/endothelial cell adhesion With phagocytosis comes a burst of oxygen con-
molecule (PECAM) 1 (CD31), which is expressed on sumption and activation of the hexose-monophos-
both the emigrating leukocyte and the endothelial cells. phate shunt. A membrane-associated NADPH oxidase,
The endothelial responses (increased blood flow from consisting of membrane and cytosolic components,
increased vasodilation and permeability) are mediated is assembled and catalyzes the reduction of oxygen to
by anaphylatoxins (e.g., C3a and C5a) as well as vaso- superoxide anion, which is then converted to hydro-
dilators such as histamine, bradykinin, serotonin, nitric gen peroxide and other toxic oxygen products (e.g.,
oxide, vascular endothelial growth factor (VEGF), and hydroxyl radical). Hydrogen peroxide + chloride +
prostaglandins E and I. Cytokines regulate some of neutrophil myeloperoxidase generate hypochlorous
these processes (e.g., TNF- induction of VEGF, inter- acid (bleach), hypochlorite, and chlorine. These prod-
feron [IFN] inhibition of prostaglandin E). ucts oxidize and halogenate microorganisms and tumor
In a healthy adult, most neutrophils leave the body cells and, when uncontrolled, can damage host tis-
by migration through the mucous membrane of the gas- sue. Strongly cationic proteins, defensins, elastase,
trointestinal tract. Normally, neutrophils spend a short cathepsins, and probably nitric oxide also participate in
time in the circulation (half-life, 67 h). Senescent neu- microbial killing. Lactoferrin chelates iron, an impor-
trophils are cleared from the circulation by macrophages tant growth factor for microorganisms, especially fungi.
in the lung and spleen. Once in the tissues, neutrophils Other enzymes, such as lysozyme and acid proteases,
46 help digest microbial debris. After 14 days in tissues, rare. In some disorders, the frequency of infection is
neutrophils die. The apoptosis of neutrophils is also variable, and patients can go for months or even years
cytokine-regulated; granulocyte colony-stimulating fac- without major infection. Aggressive management of
tor (G-CSF) and IFN- prolong their life span. Under these congenital diseases has extended the life span of
certain conditions, such as in delayed-type hypersen- patients well beyond 30 years.
sitivity, monocyte accumulation occurs within 612
h of initiation of inflammation. Neutrophils, mono- Neutropenia
cytes, microorganisms in various states of digestion,
and altered local tissue cells make up the inflammatory The consequences of absent neutrophils are dramatic.
exudate, pus. Myeloperoxidase confers the characteristic Susceptibility to infectious diseases increases sharply
SECTION II

green color to pus and may participate in turning off the when neutrophil counts fall below 1000 cells/L.
inflammatory process by inactivating chemoattractants When the absolute neutrophil count (ANC; band forms
and immobilizing phagocytic cells. and mature neutrophils combined) falls to <500 cells/
Neutrophils respond to certain cytokines (IFN-, L, control of endogenous microbial flora (e.g., mouth,
granulocyte-macrophage colony-stimulating factor [GM- gut) is impaired; when the ANC is <200/L, the local
CSF], IL-8) and produce cytokines and chemotactic inflammatory process is absent. Neutropenia can be
Cardinal Manifestations of Hematologic Disease

signals (TNF-, IL-8, macrophage inflammatory protein due to depressed production, increased peripheral
[MIP] 1) that modulate the inflammatory response. In destruction, or excessive peripheral pooling. A falling
the presence of fibrinogen, f-metleuphe or leukotriene neutrophil count or a significant decrease in the num-
B4 induces IL-8 production by neutrophils, providing ber of neutrophils below steady-state levels, together
autocrine amplification of inflammation. Chemokines with a failure to increase neutrophil counts in the set-
(chemoattractant cytokines) are small proteins produced ting of infection or other challenge, requires investiga-
by many different cell types, including endothelial tion. Acute neutropenia, such as that caused by can-
cells, fibroblasts, epithelial cells, neutrophils, and mono- cer chemotherapy, is more likely to be associated with
cytes, that regulate neutrophil, monocyte, eosinophil, increased risk of infection than neutropenia of long
and lymphocyte recruitment and activation. Chemokines duration (months to years) that reverses in response to
transduce their signals through heterotrimeric G protein infection or carefully controlled administration of endo-
linked receptors that have seven cell membrane toxin (see Laboratory Diagnosis and Management,
spanning domains, the same type of cell-surface receptor later in the chapter).
that mediates the response to the classic chemoattrac- Some causes of inherited and acquired neutropenia
tants f-metleuphe and C5a. Four major groups of che- are listed in Table 5-1. The most common neutrope-
mokines are recognized based on the cysteine structure nias are iatrogenic, resulting from the use of cytotoxic
near the N terminus: C, CC, CXC, and CXXXC. The or immunosuppressive therapies for malignancy or con-
CXC cytokines such as IL-8 mainly attract neutrophils; trol of autoimmune disorders. These drugs cause neu-
CC chemokines such as MIP-1 attract lymphocytes, tropenia because they result in decreased production
monocytes, eosinophils, and basophils; the C chemo- of rapidly growing progenitor (stem) cells of the
kine lymphotactin is T cell tropic; the CXXXC chemo- marrow. Certain antibiotics such as chloramphenicol,
kine fractalkine attracts neutrophils, monocytes, and T trimethoprimsulfamethoxazole, flucytosine, vidarabine,
cells. These molecules and their receptors not only reg- and the antiretroviral drug zidovudine may cause neutro-
ulate the trafficking and activation of inflammatory cells, penia by inhibiting proliferation of myeloid precursors.
but specific chemokine receptors serve as co-receptors Azathioprine and 6-mercaptopurine are metabolized
for HIV infection and have a role in other viral infec- by the enzyme thiopurine methyltransferase (TMPT),
tions such as West Nile infection and atherogenesis. hypofunctional polymorphisms in which can lead to
accumulation of 6-thioguanine and profound mar-
row toxicity. The marrow suppression is generally dose-
Neutrophil Abnormalities
related and dependent on continued administration of
Defects in the neutrophil life cycle can lead to dysfunc- the drug. Cessation of the offending agent and recom-
tion and compromised host defenses. Inflammation is binant human G-CSF usually reverse these forms of
often depressed, and the clinical result is often recurrent, neutropenia.
with severe bacterial and fungal infections. Aphthous Another important mechanism for iatrogenic neutro-
ulcers of mucous membranes (gray ulcers without pus) penia is the effect of drugs that serve as immune hap-
and gingivitis and periodontal disease suggest a phago- tens and sensitize neutrophils or neutrophil precursors
cytic cell disorder. Patients with congenital phagocyte to immune-mediated peripheral destruction. This form
defects can have infections within the first few days of of drug-induced neutropenia can be seen within 7 days
life. Skin, ear, upper and lower respiratory tract, and of exposure to the drug; with previous drug exposure,
bone infections are common. Sepsis and meningitis are resulting in preexisting antibodies, neutropenia may
Table 5-1 lymphocytosis may have moderate blood and bone mar- 47
Causes of Neutropenia row lymphocytosis, neutropenia, polyclonal hypergam-
Decreased Production maglobulinemia, splenomegaly, rheumatoid arthritis,
and absence of lymphadenopathy. Such patients may
Drug-inducedalkylating agents (nitrogen mustard, busul-
fan, chlorambucil, cyclophosphamide); antimetabolites
have a chronic and relatively stable course. Recurrent
(methotrexate, 6-mercaptopurine, 5-flucytosine); noncy- bacterial infections are frequent. Benign and malignant
totoxic agents (antibiotics [chloramphenicol, penicillins, forms of this syndrome occur. In some patients, a spon-
sulfonamides], phenothiazines, tranquilizers [meprobam- taneous regression has occurred even after 11 years, sug-
ate], anticonvulsants [carbamazepine], antipsychotics gesting an immunoregulatory defect as the basis for at
[clozapine], certain diuretics, anti-inflammatory agents, least one form of the disorder. Glucocorticoids, cyclo-

CHAPTER 5
antithyroid drugs, many others) sporine, and methotrexate are commonly used to man-
Hematologic diseasesidiopathic, cyclic neutropenia,
Chdiak-Higashi syndrome, aplastic anemia, infantile
age these cytopenias.
genetic disorders (see text)
Tumor invasion, myelofibrosis Hereditary neutropenias
Nutritional deficiencyvitamin B12, folate (especially with
alcoholism) Hereditary neutropenias are rare and may manifest in

Disorders of Granulocytes and Monocytes


Infectiontuberculosis, typhoid fever, brucellosis, early childhood as a profound constant neutropenia
tularemia, measles, infectious mononucleosis, malaria, or agranulocytosis. Congenital forms of neutropenia
viral hepatitis, leishmaniasis, AIDS include Kostmanns syndrome (neutrophil count <100/
Peripheral Destruction L), which is often fatal and due to mutations in the
Antineutrophil antibodies and/or splenic or lung trapping anti-apoptosis gene HAX-1; severe chronic neutropenia
Autoimmune disordersFeltys syndrome, rheumatoid (neutrophil count of 3001500/L) due to mutations in
arthritis, lupus erythematosus neutrophil elastase (ELA-2); hereditary cyclic neutrope-
Drugs as haptensaminopyrine, -methyldopa, phenyl- nia, or, more appropriately, cyclic hematopoiesis, also
butazone, mercurial diuretics, some phenothiazines due to mutations in neutrophil elastase (ELA-2); the
Granulomatosis with polyangiitis (Wegeners) cartilage-hair hypoplasia syndrome due to mutations in
Peripheral Pooling (Transient Neutropenia) the mitochondrial RNA-processing endoribonuclease
Overwhelming bacterial infection (acute endotoxemia) RMRP; Shwachman-Diamond syndrome associated
Hemodialysis with pancreatic insufficiency due to mutations in the
Cardiopulmonary bypass Shwachman-Bodian-Diamond syndrome gene SBDS;
the WHIM (warts, hypogammaglobulinemia, infections,
myelokathexis [retention of WBCs in the marrow])
syndrome, characterized by neutrophil hypersegmenta-
occur a few hours after administration of the drug. tion and bone marrow myeloid arrest due to mutations
Although any drug can cause this form of neutropenia, in the chemokine receptor CXCR4; and neutropenias
the most frequent causes are commonly used antibiot- associated with other immune defects, such as X-linked
ics, such as sulfa-containing compounds, penicillins, and agammaglobulinemia, Wiskott-Aldrich syndrome,
cephalosporins. Fever and eosinophilia may also be asso- and CD40 ligand deficiency. Mutations in the G-CSF
ciated with drug reactions, but often these signs are not receptor can develop in severe congenital neutropenia
present. Drug-induced neutropenia can be severe, but and are linked to leukemia. Absence of both myeloid
discontinuation of the sensitizing drug is sufficient for and lymphoid cells is seen in reticular dysgenesis, due to
recovery, which is usually seen within 57 days and is mutations in the nuclear genome-encoded mitochon-
complete by 10 days. Readministration of the sensitiz- drial enzyme adenylate kinase-2 (AK2).
ing drug should be avoided, since abrupt neutropenia Maternal factors can be associated with neutropenia
will often result. For this reason, diagnostic challenge in the newborn. Transplacental transfer of IgG directed
should be avoided. against antigens on fetal neutrophils can result in periph-
Autoimmune neutropenias caused by circulating eral destruction. Drugs (e.g., thiazides) ingested during
antineutrophil antibodies are another form of acquired pregnancy can cause neutropenia in the newborn by
neutropenia that results in increased destruction of neu- either depressed production or peripheral destruction.
trophils. Acquired neutropenia may also be seen with In Feltys syndromethe triad of rheumatoid arthri-
viral infections, including infection with HIV. Acquired tis, splenomegaly, and neutropenia spleen-produced anti-
neutropenia may be cyclic in nature, occurring at inter- bodies can shorten neutrophil life span, while LGLs can
vals of several weeks. Acquired cyclic or stable neu- attack marrow neutrophil precursors. Splenectomy may
tropenia may be associated with an expansion of large increase the neutrophil count in Feltys syndrome and
granular lymphocytes (LGLs), which may be T cells, lower serum neutrophil-binding IgG. Some Feltys syn-
NK cells, or NK-like cells. Patients with large granular drome patients also have neutropenia associated with an
48 increased number of LGLs. Splenomegaly with periph- neutrophilia with cell counts of 30,00050,000/L is
eral trapping and destruction of neutrophils is also seen in called a leukemoid reaction, a term often used to distin-
lysosomal storage diseases and in portal hypertension. guish this degree of neutrophilia from leukemia. In a
leukemoid reaction, the circulating neutrophils are usu-
Neutrophilia ally mature and not clonally derived.
Neutrophilia results from increased neutrophil produc-
tion, increased marrow release, or defective margination Abnormal neutrophil function
(Table 5-2). The most important acute cause of neu-
Inherited and acquired abnormalities of phagocyte
trophilia is infection. Neutrophilia from acute infec-
SECTION II

function are listed in Table 5-3. The resulting diseases


tion represents both increased production and increased
are best considered in terms of the functional defects of
marrow release. Increased production is also associated
adherence, chemotaxis, and microbicidal activity. The
with chronic inflammation and certain myeloprolifera-
distinguishing features of the important inherited disor-
tive diseases. Increased marrow release and mobilization
ders of phagocyte function are shown in Table 5-4.
of the marginated leukocyte pool are induced by glu-
cocorticoids. Release of epinephrine, as with vigorous Disorders of adhesion
Cardinal Manifestations of Hematologic Disease

exercise, excitement, or stress, will demarginate neu- Three main types of leukocyte adhesion deficiency
trophils in the spleen and lungs and double the neutro- (LAD) have been described. All are autosomal recessive
phil count in minutes. Cigarette smoking can elevate and result in the inability of neutrophils to exit the cir-
neutrophil counts above the normal range. Leukocy- culation to sites of infection, leading to leukocytosis and
tosis with cell counts of 10,00025,000/L occurs in increased susceptibility to infection (Fig. 5-8). Patients
response to infection and other forms of acute inflam- with LAD 1 have mutations in CD18, the com-
mation and results from both release of the marginated mon component of the integrins LFA-1, Mac-1, and
pool and mobilization of marrow reserves. Persistent p150,95, leading to a defect in tight adhesion between
neutrophils and the endothelium. The heterodimer
formed by CD18/CD11b (Mac-1) is also the receptor
Table 5-2 for the complement-derived opsonin C3bi (CR3). The
Causes of Neutrophilia
CD18 gene is located on distal chromosome 21q. The
severity of the defect determines the severity of clini-
Increased Production
cal disease. Complete lack of expression of the leuko-
Idiopathic cyte integrins results in a severe phenotype in which
Drug-inducedglucocorticoids, G-CSF inflammatory stimuli do not increase the expression of
Infectionbacterial, fungal, sometimes viral
leukocyte integrins on neutrophils or activated T and
Inflammationthermal injury, tissue necrosis, myocardial
and pulmonary infarction, hypersensitivity states, colla-
B cells. Neutrophils (and monocytes) from patients
gen vascular diseases with LAD 1 adhere poorly to endothelial cells and
Myeloproliferative diseasesmyelocytic leukemia, myeloid protein-coated surfaces and exhibit defective spread-
metaplasia, polycythemia vera ing, aggregation, and chemotaxis. Patients with LAD
Increased Marrow Release 1 have recurrent bacterial infections involving the skin,
oral and genital mucosa, and respiratory and intestinal
Glucocorticoids
Acute infection (endotoxin) tracts; persistent leukocytosis (resting neutrophil counts
Inflammationthermal injury of 15,00020,000/L) because cells do not marginate;
Decreased or Defective Margination
and, in severe cases, a history of delayed separation of
the umbilical stump. Infections, especially of the skin,
Drugsepinephrine, glucocorticoids, nonsteroidal
may become necrotic with progressively enlarging bor-
anti-inflammatory agents
Stress, excitement, vigorous exercise
ders, slow healing, and development of dysplastic scars.
Leukocyte adhesion deficiency type 1 (CD18); leukocyte The most common bacteria are Staphylococcus aureus
adhesion deficiency type 2 (selectin ligand, CD15s); and enteric gram-negative bacteria. LAD 2 is caused
leukocyte adhesion deficiency type 3 (Kindlin-3) by an abnormality of fucosylation of SLex (CD15s), the
Miscellaneous ligand on neutrophils that interacts with selectins on
endothelial cells and is responsible for neutrophil roll-
Metabolic disordersketoacidosis, acute renal failure,
eclampsia, acute poisoning ing along the endothelium. Infection susceptibility in
Drugslithium LAD 2 appears to be less severe than in LAD 1. LAD
Othermetastatic carcinoma, acute hemorrhage or 2 is also known as congenital disorder of glycosylation IIc
hemolysis (CDGIIc) due to mutation in a GDP-fucose transporter
(SLC35C1). LAD 3 is characterized by infection suscep-
Abbreviation: G-CSF, granulocyte colony-stimulating factor. tibility, leukocytosis, and petechial hemorrhage due to
Table 5-3 49
Types of Granulocyte and Monocyte Disorders
Cause of Indicated Dysfunction

Function Drug-Induced Acquired Inherited

Adherence- Aspirin, colchicine, alcohol, Neonatal state, hemodialysis Leukocyte adhesion deficiency types
aggregation glucocorticoids, ibuprofen, 1, 2, and 3
piroxicam
Deformability Leukemia, neonatal state, diabetes

CHAPTER 5
mellitus, immature neutrophils
Chemokinesis- Glucocorticoids (high dose), Thermal injury, malignancy, Chdiak-Higashi syndrome,
chemotaxis auranofin, colchicine (weak malnutrition, periodontal dis- neutrophil-specific granule defi-
effect), phenylbutazone, ease, neonatal state, systemic ciency, hyper IgErecurrent infection
naproxen, indomethacin, lupus erythematosus, rheuma- (Jobs) syndrome (in some patients),
IL-2 toid arthritis, diabetes mellitus, Down syndrome, -mannosidase
sepsis, influenza virus infection, deficiency, leukocyte adhesion defi-

Disorders of Granulocytes and Monocytes


herpes simplex virus infection, ciencies, Wiskott-Aldrich syndrome
acrodermatitis enteropathica, AIDS
Microbicidal Colchicine, cyclophospha- Leukemia, aplastic anemia, certain Chdiak-Higashi syndrome,
activity mide, glucocorticoids (high neutropenias, tuftsin deficiency, neutrophil-specific granule defi-
dose), TNF- blocking thermal injury, sepsis, neonatal state, ciency, chronic granulomatous dis-
antibodies diabetes mellitus, malnutrition, AIDS ease, defects in IFN- /IL-12 axis

Abbreviations: IFN, interferon; IL, interleukin; TNF-, tumor necrosis factor alpha.

Table 5-4
Inherited Disorders of Phagocyte Function: Differential Features
Clinical Manifestations Cellular or Molecular Defects Diagnosis

Chronic Granulomatous Diseases (70% X-linked, 30% Autosomal Recessive)


Severe infections of skin, ears, lungs, No respiratory burst due to the lack DHR or NBT test; no superoxide and
liver, and bone with catalase-positive of one of five NADPH oxidase sub- H2O2 production by neutrophils; immu-
microorganisms such as Staphylococ- units in neutrophils, monocytes, and noblot for NADPH oxidase compo-
cus aureus, Burkholderia cepacia, eosinophils nents; genetic detection
Aspergillus spp., Chromobacterium
violaceum; often hard to culture organ-
ism; excessive inflammation with
granulomas, frequent lymph node
suppuration; granulomas can obstruct
GI or GU tracts; gingivitis, aphthous
ulcers, seborrheic dermatitis
Chdiak-Higashi Syndrome (Autosomal Recessive)
Recurrent pyogenic infections, especially Reduced chemotaxis and Giant primary granules in neutrophils
with S. aureus; many patients get phagolysosome fusion, increased and other granule-bearing cells
lymphoma-like illness during adoles- respiratory burst activity, defective (Wrights stain); genetic detection
cence; periodontal disease; partial egress from marrow, abnormal skin
oculocutaneous albinism, nystagmus, window; defect in CHS1
progressive peripheral neuropathy,
mental retardation in some patients
Specific Granule Deficiency (Autosomal Recessive)
Recurrent infections of skin, ears, and Abnormal chemotaxis, impaired respira- Lack of secondary (specific) granules
sinopulmonary tract; delayed wound tory burst and bacterial killing, failure to in neutrophils (Wrights stain), no
healing; decreased inflammation; upregulate chemotactic and adhesion neutrophil-specific granule contents
bleeding diathesis receptors with stimulation, defect in (i.e.,lactoferrin), no defensins, plate-
transcription of granule proteins; defect let granule abnormality; genetic
in C/EBP detection

(continued)
50 Table 5-4
Inherited Disorders of Phagocyte Function: Differential Features (Continued)
Clinical Manifestations Cellular or Molecular Defects Diagnosis

Myeloperoxidase Deficiency (Autosomal Recessive)


Clinically normal except in patients with No myeloperoxidase due to pre- No peroxidase in neutrophils; genetic
underlying disease such as diabetes and posttranslational defects in detection
mellitus; then candidiasis or other fun- myeloperoxidase deficiency
gal infections
Leukocyte Adhesion Deficiency
SECTION II

Type 1: Delayed separation of umbilical Impaired phagocyte adherence, Reduced phagocyte surface expression
cord, sustained neutrophilia, recurrent aggregation, spreading, chemotaxis, of the CD18-containing integrins with
infections of skin and mucosa, gingivi- phagocytosis of C3bi-coated particles; monoclonal antibodies against LFA-1
tis, periodontal disease defective production of CD18 subunit (CD18/CD11a), Mac-1 or CR3 (CD18/
Type 2: Mental retardation, short stat- common to leukocyte integrins CD11b), p150,95 (CD18/CD11c);
ure, Bombay (hh) blood phenotype, Impaired phagocyte rolling along genetic detection
Cardinal Manifestations of Hematologic Disease

recurrent infections, neutrophilia endothelium due to defects in fucose Reduced phagocyte surface expres-
Type 3: Petechial hemorrhage, recurrent transporter sion of Sialyl-Lewisx, with monoclonal
infections Impaired signaling for integrin activation antibodies against CD15s; genetic
resulting in impaired adhesion due to detection
mutation in FERMT3 Reduced signaling for adhesion through
integrins; genetic detection
Phagocyte Activation Defects (X-linked and Autosomal Recessive)
NEMO deficiency: mild hypohidrotic Impaired phagocyte activation by IL-1, Poor in vitro response to endotoxin; lack
ectodermal dysplasia; broad-based IL-18, TLR, CD40L, TNF- leading to of NF-B activation; genetic detection
immune defect: pyogenic and encap- problems with inflammation and anti- Poor in vitro response to endotoxin;
sulated bacteria, viruses, Pneumocys- body production lack of NF-B activation by endotoxin;
tis, mycobacteria; X-linked Impaired phagocyte activation by endo- genetic detection
IRAK4 and MyD88 deficiency: sus- toxin through TLR and other pathways;
ceptibility to pyogenic bacteria such TNF- signaling preserved
as staphylococci, streptococci, clos-
tridia; resistant to candida; autosomal
recessive
Hyper IgERecurrent Infection Syndrome (Autosomal Dominant) (Jobs Syndrome)
Eczematoid or pruritic dermatitis, cold Reduced chemotaxis in some patients, Somatic and immune features involving
skin abscesses, recurrent pneumonias reduced suppressor T cell activity. lungs, skeleton, and immune system;
with S. aureus with bronchopleural Mutation in STAT3 serum IgE >2000 IU/mL; genetic testing
fistulas and cyst formation, mild eosin- Impaired T cell proliferation to mitogens Severe allergies, viral infections, high
ophilia, mucocutaneous candidiasis, IgE, eosinophilia, low IgM, progressive
characteristic facies, restrictive lung lymphopenia, genetic detection
disease, scoliosis, delayed primary
dental deciduation
DOCK8 deficiency (autosomal reces-
sive), severe eczema, atopic dermatitis,
cutaneous abscesses, HSV, HPV, and
molluscum infections, severe allergies,
cancer
Mycobacteria Susceptibility (Autosomal Dominant and Recessive Forms)
Severe extrapulmonary or disseminated Inability to kill intracellular organisms Low or very high levels of IFN- receptor
infections with bacille Calmette-Gurin due to low IFN- production or 1; functional assays of cytokine
nontuberculous mycobacteria, salmo- response; mutations in IFN- receptors, production and response; genetic
nella, histoplasmosis, coccidioidomy- IL-12 receptor, IL-12 p40, STAT1, detection
cosis, poor granuloma formation NEMO

Abbreviations: C/EBP, CCAAT/enhancer binding protein-; DHR, dihydrorhodamine (oxidation test); DOCK8, dedicator of cytokinesis 8; GI,
gastrointestinal; GU, genitourinary; HPV, human papilloma virus; HSV, herpes simplex virus; IFN, interferon; IL, interleukin; IRAK4, IL-1 receptor
associated kinase 4; LFA-1, leukocyte functionassociated antigen 1; MyD88, myeloid differentiation primary response gene 88; NADPH,
nicotinamideadenine dinueleotide phosphate; NBT, nitroblue tetrazolium (dye test); NEMO, NF-B essential modulator; NF-B, nuclear factor
B; STAT1, -3, signal transducer and activator of transcription 1, 3; TLR, Toll-like receptor; TNF, tumor necrosis factor.
impaired integrin activation caused by mutations in the lymphoma requiring bone marrow transplantation. CHS 51
gene FERMT3. neutrophils and monocytes have impaired chemotaxis
and abnormal rates of microbial killing due to slow rates
Disorders of neutrophil granules
of fusion of the lysosomal granules with phagosomes. NK
The most common neutrophil defect is myeloperoxi- cell function is also impaired. CHS patients may develop
dase deficiency, a primary granule defect inherited as a severe disabling peripheral neuropathy in adulthood that
an autosomal recessive trait; the incidence is 1 in 2000 can lead to bed confinement.
persons. Isolated myeloperoxidase deficiency is not asso- Specific granule deficiency is a rare autosomal reces-
ciated with clinically compromised defenses, presum- sive disease in which the production of secondary gran-
ably because other defense systems such as hydrogen

CHAPTER 5
ules and their contents, as well as the primary granule
peroxide generation are amplified. Microbicidal activity component defensins, is defective. The defect in bacte-
of neutrophils is delayed but not absent. Myeloperoxi- rial killing leads to severe bacterial infections. One type
dase deficiency may make other acquired host defense of specific granule deficiency is due to a mutation in
defects more serious. An acquired form of myeloperoxi- the CCAAT/enhancer binding protein-, a regulator of
dase deficiency occurs in myelomonocytic leukemia and expression of granule components.
acute myeloid leukemia.

Disorders of Granulocytes and Monocytes


Chdiak-Higashi syndrome (CHS) is a rare disease Chronic granulomatous disease
with autosomal recessive inheritance due to defects in the Chronic granulomatous disease (CGD) is a group
lysosomal transport protein LYST, encoded by the gene of disorders of granulocyte and monocyte oxidative
CHS1 at 1q42. This protein is required for normal pack- metabolism. Although CGD is rare, with an incidence
aging and disbursement of granules. Neutrophils (and all of 1 in 200,000 individuals, it is an important model of
cells containing lysosomes) from patients with CHS char- defective neutrophil oxidative metabolism. Most often
acteristically have large granules (Fig. 5-9), making it a CGD is inherited as an X-linked recessive trait; 30%
systemic disease. Patients with CHS have nystagmus, par- of patients inherit the disease in an autosomal recessive
tial oculocutaneous albinism, and an increased number pattern. Mutations in the genes for the five proteins
of infections resulting from many bacterial agents. Some that assemble at the plasma membrane account for all
CHS patients develop an accelerated phase in child- patients with CGD. Two proteins (a 91-kDa protein,
hood with a hemophagocytic syndrome and an aggressive abnormal in X-linked CGD, and a 22-kDa protein,
absent in one form of autosomal recessive CGD) form
the heterodimer cytochrome b-558 in the plasma mem-
brane. Three other proteins (40-, 47-, and 67-kDa,
abnormal in the other autosomal recessive forms of
CGD) are cytoplasmic in origin and interact with the
cytochrome after cell activation to form NADPH oxi-
dase, required for hydrogen peroxide production. Leuko-
cytes from patients with CGD have severely diminished
hydrogen peroxide production. The genes involved in
each of the defects have been cloned and sequenced
and the chromosome locations identified. Patients with
CGD characteristically have increased numbers of infec-
tions due to catalase-positive microorganisms (organ-
isms that destroy their own hydrogen peroxide). When
patients with CGD become infected, they often have
extensive inflammatory reactions, and lymph node
suppuration is common despite the administration of
appropriate antibiotics. Aphthous ulcers and chronic
inflammation of the nares are often present. Granulo-
mas are frequent and can obstruct the gastrointestinal or
genitourinary tracts. The excessive inflammation reflects
failure to downregulate inflammation, reflecting failure
Figure 5-9 to inhibit the synthesis, degradation of, or response to
Chdiak-Higashi syndrome. The granulocytes contain huge chemoattractants or residual antigens, leading to persistent
cytoplasmic granules formed from aggregation and fusion of neutrophil accumulation. Impaired killing of intracellu-
azurophilic and specific granules (arrows). Large abnormal lar microorganisms by macrophages may lead to persis-
granules are found in other granule-containing cells through- tent cell-mediated immune activation and granuloma
out thebody. formation. Autoimmune complications such as immune
52 thrombocytopenic purpura and juvenile rheumatoid (transferrin, fibronectin, transcobalamin II), nucleosides,
arthritis are also increased in CGD. In addition, discoid and cytokines (TNF-; IL-1, -8, -12, -18). IL-1 has
lupus is more common in X-linked carriers. Late com- many functions, including initiating fever in the hypo-
plications, including nodular regenerative hyperplasia thalamus, mobilizing leukocytes from the bone mar-
and portal hypertension, are increasingly recognized in row, and activating lymphocytes and neutrophils.
long-term survivors of severe CGD. TNF- is a pyrogen that duplicates many of the actions
of IL-1 and plays an important role in the pathogenesis
Disorders of phagocyte activation
of gram-negative shock. TNF- stimulates production
Phagocytes depend on cell-surface stimulation to of hydrogen peroxide and related toxic oxygen species
induce signals that evoke multiple levels of the inflam- by macrophages and neutrophils. In addition, TNF-
SECTION II

matory response, including cytokine synthesis, chemo- induces catabolic changes that contribute to the pro-
taxis, and antigen presentation. Mutations affecting the found wasting (cachexia) associated with many chronic
major pathway that signals through NF-B have been diseases.
noted in patients with a variety of infection susceptibil- Other macrophage-secreted products include reac-
ity syndromes. If the defects are at a very late stage of tive oxygen and nitrogen metabolites, bioactive lipids
signal transduction, in the protein critical for NF-B (arachidonic acid metabolites and platelet-activating fac-
Cardinal Manifestations of Hematologic Disease

activation known as the NF-B essential modulator tors), chemokines, CSFs, and factors stimulating fibro-
(NEMO), then affected males develop ectodermal dys- blast and vessel proliferation. Macrophages help regulate
plasia and severe immune deficiency with susceptibil- the replication of lymphocytes and participate in the
ity to bacteria, fungi, mycobacteria, and viruses. If the killing of tumors, viruses, and certain bacteria (Myco-
defects in NF-B activation are closer to the cell-surface bacterium tuberculosis and Listeria monocytogenes). Macro-
receptors, in the proteins transducing Toll-like recep- phages are key effector cells in the elimination of intra-
tor signals, IL-1 receptorassociated kinase 4 (IRAK4), cellular microorganisms. Their ability to fuse to form
and myeloid differentiation primary response gene 88 giant cells that coalesce into granulomas in response to
(MyD88), then children have a marked susceptibility to some inflammatory stimuli is important in the elimina-
pyogenic infections early in life but develop resistance to tion of intracellular microbes and is under the control of
infection later. IFN-. Nitric oxide induced by IFN- is an important
effector against intracellular parasites, including tubercu-
losis and Leishmania.
Mononuclear Phagocytes Macrophages play an important role in the immune
response. They process and present antigen to lympho-
The mononuclear phagocyte system is composed of cytes and secrete cytokines that modulate and direct
monoblasts, promonocytes, and monocytes in addition lymphocyte development and function. Macrophages
to the structurally diverse tissue macrophages that make participate in autoimmune phenomena by removing
up what was previously referred to as the reticuloendo- immune complexes and other substances from the cir-
thelial system. Macrophages are long-lived phagocytic culation. Polymorphisms in macrophage receptors for
cells capable of many of the functions of neutrophils. immunoglobulin (FcRII) determine susceptibility to
They are also secretory cells that participate in many some infections and autoimmune diseases. In wound
immunologic and inflammatory processes distinct from healing, they dispose of senescent cells, and they con-
neutrophils. Monocytes leave the circulation by diape- tribute to atheroma development. Macrophage elastase
desis more slowly than neutrophils and have a half-life mediates development of emphysema from cigarette
in the blood of 1224 h. smoking.
After blood monocytes arrive in the tissues, they dif-
ferentiate into macrophages (big eaters) with specialized
Disorders of the Mononuclear
functions suited for specific anatomic locations. Mac-
Phagocyte System
rophages are particularly abundant in capillary walls of
the lung, spleen, liver, and bone marrow, where they Many disorders of neutrophils extend to mononu-
function to remove microorganisms and other noxious clear phagocytes. Thus, drugs that suppress neutrophil
elements from the blood. Alveolar macrophages, liver production in the bone marrow can cause monocyto-
Kupffer cells, splenic macrophages, peritoneal macro- penia. Transient monocytopenia occurs after stress or
phages, bone marrow macrophages, lymphatic macro- glucocorticoid administration. Monocytosis is associ-
phages, brain microglial cells, and dendritic macrophages ated with tuberculosis, brucellosis, subacute bacterial
all have specialized functions. Macrophage-secreted endocarditis, Rocky Mountain spotted fever, malaria,
products include lysozyme, neutral proteases, acid and visceral leishmaniasis (kala azar). Monocytosis also
hydrolases, arginase, complement components, enzyme occurs with malignancies, leukemias, myeloprolifera-
inhibitors (plasmin, 2-macroglobulin), binding proteins tive syndromes, hemolytic anemias, chronic idiopathic
neutropenias, and granulomatous diseases such as and neutrophils. In certain diseases, such as AIDS, 53
sarcoidosis, regional enteritis, and some collagen vascu- IFN- production may be deficient, whereas in other
lar diseases. Patients with LAD, hyperimmunoglobulin diseases, such as T cell lymphomas, excessive release of
Erecurrent infection (Jobs) syndrome, CHS, and IFN- may be associated with erythrophagocytosis by
CGD all have defects in the mononuclear phagocyte splenic macrophages.
system. Autoinflammatory diseases are characterized by abnor-
Monocyte cytokine production or response is mal cytokine regulation, leading to excess inflammation in
impaired in some patients with disseminated nontuber- the absence of infection. These diseases can mimic infec-
culous mycobacterial infection who are not infected tious or immunodeficient syndromes. Gain-of-function

CHAPTER 5
with HIV. Genetic defects in the pathways regulated by mutations in the TNF- receptor cause TNF- recep-
IFN- and IL-12 lead to impaired killing of intracellular torassociated periodic syndrome (TRAPS), which is
bacteria, mycobacteria, salmonellae, and certain viruses characterized by recurrent fever in the absence of infec-
(Fig. 5-10). tion, due to persistent stimulation of the TNF- recep-
Certain viral infections impair mononuclear phago- tor. Diseases with abnormal IL-1 regulation leading to
cyte function. For example, influenza virus infection fever include familial Mediterranean fever due to muta-
causes abnormal monocyte chemotaxis. Mononuclear tions in PYRIN. Mutations in cold-induced autoinflamma-

Disorders of Granulocytes and Monocytes


phagocytes can be infected by HIV using CCR5, the tory syndrome 1 (CIAS1) lead to neonatal-onset multi-
chemokine receptor that acts as a co-receptor with CD4 system autoinflammatory disease, familial cold urticaria,
for HIV. T lymphocytes produce IFN-, which induces and Muckle-Wells syndrome. The syndrome of pyo-
FcR expression and phagocytosis and stimulates hydro- derma gangrenosum, acne, and sterile pyogenic arthritis
gen peroxide production by mononuclear phagocytes (PAPA syndrome) is caused by mutations in CD2BP1.
In contrast to these syndromes of overexpression of
proinflammatory cytokines, blockade of TNF- by the
antagonists infliximab, adalimumab, certolizumab, or
IL-2
IL-2R
etanercept has been associated with severe infections
due to tuberculosis, nontuberculous mycobacteria, and
fungi.
IFN- Monocytopenia occurs with acute infections, with
T/NK
1
stress, and after treatment with glucocorticoids. Mono-
IL-12R cytopenia also occurs in aplastic anemia, hairy cell leu-
2 kemia, and acute myeloid leukemia and as a direct result
?
18
IFN-R
of myelotoxic drugs.
IL-15

STAT1
1 2

IL-12 AFB Eosinophils


Salm. TNF-
NEMO
Eosinophils and neutrophils share similar morphol-
NRAMP1 ogy, many lysosomal constituents, phagocytic capacity,
Macrophage
TNF-R and oxidative metabolism. Eosinophils express a spe-
CD14 TLR cific chemoattractant receptor and respond to a spe-
cific chemokine, eotaxin, but little is known about their
Figure 5-10 required role. Eosinophils are much longer lived than
Lymphocytemacrophage interactions underlying resis-
neutrophils, and unlike neutrophils, tissue eosinophils
tance to mycobacteria and other intracellular parasites
can recirculate. During most infections, eosinophils
such as Salmonella. Mycobacteria infect macrophages, lead-
appear unimportant. However, in invasive helminthic
ing to the production of IL-12, which activates T or NK cells
infections, such as hookworm, schistosomiasis, strongy-
through its receptor, leading to production of IL-2 and IFN-.
IFN- acts through its receptor on macrophages to upregu-
loidiasis, toxocariasis, trichinosis, filariasis, echinococco-
late TNF- and IL-12 and kill intracellular parasites. Mutant
sis, and cysticercosis, the eosinophil plays a central role
forms of the cytokines and receptors shown in large type in host defense. Eosinophils are associated with bron-
have been found in severe cases of nontuberculous myco- chial asthma, cutaneous allergic reactions, and other
bacterial infection and salmonellosis. AFB, acid-fast bacilli; hypersensitivity states.
IFN, interferon; IL, interleukin; NEMO, NF-B essential mod- The distinctive feature of the red-staining (Wrights
ulator; NK, natural killer; STAT1, signal transducer and acti- stain) eosinophil granule is its crystalline core consisting
vator of transcription 1; TLR, Toll-like receptor; TNF, tumor of an arginine-rich protein (major basic protein) with
necrosis factor. histaminase activity, important in host defense against
54 parasites. Eosinophil granules also contain a unique The idiopathic hypereosinophilic syndrome repre-
eosinophil peroxidase that catalyzes the oxidation of sents a heterogeneous group of disorders with the com-
many substances by hydrogen peroxide and may facili- mon feature of prolonged eosinophilia of unknown
tate killing of microorganisms. cause and organ system dysfunction, including the
Eosinophil peroxidase, in the presence of hydrogen heart, central nervous system, kidneys, lungs, gastroin-
peroxide and halide, initiates mast cell secretion in vitro testinal tract, and skin. The bone marrow is involved
and thereby promotes inflammation. Eosinophils con- in all affected individuals, but the most severe com-
tain cationic proteins, some of which bind to heparin plications involve the heart and central nervous sys-
and reduce its anticoagulant activity. Eosinophil-derived tem. Clinical manifestations and organ dysfunction are
neurotoxin and eosinophil cationic protein are ribonu- highly variable. Eosinophils are found in the involved
SECTION II

cleases that can kill respiratory syncytial virus. Eosinophil tissues and likely cause tissue damage by local deposi-
cytoplasm contains Charcot-Leyden crystal protein, a tion of toxic eosinophil proteins such as eosinophil
hexagonal bipyramidal crystal first observed in a patient cationic protein and major basic protein. In the heart,
with leukemia and then in sputum of patients with the pathologic changes lead to thrombosis, endocar-
asthma; this protein is lysophospholipase and may func- dial fibrosis, and restrictive endomyocardiopathy. The
tion to detoxify certain lysophospholipids. damage to tissues in other organ systems is similar.
Cardinal Manifestations of Hematologic Disease

Several factors enhance the eosinophils function in Some cases are due to mutations involving the platelet-
host defense. T cellderived factors enhance the ability derived growth factor receptor, and these are extremely
of eosinophils to kill parasites. Mast cellderived eosino- sensitive to the tyrosine kinase inhibitor imatinib.
phil chemotactic factor of anaphylaxis (ECFa) increases Glucocorticoids, hydroxyurea, and IFN- each have
the number of eosinophil complement receptors and been used successfully, as have therapeutic antibodies
enhances eosinophil killing of parasites. Eosinophil against IL-5. Cardiovascular complications are managed
CSFs (e.g., IL-5) produced by macrophages increase aggressively.
eosinophil production in the bone marrow and activate The eosinophiliamyalgia syndrome is a multisystem
eosinophils to kill parasites. disease, with prominent cutaneous, hematologic, and
visceral manifestations, that frequently evolves into a
chronic course and can occasionally be fatal. The syn-
Eosinophilia drome is characterized by eosinophilia (eosinophil
count >1000/L) and generalized disabling myalgias
Eosinophilia is the presence of >500 eosinophils per L without other recognized causes. Eosinophilic fasci-
of blood and is common in many settings besides para- itis, pneumonitis, and myocarditis; neuropathy culmi-
site infection. Significant tissue eosinophilia can occur nating in respiratory failure; and encephalopathy may
without an elevated blood count. A common cause of occur. The disease is caused by ingesting contaminants
eosinophilia is allergic reaction to drugs (iodides, aspi- in l-tryptophancontaining products. Eosinophils,
rin, sulfonamides, nitrofurantoin, penicillins, and cepha- lymphocytes, macrophages, and fibroblasts accumulate
losporins). Allergies such as hay fever, asthma, eczema, in the affected tissues, but their role in pathogenesis is
serum sickness, allergic vasculitis, and pemphigus are unclear. Activation of eosinophils and fibroblasts and
associated with eosinophilia. Eosinophilia also occurs the deposition of eosinophil-derived toxic proteins in
in collagen vascular diseases (e.g., rheumatoid arthri- affected tissues may contribute. IL-5 and transform-
tis, eosinophilic fasciitis, allergic angiitis, and periarteri- ing growth factor have been implicated as potential
tis nodosa) and malignancies (e.g., Hodgkins disease; mediators. Treatment is withdrawal of products con-
mycosis fungoides; chronic myeloid leukemia; and can- taining l-tryptophan and the administration of gluco-
cer of the lung, stomach, pancreas, ovary, or uterus), corticoids. Most patients recover fully, remain stable, or
as well as in Jobs syndrome, DOCK8 deficiency (dis- show slow recovery, but the disease can be fatal in up
cussed later), and CGD. Eosinophilia is commonly to 5% of patients.
present in helminthic infections. IL-5 is the dominant
eosinophil growth factor. Therapeutic administration
of the cytokines IL-2 and GM-CSF frequently leads to
Eosinopenia
transient eosinophilia. The most dramatic hypereosino-
philic syndromes are Loefflers syndrome, tropical pul- Eosinopenia occurs with stress, such as acute bacterial
monary eosinophilia, Loefflers endocarditis, eosinophilic infection, and after treatment with glucocorticoids. The
leukemia, and idiopathic hypereosinophilic syndrome mechanism of eosinopenia of acute bacterial infection is
(50,000100,000/L). IL-5 is the dominant eosinophil unknown but is independent of endogenous glucocor-
growth factor and can be specifically inhibited with the ticoids, since it occurs in animals after total adrenalec-
monoclonal antibody mepolizumab. tomy. There is no known adverse effect of eosinopenia.
microscopically (NBT) or by flow cytometry (DHR). 55
Hyperimmunoglobulin E Qualitative studies of superoxide and hydrogen perox-
Recurrent Infection Syndrome ide production may further define neutrophil oxidative
The hyperimmunoglobulin Erecurrent infection syn- function.
drome, or Jobs syndrome, is a rare multisystem disease Patients with leukopenias or leukocyte dysfunction
in which the immune and somatic systems are affected, often have delayed inflammatory responses. Therefore,
including neutrophils, monocytes, T cells, B cells, and clinical manifestations may be minimal despite over-
osteoclasts. Autosomal dominant mutations in signal whelming infection, and unusual infections must always
transducer and activator of transcription 3 (STAT3) be suspected. Early signs of infection demand prompt,

CHAPTER 5
lead to inhibition of normal STAT signaling with aggressive culturing for microorganisms, use of antibiot-
broad and profound effects. Patients have characteris- ics, and surgical drainage of abscesses. Prolonged courses
tic facies with broad noses, kyphoscoliosis and osteo- of antibiotics are often required. In patients with CGD,
porosis, and eczema. The primary teeth erupt normally prophylactic antibiotics (trimethoprimsulfamethoxa-
but do not deciduate, often requiring extraction. zole) and antifungals (itraconazole) markedly diminish
Patients develop recurrent sinopulmonary and cuta- the frequency of life-threatening infections. Glucocor-
ticoids may relieve gastrointestinal or genitourinary

Disorders of Granulocytes and Monocytes


neous infections that tend to be much less inflamed
than appropriate for the degree of infection and have tract obstruction by granulomas in patients with CGD.
been referred to as cold abscesses. Characteristi- Although TNF- blocking agents may markedly relieve
cally, pneumonias cavitate, leading to pneumatoceles. inflammatory bowel symptoms, extreme caution must
Coronary artery aneurysms are common, as are cere- be exercised in their use in CGD inflammatory bowel
bral demyelinated plaques that accumulate with age. disease, as it profoundly increases these patients already
Importantly, IL-17producing cells, which are thought heightened susceptibility to infection. Recombinant
responsible for protection against extracellular and human IFN-, which nonspecifically stimulates phago-
mucosal infections, are profoundly reduced in Jobs cytic cell function, reduces the frequency of infections
syndrome. Despite very high IgE levels, these patients in patients with CGD by 70% and reduces the severity
do not have elevated levels of allergy. An important of infection. This effect of IFN- in CGD is additive to
syndrome with clinical overlap with STAT3 defi- the effect of prophylactic antibiotics. The recommended
ciency is due to autosomal recessive defects in dedica- dose is 50 g/m2 subcutaneously three times weekly.
tor of cytokinesis 8 (DOCK8). In DOCK8 deficiency, IFN- has also been used successfully in the treatment
IgE elevation is joined to severe allergy, viral suscepti- of leprosy, nontuberculous mycobacteria, and visceral
bility, and increased rates of cancer. leishmaniasis.
Rigorous oral hygiene reduces but does not eliminate
the discomfort of gingivitis, periodontal disease, and
aphthous ulcers; chlorhexidine mouthwash and tooth
Laboratory Diagnosis and brushing with a hydrogen peroxidesodium bicarbon-
Management ate paste helps many patients. Oral antifungal agents
(fluconazole, itraconazole, voriconazole, posaconazole)
Initial studies of WBC and differential and often a bone have reduced mucocutaneous candidiasis in patients
marrow examination may be followed by assessment with Jobs syndrome. Androgens, glucocorticoids, lith-
of bone marrow reserves (steroid challenge test), mar- ium, and immunosuppressive therapy have been used to
ginated circulating pool of cells (epinephrine challenge restore myelopoiesis in patients with neutropenia due to
test), and marginating ability (endotoxin challenge test) impaired production. Recombinant G-CSF is useful in
(Fig. 5-7). In vivo assessment of inflammation is possible the management of certain forms of neutropenia due to
with a Rebuck skin window test or an in vivo skin blis- depressed neutrophil production, especially those related
ter assay, which measures the ability of leukocytes and to cancer chemotherapy. Patients with chronic neutro-
inflammatory mediators to accumulate locally in the penia with evidence of a good bone marrow reserve
skin. In vitro tests of phagocyte aggregation, adherence, need not receive prophylactic antibiotics. Patients
chemotaxis, phagocytosis, degranulation, and microbi- with chronic or cyclic neutrophil counts <500/L
cidal activity (for S. aureus) may help pinpoint cellular may benefit from prophylactic antibiotics and G-CSF
or humoral lesions. Deficiencies of oxidative metabo- during periods of neutropenia. Oral trimethoprim
lism are detected with either the nitroblue tetrazolium sulfamethoxazole (160/800 mg) twice daily can prevent
(NBT) dye test or the dihydrorhodamine (DHR) oxi- infection. Increased numbers of fungal infections are
dation test. These tests are based on the ability of prod- not seen in patients with CGD on this regimen. Oral
ucts of oxidative metabolism to alter the oxidation quinolones such as levofloxacin and ciprofloxacin are
states of reporter molecules so that they can be detected alternatives.
56 In the setting of cytotoxic chemotherapy with severe, years without a life-threatening infection, there may still
persistent neutropenia, trimethoprimsulfamethoxazole be delayed effects of prolonged antimicrobials and other
prevents Pneumocystis jirovecii pneumonia. These patients inflammatory complications. Cure of most congenital
and patients with phagocytic cell dysfunction should phagocyte defects is possible by bone marrow transplan-
avoid heavy exposure to airborne soil, dust, or decay- tation, and rates of success are improving (Chap. 30).
ing matter (mulch, manure), which are often rich in The identification of specific gene defects in patients
Nocardia spp. and the spores of Aspergillus spp. and other with LAD 1, CGD, and other immunodeficiencies has
fungi. Restriction of activities or social contact has no led to gene therapy trials in a number of genetic WBC
proven role in reducing risk of infection. disorders.
Although aggressive medical care for many patients
SECTION II

with phagocytic disorders can allow them to go for


Cardinal Manifestations of Hematologic Disease
CHAPTER 6

ATLAS OF HEMATOLOGY AND ANALYSIS OF


PERIPHERAL BLOOD SMEARS

Dan L. Longo

Some of the relevant findings in peripheral blood, can be associated with falsely low automated plate-
enlarged lymph nodes, and bone marrow are illustrated let counts. Similarly, neutrophil fragmentation can be a
in this chapter. Systematic histologic examination of the source of falsely elevated automated platelet counts.
bone marrow and lymph nodes is beyond the scope of Next one examines the red blood cells. One can
a general medicine textbook. However, every internist gauge their size by comparing the red cell with the
should know how to examine a peripheral blood smear. nucleus of a small lymphocyte. Both are normally about
The examination of a peripheral blood smear is 8 m wide. Red cells that are smaller than the small
one of the most informative exercises a physician can lymphocyte nucleus may be microcytic; those larger
perform. Although advances in automated technology than the small lymphocyte nucleus may be macrocytic.
have made the examination of a peripheral blood smear Macrocytic cells also tend to be more oval than spheri-
by a physician seem less important, the technology is not cal in shape and are sometimes called macroovalocytes.
a completely satisfactory replacement for a blood smear The automated mean corpuscular volume (MCV) can
interpretation by a trained medical professional who also assist in making a classification. However, some patients
knows the patients clinical history, family history, social may have both iron and vitamin B12 deficiency, which
history, and physical findings. It is useful to ask the labo- will produce an MCV in the normal range but wide
ratory to generate a Wrights-stained peripheral blood variation in red cell size. When the red cells vary greatly
smear and examine it. in size, anisocytosis is said to be present. When the red
The best place to examine blood cell morphology is cells vary greatly in shape, poikilocytosis is said to be pres-
the feathered edge of the blood smear where red cells lie ent. The electronic cell counter provides an indepen-
in a single layer, side by side, just barely touching one dent assessment of variability in red cell size. It measures
another but not overlapping. The authors approach is the range of red cell volumes and reports the results as
to look at the smallest cellular elements, the platelets, red cell distribution width (RDW). This value is cal-
first and work his way up in size to red cells and then culated from the MCV; thus, cell width is not being
white cells. measured but cell volume is. The term is derived from
Using an oil immersion lens that magnifies the cells the curve displaying the frequency of cells at each vol-
100-fold, one counts the platelets in five to six fields, ume, also called the distribution. The width of red
averages the number per field, and multiplies by 20,000 cell volume distribution curve is what determines the
to get a rough estimate of the platelet count. The plate- RDW. The RDW is calculated as follows: RDW =
lets are usually 12 m in diameter and have a blue (standard deviation of MCV mean MCV) 100. In
granulated appearance. There is usually 1 platelet for the presence of morphologic anisocytosis, RDW (nor-
every 20 or so red cells. Of course, the automated coun- mally 1114%) increases to 1518%. The RDW is
ter is much more accurate, but gross disparities between useful in at least two clinical settings. In patients with
the automated and manual counts should be assessed. microcytic anemia, the differential diagnosis is generally
Large platelets may be a sign of rapid platelet turnover, between iron deficiency and thalassemia. In thalas-
as young platelets are often larger than old ones; alter- semia, the small red cells are generally of uniform size
natively, certain rare inherited syndromes can produce with a normal small RDW. In iron deficiency, the size
large platelets. Platelet clumping visible on the smear variability and the RDW are large. In addition, a large
57
58 RDW can suggest a dimorphic anemia when a chronic in iron deficiency, myelodysplastic syndromes, megalo-
atrophic gastritis can produce both vitamin B12 malab- blastic anemia, and thalassemias. Stomatocytes are red cells
sorption to produce macrocytic anemia and blood loss in which the area of central pallor takes on the morphol-
to produce iron deficiency. In such settings, RDW is ogy of a slit instead of the usual round shape. Stomato-
also large. An elevated RDW also has been reported as cytes can indicate an inherited red cell membrane defect
a risk factor for all-cause mortality in population-based and also can be seen in alcoholism. Target cells have an
studies (Patel KV, Ferrucci L, Ershler WB, et al: Red area of central pallor that contains a dense center, or bulls
blood cell distribution width and the risk of death in eye. These cells are seen classically in thalassemia, but
middle-aged and older adults. Arch Intern Med 169:515, they are also present in iron deficiency, cholestatic liver
2009), a finding that is unexplained currently. disease, and some hemoglobinopathies. They also can be
SECTION II

After red cell size is assessed, one examines the hemo- generated artifactually by improper slide making.
globin content of the cells. They are either normal in One last feature of the red cells to assess before mov-
color (normochromic) or pale in color (hypochromic). They ing to the white blood cells is the distribution of the
are never hyperchromic. If more than the normal red cells on the smear. In most individuals, the cells lie
amount of hemoglobin is made, the cells get larger side by side in a single layer. Some patients have red cell
they do not become darker. In addition to hemoglobin clumping (called agglutination) in which the red cells pile
Cardinal Manifestations of Hematologic Disease

content, the red cells are examined for inclusions. Red upon one another; it is seen in certain paraproteinemias
cell inclusions are the following: and autoimmune hemolytic anemias. Another abnormal
distribution involves red cells lying in single cell rows
1. Basophilic stipplingdiffuse fine or coarse blue dots
on top of one another like stacks of coins. This is called
in the red cell usually representing RNA residue
rouleaux formation and reflects abnormal serum protein
especially common in lead poisoning
levels.
2. Howell-Jolly bodiesdense blue circular inclusions
Finally, one examines the white blood cells. Three
that represent nuclear remnantstheir presence
types of granulocytes are usually present: neutrophils,
implies defective splenic function
eosinophils, and basophils, in decreasing frequency. Neu-
3. Nucleired cells may be released or pushed out of
trophils are generally the most abundant white cell. They
the marrow prematurely before nuclear extrusion
are round, are 1014 m wide, and contain a lobulated
often implies a myelophthisic process or a vigorous
nucleus with two to five lobes connected by a thin chro-
narrow response to anemia, usually hemolytic anemia
matin thread. Bands are immature neutrophils that have
4. Parasitesred cell parasites include malaria and babesia
not completed nuclear condensation and have a U-shaped
5. Polychromatophiliathe red cell cytoplasm has a bluish
nucleus. Bands reflect a left shift in neutrophil maturation
hue, reflecting the persistence of ribosomes still actively
in an effort to make more cells more rapidly. Neutrophils
making hemoglobin in a young red cell
can provide clues to a variety of conditions. Vacuolated
Vital stains are necessary to see precipitated hemoglo- neutrophils may be a sign of bacterial sepsis. The presence
bin called Heinz bodies. of 1- to 2-m blue cytoplasmic inclusions, called Dhle
Red cells can take on a variety of different shapes. bodies, can reflect infections, burns, or other inflamma-
All abnormally shaped red cells are poikilocytes. Small tory states. If the neutrophil granules are larger than nor-
red cells without the central pallor are spherocytes; they mal and stain a darker blue, toxic granulations are said to
can be seen in hereditary spherocytosis, hemolytic be present, and they also suggest a systemic inflammation.
anemias of other causes, and clostridial sepsis. Dacrocytes The presence of neutrophils with more than five nuclear
are teardrop-shaped cells that can be seen in hemolytic lobes suggests megaloblastic anemia. Large misshapen
anemias, severe iron deficiency, thalassemias, myelofi- granules may reflect the inherited Chdiak-Higashi
brosis, and myelodysplastic syndromes. Schistocytes are syndrome.
helmet-shaped cells that reflect microangiopathic hemo- Eosinophils are slightly larger than neutrophils, have
lytic anemia or fragmentation on an artificial heart valve. bilobed nuclei, and contain large red granules. Diseases
Echinocytes are spiculated red cells with the spikes evenly of eosinophils are associated with too many of them
spaced; they can represent an artifact of abnormal drying rather than any morphologic or qualitative change. They
of the blood smear or reflect changes in stored blood. normally total less than one-thirtieth the number of neu-
They also can be seen in renal failure and malnutrition trophils. Basophils are even more rare than eosinophils
and are often reversible. Acanthocytes are spiculated red in the blood. They have large dark blue granules and
cells with the spikes irregularly distributed. This process may be increased as part of chronic myeloid leukemia.
tends to be irreversible and reflects underlying renal dis- Lymphocytes can be present in several morphologic
ease, abetalipoproteinemia, or splenectomy. Elliptocytes forms. Most common in healthy individuals are small
are elliptical-shaped red cells that can reflect an inherited lymphocytes with a small dark nucleus and scarce cyto-
defect in the red cell membrane, but they also are seen plasm. In the presence of viral infections, more of the
lymphocytes are larger, about the size of neutrophils, a variety of shapes but usually appears to be folded; the 59
with abundant cytoplasms and a less condensed nuclear cytoplasm is gray.
chromatin. These cells are called reactive lymphocytes. Abnormal cells may appear in the blood. Most
About 1% of lymphocytes are larger and contain blue often the abnormal cells originate from neoplasms of
granules in a light blue cytoplasm; they are called large bone marrowderived cells, including lymphoid cells,
granular lymphocytes. In chronic lymphoid leukemia, the myeloid cells, and occasionally red cells. More rarely,
small lymphocytes are increased in number, and many other types of tumors can get access to the bloodstream,
of them are ruptured in making the blood smear, leav- and rare epithelial malignant cells may be identified.
ing a smudge of nuclear material without a surround- The chances of seeing such abnormal cells is increased
ing cytoplasm or cell membrane; they are called smudge by examining blood smears made from buffy coats, the

CHAPTER 6
cells and are rare in the absence of chronic lymphoid layer of cells that is visible on top of sedimenting red
leukemia. cells when blood is left in the test tube for an hour.
Monocytes are the largest white blood cells, ranging Smears made from finger sticks may include rare endo-
from 15 to 22 m in diameter. The nucleus can take on thelial cells.

Atlas of Hematology and Analysis of Peripheral Blood Smears


Figure 6-1 Figure 6-3
Normal peripheral blood smear. Small lymphocyte in cen- Hypochromic microcytic anemia of iron deficiency. Small
ter of field. Note that the diameter of the red blood cell is lymphocyte in field helps assess the red blood cell size.
similar to the diameter of the small lymphocyte nucleus.

Figure 6-2 Figure 6-4


Reticulocyte count preparation. This new methylene blue Iron deficiency anemia next to normal red blood cells.
stained blood smear shows large numbers of heavily stained Microcytes (right panel) are smaller than normal red blood
reticulocytes (the cells containing the dark bluestaining RNA cells (cell diameter <7 m) (left panel) and may or may not be
precipitates). poorly hemoglobinized (hypochromic).
60
SECTION II

Figure 6-5 Figure 6-8


Polychromatophilia. Note large red cells with light purple Spherocytosis. Note small hyperchromatic cells without the
Cardinal Manifestations of Hematologic Disease

coloring. usual clear area in the center.

Figure 6-6 Figure 6-9


Macrocytosis. These cells are both larger than normal (mean Rouleaux formation. Small lymphocyte in center of field.
corpuscular volume >100) and somewhat oval in shape. These red cells align themselves in stacks and are related to
Some morphologists call these cells macroovalocytes. increased serum protein levels.

Figure 6-7
Hypersegmented neutrophils. Hypersegmented neutro-
phils (multilobed polymorphonuclear leukocytes) are larger Figure 6-10
than normal neutrophils with five or more segmented nuclear Red cell agglutination. Small lymphocyte and segmented
lobes. They are commonly seen with folic acid or vitamin B12 neutrophil in the upper left center. Note the irregular collec-
deficiency. tions of aggregated red cells.
61

CHAPTER 6
Figure 6-14
Elliptocytosis. Small lymphocyte in center of field. Elliptical
Figure 6-11 shape of red cells related to weakened membrane structure,
Fragmented red cells. Heart valve hemolysis. usually due to mutations in spectrin.

Atlas of Hematology and Analysis of Peripheral Blood Smears


Figure 6-15
Stomatocytosis. Red cells characterized by a wide trans-
verse slit or stoma. This often is seen as an artifact in a
dehydrated blood smear. These cells can be seen in hemo-
lytic anemias and in conditions in which the red cells are
Figure 6-12
overhydrated or dehydrated.
Sickle cells. Homozygous sickle cell disease. A nucleated
red cell and neutrophil are also in the field.

Figure 6-16
Acanthocytosis. Spiculated red cells are of two types: acan-
thocytes are contracted dense cells with irregular membrane
projections that vary in length and width; echinocytes have
small, uniform, and evenly spaced membrane projections.
Figure 6-13 Acanthocytes are present in severe liver disease, in patients
Target cells. Target cells are recognized by the bulls-eye with abetalipoproteinemia, and in rare patients with McLeod
appearance of the cell. Small numbers of target cells are blood group. Echinocytes are found in patients with severe
seen with liver disease and thalassemia. Larger numbers are uremia, in glycolytic red cell enzyme defects, and in microan-
typical of hemoglobin C disease. giopathic hemolytic anemia.
62
SECTION II

Figure 6-17
Howell-Jolly bodies. Howell-Jolly bodies are tiny nuclear
remnants that normally are removed by the spleen. They Figure 6-20
appear in the blood after splenectomy (defect in removal) and Reticulin stain of marrow myelofibrosis. Silver stain of a
Cardinal Manifestations of Hematologic Disease

with maturation/dysplastic disorders (excess production). myelofibrotic marrow showing an increase in reticulin fibers
(black-staining threads).

Figure 6-18
Teardrop cells and nucleated red blood cells characteris-
tic of myelofibrosis. A teardrop-shaped red blood cell (left Figure 6-21
panel) and a nucleated red blood cell (right panel) as typically Stippled red cell in lead poisoning. Mild hypochromia.
seen with myelofibrosis and extramedullary hematopoiesis. Coarsely stippled red cell.

Figure 6-19 Figure 6-22


Myelofibrosis of the bone marrow. Total replacement of Heinz bodies. Blood mixed with hypotonic solution of crys-
marrow precursors and fat cells by a dense infiltrate of retic- tal violet. The stained material is precipitates of denatured
ulin fibers and collagen (H&E stain). hemoglobin within cells.
63

CHAPTER 6
Figure 6-23
Giant platelets. Giant platelets, together with a marked
increase in the platelet count, are seen in myeloproliferative Figure 6-26
disorders, especially primary thrombocythemia. Normal eosinophils. The film was prepared from the buffy

Atlas of Hematology and Analysis of Peripheral Blood Smears


coat of the blood from a normal donor. E, eosinophil; L, lym-
phocyte. N, neutrophil;

Figure 6-27
Figure 6-24
Normal basophil. The film was prepared from the buffy coat
Normal granulocytes. The normal granulocyte has a seg-
of the blood from a normal donor. B, basophil; L, lymphocyte.
mented nucleus with heavy, clumped chromatin; fine neutro-
philic granules are dispersed throughout the cytoplasm.

Figure 6-25 Figure 6-28


Normal monocytes. The film was prepared from the buffy Pelger-Het anomaly. In this benign disorder, the major-
coat of the blood from a normal donor. L, lymphocyte; M ity of granulocytes are bilobed. The nucleus frequently has a
monocyte; N, neutrophil. spectacle-like, or pince-nez, configuration.
64
SECTION II

Figure 6-29
Figure 6-32
Dhle body. Neutrophil band with Dhle body. The neutro-
Aplastic anemia bone marrow. Normal hematopoietic pre-
phil with a sausage-shaped nucleus in the center of the field
cursor cells are virtually absent, leaving behind fat cells, retic-
is a band form. Dhle bodies are discrete, blue-staining non-
Cardinal Manifestations of Hematologic Disease

uloendothelial cells, and the underlying sinusoidal structure.


granular areas found in the periphery of the cytoplasm of the
neutrophil in infections and other toxic states. They represent
aggregates of rough endoplasmic reticulum.

Figure 6-30
Chdiak-Higashi disease. Note giant granules in neutrophil.
Figure 6-33
Metastatic cancer in the bone marrow. Marrow biopsy
specimen infiltrated with metastatic breast cancer and reac-
tive fibrosis (H&E stain).

Figure 6-31
Normal bone marrow. Low-power view of normal adult mar-
row (H&E stain), showing a mix of fat cells (clear areas) and
hematopoietic cells. The percentage of the space that con-
sists of hematopoietic cells is referred to as marrow cellularity.
In adults, normal marrow cellularity is 3540%. If demands for
increased marrow production occur, cellularity may increase Figure 6-34
to meet the demand. As people age, the marrow cellularity Lymphoma in the bone marrow. Nodular (follicular) lym-
decreases and the marrow fat increases. Patients >70 years phoma infiltrate in a marrow biopsy specimen. Note the
old may have a 2030% marrow cellularity. characteristic paratrabecular location of the lymphoma cells.
65

A B

CHAPTER 6
C D
Figure 6-35
Erythroid hyperplasia of the marrow. Marrow aspirate Figure 6-38
specimen with a myeloid/erythroid ratio (M/E ratio) of 1:12, Prussian blue staining of marrow iron stores. Iron stores

Atlas of Hematology and Analysis of Peripheral Blood Smears


typical for a patient with a hemolytic anemia or one recover- can be graded on a scale of 0 to 4+. A: a marrow with
ing from blood loss. excess iron stores (>4+); B: normal stores (23+); C: minimal
stores (1+); and D: absent iron stores (0).

Figure 6-39
Figure 6-36
Ringed sideroblast. An orthochromatic normoblast with
Myeloid hyperplasia of the marrow. Marrow aspirate speci-
a collar of blue granules (mitochondria encrusted with iron)
men showing a myeloid/erythroid ratio of 3:1, suggesting
surrounding the nucleus.
either a loss of red blood cell precursors or an expansion of
myeloid elements.

Figure 6-37
Megaloblastic erythropoiesis. High-power view of megalo-
blastic red blood cell precursors from a patient with a mac-
rocytic anemia. Maturation is delayed, with late normoblasts Figure 6-40
showing a more immature-appearing nucleus with a lattice- Acute myeloid leukemia. Leukemic myeloblast with an Auer
like pattern with normal cytoplasmic maturation. rod. Note two to four large, prominent nucleoli in each cell.
66
SECTION II

Figure 6-44
Burkitts leukemia, acute lymphoblastic leukemia.
Cardinal Manifestations of Hematologic Disease

Figure 6-41
Acute promyelocytic leukemia. Note prominent cytoplas-
mic granules in the leukemia cells.

Figure 6-42 Figure 6-45


Acute erythroleukemia. Note giant dysmorphic erythro- Chronic myeloid leukemia in the peripheral blood.
blasts; two are binucleate, and one is multinucleate.

Figure 6-43 Figure 6-46


Acute lymphoblastic leukemia. Chronic lymphoid leukemia in the peripheral blood.
67

CHAPTER 6
Figure 6-50
Figure 6-47 Diffuse large B cell lymphoma in a lymph node. The neo-
Szarys syndrome. Lymphocytes with frequently convo- plastic cells are heterogeneous but predominantly large cells

Atlas of Hematology and Analysis of Peripheral Blood Smears


luted nuclei (Szary cells) in a patient with advanced mycosis with vesicular chromatin and prominent nucleoli.
fungoides.

Figure 6-51
Figure 6-48 Burkitts lymphoma in a lymph node. Burkitts lymphoma
Adult T cell leukemia. Peripheral blood smear showing with starry-sky appearance. The lighter areas are macro-
leukemia cells with typical flower-shaped nucleus. phages attempting to clear dead cells.

Figure 6-49
Follicular lymphoma in a lymph node. The normal nodal Figure 6-52
architecture is effaced by nodular expansions of tumor cells. Erythrophagocytosis accompanying aggressive lym-
Nodules vary in size and contain predominantly small lym- phoma. The central macrophage is ingesting red cells, neu-
phocytes with cleaved nuclei along with variable numbers of trophils, and platelets. (Courtesy of Dr. Kiyomi Tsukimori,
larger cells with vesicular chromatin and prominent nucleoli. Kyushu University, Fukuoka, Japan.)
68
SECTION II

Figure 6-53
Hodgkins disease. A Reed-Sternberg cell is present near
the center of the field; a large cell with a bilobed nucleus and
prominent nucleoli giving an owls eyes appearance. The
Cardinal Manifestations of Hematologic Disease

majority of the cells are normal lymphocytes, neutrophils, Figure 6-56


and eosinophils that form a pleiomorphic cellular infiltrate. Multiple myeloma.

Figure 6-54
Lacunar cell; Reed-Sternberg cell variant in nodular scle-
rosing Hodgkins disease. High-power view of single mono-
nuclear lacunar cell with retracted cytoplasm in a patient with
nodular sclerosing Hodgkins disease.

Figure 6-57
Color serum in hemoglobinemia. The distinctive red color-
ation of plasma (hemoglobinemia) in a spun blood sample in
a patient with intravascular hemolysis.

Acknowledgment

Figures in this chapter were borrowed from Lichtman M,


et al (eds): Williams Hematology, 7th edition. New York,
McGraw-Hill, 2005; Hillman RS, Ault KA: Hematology in
General Practice, 4th edition. New York, McGraw-Hill, 2005.

Figure 6-55
Normal plasma cell.
SECTION III

Anemias
chApter 7

IRON DEFICIENCY AND OTHER


HYPOPROLIFERATIVE ANEMIAS

John W. Adamson

Anemias associated with normocytic and normochromic red Table 7-1. Without iron, cells lose their capacity for
cells and an inappropriately low reticulocyte response electron transport and energy metabolism. In erythroid
(reticulocyte index <22.5) are hypoproliferative anemias. cells, hemoglobin synthesis is impaired, resulting in ane-
This category includes early iron deficiency (before mia and reduced O2 delivery to tissue.
hypochromic microcytic red cells develop), acute and
chronic inflammation (including many malignancies), ThE IrON CyClE IN huMaNS
renal disease, hypometabolic states such as protein mal-
nutrition and endocrine deficiencies, and anemias from Figure 7-1 outlines the major pathways of internal
marrow damage. Marrow damage states are discussed in iron exchange in humans. Iron absorbed from the diet
Chap. 11. or released from stores circulates in the plasma bound
Hypoproliferative anemias are the most common to transferrin, the iron transport protein. Transferrin
anemias, and anemia associated with chronic inflam- is a bilobed glycoprotein with two iron binding sites.
mation is the most common of these. The anemia of Transferrin that carries iron exists in two forms
inflammation, similar to iron deficiency, is related in monoferric (one iron atom) or diferric (two iron atoms).
part to abnormal iron metabolism. The anemias asso- The turnover (half-clearance time) of transferrin-bound
ciated with renal disease, inflammation, cancer, and iron is very rapidtypically 6090 min. Because almost
hypometabolic states are characterized by an abnormal all of the iron transported by transferrin is delivered to
erythropoietin response to the anemia. the erythroid marrow, the clearance time of transferrin-
bound iron from the circulation is affected most by the
plasma iron level and the erythroid marrow activity.
When erythropoiesis is markedly stimulated, the pool of
Iron metABolIsm erythroid cells requiring iron increases, and the clearance
time of iron from the circulation decreases. The half-
Iron is a critical element in the function of all cells, clearance time of iron in the presence of iron deficiency
although the amount of iron required by individual tis- is as short as 1015 min. With suppression of erythro-
sues varies during development. At the same time, the poiesis, the plasma iron level typically increases, and the
body must protect itself from free iron, which is highly
toxic in that it participates in chemical reactions that Table 7-1
generate free radicals such as singlet O2 or OH. Conse-
BODy IrON DISTrIBuTION
quently, elaborate mechanisms have evolved that allow
iron to be made available for physiologic functions IrON CONTENT, mg
while at the same time conserving this element and
aDulT MalE, aDulT fEMalE,
handling it in such a way that toxicity is avoided. 80 kg 60 kg
The major role of iron in mammals is to carry O2
as part of hemoglobin. O2 is also bound by myoglobin Hemoglobin 2500 1700
Myoglobin/enzymes 500 300
in muscle. Iron is a critical element in iron-containing Transferrin iron 3 3
enzymes, including the cytochrome system in mito- Iron stores 6001000 0300
chondria. Iron distribution in the body is shown in
70
receptor protein. Within the erythroid cell, iron in 71
excess of the amount needed for hemoglobin synthe-
RE
stor
sis binds to a storage protein, apoferritin, forming ferritin.
es
This mechanism of iron exchange also takes place in
Circulating other cells of the body expressing transferrin receptors,
thro
id erythrocytes RE especially liver parenchymal cells, where the iron can be
Ery rrow cell
m a s incorporated into heme-containing enzymes or stored.
The iron incorporated into hemoglobin subsequently
Transferrin
iron enters the circulation as new red cells are released from
Gut Extravascular the bone marrow. The iron is then part of the red cell
exchange
mass and will not become available for reutilization
until the red cell dies.
Parenchyma In a normal individual, the average red cell life span
(Liver)
is 120 days. Thus, 0.81% of red cells turn over each
day. At the end of its life span, a red cell is recognized
as senescent by the cells of the reticuloendothelial (RE) sys-
Figure 7-1
tem, and the cell undergoes phagocytosis. Once within

CHAPTER 7
Internal iron exchange. Normally approximately 80% of
the RE cell, the hemoglobin from the ingested red
iron passing through the plasma transferrin pool is recycled cell is broken down, the globin and other proteins are
from broken-down red cells. Absorption of approximately returned to the amino acid pool, and the iron is shuttled
1 mg/d is required from the diet in men and 1.4 mg/d in back to the surface of the RE cell, where it is presented
women to maintain homeostasis. As long as transferrin satu- to circulating transferrin. It is the efficient and highly
ration is maintained between 2060% and erythropoiesis is conserved recycling of iron from senescent red cells

Iron Deficiency and Other Hypoproliferative Anemias


not increased, use of iron stores is not required. However, in that supports steady state (and even mildly accelerated)
the event of blood loss, dietary iron deficiency, or inadequate erythropoiesis.
iron absorption, up to 40 mg/d of iron can be mobilized from Because each milliliter of red cells contains 1 mg of
stores. RE, reticuloendothelial. elemental iron, the amount of iron needed to replace
red cells lost through senescence amounts to 20mg/d
(assuming an adult with a red cell mass of 2 L). Any
additional iron required for daily red cell production
half-clearance time may be prolonged to several hours. comes from the diet. Normally, an adult male will
Normally, the iron bound to transferrin turns over 68 need to absorb at least 1 mg of elemental iron daily
times per day. Assuming a normal plasma iron level of to meet needs, while females in the childbearing years
80100 g/dL, the amount of iron passing through the will need to absorb an average of 1.4 mg/d. However,
transferrin pool is 2024 mg/d. to achieve a maximum proliferative erythroid mar-
The irontransferrin complex circulates in the plasma row response to anemia, additional iron must be
until it interacts with specific transferrin receptors on the available. With markedly stimulated erythropoiesis,
surface of marrow erythroid cells. Diferric transfer- demands for iron are increased by as much as six- to
rin has the highest affinity for transferrin receptors; eightfold. With extravascular hemolytic anemia, the
apotransferrin (not carrying iron) has very little affin- rate of red cell destruction is increased, but the iron
ity. Although transferrin receptors are found on cells in recovered from the red cells is efficiently reutilized
many tissues within the bodyand all cells at some time for hemoglobin synthesis. In contrast, with intravas-
during development will display transferrin receptors cular hemolysis or blood loss anemia, the rate of red
the cell having the greatest number of receptors cell production is limited by the amount of iron that
(300,000 to 400,000/cell) is the developing erythroblast. can be mobilized from stores. Typically, the rate of
Once the iron-bearing transferrin interacts with its mobilization under these circumstances will not sup-
receptor, the complex is internalized via clathrin-coated port red cell production more than 2.5 times normal.
pits and transported to an acidic endosome, where the If the delivery of iron to the stimulated marrow is sub-
iron is released at the low pH. The iron is then made optimal, the marrows proliferative response is blunted,
available for heme synthesis while the transferrin and hemoglobin synthesis is impaired. The result is a
receptor complex is recycled to the surface of the cell, hypoproliferative marrow accompanied by microcytic,
where the bulk of the transferrin is released back into hypochromic anemia.
circulation and the transferrin receptor reanchors into Whereas blood loss or hemolysis places a demand
the cell membrane. At this point, a certain amount of on the iron supply, inflammatory conditions interfere
the transferrin receptor protein may be released into with iron release from stores and can result in a rapid
circulation and can be measured as soluble transferrin decrease in the serum iron (discussed later).
72 Nutritional Iron Balance At the brush border of the absorptive cell, the ferric
iron is converted to the ferrous form by a ferrireduc-
The balance of iron in humans is tightly controlled and tase. Transport across the membrane is accomplished by
designed to conserve iron for reutilization. There is divalent metal transporter type 1 (DMT-1, also known
no regulated excretory pathway for iron, and the only as natural resistance macrophage-associated protein type
mechanisms by which iron is lost are blood loss (via gas- 2 [Nramp 2] or DCT-1). DMT-1 is a general cation
trointestinal [GI] bleeding, menses, or other forms of transporter. Once inside the gut cell, iron may be stored
bleeding) and the loss of epithelial cells from the skin, as ferritin or transported through the cell to be released
gut, and genitourinary tract. Normally, the only route at the basolateral surface to plasma transferrin through
by which iron comes into the body is via absorption the membrane-embedded iron exporter, ferroportin.
from food or from medicinal iron taken orally. Iron The function of ferroportin is negatively regulated by
may also enter the body through red-cell transfusions or hepcidin, the principal iron regulatory hormone. In
injection of iron complexes. The margin between the the process of release, iron interacts with another fer-
amount of iron available for absorption and the require- roxidase, hephaestin, which oxidizes the iron to the fer-
ment for iron in growing infants and adult females is ric form for transferrin binding. Hephaestin is similar to
narrow; this accounts for the great prevalence of iron ceruloplasmin, the copper-carrying protein.
deficiency worldwidecurrently estimated at one-half Iron absorption is influenced by a number of physi-
billion people.
SECTION III

ologic states. Erythroid hyperplasia stimulates iron


The amount of iron required from the diet to absorption even in the face of normal or increased iron
replace losses averages approximately 10% of body iron stores, and hepcidin levels are inappropriately low.
content a year in men and 15% in women of childbear- The molecular mechanism underlying this relation-
ing age. Dietary iron content is closely related to total ship is not known. Thus, patients with anemias asso-
caloric intake (~6 mg of elemental iron per 1000 calo- ciated with high levels of ineffective erythropoiesis
ries). Iron bioavailability is affected by the nature of the
Anemias

absorb excess amounts of dietary iron. Over time, this


foodstuff, with heme iron (e.g., red meat) being most may lead to iron overload and tissue damage. In iron
readily absorbed. In the United States, the average iron deficiency, hepcidin levels are low, and iron is much
intake in an adult male is 15 mg/d with 6% absorption; more efficiently absorbed; the contrary is true in states
for the average female, the daily intake is 11 mg/d with of secondary iron overload. A normal individual can
12% absorption. An individual with iron deficiency can reduce iron absorption in situations of excessive intake
increase iron absorption to approximately 20% of the or medicinal iron intake; however, while the percent-
iron present in a meat-containing diet but only 510% age of iron absorbed goes down, the absolute amount
of the iron in a vegetarian diet. As a result, one-third goes up. This accounts for the acute iron toxicity occa-
of the female population in the United States has vir- sionally seen when children ingest large numbers of iron
tually no iron stores. Vegetarians are at an additional tablets. Under these circumstances, the amount of iron
disadvantage because certain foodstuffs that include absorbed exceeds the transferrin binding capacity of the
phytates and phosphates reduce iron absorption by plasma, resulting in free iron that affects critical organs
approximately 50%. When ionizable iron salts are given such as cardiac muscle cells.
together with food, the amount of iron absorbed is
reduced. When the percentage of iron absorbed from
individual food items is compared with the percentage
for an equivalent amount of ferrous salt, iron in vegeta- Iron-Deficiency Anemia
bles is only about one-twentieth as available, egg iron
one-eighth, liver iron one-half, and heme iron one-half Iron deficiency is one of the most prevalent
to two-thirds. forms of malnutrition. Globally, 50% of anemia
Infants, children, and adolescents may be unable to is attributable to iron deficiency and accounts
maintain normal iron balance because of the demands of for approximately 841,000 deaths annually worldwide.
body growth and lower dietary intake of iron. During Africa and parts of Asia bear 71% of the global mor-
the last two trimesters of pregnancy, daily iron require- tality burden; North America represents only 1.4% of
ments increase to 56 mg. That is the reason why iron the total morbidity and mortality associated with iron
supplements are strongly recommended for pregnant deficiency.
women in developed countries.
Iron absorption takes place largely in the proxi-
mal small intestine and is a carefully regulated process. Stages of Iron Deficiency
For absorption, iron must be taken up by the luminal The progression to iron deficiency can be divided into
cell. That process is facilitated by the acidic contents three stages (Fig. 7-2). The first stage is negative iron
of the stomach, which maintains the iron in solution. balance, in which the demands for (or losses of) iron
Negative Iron- Iron- despite the dwindling iron stores. Once the transfer- 73
iron deficient deficiency
Normal balance erythropoiesis anemia rin saturation falls to 1520%, hemoglobin synthe-
Iron stores sis becomes impaired. This is a period of iron-deficient
Erythron iron
erythropoiesis. Careful evaluation of the peripheral blood
smear reveals the first appearance of microcytic cells,
Marrow iron
13+ 0-1+ 0 0 and if the laboratory technology is available, one finds
stores
hypochromic reticulocytes in circulation. Gradually, the
Serum ferritin
(g/L)
50200 <20 <15 <15 hemoglobin and hematocrit begin to fall, reflecting iron-
deficiency anemia. The transferrin saturation at this point
TIBC (g/dL) 300360 >360 >380 >400 is 1015%.
When moderate anemia is present (hemoglobin
SI (g/dL) 50150 NL <50 <30
1013 g/dL), the bone marrow remains hypoprolifera-
tive. With more severe anemia (hemoglobin 78 g/dL),
Saturation (%) 3050 NL <20 <10
hypochromia and microcytosis become more promi-
Marrow
4060 NL <10 <10 nent, target cells and misshapen red cells (poikilocytes)
sideroblasts (%)
RBC
appear on the blood smear as cigar- or pencil-shaped
protoporphyrin 3050 NL >100 >200 forms, and the erythroid marrow becomes increasingly

CHAPTER 7
(g/dL)
ineffective. Consequently, with severe prolonged iron-
RBC Microcytic
Microcytic/
morphology
NL NL NL
hypochromic deficiency anemia, erythroid hyperplasia of the marrow
develops rather than hypoproliferation.
Figure 7-2
Laboratory studies in the evolution of iron deficiency.
Measurements of marrow iron stores, serum ferritin, and total Causes of Iron Deficiency
iron-binding capacity (TIBC) are sensitive to early iron-store

Iron Deficiency and Other Hypoproliferative Anemias


Conditions that increase demand for iron, increase iron
depletion. Iron-deficient erythropoiesis is recognized from
loss, or decrease iron intake or absorption can produce
additional abnormalities in the serum iron (SI), percent trans-
iron deficiency (Table 7-2).
ferrin saturation, the pattern of marrow sideroblasts, and the
red cell protoporphyrin level. Patients with iron-deficiency
anemia demonstrate all the same abnormalities plus hypo- Clinical Presentation of Iron
chromic microcytic anemia. NL, normal; RBC, red blood Deficiency
cell. (From RS Hillman, CA Finch: Red Cell Manual, 7th ed.
Philadelphia, Davis, 1996, with permission.) Certain clinical conditions carry an increased likelihood
of iron deficiency. Pregnancy, adolescence, periods
exceed the bodys ability to absorb iron from the diet. of rapid growth, and an intermittent history of blood
This stage results from a number of physiologic mech- loss of any kind should alert the clinician to possible
anisms, including blood loss, pregnancy (in which the iron deficiency. A cardinal rule is that the appearance
demands for red cell production by the fetus outstrip the of iron deficiency in an adult male means GI blood loss
mothers ability to provide iron), rapid growth spurts in
an adolescent, or inadequate dietary iron intake. Blood Table 7-2
loss in excess of 1020 mL of red cells per day is greater Causes of Iron Deficiency
than the amount of iron that the gut can absorb from a Increased Demand for Iron
normal diet. Under these circumstances, the iron defi-
Rapid growth in infancy or adolescence
cit must be made up by mobilization of iron from RE Pregnancy
storage sites. During this period, iron storesreflected Erythropoietin therapy
by the serum ferritin level or the appearance of stainable Increased Iron Loss
iron on bone marrow aspirationsdecrease. As long as
Chronic blood loss
iron stores are present and can be mobilized, the serum Menses
iron, total iron-binding capacity (TIBC), and red cell Acute blood loss
protoporphyrin levels remain within normal limits. At Blood donation
this stage, red cell morphology and indices are normal. Phlebotomy as treatment for polycythemia vera
When iron stores become depleted, the serum iron Decreased Iron Intake or Absorption
begins to fall. Gradually, the TIBC increases, as do Inadequate diet
red cell protoporphyrin levels. By definition, marrow Malabsorption from disease (sprue, Crohns disease)
iron stores are absent when the serum ferritin level is Malabsorption from surgery (postgastrectomy)
<15 g/L. As long as the serum iron remains within Acute or chronic inflammation
the normal range, hemoglobin synthesis is unaffected
74 until proven otherwise. Signs related to iron deficiency >200
depend on the severity and chronicity of the anemia in
addition to the usual signs of anemiafatigue, pallor, 100
and reduced exercise capacity. Cheilosis (fissures at the
Normal
corners of the mouth) and koilonychia (spooning of the

Serum ferritin, g/L


males
75
fingernails) are signs of advanced tissue iron deficiency.
The diagnosis of iron deficiency is typically based on Normal
females
laboratory results. 50

25
Laboratory Iron Studies Iron deficiency
12
Serum iron and total iron-binding capacity 0
Store depletion

The serum iron level represents the amount of circulat- 0 10 20 30 40 50 60 70 80


ing iron bound to transferrin. The TIBC is an indirect Age, years
measure of the circulating transferrin. The normal range
Figure 7-3
for the serum iron is 50150 g/dL; the normal range for
Serum ferritin levels as a function of sex and age. Iron
TIBC is 300360 g/dL. Transferrin saturation, which is
SECTION III

store depletion and iron deficiency are accompanied by


normally 2550%, is obtained by the following formula:
a decrease in serum ferritin level below 20 g/L. (From RS
serum iron 100 TIBC. Iron-deficiency states are
Hillman et al: Hematology in Clinical Practice, 5th ed. New York,
associated with saturation levels below 20%. There is a McGraw-Hill, 2010, with permission.)
diurnal variation in the serum iron. A transferrin satura-
tion >50% indicates that a disproportionate amount of
the iron bound to transferrin is being delivered to non- indicator of iron overload than the marrow iron stain.
Anemias

erythroid tissues. If this persists for an extended time, However, in addition to storage iron, the marrow iron
tissue iron overload may occur. stain provides information about the effective delivery
of iron to developing erythroblasts. Normally, when the
marrow smear is stained for iron, 2040% of developing
Serum ferritin erythroblastscalled sideroblastswill have visible ferri-
Free iron is toxic to cells, and the body has established tin granules in their cytoplasms. This represents iron in
an elaborate set of protective mechanisms to bind iron excess of that needed for hemoglobin synthesis. In states
in various tissue compartments. Within cells, iron is in which release of iron from storage sites is blocked,
stored complexed to protein as ferritin or hemosiderin. RE iron will be detectable, and there will be few or no
Apoferritin binds to free ferrous iron and stores it in the sideroblasts. In the myelodysplastic syndromes, mito-
ferric state. As ferritin accumulates within cells of the chondrial dysfunction can occur, and accumulation
RE system, protein aggregates are formed as hemosid- of iron in mitochondria appears in a necklace fashion
erin. Iron in ferritin or hemosiderin can be extracted around the nucleus of the erythroblast. Such cells are
for release by the RE cells, although hemosiderin is referred to as ringed sideroblasts.
less readily available. Under steady-state conditions,
the serum ferritin level correlates with total body iron Red cell protoporphyrin levels
stores; thus, the serum ferritin level is the most conve-
nient laboratory test to estimate iron stores. The nor- Protoporphyrin is an intermediate in the pathway to
mal value for ferritin varies according to the age and heme synthesis. Under conditions in which heme syn-
gender of the individual (Fig. 7-3). Adult males have thesis is impaired, protoporphyrin accumulates within
serum ferritin values averaging 100 g/L, while adult
females have levels averaging 30 g/L. As iron stores Table 7-3
are depleted, the serum ferritin falls to <15 g/L. Such
Iron Store Measurements
levels are diagnostic of absent body iron stores.
MARROW IRON SERUM
IRON STORES STAIN, 0-4+ FERRITIN, lg/L
Evaluation of bone marrow iron stores 0 0 <15
1300 mg Trace to 1+ 1530
Although RE cell iron stores can be estimated from
300800 mg 2+ 3060
the iron stain of a bone marrow aspirate or biopsy, the 8001000 mg 3+ 60150
measurement of serum ferritin has largely supplanted 12 g 4+ >150
bone marrow aspirates for determination of storage Iron overload >5001000
iron (Table 7-3). The serum ferritin level is a better
the red cell. This reflects an inadequate iron supply to the anemia of chronic inflammation is normocytic and 75
erythroid precursors to support hemoglobin synthe- normochromic. The iron values usually make the dif-
sis. Normal values are <30 g/dL of red cells. In iron ferential diagnosis clear, as the ferritin level is normal
deficiency, values in excess of 100 g/dL are seen. The or increased and the percent transferrin saturation and
most common causes of increased red cell protopor- TIBC are typically below normal.
phyrin levels are absolute or relative iron deficiency and Finally, the myelodysplastic syndromes represent
lead poisoning. the third and least common condition. Occasionally,
patients with myelodysplasia have impaired hemoglobin
Serum levels of transferrin receptor protein synthesis with mitochondrial dysfunction, resulting in
impaired iron incorporation into heme. The iron values
Because erythroid cells have the highest numbers of again reveal normal stores and more than an adequate
transferrin receptors of any cell in the body and because supply to the marrow, despite the microcytosis and
transferrin receptor protein (TRP) is released by cells hypochromia.
into the circulation, serum levels of TRP reflect the
total erythroid marrow mass. Another condition in
which TRP levels are elevated is absolute iron defi-
Treatment Iron-Deficiency Anemia
ciency. Normal values are 49 g/L determined by

CHAPTER 7
immunoassay. This laboratory test is becoming increas- The severity and cause of iron-deficiency anemia will
ingly available and, along with the serum ferritin, has determine the appropriate approach to treatment. As
been proposed to distinguish between iron deficiency an example, symptomatic elderly patients with severe
and the anemia of chronic inflammation (discussed later). iron-deficiency anemia and cardiovascular instability
may require red cell transfusions. Younger individuals
who have compensated for their anemia can be treated
Differential Diagnosis

Iron Deficiency and Other Hypoproliferative Anemias


more conservatively with iron replacement. The foremost
Other than iron deficiency, only three conditions need issue for the latter patient is the precise identification of
to be considered in the differential diagnosis of a hypo- the cause of the iron deficiency.
chromic microcytic anemia (Table 7-4). The first is an For the majority of cases of iron deficiency (pregnant
inherited defect in globin chain synthesis: the thalasse- women, growing children and adolescents, patients
mias. These are differentiated from iron deficiency most with infrequent episodes of bleeding, and those with
readily by serum iron values; normal or increased serum inadequate dietary intake of iron), oral iron therapy will
iron levels and transferrin saturation are characteristic of suffice. For patients with unusual blood loss or malab-
the thalassemias. In addition, the red blood cell distribu- sorption, specific diagnostic tests and appropriate ther-
tion width (RDW) index is generally small in thalasse- apy take priority. Once the diagnosis of iron-deficiency
mia and elevated in iron deficiency. anemia and its cause is made, there are three major
The second condition is the anemia of chronic therapeutic approaches.
inflammation with inadequate iron supply to the ery- Red Cell Transfusion Transfusion therapy
throid marrow. The distinction between true iron-defi- is reserved for individuals who have symptoms of ane-
ciency anemia and the anemia associated with chronic mia, cardiovascular instability, continued and excessive
inflammation is among the most common diagnostic blood loss from whatever source, and require immediate
problem encountered by clinicians (see below). Usually intervention. The management of these patients is less

Table 7-4
Diagnosis of Microcytic Anemia
TEST Iron Deficiency Inflammation Thalassemia Sideroblastic Anemia

Smear Micro/hypo Normal micro/hypo Micro/hypo with targeting Variable


SI <30 <50 Normal to high Normal to high
TIBC >360 <300 Normal Normal
Percent saturation <10 1020 3080 3080
Ferritin (g/L) <15 30200 50300 50300
Hemoglobin pattern Normal Normal Abnormal with Normal
on electrophoresis thalassemia; can be normal
with thalassemia

Abbreviations: SI, serum iron; TIBC, total iron-binding capacity.


76 related to the iron deficiency than it is to the conse- after correction of the anemia will be necessary to
quences of the severe anemia. Not only do transfusions achieve this.
correct the anemia acutely, but the transfused red cells Of the complications of oral iron therapy, GI dis-
also provide a source of iron for reutilization, assuming tress is the most prominent and is seen in 1520% of
they are not lost through continued bleeding. Trans- patients. Abdominal pain, nausea, vomiting, or con-
fusion therapy will stabilize the patient while other stipation may lead to noncompliance. Although small
options are reviewed. doses of iron or iron preparations with delayed release
Oral Iron Therapy In an asymptomatic may help somewhat, the GI side effects are a major
patient with established iron-deficiency anemia, treat- impediment to the effective treatment of a number of
ment with oral iron is usually adequate. Multiple prep- patients.
arations are available, ranging from simple iron salts The response to iron therapy varies, depending on
to complex iron compounds designed for sustained the erythropoietin stimulus and the rate of absorp-
release throughout the small intestine (Table 7-5). tion. Typically, the reticulocyte count should begin to
Although the various preparations contain different increase within 47 days after initiation of therapy and
amounts of iron, they are generally all absorbed well peak at 11 weeks. The absence of a response may be
and are effective in treatment. Some come with other due to poor absorption, noncompliance (which is com-
SECTION III

compounds designed to enhance iron absorption, such mon), or a confounding diagnosis. A useful test in the
as ascorbic acid. It is not clear whether the benefits of clinic to determine the patients ability to absorb iron is
such compounds justify their costs. Typically, for iron the iron tolerance test. Two iron tablets are given to the
replacement therapy, up to 300 mg of elemental iron patient on an empty stomach, and the serum iron is
per day is given, usually as three or four iron tablets measured serially over the subsequent 2 hours. Normal
(each containing 5065 mg elemental iron) given over absorption will result in an increase in the serum iron
of at least 100 g/dL. If iron deficiency persists despite
Anemias

the course of the day. Ideally, oral iron preparations


should be taken on an empty stomach, since food may adequate treatment, it may be necessary to switch to
inhibit iron absorption. Some patients with gastric dis- parenteral iron therapy.
ease or prior gastric surgery require special treatment
with iron solutions, as the retention capacity of the Parenteral Iron Therapy Intravenous
stomach may be reduced. The retention capacity is nec- iron can be given to patients who are unable to tolerate
essary for dissolving the shell of the iron tablet before oral iron; whose needs are relatively acute; or who need
the release of iron. A dose of 200300 mg of elemental iron on an ongoing basis, usually due to persistent GI
iron per day should result in the absorption of iron up to blood loss. Parenteral iron use has been increasing rap-
50 mg/d. This supports a red cell production level of two idly in the last several years with the recognition that
to three times normal in an individual with a normally recombinant erythropoietin (EPO) therapy induces a
functioning marrow and appropriate erythropoietin large demand for irona demand that frequently can-
stimulus. However, as the hemoglobin level rises, eryth- not be met through the physiologic release of iron from
ropoietin stimulation decreases, and the amount of iron RE sources or oral iron absorption. The safety of paren-
absorbed is reduced. The goal of therapy in individuals teral ironparticularly iron dextranhas been a con-
with iron-deficiency anemia is not only to repair the cern. The serious adverse reaction rate to intravenous
anemia but also to provide stores of at least 0.51 g of high-molecular weight iron dextran is 0.7%. Fortunately,
iron. Sustained treatment for a period of 612 months newer iron complexes are available in the United States,
such as sodium ferric gluconate (Ferrlecit) and iron
sucrose (Venofer) that have much lower rates of adverse
Table 7-5 effects.
Oral Iron Preparations Parenteral iron is used in two ways: one is to admin-
ister the total dose of iron required to correct the hemo-
Tablet (Iron Elixir (Iron
Generic Name Content), mg Content), mg in 5 mL globin deficit and provide the patient with at least
500 mg of iron stores; the second is to give repeated
Ferrous sulfate 325 (65) 300 (60)
small doses of parenteral iron over a protracted period.
195 (39) 90 (18)
Extended release 525 (105) The latter approach is common in dialysis centers, where
Ferrous fumarate 325 (107) it is not unusual for 100 mg of elemental iron to be given
195 (64) 100 (33) weekly for 10 weeks to augment the response to recom-
Ferrous gluconate 325 (39) 300 (35) binant EPO therapy. The amount of iron needed by an
Polysaccharide 150 (150) 100 (100)
iron
individual patient is calculated by the following formula:
50 (50) Body weight (kg) 2.3 (15patients hemoglobin, g/dL) +
500 or 1000 mg (for stores).
the differential diagnosis of iron deficiency because many 77
In administering intravenous iron dextran, anaphy- of the features of the anemia are brought about by inade-
laxis is a concern. Anaphylaxis is much rarer with the quate iron delivery to the marrow, despite the presence of
newer preparations. The factors that have correlated normal or increased iron stores. This is reflected by a low
with an anaphylactic-like reaction include a history of serum iron, increased red cell protoporphyrin, a hypop-
multiple allergies or a prior allergic reaction to dextran roliferative marrow, transferrin saturation in the range of
(in the case of iron dextran). Generalized symptoms 1520%, and a normal or increased serum ferritin. The
appearing several days after the infusion of a large dose serum ferritin values are often the most distinguishing fea-
of iron can include arthralgias, skin rash, and low-grade tures between true iron-deficiency anemia and the iron-
fever. These may be dose-related, but they do not pre- restricted erythropoiesis associated with inflammation.
clude the further use of parenteral iron in the patient. Typically, serum ferritin values increase threefold over
To date, patients with sensitivity to iron dextran have basal levels in the face of inflammation. These changes are
been safely treated with iron gluconate. If a large dose due to the effects of inflammatory cytokines and hepcidin,
of iron dextran is to be given (>100 mg), the iron prepa- the key iron regulatory hormone, acting at several levels
ration should be diluted in 5% dextrose in water or 0.9% of erythropoiesis (Fig. 7-4).
NaCl solution. The iron solution can then be infused Interleukin 1 (IL-1) directly decreases EPO produc-
over a 60- to 90-minute period (for larger doses) or at tion in response to anemia. IL-1, acting through acces-

CHAPTER 7
a rate convenient for the attending nurse or physician. sory cell release of interferon (IFN-), suppresses the
Although a test dose (25 mg) of parenteral iron dextran response of the erythroid marrow to EPOan effect
is recommended, in reality, a slow infusion of a larger that can be overcome by EPO administration in vitro
dose of parenteral iron solution will afford the same and in vivo. In addition, tumor necrosis factor (TNF),
kind of early warning as a separately injected test dose. acting through the release of IFN- by marrow stromal
Early in the infusion of iron, if chest pain, wheezing, a fall cells, also suppresses the response to EPO. Hepcidin,
in blood pressure, or other systemic symptoms occur,

Iron Deficiency and Other Hypoproliferative Anemias


made by the liver, is increased in inflammation and
the infusion of iron should be stopped immediately. acts to suppress iron absorption and iron release from
storage sites. The overall result is a chronic hypopro-
liferative anemia with classic changes in iron metabo-
Other Hypoproliferative Anemias lism. The anemia is further compounded by a mild to
moderate shortening in red cell survival.
In addition to mild to moderate iron-deficiency anemia, With chronic inflammation, the primary disease will
the hypoproliferative anemias can be divided into four determine the severity and characteristics of the anemia.
categories: (1) chronic inflammation, (2) renal disease, For example, many patients with cancer also have ane-
(3) endocrine and nutritional deficiencies (hypometa- mia that is typically normocytic and normochromic. In
bolic states), and (4) marrow damage (Chap. 11). With
chronic inflammation, renal disease, or hypometabo-
Neoplasms
lism, endogenous EPO production is inadequate for the Bacterial infections
degree of anemia observed. For the anemia of chronic TNF
inflammation, the erythroid marrow also responds inad- Interferon
equately to stimulation, due in part to defective iron
reutilization. As a result of the lack of adequate EPO
EPO Marrow
stimulation, an examination of the peripheral blood + BFU/CFU-E
RBC
smear will disclose only an occasional polychromato- Iron

philic (shift) reticulocyte. In cases of iron deficiency


Interferon
or marrow damage, appropriate elevations in endog-
enous EPO levels are typically found, and shift reticulo- IL-1
cytes will be present on the blood smear. Rheumatoid arthritis

Figure 7-4
Anemia of Acute and Chronic Suppression of erythropoiesis by inflammatory cytokines.
Inflammation/Infection (The Anemia Through the release of tumor necrosis factor (TNF) and
of Inflammation) interferon (IFN-), neoplasms and bacterial infections sup-
press erythropoietin (EPO) production and the proliferation
The anemia of inflammationwhich encompasses of erythroid progenitors (erythroid burst-forming units and
inflammation, infection, tissue injury, and conditions erythroid colony-forming units [BFU/CFU-E]). The mediators
(e.g., cancer) associated with the release of proinflam- in patients with vasculitis and rheumatoid arthritis include
matory cytokinesis one of the most common forms of interleukin 1 (IL-1) and IFN- The red arrows indicate sites of
anemia seen clinically. It is the most important anemia in inflammatory cytokine inhibitory effects.
78 contrast, patients with long-standing active rheumatoid renal failure, the correlation between the anemia and
arthritis or chronic infections such as tuberculosis will renal function is weaker. Patients with the hemolytic-
have a microcytic, hypochromic anemia. In both cases, uremic syndrome increase erythropoiesis in response
the bone marrow is hypoproliferative, but the differ- to the hemolysis, despite renal failure requiring dialysis.
ences in red cell indices reflect differences in the avail- Polycystic kidney disease also shows a smaller degree
ability of iron for hemoglobin synthesis. Occasionally, of EPO deficiency for a given level of renal failure. By
conditions associated with chronic inflammation are also contrast, patients with diabetes or myeloma have more
associated with chronic blood loss. Under these circum- severe EPO deficiency for a given level of renal failure.
stances, a bone marrow aspirate stained for iron may be Assessment of iron status provides information to distin-
necessary to rule out absolute iron deficiency. How- guish the anemia of CKD from the other forms of hypop-
ever, the administration of iron in this case will correct roliferative anemia (Table 7-6) and to guide management.
the iron deficiency component of the anemia and leave Patients with the anemia of CKD usually present with
the inflammatory component unaffected. normal serum iron, TIBC, and ferritin levels. However,
The anemia associated with acute infection or those maintained on chronic hemodialysis may develop
inflammation is typically mild but becomes more pro- iron deficiency from blood loss through the dialysis proce-
nounced over time. Acute infection can produce a dure. Iron must be replenished in these patients to ensure
decrease in hemoglobin levels of 23 g/dL within 1 an adequate response to EPO therapy (discussed later).
SECTION III

or 2 days; this is largely related to the hemolysis of red


cells near the end of their natural life span. The fever
and cytokines released exert a selective pressure against Anemia in Hypometabolic States
cells with more limited capacity to maintain the red Patients who are starving, particularly for protein, and
cell membrane. In most individuals, the mild anemia is those with a variety of endocrine disorders that produce
reasonably well tolerated, and symptoms, if present, are lower metabolic rates may develop a mild to moderate
Anemias

associated with the underlying disease. Occasionally, in hypoproliferative anemia. The release of EPO from the
patients with preexisting cardiac disease, moderate ane- kidney is sensitive to the need for O2, not just O2 lev-
mia (hemoglobin 1011 g/dL) may be associated with els. Thus, EPO production is triggered at lower levels
angina, exercise intolerance, and shortness of breath. The of blood O2 content in disease states (e.g., hypothyroid-
erythropoietic profile that distinguishes the anemia of ism and starvation) where metabolic activity, and thus
inflammation from the other causes of hypoproliferative O2 demand, is decreased.
anemias is shown in Table 7-6.
Endocrine deficiency states
Anemia of Chronic Kidney Disease
The difference in the levels of hemoglobin between
(Ckd)
men and women is related to the effects of androgen
Progressive CKD is usually associated with a moder- and estrogen on erythropoiesis. Testosterone and ana-
ate to severe hypoproliferative anemia; the level of the bolic steroids augment erythropoiesis; castration and
anemia correlates with the stage of CKD. Red cells are estrogen administration to males decrease erythropoi-
typically normocytic and normochromic, and reticulo- esis. Patients who are hypothyroid or have deficits in
cytes are decreased. The anemia is primarily due to a pituitary hormones also may develop a mild anemia.
failure of EPO production by the diseased kidney and a Pathogenesis may be complicated by other nutritional
reduction in red cell survival. In certain forms of acute deficiencies because iron and folic acid absorption can

Table 7-6
Diagnosis of Hypoproliferative Anemias
Tests Iron Deficiency Inflammation Renal Disease Hypometabolic States

Anemia Mild to severe Mild Mild to severe Mild


MCV (fL) 6090 8090 90 90
Morphology Normo-microcytic Normocytic Normocytic Normocytic
SI <30 <50 Normal Normal
TIBC >360 <300 Normal Normal
Saturation (%) <10 1020 Normal Normal
Serum ferritin (g/L) <15 30200 115150 Normal
Iron stores 0 24+ 14+ Normal

Abbreviations: MCV, mean corpuscular volume; SI, serum iron; TIBC, total iron-binding capacity.
be affected by these disorders. Usually, correction of the 79
hormone deficiency reverses the anemia. treatment. The two major forms of treatment are trans-
Anemia may be more severe in Addisons disease, fusions and EPO.
depending on the level of thyroid and androgen hor- Transfusions Thresholds for transfusion should
mone dysfunction; however, anemia may be masked be altered based on the patients symptoms. In gen-
by decreases in plasma volume. Once such patients are eral, patients without serious underlying cardiovascu-
given cortisol and volume replacement, the hemoglo- lar or pulmonary disease can tolerate hemoglobin levels
bin level may fall rapidly. Mild anemia complicating above 8 g/dL and do not require intervention until the
hyperparathyroidism may be due to decreased EPO hemoglobin falls below that level. Patients with more
production as a consequence of the renal effects of physiologic compromise may need to have their hemo-
hypercalcemia or to impaired proliferation of erythroid globin levels kept above 11 g/dL. A typical unit of packed
progenitors. red cells increases the hemoglobin level by 1 g/dL.
Transfusions are associated with certain infectious risks
Protein starvation (Chap. 12), and chronic transfusions can produce iron
overload. Importantly, the liberal use of blood has been
Decreased dietary intake of protein may lead to mild to
associated with increased morbidity and mortality, par-
moderate hypoproliferative anemia; this form of ane-
ticularly in the intensive care setting. Therefore, in the

CHAPTER 7
mia may be prevalent in the elderly. The anemia can
absence of documented tissue hypoxia, a conservative
be more severe in patients with a greater degree of star-
approach to the use of red cell transfusions is preferable.
vation. In marasmus, where patients are both protein
and calorie deficient, the release of EPO is impaired in Erythropoietin (EPO) EPO is particularly
proportion to the reduction in metabolic rate; however, useful in anemias in which endogenous EPO levels
the degree of anemia may be masked by volume deple- are inappropriately low, such as CKD or the anemia
tion and becomes apparent after refeeding. Deficiencies of chronic inflammation. Iron status must be evalu-

Iron Deficiency and Other Hypoproliferative Anemias


in other nutrients (iron, folate) may also complicate the ated and iron repleted to obtain optimal effects from
clinical picture but may not be apparent at diagnosis. EPO. In patients with CKD, the usual dose of EPO is
Changes in the erythrocyte indices on refeeding should 50150 U/kg three times a week intravenously. Hemo-
prompt evaluation of iron, folate, and B12 status. globin levels of 1012 g/dL are usually reached within
46 weeks if iron levels are adequate; 90% of these
patients respond. Once a target hemoglobin level is
Anemia in liver disease
achieved, the EPO dose can be decreased. A decrease
A mild hypoproliferative anemia may develop in in hemoglobin level occurring in the face of EPO ther-
patients with chronic liver disease from nearly any apy usually signifies the development of an infection
cause. The peripheral blood smear may show spur or iron depletion. Aluminum toxicity and hyperpara-
cells and stomatocytes from the accumulation of excess thyroidism can also compromise the EPO response.
cholesterol in the membrane from a deficiency of lec- When an infection intervenes, it is best to interrupt
ithin-cholesterol acyltransferase. Red cell survival is the EPO therapy and rely on transfusion to correct the
shortened, and the production of EPO is inadequate to anemia until the infection is adequately treated. The
compensate. In alcoholic liver disease, nutritional defi- dose needed to correct the anemia in patients with
ciencies are common and complicate the management. cancer is higher, up to 300 U/kg three times a week,
Folate deficiency from inadequate intake, as well as iron and only approximately 60% of patients respond.
deficiency from blood loss and inadequate intake, can Because of evidence that tumor progression may
alter the red cell indices. result from EPO administration, the risks and benefits
of using EPO in patients with chemotherapy-induced
anemia must be weighed carefully, and the target
Treatment Hypoproliferative Anemias hemoglobin should be that necessary to avoid trans-
fusions.
Many patients with hypoproliferative anemias experi- Longer-acting preparations of EPO can reduce the
ence recovery of normal hemoglobin levels when the frequency of injections. Darbepoetin alfa, a molecularly
underlying disease is appropriately treated. For those in modified EPO with additional carbohydrate, has a half-
whom such reversals are not possiblesuch as patients life in the circulation that is three to four times longer
with end-stage kidney disease, cancer, and chronic than recombinant human EPO, permitting weekly or
inflammatory diseasessymptomatic anemia requires every other week dosing.
CHapter 8

DISORDERS OF HEMOGLOBIN

Edward J. Benz, Jr.

Hemoglobin is critical for normal oxygen delivery to The tetrameric quaternary structure of HbA contains two
tissues; it is also present in erythrocytes in such high dimers. Numerous tight interactions (i.e., 11
concentrations that it can alter red cell shape, deform- contacts) hold the and chains together. The com-
ability, and viscosity. Hemoglobinopathies are disor- plete tetramer is held together by interfaces (i.e., 12
ders affecting the structure, function, or production of contacts) between the -like chain of one dimer and
hemoglobin. These conditions are usually inherited and the non- chain of the other dimer.
range in severity from asymptomatic laboratory abnor- The hemoglobin tetramer is highly soluble, but
malities to death in utero. Different forms may present individual globin chains are insoluble. Unpaired globin
as hemolytic anemia, erythrocytosis, cyanosis, or vaso- precipitates, forming inclusions that damage the cell.
occlusive stigmata. Normal globin chain synthesis is balanced so that each
newly synthesized or non- globin chain will have an
available partner with which to pair.
Solubility and reversible oxygen binding are the
properties of tHe Human key properties deranged in hemoglobinopathies. Both
Hemoglobins depend most on the hydrophilic surface amino acids,
the hydrophobic amino acids lining the heme pocket,
Hemoglobin structure a key histidine in the F helix, and the amino acids
Different hemoglobins are produced during embryonic, forming the 11 and 12 contact points. Mutations
fetal, and adult life (Fig. 8-1). Each consists of a tetra- in these strategic regions tend to be the ones that alter
mer of globin polypeptide chains: a pair of -like chains oxygen affinity or solubility.
141 amino acids long and a pair of -like chains 146
amino acids long. The major adult hemoglobin, HbA,
has the structure 22. HbF (22) predominates dur- Chromosomes
ing most of gestation, and HbA2 (22) is minor adult 0 Kilobases 50
hemoglobin. Embryonic hemoglobins need not be 2 1
considered here. 16 3'
Each globin chain enfolds a single heme moiety,
consisting of a protoporphyrin IX ring complexed with G A
11 3'
a single iron atom in the ferrous state (Fe2+). Each heme
moiety can bind a single oxygen molecule; a mol- Polypeptide
ecule of hemoglobin can transport up to four oxygen chains
molecules. 22 22 22
The amino acid sequences of the various globins
Hemoglobins F A2 A
are highly homologous to one another. Each has a
highly helical secondary structure. Their globular tertiary Figure 8-1
structures cause the exterior surfaces to be rich in polar the globin genes. The -like genes (,) are encoded on
(hydrophilic) amino acids that enhance solubility and chromosome 16; the -like genes (,,,) are encoded
the interior to be lined with nonpolar groups, form- on chromosome 11. The and genes encode embryonic
ing a hydrophobic pocket into which heme is inserted. globins.
80
Function of Hemoglobin because the latter is a weaker acid (Fig. 8-2). Thus, 81
hemoglobin has a lower oxygen affinity at low pH.
To support oxygen transport, hemoglobin must bind The major small molecule that alters oxygen affinity in
O2 efficiently at the partial pressure of oxygen (PO2) humans is 2,3-bisphosphoglycerate (2,3-BPG, formerly
of the alveolus, retain it, and release it to tissues at the 2,3-DPG), which lowers oxygen affinity when bound
PO2 of tissue capillary beds. Oxygen acquisition and to hemoglobin. HbA has a reasonably high affinity for
delivery over a relatively narrow range of oxygen ten- 2,3-BPG. HbF does not bind 2,3-BPG, so it tends to
sions depend on a property inherent in the tetrameric have a higher oxygen affinity in vivo. Hemoglobin
arrangement of heme and globin subunits within the also binds nitric oxide reversibly; this interaction influ-
hemoglobin molecule called cooperativity or hemeheme ences vascular tone, but its clinical relevance remains
interaction. controversial.
At low oxygen tensions, the hemoglobin tetramer is Proper oxygen transport depends on the tetrameric
fully deoxygenated (Fig. 8-2). Oxygen binding begins structure of the proteins, the proper arrangement of the
slowly as O2 tension rises. However, as soon as some charged amino acids, and interaction with protons or
oxygen has been bound by the tetramer, an abrupt 2,3-BPG.
increase occurs in the slope of the curve. Thus, hemo-
globin molecules that have bound some oxygen develop
a higher oxygen affinity, greatly accelerating their ability

CHAPTER 8
Developmental Biology of Human
to combine with more oxygen. This S-shaped oxygen
Hemoglobins
equilibrium curve (Fig. 8-2), along which substantial
amounts of oxygen loading and unloading can occur over Red cells first appearing at about 6 weeks after concep-
a narrow range of oxygen tensions, is physiologically tion contain the embryonic hemoglobins Hb Portland
more useful than the high-affinity hyperbolic curve of (22), Hb Gower I (22), and Hb Gower II (22). At
individual monomers. 1011 weeks, fetal hemoglobin (HbF; 22) becomes

Disorders of Hemoglobin
Oxygen affinity is modulated by several factors. predominant. The switch to nearly exclusive synthe-
The Bohr effect is the ability of hemoglobin to deliver sis of adult hemoglobin (HbA; 22) occurs at about
more oxygen to tissues at low pH. It arises from the 38 weeks (Fig. 8-1). Fetuses and newborns therefore
stabilizing action of protons on deoxyhemoglobin, require -globin but not -globin for normal gestation.
which binds protons more readily than oxyhemoglobin A major advance in understanding the HbF-to-HbA

pH Less Oxyhemoglobin
2,3-BPG O2
T delivered
Percent saturation of hemoglobin

100
Salt bridges
Oxygen 2,3-BPG
75

50 pH More
P 50 2,3-BPG O2
delivered Heme
T
25

0 Deoxyhemoglobin
Oxyhemoglobin Deoxyhemoglobin
0 25 50 75 100
Tissue PO (mm Hg)
2

Figure 8-2
Hemoglobinoxygen dissociation curve. The hemoglo- and 2,3-BPG and CO2 bound. Deoxyhemoglobin does not
bin tetramer can bind up to four molecules of oxygen in the bind oxygen efficiently until the cell returns to conditions of
iron-containing sites of the heme molecules. As oxygen is higher pH, the most important modulator of O2 affinity (Bohr
bound, 2,3-BPG and CO2 are expelled. Salt bridges are bro- effect). When acid is produced in the tissues, the dissocia-
ken, and each of the globin molecules changes its confor- tion curve shifts to the right, facilitating oxygen release and
mation to facilitate oxygen binding. Oxygen release to the CO2 binding. Alkalosis has the opposite effect, reducing
tissues is the reverse process, salt bridges being formed oxygen delivery.
82 transition has been the demonstration that the transcrip- protein (AHSP), that enhances the folding and solubility
tion factor Bcl11a plays a pivotal role in its regulation. of globin, which is otherwise easily denatured, lead-
Small amounts of HbF are produced during postnatal ing to insoluble precipitates. These precipitates play an
life. A few red cell clones called F cells are progeny of important role in the thalassemia syndromes and certain
a small pool of immature committed erythroid precur- unstable hemoglobin disorders. Polymorphic variation
sors (BFU-e) that retain the ability to produce HbF. in the amounts and/or functional capacity of AHSP
Profound erythroid stresses, such as severe hemolytic might explain some of the clinical variability seen in
anemias, bone marrow transplantation, or cancer che- patients inheriting identical thalassemia mutations.
motherapy, cause more of the F-potent BFU-e to be
recruited. HbF levels thus tend to rise in some patients
with sickle cell anemia or thalassemia. This phenom-
enon probably explains the ability of hydroxyurea to Classification of
increase levels of HbF in adults. Agents such as butyrate Hemoglobinopathies
and histone deacetylase inhibitors can also activate fetal
globin genes partially after birth. There are five major classes of hemoglobinopathies
(Table 8-1). Structural hemoglobinopathies occur when
mutations alter the amino acid sequence of a globin
Section Iii

Genetics and Biosynthesis of chain, altering the physiologic properties of the variant
HumanHemoglobin hemoglobins and producing the characteristic clini-
cal abnormalities. The most clinically relevant vari-
The human hemoglobins are encoded in two tightly ant hemoglobins polymerize abnormally, as in sickle
linked gene clusters; the -like globin genes are clus- cell anemia, or exhibit altered solubility or oxygen-
tered on chromosome 16, and the -like genes on binding affinity. Thalassemia syndromes arise from mutations
chromosome 11 (Fig. 8-1). The -like cluster consists
Anemias

of two -globin genes and a single copy of the gene.


The non- gene cluster consists of a single gene, Table 8-1
the G and A fetal globin genes, and the adult and
Classification of Hemoglobinopathies
genes.
Important regulatory sequences flank each gene. I. Structural hemoglobinopathieshemoglobins with
altered amino acid sequences that result in deranged
Immediately upstream are typical promoter elements
function or altered physical or chemical properties
needed for the assembly of the transcription initiation A.Abnormal hemoglobin polymerizationHbS,
complex. Sequences in the 5 flanking region of the hemoglobin sickling
and the genes appear to be crucial for the correct B. Altered O2 affinity
developmental regulation of these genes, while ele- 1. High affinitypolycythemia
ments that function like classic enhancers and silencers 2. Low affinitycyanosis, pseudoanemia
are in the 3 flanking regions. The locus control region C. Hemoglobins that oxidize readily
1. Unstable hemoglobinshemolytic anemia,
(LCR) elements located far upstream appear to control
jaundice
the overall level of expression of each cluster. These 2. M hemoglobinsmethemoglobinemia, cyanosis
elements achieve their regulatory effects by interacting II Thalassemiasdefective biosynthesis of globin chains
with trans-acting transcription factors. Some of these A. Thalassemias
factors are ubiquitous (e.g., Sp1 and YY1), while others B. Thalassemias
are more or less limited to erythroid cells or hemato- C. , , Thalassemias
poietic cells (e.g., GATA-1, NFE-2, and EKLF). The III. Thalassemic hemoglobin variantsstructurally
LCR controlling the -globin gene cluster is modulated abnormal Hb associated with co-inherited thalassemic
phenotype
by a SWI/SNF-like protein called ATRX; this protein A. HbE
appears to influence chromatin remodeling and DNA B. Hb Constant Spring
methylation. The association of thalassemia with C. Hb Lepore
mental retardation and myelodysplasia in some fami- IV. Hereditary persistence of fetal hemoglobin
lies appears to be related to mutations in the ATRX persistence of high levels of HbF into adult life
pathway. This pathway also modulates genes specifi- V. Acquired hemoglobinopathies
cally expressed during erythropoiesis, such as those that A. Methemoglobin due to toxic exposures
B. Sulfhemoglobin due to toxic exposures
encode the enzymes for heme biosynthesis. Normal
C. Carboxyhemoglobin
red blood cell (RBC) differentiation requires the coor- D. HbH in erythroleukemia
dinated expression of the globin genes with the genes E. Elevated HbF in states of erythroid stress and bone
responsible for heme and iron metabolism. RBC pre- marrow dysplasia
cursors contain a protein, -hemoglobin-stabilizing
that impair production or translation of globin mRNA, are symptomatic in utero and after birth because nor- 83
leading to deficient globin chain biosynthesis. Clinical mal function of the -globin gene is required through-
abnormalities are attributable to the inadequate supply out gestation and adult life. In contrast, infants with
of hemoglobin and the imbalances in the production of -globin hemoglobinopathies tend to be asymptom-
individual globin chains, leading to premature destruc- atic until 39 months of age, when HbA has largely
tion of erythroblasts and RBC. Thalassemic hemoglobin replaced HbF. Prevention or partial reversion of the
variants combine features of thalassemia (e.g., abnormal switch should thus be an effective therapeutic strategy
globin biosynthesis) and of structural hemoglobinopa- for -chain hemoglobinopathies.
thies (e.g., an abnormal amino acid sequence). Heredi-
tary persistence of fetal hemoglobin (HPFH) is characterized
by synthesis of high levels of fetal hemoglobin in adult
life. Acquired hemoglobinopathies include modifications of Detection and Characterization
the hemoglobin molecule by toxins (e.g., acquired met- of HemoglobinopathiesGeneral
hemoglobinemia) and clonal abnormalities of hemo- Methods
globin synthesis (e.g., high levels of HbF production
in preleukemia and thalassemia in myeloproliferative Electrophoretic techniques are widely used for hemo-
disorders). globin analysis. Electrophoresis at pH 8.6 on cellulose

CHAPTER 8
acetate membranes is especially simple, inexpensive, and
reliable for initial screening. Agar gel electrophoresis
Epidemiology at pH 6.1 in citrate buffer is often used as a comple-
mentary method because each method detects different
Hemoglobinopathies are especially common in variants. Some important variants are electrophoreti-
areas in which malaria is endemic. This cluster- cally silent. These mutant hemoglobins can usually be
ing of hemoglobinopathies is assumed to reflect characterized by more specialized techniques such as

Disorders of Hemoglobin
a selective survival advantage for the abnormal RBC, isoelectric focusing and/or high-pressure liquid chro-
which presumably provide a less hospitable environ- matography (HPLC), which is rapidly replacing electro-
ment during the obligate RBC stages of the parasitic life phoresis for initial analysis.
cycle. Very young children with thalassemia are more Quantitation of the hemoglobin profile is often
susceptible to infection with the nonlethal Plasmodium desirable. HbA2 is frequently elevated in -thalassemia
vivax. Thalassemia might then favor a natural protection trait and depressed in iron deficiency. HbF is elevated
against infection with the more lethal P. falciparum. in HPFH, some -thalassemia syndromes, and occa-
Thalassemias are the most common genetic disorders sional periods of erythroid stress or marrow dysplasia.
in the world, affecting nearly 200 million people world- For characterization of sickle cell trait, sickle thalassemia
wide. About 15% of American blacks are silent carri- syndromes, or HbSC disease, and for monitoring the
ers for thalassemia; -thalassemia trait (minor) occurs progress of exchange transfusion therapy to lower the
in 3% of American blacks and in 115% of persons of percentage of circulating HbS, quantitation of individ-
Mediterranean origin. -Thalassemia has a 1015% ual hemoglobins is also required. In most laboratories,
incidence in individuals from the Mediterranean and quantitation is performed only if the test is specifically
Southeast Asia and 0.8% in American blacks. The num- ordered. Complete characterization, including amino
ber of severe cases of thalassemia in the United States acid sequencing or gene cloning and sequencing, is
is about 1000. Sickle cell disease is the most common readily available from several reference laboratories.
structural hemoglobinopathy, occurring in heterozy- Because some variants can comigrate with HbA or
gous form in 8% of American blacks and in homozy- HbS (sickle hemoglobin), electrophoretic assessment
gous form in 1 in 400. Between 2 and 3% of American should always be regarded as incomplete unless func-
blacks carry a hemoglobin C allele. tional assays for hemoglobin sickling, solubility, or
oxygen affinity are also performed, as dictated by the
clinical presentation. The best sickling assays involve
Inheritance and Ontogeny
measurement of the degree to which the hemoglobin
Hemoglobinopathies are autosomal codominant traits sample becomes insoluble, or gelated, as it is deoxy-
compound heterozygotes who inherit a different abnor- genated (i.e., sickle solubility test). Unstable hemoglo-
mal mutant allele from each parent exhibit composite bins are detected by their precipitation in isopropanol
features of each. For example, patients inheriting or after heating to 50C. High-O2 affinity and low-
sickle thalassemia exhibit features of thalassemia O2 affinity variants are detected by quantitating the
and sickle cell anemia. The chain is present in HbA, P50, the partial pressure of oxygen at which the hemo-
HbA2, and HbF; -chain mutations thus cause abnor- globin sample becomes 50% saturated with oxygen.
malities in all three. The -globin hemoglobinopathies Direct tests for the percentage carboxyhemoglobin and
84 methemoglobin, employing spectrophotometric tech- or frank infarction in the spleen, central nervous system,
niques, can readily be obtained from most clinical labo- bones, liver, kidneys, and lungs (Fig. 8-3).
ratories on an urgent basis. Several sickle syndromes occur as the result of
Laboratory evaluation remains an adjunct rather than inheritance of HbS from one parent and another
the primary diagnostic aid. Diagnosis is best established hemoglobinopathy, such as thalassemia or HbC
by recognition of a characteristic history, physical find- (226 GluLys), from the other parent. The prototype
ings, peripheral blood smear morphology, and abnor- disease, sickle cell anemia, is the homozygous state for
malities of the complete blood cell count (e.g., profound HbS (Table 8-2).
microcytosis with minimal anemia in thalassemia trait).
Clinical manifestations of sickle cell anemia
Most patients with sickling syndromes have hemolytic
Structurally Abnormal anemia, with hematocrits from 15 to 30%, and sig-
Hemoglobins nificant reticulocytosis. Anemia was once thought to
Sickle Cell Syndromes exert protective effects against vasoocclusion by reduc-
ing blood viscosity. However, natural history and drug
The sickle cell syndromes are caused by a muta- therapy trials suggest that an increase in the hematocrit
tion in the -globin gene that changes the sixth amino
Section Iii

and feedback inhibition of reticulocytosis might be ben-


acid from glutamic acid to valine. HbS (226 GluVa1) eficial, even at the expense of increased blood viscos-
polymerizes reversibly when deoxygenated to form a ity. The role of adhesive reticulocytes in vasoocclusion
gelatinous network of fibrous polymers that stiffen the might account for these paradoxical effects.
RBC membrane, increase viscosity, and cause dehy- Granulocytosis is common. The white count can
dration due to potassium leakage and calcium influx fluctuate substantially and unpredictably during and
(Fig. 8-3). These changes also produce the sickle shape. between painful crises, infectious episodes, and other
Anemias

Sickled cells lose the pliability needed to traverse small intercurrent illnesses.
capillaries. They possess altered sticky membranes that Vasoocclusion causes protean manifestations. Inter-
are abnormally adherent to the endothelium of small mittent episodes of vasoocclusion in connective and
venules. These abnormalities provoke unpredictable epi- musculoskeletal structures produce ischemia manifested
sodes of microvascular vasoocclusion and premature RBC by acute pain and tenderness, fever, tachycardia, and
destruction (hemolytic anemia). Hemolysis occurs because anxiety. These recurrent episodes, called painful crises,
the spleen destroys the abnormal RBC. The rigid adher- are the most common clinical manifestation. Their
ent cells clog small capillaries and venules, causing tissue frequency and severity vary greatly. Pain can develop
ischemia, acute pain, and gradual end-organ damage. This almost anywhere in the body and may last from a few
venoocclusive component usually dominates the clinical hours to 2 weeks. Repeated crises requiring hospital-
course. Prominent manifestations include episodes of isch- ization (>3 per year) correlate with reduced survival
emic pain (i.e., painful crises) and ischemic malfunction in adult life, suggesting that these episodes are associ-
ated with accumulation of chronic end-organ damage.
Provocative factors include infection, fever, excessive exer-
cise, anxiety, abrupt changes in temperature, hypoxia,
and hypertonic dyes.
Arterial PO2 oxy Hbs Capillary venous PO2
(soluble) deoxy Hbs (polymerized) Repeated micro-infarction can destroy tissues having
microvascular beds prone to sickling. Thus, the spleen
is frequently lost within the first 1836 months of life,
Stiff, viscous sickle cell
Membrane changes causing susceptibility to infection, particularly by pneu-
Ca2 influx, K leakage
mococci. Acute venous obstruction of the spleen (splenic
sequestration crisis), a rare occurrence in early childhood,
Capillary venule occlusion Shortened red cell survival
may require emergency transfusion and/or splenectomy
(hemolytic anemia) to prevent trapping of the entire arterial output in the
obstructed spleen. Occlusion of retinal vessels can pro-
duce hemorrhage, neovascularization, and eventual
Microinfarction Anemia, jaundice, detachments. Renal papillary necrosis invariably pro-
Ischemic tissue pain gallstones, leg ulcers
Ischemic organ malfunction duces isosthenuria. More widespread renal necrosis leads
Autoinfarction of spleen
to renal failure in adults, a common late cause of death.
Bone and joint ischemia can lead to aseptic necro-
Figure 8-3 sis, especially of the femoral or humeral heads; chronic
Pathophysiology of sickle cell crisis. arthropathy; and unusual susceptibility to osteomyelitis,
Table 8-2 85
Clinical Features of Sickle Hemoglobinopathies
Hemoglobin Level Hemoglobin
Condition Clinical Abnormalities g/L (g/dl) MCV, fl Electrophoresis

Sickle cell trait None; rare painless hematuria Normal Normal Hb S/A:40/60
Sickle cell anemia Vasoocclusive crises with infarction of 70100 (710) 80100 Hb S/A:100/0
spleen, brain, marrow, kidney, lung; Hb F:225%
aseptic necrosis of bone; gallstones;
priapism; ankle ulcers
S/ thalassemia Vasoocclusive crises; aseptic necrosis of 70100 (710) 6080 Hb S/A:100/0
bone Hb F:110%
S/+ thalassemia Rare crises and aseptic necrosis 100140 (1014) 7080 Hb S/A:60/40
Hemoglobin SC Rare crises and aseptic necrosis; 100140 (1014) 80100 Hb S/A:50/0
painless hematuria Hb C:50%

CHAPTER 8
which may be caused by organisms, such as Salmonella, tend to have similar, slightly milder, symptoms, per-
rarely encountered in other settings. The handfoot haps because of the ameliorating effects of production
syndrome is caused by painful infarcts of the digits and of other hemoglobins within the RBC. Hemoglobin
dactylitis. Stroke is especially common in children; SC disease, one of the more common variants of sickle
a small subset tend to experience repeated episodes. cell anemia, is frequently marked by lesser degrees of
Stroke is less common in adults and is often hemor- hemolytic anemia and a greater propensity for the devel-

Disorders of Hemoglobin
rhagic. A particularly painful complication in males is opment of retinopathy and aseptic necrosis of bones.
priapism, due to infarction of the penile venous outflow In most respects, however, the clinical manifestations
tracts; permanent impotence is a frequent consequence. resemble sickle cell anemia. Some rare hemoglobin vari-
Chronic lower leg ulcers probably arise from ischemia ants actually aggravate the sickling phenomenon.
and superinfection in the distal circulation. The clinical variability in different patients inherit-
Acute chest syndrome is a distinctive manifestation ing the same disease-causing mutation (sickle hemo-
characterized by chest pain, tachypnea, fever, cough, globin) has made sickle cell disease the focus of efforts
and arterial oxygen desaturation. It can mimic pneu- to identify modifying genetic polymorphisms in other
monia, pulmonary emboli, bone marrow infarction and genes that might account for the heterogeneity. The
embolism, myocardial ischemia, or in situ lung infarc- complexity of the data obtained thus far has dampened
tion. Acute chest syndrome is thought to reflect in situ the expectation that genomewide analysis will yield
sickling within the lung, producing pain and temporary individualized profiles that predict a patients clinical
pulmonary dysfunction. Often it is difficult or impos- course. Nevertheless, a number of interesting patterns
sible to distinguish among other possibilities. Pulmonary have emerged from these modifying gene analyses. For
infarction and pneumonia are the most frequent under- example, genes affecting the inflammatory response or
lying or concomitant conditions in patients with this cytokine expression appear to be modifying candidates.
syndrome. Repeated episodes of acute chest pain cor- Genes that affect transcriptional regulation of lympho-
relate with reduced survival. Acutely, reduction in arte- cytes may be involved. Thus, it appears likely that key
rial oxygen saturation is especially ominous because it polymorphic changes in the patients inflammatory
promotes sickling on a massive scale. Chronic acute or response to the damages provoked by sickle red cells or
subacute pulmonary crises lead to pulmonary hyperten- in the response to chronic or recurrent infections may
sion and cor pulmonale, an increasingly common cause prove to be important for prognosticating the clinical
of death as patients survive longer. Considerable con- severity of disease.
troversy exists about the possible role played by free
plasma HbS in scavenging NO2, thus raising pulmonary
Clinical manifestations of sickle cell trait
vascular tone. Trials of sildenafil to restore NO2 levels
were terminated because of adverse effects. Sickle cell trait is usually asymptomatic. Anemia and
Sickle cell syndromes are remarkable for their clinical painful crises are rare. An uncommon but highly dis-
heterogeneity. Some patients remain virtually asymptom- tinctive symptom is painless hematuria often occurring
atic into or even through adult life, while others have in adolescent males, probably due to papillary necro-
repeated crises requiring hospitalization from early child- sis. Isosthenuria is a more common manifestation of
hood. Patients with sickle thalassemia and sickle-HbE the same process. Sloughing of papillae with urethral
86 obstruction has been reported, as have isolated cases of
massive sickling or sudden death due to exposure to Treatment Sickle Cell Syndromes
high altitudes or extremes of exercise and dehydration.
Patients with sickle cell syndromes require ongoing
Avoidance of dehydration and extreme physical stress
continuity of care. Familiarity with the pattern of symp-
should be advised.
toms provides the best safeguard against excessive use
of the emergency department, hospitalization, and
Diagnosis habituation to addictive narcotics. Additional preven-
Sickle cell syndromes are suspected on the basis of hemo- tive measures include regular slit-lamp examinations
lytic anemia, RBC morphology (Fig. 8-4), and inter- to monitor development of retinopathy; antibiotic pro-
mittent episodes of ischemic pain. Diagnosis is confirmed phylaxis appropriate for splenectomized patients dur-
by hemoglobin electrophoresis and the sickling tests ing dental or other invasive procedures; and vigorous
already discussed. Thorough characterization of the exact oral hydration during or in anticipation of periods of
hemoglobin profile of the patient is important because extreme exercise, exposure to heat or cold, emotional
sickle thalassemia and hemoglobin SC disease have dis- stress, or infection. Pneumococcal and Haemophilus
tinct prognoses and clinical features. Diagnosis is usually influenzae vaccines are less effective in splenecto-
established in childhood, but occasional patients, often mized individuals. Thus, patients with sickle cell anemia
should be vaccinated early in life.
Section Iii

with compound heterozygous states, do not develop


symptoms until the onset of puberty, pregnancy, or The management of acute painful crisis includes vig-
early adult life. Genotyping of family members and orous hydration, thorough evaluation for underlying
potential parental partners is critical for genetic counsel- causes (e.g., infection), and aggressive analgesia admin-
ing. Details of the childhood history establish prognosis istered by a standing order and/or patient-controlled
and need for aggressive or experimental therapies. Fac- analgesia (PCA) pump. Morphine (0.10.15 mg/kg every
tors associated with increased morbidity and reduced 34 h) should be used to control severe pain. Bone
Anemias

survival are more than three crises requiring hospitaliza- pain may respond as well to ketorolac (3060 mg ini-
tion per year, chronic neutrophilia, a history of splenic tial dose, then 1530 mg every 68 h). Inhalation of
sequestration or handfoot syndrome, and second epi- nitrous oxide can provide short-term pain relief, but
sodes of acute chest syndrome. Patients with a history of great care must be exercised to avoid hypoxia and
cerebrovascular accidents are at higher risk for repeated respiratory depression. Nitrous oxide also elevates O2
episodes and require partial exchange transfusion and affinity, reducing O2 delivery to tissues. Its use should
especially close monitoring using Doppler carotid flow be restricted to experts. Many crises can be managed
measurements. Patients with severe or repeated epi- at home with oral hydration and oral analgesia. Use
sodes of acute chest syndrome may need lifelong trans- of the emergency department should be reserved for
fusion support, utilizing partial exchange transfusion, if especially severe symptoms or circumstances in which
possible. other processes, e.g., infection, are strongly suspected.
Nasal oxygen should be employed as appropriate
to protect arterial saturation. Most crises resolve in
17 days. Use of blood transfusion should be reserved
for extreme cases: transfusions do not shorten the
duration of thecrisis.
No tests are definitive to diagnose acute painful
crises. Critical to good management is an approach that
recognizes that most patients reporting crisis symptoms
do indeed have crisis or another significant medical
problem. Diligent diagnostic evaluation for underlying
causes is imperative, even though these are found infre-
quently. In adults, the possibility of aseptic necrosis or
sickle arthropathy must be considered, especially if pain
and immobility become repeated or chronic at a single
site. Nonsteroidal anti-inflammatory agents are often
effective for sickle cell arthropathy.
Acute chest syndrome is a medical emergency that
Figure 8-4
may require management in an intensive care unit.
Sickle cell anemia. The elongated and crescent-shaped red
blood cells seen on this smear represent circulating irrevers-
Hydration should be monitored carefully to avoid the
ibly sickled cells. Target cells and a nucleated red blood cell development of pulmonary edema, and oxygen therapy
are also seen. should be especially vigorous for protection of arterial
saturation. Diagnostic evaluation for pneumonia and opulation. Children who do suffer a cerebrovascular
p 87
pulmonary embolism should be especially thorough, accident should be maintained for at least 35 years on
since these may occur with atypical symptoms. Critical a program of vigorous exchange transfusion, as the risk
interventions are transfusion to maintain a hematocrit of second strokes is extremely high.
>30, and emergency exchange transfusion if arterial Gene therapy for sickle cell anemia is being inten-
saturation drops to <90%. As patients with sickle cell sively pursued, but no safe measures are currently
syndrome increasingly survive into their fifth and sixth available. Agents blocking RBC dehydration or vascu-
decades, end-stage renal failure and pulmonary hyper- lar adhesion, such as clotrimazole or magnesium, may
tension are becoming increasingly prominent causes of have value as an adjunct to hydroxyurea therapy, pend-
end-stage morbidity. A sickle cell cardiomyopathy and/ ing the completion of ongoing trials. Combinations of
or premature coronary artery disease may compromise clotrimazole and magnesium are being evaluated.
cardiac function in later years. Sickle cell patients have
received kidney transplants, but they often experience
an increase in the frequency and severity of crises, pos- Unstable Hemoglobins
sibly due to increased infection as a consequence of
immunosuppression. Amino acid substitutions that reduce solubility or increase
The most significant advance in the therapy of sickle susceptibility to oxidation produce unstable hemoglo-

CHAPTER 8
cell anemia has been the introduction of hydroxyurea bins that precipitate, forming inclusion bodies injurious
as a mainstay of therapy for patients with severe symp- to the RBC membrane. Representative mutations are
toms. Hydroxyurea (1030 mg/kg per day) increases those that interfere with contact points between the
fetal hemoglobin and may also exert beneficial affects and subunits (e.g., Hb Philly [35TyrPhe]), alter the
on RBC hydration, vascular wall adherence, and suppres- helical segments (e.g., Hb Genova [28LeuPro]), or dis-
sion of the granulocyte and reticulocyte counts; dos- rupt interactions of the hydrophobic pockets of the

Disorders of Hemoglobin
age is titrated to maintain a white cell count between globin subunits with heme (e.g., Hb Koln [98ValMet])
5000 and 8000 per L. White cells and reticulocytes may (Table 8-3). The inclusions, called Heinz bodies, are
play a major role in the pathogenesis of sickle cell crisis, clinically detectable by staining with supravital dyes
and their suppression may be an important benefit of such as crystal violet. Removal of these inclusions by
hydroxyurea therapy. the spleen generates pitted, rigid cells that have short-
Hydroxyurea should be considered in patients expe- ened life spans, producing hemolytic anemia of vari-
riencing repeated episodes of acute chest syndrome or able severity, sometimes requiring chronic transfusion
with more than three crises per year requiring hospi-
talization. The utility of this agent for reducing the inci-
dence of other complications (priapism, retinopathy)
Table 8-3
is under evaluation, as are the long-term side effects.
Hydroxyurea offers broad benefits to most patients Representative Abnormal Hemoglobins With
Altered Synthesis or Function
whose disease is severe enough to impair their func-
tional status, and it may improve survival. HbF levels Main Clinical
increase in most patients within a few months. Designation Mutation Population Effectsa

The antitumor drug 5-azacytidine was the first agent Sickle or S 6GluVal African Anemia, ischemic
found to elevate HbF. It never achieved widespread infarcts
use because of concerns about acute toxicity and car- C 6GluLys African Mild anemia; inter-
cinogenesis. However, low doses of the related agent acts with HbS
5-deoxyazacytidine (decitabine) can elevate HbF with E 26GluLys Southeast Microcytic anemia,
more acceptable toxicity. Asian splenomegaly,
Bone marrow transplantation can provide defini- thalassemic
tive cures but is known to be effective and safe only in phenotype
children. Partially myeloablative conditioning regimens Kln 98ValMet Sporadic Hemolytic anemia,
(mini transplants) may allow more widespread use of Heinz bodies
this modality. Prognostic features justifying bone mar- when splenecto-
row transplant are the presence of repeated crises early mized
in life, a high neutrophil count, or the development of Yakima 99AspHis Sporadic Polycythemia
handfoot syndrome. Children at risk for stroke can Kansas 102AsnLys Sporadic Mild anemia
now be identified through the use of Doppler ultra- M. Iwata 87HisTyr
Sporadic Methemoglobinemia
sound techniques. Prophylactic exchange transfusion
appears to substantially reduce the risk of stroke in this a
See text for details.
88 support. Splenectomy may be needed to correct the Diagnosis and Management of
anemia. Leg ulcers and premature gallbladder disease Patients with Unstable Hemoglobins,
due to bilirubin loading are frequent stigmata. High-Affinity Hemoglobins, and
Unstable hemoglobins occur sporadically, often by Methemoglobinemia
spontaneous new mutations. Heterozygotes are often
symptomatic because a significant Heinz body burden Unstable hemoglobin variants should be suspected in
can develop even when the unstable variant accounts patients with nonimmune hemolytic anemia, jaundice,
for only a portion of the total hemoglobin. Symptom- splenomegaly, or premature biliary tract disease. Severe
atic unstable hemoglobins tend to be -globin variants hemolysis usually presents during infancy as neonatal
because sporadic mutations affecting only one of the jaundice or anemia. Milder cases may present in adult
four globins would generate only 2030% abnormal life with anemia or only as unexplained reticulocytosis,
hemoglobin. hepatosplenomegaly, premature biliary tract disease, or
leg ulcers. Because spontaneous mutation is common,
family history of anemia may be absent. The periph-
Hemoglobins with Altered eral blood smear often shows anisocytosis, abundant
OxygenAffinity cells with punctate inclusions, and irregular shapes (i.e.,
poikilocytosis).
High-affinity hemoglobins (e.g., Hb Yakima [99AspHis]) The two best tests for diagnosing unstable hemoglo-
Section Iii

bind oxygen more readily but deliver less O2 to bins are the Heinz body preparation and the isopropa-
tissues at normal capillary PO2 levels (Fig. 8-2). Mild nol or heat stability test. Many unstable Hb variants are
tissue hypoxia ensues, stimulating RBC production and electrophoretically silent. A normal electrophoresis does
erythrocytosis (Table 8-3). In extreme cases, the hemat- not rule out the diagnosis.
ocrits can rise to 6065%, increasing blood viscosity and Severely affected patients may require transfusion
producing typical symptoms (headache, somnolence, support for the first 3 years of life because splenec-
Anemias

or dizziness). Phlebotomy may be required. Typical tomy before age 3 years is associated with a significantly
mutations alter interactions within the heme pocket higher immune deficit. Splenectomy is usually effective
or disrupt the Bohr effect or salt-bond site. Mutations thereafter, but occasional patients may require lifelong
that impair the interaction of HbA with 2,3-BPG can transfusion support. After splenectomy, patients can
increase O2 affinity because 2,3-BPG binding lowers O2 develop cholelithiasis and leg ulcers, hypercoagulable
affinity. states, and susceptibility to overwhelming sepsis. Sple-
Low-affinity hemoglobins (e.g., Hb Kansas [102AsnLys]) nectomy should be avoided or delayed unless it is the
bind sufficient oxygen in the lungs, despite their lower only alternative. Precipitation of unstable hemoglobins
oxygen affinity, to achieve nearly full saturation. At is aggravated by oxidative stress, e.g., infection and anti-
capillary oxygen tensions, they lose sufficient amounts malarial drugs, which should be avoided when possible.
of oxygen to maintain homeostasis at a low hematocrit High-O2 affinity hemoglobin variants should be sus-
(Fig. 8-2) (pseudoanemia). Capillary hemoglobin desatu- pected in patients with erythrocytosis. The best test
ration can also be sufficient to produce clinically appar- for confirmation is measurement of the P50. A high-
ent cyanosis. Despite these findings, patients usually O2 affinity Hb causes a significant left shift (i.e., lower
require no specific treatment. numeric value of the P50); confounding conditions, e.g.,
tobacco smoking or carbon monoxide exposure, can
also lower the P50.
Methemoglobinemias High-affinity hemoglobins are often asymptom-
Methemoglobin is generated by oxidation of the heme atic; rubor or plethora may be telltale signs. When the
iron moieties to the ferric state, causing a characteristic hematocrit reaches to 5560%, symptoms of high blood
bluish-brown muddy color resembling cyanosis. Methe- viscosity and sluggish flow (e.g., headache, lethargy,
moglobin has such high oxygen affinity that virtually no dizziness) may be present. These persons may benefit
oxygen is delivered. Levels >5060% are often fatal. from judicious phlebotomy. Erythrocytosis represents
Congenital methemoglobinemia arises from globin an appropriate attempt to compensate for the impaired
mutations that stabilize iron in the ferric state (e.g., oxygen delivery by the abnormal variant. Overzeal-
HbM Iwata [87HisTyr], Table 8-3) or from mutations ous phlebotomy may stimulate increased erythropoiesis
that impair the enzymes that reduce methemoglobin to or aggravate symptoms by thwarting this compensa-
hemoglobin (e.g., methemoglobin reductase, NADP tory mechanism. The guiding principle of phlebotomy
diaphorase). Acquired methemoglobinemia is caused should be to improve oxygen delivery by reduc-
by toxins that oxidize heme iron, notably nitrate and ing blood viscosity and increasing blood flow rather
nitrite-containing compounds, including drugs com- than restoration of a normal hematocrit. Modest iron
monly used in cardiology and anesthesiology. deficiency may aid in control.
Low-affinity hemoglobins should be considered in 89
patients with cyanosis or a low hematocrit with no
other reason apparent after thorough evaluation. The
P50 test confirms the diagnosis. Counseling and reassur-
ance are the interventions of choice.
Methemoglobin should be suspected in patients with
hypoxic symptoms who appear cyanotic but have a
PaO2 sufficiently high that hemoglobin should be fully
saturated with oxygen. A history of nitrite or other oxi-
dant ingestions may not always be available; some expo-
sures may be inapparent to the patient, and others may
result from suicide attempts. The characteristic muddy
appearance of freshly drawn blood can be a critical clue.
The best diagnostic test is methemoglobin assay, which
Figure 8-5
is usually available on an emergency basis. -Thalassemia intermedia. Microcytic and hypochromic
Methemoglobinemia often causes symptoms of cere- red blood cells are seen that resemble the red blood cells of
bral ischemia at levels >15%; levels >60% are usually severe iron deficiency anemia. Many elliptical and teardrop-

CHAPTER 8
lethal. Intravenous injection of 1 mg/kg of methylene shaped red blood cells are noted.
blue is effective emergency therapy. Milder cases and
follow-up of severe cases can be treated orally with
methylene blue (60 mg three to four times each day) or
form toxic inclusion bodies that kill developing erythro-
ascorbic acid (300600 mg/d).
blasts in the marrow. Few of the proerythroblasts begin-
ning erythroid maturation survive. The surviving RBCs

Disorders of Hemoglobin
bear a burden of inclusion bodies that are detected in
Thalassemia Syndromes the spleen, shortening the RBC life span and producing
severe hemolytic anemia. The resulting profound ane-
The thalassemia syndromes are inherited disorders of - mia stimulates erythropoietin release and compensatory
or -globin biosynthesis. The reduced supply of globin erythroid hyperplasia, but the marrow response is sabo-
diminishes production of hemoglobin tetramers, causing taged by the ineffective erythropoiesis. Anemia persists.
hypochromia and microcytosis. Unbalanced accumula- Erythroid hyperplasia can become exuberant and pro-
tion of and subunits occurs because the synthesis of duce masses of extramedullary erythropoietic tissue in
the unaffected globins proceeds at a normal rate. Unbal- the liver and spleen.
anced chain accumulation dominates the clinical pheno- Massive bone marrow expansion deranges growth
type. Clinical severity varies widely, depending on the and development. Children develop characteristic
degree to which the synthesis of the affected globin is chipmunk facies due to maxillary marrow hyperpla-
impaired, altered synthesis of other globin chains, and sia and frontal bossing. Thinning and pathologic fracture
coinheritance of other abnormal globin alleles. of long bones and vertebrae may occur due to cortical
invasion by erythroid elements and profound growth
retardation. Hemolytic anemia causes hepatospleno-
Clinical Manifestations of megaly, leg ulcers, gallstones, and high-output conges-
a-Thalassemia Syndromes tive heart failure. The conscription of caloric resources
Mutations causing thalassemia can affect any step in the to support erythropoiesis leads to inanition; suscepti-
pathway of globin gene expression: transcription, pro- bility to infection; endocrine dysfunction; and in the
cessing of the mRNA precursor, translation, and post- most severe cases, death during the first decade of life.
translational metabolism of the -globin polypeptide Chronic transfusions with RBCs improve oxygen deliv-
chain. The most common forms arise from mutations ery, suppress the excessive ineffective erythropoiesis,
that derange splicing of the mRNA precursor or prema- and prolong life, but the inevitable side effects, notably
turely terminate translation of the mRNA. iron overload, usually prove fatal by age 30 years.
Hypochromia and microcytosis characterize all Severity is highly variable. Known modulating fac-
forms of thalassemia because of the reduced amounts tors are those that ameliorate the burden of unpaired
of hemoglobin tetramers (Fig. 8-5). In heterozygotes -globin inclusions. Alleles associated with milder
(-thalassemia trait), this is the only abnormality seen. synthetic defects and coinheritance of -thalassemia
Anemia is minimal. In more severe homozygous states, trait reduce clinical severity by reducing accumulation
unbalanced - and -globin accumulation causes accu- of excess globin. HbF persists to various degrees in
mulation of highly insoluble unpaired chains. They thalassemias. -Globin gene chains can substitute for
90 Table 8-4
The ` Thalassemias
Hemoglobin Level,
Condition Hemoglobin A, % Hemoglobin H (a4), % g/L (g/dL) MCV, fL

Normal 97 0 150 (15) 90


Silent thalassemia: / 98100 0 150 (15) 90
Thalassemia trait: 8595 Rare red blood cell 120130 (1213) 7080
/ homozygous -thal-2a inclusions
or
/ heterozygous -thal-1a
Hemoglobin H disease: 7095 530 60100 (610) 6070
/ heterozygous -thal-1/-thal-2
Hydrops fetalis: 0 510b Fatal in utero or at
/ homozygous -thal-1 birth

a
When both alleles on one chromosome are deleted, the locus is called -thal-1; when only a single allele on one chromosome is deleted,
the locus is called -thal-2.
Section Iii

b
9095% of the hemoglobin is hemoglobin Barts (tetramers of chains).

chains, generating more hemoglobin and reducing the moderately severe hemolytic anemia but milder ineffec-
burden of -globin inclusions. The terms -thalassemia tive erythropoiesis. Survival into midadult life without
major and -thalassemia intermedia are used to reflect the transfusions is common.
The homozygous state for the -thalassemia-1
Anemias

clinical heterogeneity. Patients with -thalassemia major


require intensive transfusion support to survive. Patients cis deletion (hydrops fetalis) causes total absence of
with -thalassemia intermedia have a somewhat milder -globin synthesis. No physiologically useful hemoglo-
phenotype and can survive without transfusion. The bin is produced beyond the embryonic stage. Excess
terms -thalassemia minor and -thalassemia trait describe globin forms tetramers called Hb Barts (4), which has
asymptomatic heterozygotes for thalassemia. a very high oxygen affinity. It delivers almost no O2
to fetal tissues, causing tissue asphyxia, edema (hydrops
Thalassemia Syndromes fetalis), congestive heart failure, and death in utero.
-Thalassemia-2 trait is common (1520%) among peo-
The four classic thalassemias, most common in ple of African descent. The cis -thalassemia-1 deletion
Asians, are -thalassemia-2 trait, in which one of the is almost never seen, however. Thus, -thalassemia-2
four -globin loci is deleted; -thalassemia-1 trait, with and the trans form of -thalassemia-1 are very common,
two deleted loci; HbH disease, with three loci deleted; but HbH disease and hydrops fetalis are rare.
and hydrops fetalis with Hb Barts, with all four loci It has been known for some time that some patients
deleted (Table 8-4). Nondeletion forms of thalasse- with myelodysplasia or erythroleukemia produce RBC
mia also exist. clones containing HbH. This phenomenon is due to
-Thalassemia-2 trait is an asymptomatic, silent carrier mutations in the ATRX pathway that affect the LCR
state. -Thalassemia-1 trait resembles -thalassemia minor. of the -globin gene cluster.
Offspring doubly heterozygous for -thalassemia-2 and
-thalassemia-1 exhibit a more severe phenotype called
Diagnosis and Management of
HbH disease. Heterozygosity for a deletion that removes
Thalassemias
both genes from the same chromosome (cis deletion)
is common in Asians and in those from the Mediterra- The diagnosis of -thalassemia major is readily made
nean region, as is homozygosity for -thalassemia-2 (trans during childhood on the basis of severe anemia accom-
deletion). Both produce asymptomatic hypochromia and panied by the characteristic signs of massive ineffective
microcytosis. erythropoiesis: hepatosplenomegaly; profound micro-
In HbH disease, HbA production is only 2530% nor- cytosis; a characteristic blood smear (Fig. 8-5); and
mal. Fetuses accumulate some unpaired chains (Hb elevated levels of HbF, HbA2, or both. Many patients
Barts; -chain tetramers). In adults, unpaired chains require chronic hypertransfusion therapy designed to
accumulate and are soluble enough to form 4 tetramers maintain a hematocrit of at least 2730% so that eryth-
called HbH. HbH forms few inclusions in erythroblasts ropoiesis is suppressed. Splenectomy is required if the
and precipitates in circulating RBC. Patients with HbH annual transfusion requirement (volume of RBCs per
disease have thalassemia intermedia characterized by kilogram of body weight per year) increases by >50%.
Folic acid supplements may be useful. Vaccination with Hemoglobin Lepore 91
Pneumovax in anticipation of eventual splenectomy is
advised, as is close monitoring for infection, leg ulcers, Hb Lepore (2[]2) arises by an unequal crossover and
and biliary tract disease. Many patients develop endo- recombination event that fuses the proximal end of the
crine deficiencies as a result of iron overload. Early -gene with the distal end of the closely linked -gene.
endocrine evaluation is required for glucose intolerance, It is common in the Mediterranean basin. The result-
thyroid dysfunction, and delayed onset of puberty or ing chromosome contains only the fused gene. The
secondary sexual characteristics. Lepore () globin is synthesized poorly because the
Patients with -thalassemia intermedia exhibit similar fused gene is under the control of the weak -globin
stigmata but can survive without chronic hypertransfu- promoter. Hb Lepore alleles have a phenotype like
sion. Management is particularly challenging because thalassemia, except for the added presence of 220%
a number of factors can aggravate the anemia, includ- Hb Lepore. Compound heterozygotes for Hb Lepore
ing infection, onset of puberty, and development of and a classic -thalassemia allele may also have severe
splenomegaly and hypersplenism. Some patients may thalassemia.
eventually benefit from splenectomy. The expanded
erythron can cause absorption of excessive dietary iron
and hemosiderosis, even without transfusion. Hemoglobin E

CHAPTER 8
-Thalassemia minor (i.e., thalassemia trait) usually HbE (i.e., 2226GluLys) is extremely common
presents as profound microcytosis and hypochromia in Cambodia, Thailand, and Vietnam. The gene
with target cells but only minimal or mild anemia. The has become far more prevalent in the United
mean corpuscular volume is rarely >75 fL; the hemato- States as a result of immigration of Asian persons, espe-
crit is rarely <3033%. Hemoglobin analysis classically cially in California, where HbE is the most common
reveals an elevated HbA2 (3.57.5%), but some forms variant detected. HbE is mildly unstable but not enough

Disorders of Hemoglobin
are associated with normal HbA2 and/or elevated HbF. to affect RBC life span significantly. Heterozygotes
Genetic counseling and patient education are essential. resemble individuals with a mild -thalassemia trait.
Patients with -thalassemia trait should be warned that Homozygotes have somewhat more marked abnormali-
their blood picture resembles iron deficiency and can ties but are asymptomatic. Compound heterozygotes for
be misdiagnosed. They should eschew empirical use of HbE and a -thalassemia gene can have -thalassemia
iron, yet iron deficiency can develop during pregnancy intermedia or -thalassemia major, depending on the
or from chronic bleeding. severity of the coinherited thalassemic gene.
Persons with -thalassemia trait may exhibit mild The E allele contains a single base change in codon
hypochromia and microcytosis usually without anemia. 26 that causes the amino acid substitution. However,
HbA2 and HbF levels are normal. Affected individuals this mutation activates a cryptic RNA splice site, gener-
usually require only genetic counseling. HbH disease ating a structurally abnormal globin mRNA that cannot
resembles -thalassemia intermedia, with the added be translated from about 50% of the initial pre-mRNA
complication that the HbH molecule behaves like mod- molecules. The remaining 4050% are normally spliced
erately unstable hemoglobin. Patients with HbH dis- and generate functional mRNA that is translated into
ease should undergo splenectomy if excessive anemia E-globin because the mature mRNA carries the base
or a transfusion requirement develops. Oxidative drugs change that alters codon 26.
should be avoided. Iron overload leading to death can Genetic counseling of the persons at risk for HbE
occur in more severely affected patients. should focus on the interaction of HbE with thalas-
semia rather than HbE homozygosity, a condition asso-
ciated with asymptomatic microcytosis, hypochromia,
Prevention
and hemoglobin levels rarely <100 g/L (<10 g/dL).
Antenatal diagnosis of thalassemia syndromes is now
widely available. DNA diagnosis is based on polymerase
chain reaction amplification of fetal DNA, obtained by Hereditary Persistence of Fetal
amniocentesis or chorionic villus biopsy followed by Hemoglobin
hybridization to allele-specific oligonucleotides probes
or direct DNA sequencing. HPFH is characterized by continued synthesis of high
levels of HbF in adult life. No deleterious effects are
apparent, even when all of the hemoglobin produced is
Thalassemic Structural Variants HbF. These rare patients demonstrate convincingly that
prevention or reversal of the fetal to adult hemoglobin
Thalassemic structural variants are characterized by both switch would provide effective therapy for sickle cell
defective synthesis and abnormal structure. anemia and thalassemia.
92 Acquired Hemoglobinopathies Desferoxamine is relatively nontoxic. Occasional cata-
The two most important acquired hemoglobinopa- racts; deafness; and local skin reactions, including urti-
thies are carbon monoxide poisoning and methemo- caria, occur. Skin reactions can usually be managed with
globinemia (discussed earlier). Carbon monoxide has antihistamines. Negative iron balance can be achieved,
a higher affinity for hemoglobin than does oxygen; it even in the face of a high transfusion requirement, but
can replace oxygen and diminish O2 delivery. Chronic this alone does not prevent long-term morbidity and
elevation of carboxyhemoglobin levels to 10 or 15%, as mortality in chronically transfused patients. Irreversible
occurs in smokers, can lead to secondary polycythemia. end-organ deterioration develops at relatively modest
Carboxyhemoglobin is cherry red in color and masks levels of iron overload, even if symptoms do not appear
the development of cyanosis usually associated with for many years thereafter. To enjoy a significant survival
poor O2 delivery to tissues. advantage, chelation must begin before 58 years of
Abnormalities of hemoglobin biosynthesis have also age in -thalassemia major.
been described in blood dyscrasias. In some patients with Deferasirox is an oral iron-chelating agent. Single
myelodysplasia, erythroleukemia, or myeloproliferative daily doses of 20 to 30 mg/kg deferasirox produced
disorders, elevated HbF or a mild form of HbH dis- reductions in liver iron concentration comparable to
ease may also be seen. The abnormalities are not severe desferoxamine in long-term transfused adult and pedi-
enough to alter the course of the underlying disease. atric patients. Deferasirox produces some elevations
Section Iii

in liver enzymes and slight but persistent increases in


serum creatinine without apparent clinical consequence.
Treatment Transfusional Hemosiderosis Other toxicities are similar to those of desferoxamine. Its
toxicity profile is acceptable, although long-term effects
Chronic blood transfusion can lead to bloodborne infec- are still being evaluated.
tion, alloimmunization, febrile reactions, and lethal iron
Anemias

overload (Chap. 12). A unit of packed RBCs contains


250300 mg iron (1 mg/mL). The iron assimilated by a
single transfusion of two units of packed RBCs is thus Experimental Therapies
equal to a 1- to 2-year intake of iron. Iron accumulates
in chronically transfused patients because no mecha- Bone Marrow Transplantation, Gene
nisms exist for increasing iron excretion: an expanded Therapy, and Manipulation of HBF
erythron causes especially rapid development of iron
Bone marrow transplantation provides stem cells able
overload because accelerated erythropoiesis promotes
to express normal hemoglobin; it has been used in
excessive absorption of dietary iron. Vitamin C should
a large number of patients with thalassemia and a
not be supplemented because it generates free radicals
smaller number of patients with sickle cell anemia.
in iron excess states.
Early in the course of disease, before end-organ dam-
Patients who receive >100 units of packed RBCs usually
age occurs, transplantation is curative in 8090% of
develop hemosiderosis. The ferritin level rises, followed
patients. In highly experienced centers, the treatment-
by early endocrine dysfunction (glucose intolerance and
related mortality rate is <10%. Since survival into adult
delayed puberty), cirrhosis, and cardiomyopathy. Liver
life is possible with conventional therapy, the decision
biopsy shows both parenchymal and reticuloendothelial
to transplant is best made in consultation with special-
iron. The superconducting quantum-interference device
ized centers.
(SQUID) is accurate at measuring hepatic iron but is not
Gene therapy of thalassemia and sickle cell disease has
widely available. Cardiac toxicity is often insidious. Early
proved to be an elusive goal. Uptake of gene vectors
development of pericarditis is followed by dysrhythmia
into the nondividing hematopoietic stem cells has been
and pump failure. The onset of heart failure is ominous,
inefficient. Lentiviral-type vectors that can transduce
often presaging death within a year.
nondividing cells may solve this problem.
The decision to start long-term transfusion support
Reestablishing high levels of fetal hemoglobin synthe-
should also prompt one to institute therapy with iron-
sis should ameliorate the symptoms of chain hemoglo-
chelating agents. Desferoxamine (Desferal) is for paren-
binopathies. Cytotoxic agents such as hydroxyurea and
teral use. Its iron-binding kinetics require chronic slow
cytarabine promote high levels of HbF synthesis, probably
infusion via a metering pump. The constant presence of
by stimulating proliferation of the primitive HbF-produc-
the drug improves the efficiency of chelation and pro-
ing progenitor cell population (i.e., F cell progenitors).
tects tissues from occasional releases of the most toxic
Unfortunately, this regimen has not yet been effective in
fraction of ironlow-molecular-weight ironwhich
thalassemia. Butyrates stimulate HbF production but
may not be sequestered by protective proteins.
only transiently. Pulsed or intermittent administration
has been found to sustain HbF induction in the majority affect RBC counts more dramatically. These hypoplas- 93
of patients with sickle cell disease. It is unclear whether tic crises are usually transient and self-correcting before
butyrates will have similar activity in patients with intervention is required.
thalassemia. Aplastic crisis refers to a profound cessation of
erythroid activity in patients with chronic hemolytic
anemias. It is associated with a rapidly falling hemato-
Aplastic and Hypoplastic crit. Episodes are usually self-limited. Aplastic crises are
Crisis in Patients with caused by infection with a particular strain of parvovi-
Hemoglobinopathies rus, B19A. Children infected with this virus usually
develop permanent immunity. Aplastic crises do not
Patients with hemolytic anemias sometimes exhibit an often recur and are rarely seen in adults. Management
alarming decline in hematocrit during and immediately requires close monitoring of the hematocrit and reticu-
after acute illnesses. Bone marrow suppression occurs locyte count. If anemia becomes symptomatic, transfusion
in almost everyone during acute inflammatory illnesses. support is indicated. Most crises resolve spontaneously
In patients with short RBC life spans, suppression can within 12 weeks.

CHAPTER 8
Disorders of Hemoglobin
chaPter 9

MEGALOBLASTIC ANEMIAS

A. Victor Hoffbrand

The megaloblastic anemias are a group of disorders Adult daily losses (mainly in the urine and feces) are
characterized by the presence of distinctive morpho- 13 g (0.1% of body stores), and as the body does
logic appearances of the developing red cells in the bone not have the ability to degrade cobalamin, daily require-
marrow. The marrow is usually cellular, and the anemia ments are also about 13 g. Body stores are of the
is based on ineffective erythropoiesis. The cause is usu- order of 23 mg, sufficient for 34 years if supplies are
ally a deficiency of either cobalamin (vitamin B12) or completely cut off.
folate, but megaloblastic anemia may occur because of
genetic or acquired abnormalities that affect the metabo-
lism of these vitamins or because of defects in DNA syn- Absorption
thesis not related to cobalamin or folate (Table 9-1). Two mechanisms exist for cobalamin absorption. One
Cobalamin and folate absorption and metabolism are is passive, occurring equally through buccal, duodenal,
described next, followed by the biochemical basis, clini- and ileal mucosa; it is rapid but extremely inefficient,
cal and laboratory features, causes, and treatment of with <1% of an oral dose being absorbed by this pro-
megaloblastic anemia. cess. The normal physiologic mechanism is active; it
occurs through the ileum and is efficient for small (a few
micrograms) oral doses of cobalamin, and it is medi-
coBalamin ated by gastric intrinsic factor (IF). Dietary cobalamin
is released from protein complexes by enzymes in the
Cobalamin (vitamin B12) exists in a number of differ- stomach, duodenum, and jejunum; it combines rapidly
ent chemical forms. All have a cobalt atom at the cen- with a salivary glycoprotein that belongs to the family of
ter of a corrin ring. In nature, the vitamin is mainly in cobalamin-binding proteins known as HC (HCs). In the
the 2-deoxyadenosyl (ado) form, which is located in
mitochondria. It is the cofactor for the enzyme methyl-
malonyl coenzyme A (CoA) mutase. The other major
natural cobalamin is methylcobalamin, the form in Table 9-1
human plasma and in cell cytoplasm. It is the cofactor caUses of megaloBlastic anemia
for methionine synthase. There are also minor amounts
Cobalamin deficiency or abnormalities of cobalamin
of hydroxocobalamin to which methyl- and adocobala- metabolism (see Tables 9-3 and 9-4)
min are converted rapidly by exposure to light. Folate deficiency or abnormalities of folate metabolism
(see Table 9-5)
Therapy with antifolate drugs (e.g., methotrexate)
Dietary sources and requirements Independent of either cobalamin or folate deficiency and
Cobalamin is synthesized solely by microorganisms. refractory to cobalamin and folate therapy:
Some cases of acute myeloid leukemia, myelodysplasia
Ruminants obtain cobalamin from the foregut, but the
Therapy with drugs interfering with synthesis of DNA
only source for humans is food of animal origin, e.g., (e.g., cytosine arabinoside, hydroxyurea,
meat, fish, and dairy products. Vegetables, fruits, and 6-mercaptopurine, azidothymidine [AZT])
other foods of nonanimal origin are free from cobala- Orotic aciduria (responds to uridine)
min unless they are contaminated by bacteria. A nor- Thiamine-responsive
mal Western diet contains 530 g of cobalamin daily.
94
intestine, the HC is digested by pancreatic trypsin and The TC II cobalamin is internalized by endocytosis via 95
the cobalamin is transferred to IF. clathrin-coated pits; the complex is degraded, but the
IF (gene at chromosome 11q13 coding for 9 exons) receptor probably is recycled to the cell membrane as
is produced in the gastric parietal cells of the fundus is the case for transferrin. Export of free cobalamin is
and body of the stomach, and its secretion parallels that via the ATP-binding cassette drug transporter alias mul-
of hydrochloric acid. Normally, there is a vast excess tidrug resistance protein 1.
of IF. The IFcobalamin complex passes to the ileum,
where IF attaches to a specific receptor (cubilin) on the
microvillus membrane of the enterocytes. Cubilin also
is present in yolk sac and renal proximal tubular epithe- Folate
lium. Cubulin appears to traffic by means of amnionless
(AMN), an endocytic receptor protein that directs sub- Dietary folate
localization and endocytosis of cubulin with its ligand Folic (pteroylglutamic) acid is a yellow, crystalline,
IFcobalamin complex. The cobalaminIF complex water-soluble substance. It is the parent compound of
enters the ileal cell, where IF is destroyed. After a delay a large family of natural folate compounds, which dif-
of about 6 h, the cobalamin appears in portal blood fer from it in three respects: (1) they are partly or
attached to transcobalamin (TC) II. completely reduced to di- or tetrahydrofolate (THF)

CHAPTER 9
Between 0.5 and 5 g of cobalamin enters the bile derivatives, (2) they usually contain a single carbon
each day. This binds to IF, and a major portion of unit (Table 9-2), and (3) 7090% of natural folates are
biliary cobalamin normally is reabsorbed together folate-polyglutamates.
with cobalamin derived from sloughed intestinal cells. Most foods contain some folate. The highest con-
Because of the appreciable amount of cobalamin under- centrations are found in liver, yeast, spinach, other
going enterohepatic circulation, cobalamin deficiency greens, and nuts (>100 g/100 g). The total folate

Megaloblastic Anemias
develops more rapidly in individuals who malabsorb content of an average Western diet is 250 g daily,
cobalamin than it does in vegans, in whom reabsorption but the amount varies widely according to the type of
of biliary cobalamin is intact. food eaten and the method of cooking. Folate is eas-
ily destroyed by heating, particularly in large volumes of
Transport water. Total-body folate in an adult is 10 mg, with the
liver containing the largest store. Daily adult require-
Two main cobalamin transport proteins exist in human ments are 100 g, so stores are sufficient for only 34
plasma; they both bind cobalaminone molecule for months in normal adults, and severe folate deficiency
one molecule. One HC, known as TC I, is closely may develop rapidly.
related to other cobalamin-binding HCs in milk, gas-
tric juice, bile, saliva, and other fluids. The gene TCNL
is at chromosome 11q11-q12.3 and has 9 exons. These
Absorption
HCs differ from each other only in the carbohydrate
moiety of the molecule. TC I is derived primarily Folates are absorbed rapidly from the upper small
from the specific granules in neutrophils. Normally, it intestine. The absorption of folate polyglutamates is
is about two-thirds saturated with cobalamin, which it less efficient than that of monoglutamates; on aver-
binds tightly. TC I does not enhance cobalamin entry age, 50% of food folate is absorbed. Polyglutamate
into tissues. Glycoprotein receptors on liver cells are forms are hydrolyzed to the monoglutamate deriva-
involved in the removal of TC I from plasma, and TC tives either in the lumen of the intestine or within the
I may play a role in the transport of cobalamin ana- mucosa. All dietary folates are converted to 5-methyl-
logues (which it binds more effectively than IF) to the THF (5-MTHF) within the small-intestinal mucosa
liver for excretion in bile. before entering portal plasma. The monoglutamates
The other major cobalamin transport protein in are actively transported across the enterocyte by a
plasma is TC II. The gene is on chromosome 22q11- carrier-mediated mechanism. Pteroylglutamic acid at
q13.1. As for IF and HC, there are 9 exons. The three doses >400 g is absorbed largely unchanged and con-
proteins are likely to have a common ancestral ori- verted to natural folates in the liver. Lower doses are
gin. TC II is synthesized by liver and by other tissues, converted to 5-MTHF during absorption through the
including macrophages, ileum, and vascular endo- intestine.
thelium. It normally carries only 2060 ng of cobala- About 6090 g of folate enters the bile each day
min per liter of plasma and readily gives up cobalamin and is excreted into the small intestine. Loss of this
to marrow, placenta, and other tissues, which it enters folate, together with the folate of sloughed intestinal
by receptor-mediated endocytosis involving the TC II cells, accelerates the speed with which folate deficiency
receptor and megalin (encoded by the LRP-2 gene). develops in malabsorption conditions.
96 Table 9-2
Biochemical Reactions of Folate Coenzymes
Coenzyme Form of Single Carbon
Reaction Folate Involved Unit Transferred Importance

Formate activation THF CHO Generation of 10-formyl-THF


Purine synthesis
Formation of glycinamide 5,10-MethyleneTHF CHO Formation of purines needed for DNA, RNA syn-
ribonucleotide thesis, but reactions probably not rate-limiting
Formylation of amino- 10-Formyl (CHO)THF
imidazole carboxamide
ribonucleotide (AICAR)
Pyrimidine synthesis
Methylation of deoxyuridine 5,10-MethyleneTHF CH3 Rate limiting in DNA synthesis
monophosphate (dUMP) to Oxidizes THF to DHF
thymidine monophosphate
(dTMP)
Some breakdown of folate at the C-9N-10
Section Iii

bond
Amino acid interconversion
Serineglycine interconversion THF =CH2 Entry of single carbon units into active pool
Homocysteine to methionine 5-Methyl(M)THF CH3 Demethylation of 5-MTHF to THF; also requires
cobalamin, flavine adenine dinucleotide, ATP,
and adenosylmethionine
Forminoglutamic acid to THF HNCH=
Anemias

glutamic acid in histidine


catabolism

Abbreviations: ATP, adenosine triphosphate; DHF, dihydrofolate; THF, tetrahydrofolate.

Transport involved in purine synthesis and one in pyrimidine syn-


thesis necessary for DNA and RNA replication. Folate
Folate is transported in plasma; about one-third is is also a coenzyme for methionine synthesis, in which
loosely bound to albumin, and two-thirds is unbound. methylcobalamin is also involved and in which THF is
In all body fluids (plasma, cerebrospinal fluid, milk, bile) regenerated. THF is the acceptor of single carbon units
folate is largely, if not entirely, 5-MTHF in the mono- newly entering the active pool via conversion of ser-
glutamate form. Two types of folate-binding protein ine to glycine. Methionine, the other product of the
are involved in the entry of MTHF into cells. A high- methionine synthase reaction, is the precursor for S-
affinity proton-coupled folate receptor (PCFT/HCPI) adenosylmethionine (SAM), the universal methyl donor
takes folate into cells by endocytosis and is internalized involved in >100 methyltransferase reactions (Fig. 9-1).
by clathrin-coated pits or in a vesicle (caveola), which is During thymidylate synthesis, 5,10-methylene-THF
then acidified, releasing folate. It accounts for the bulk is oxidized to DHF (dihydrofolate). The enzyme DHF
of folate absorption. Folate then is carried by the mem- reductase converts this to THF. The drugs methotrexate,
brane folate transporter into the cytoplasm. The high- pyrimethamine, and (mainly in bacteria) trimethoprim
affinity receptor is attached to the outer surface of the inhibit DHF reductase and so prevent formation of
cell membrane by glycosyl phosphatidylinositol link- active THF coenzymes from DHF. A small fraction of
ages. It may be involved in transport of oxidized folates the folate coenzyme is not recycled during thymidylate
and folate breakdown products to the liver for excretion synthesis but is degraded.
in bile. An independent low-affinity reduced-folate car-
rier also mediates uptake of physiologic folates into cells
but also regulates the uptake of methotrexate.
Biochemical Basis of
Biochemical functions Megaloblastic Anemia
Folates (as the intracellular polyglutamate derivatives) The common feature of all megaloblastic anemias is a
act as coenzymes in the transfer of single-carbon units defect in DNA synthesis that affects rapidly dividing
(Fig. 9-1 and Table 9-2). Two of these reactions are cells in the bone marrow. All conditions that give rise
Methylated product
97
Substrate
(e.g., methylated lipids, myelin Methyltransferases
basic protein, DOPA, DNA)
GSH

Pyruvate S-Adenosylhomocysteine S-Adenosylmethionine


Cysteine (SAH) (SAM)

THE METHYLATION
Cystathionine ATP
CYCLE
Cystathionine
synthase
vitamin B6 Homocysteine Methionine
Polyglutamate
synthase
+ glutamates
Methionine synthase
methylcobalamin
Tetrahydrofolate

CHAPTER 9
Cell 5-Methyl
tetrahydrofolate Serine DHF
Glycine reductase
Purines
5,10-Methylene-
tetrahydrofolate Formate
reductase Dihydrofolate

Megaloblastic Anemias
5, 10-Methylene 10-Formyl
tetrahydrofolate tetrahydrofolate
DNA CYCLE
(CELL REPLICATION)

5-Methyl
tetrahydrofolate
(monoglutamate) Deoxyuridine Deoxythymidine
monophosphate monophosphate

Folic acid Folic acid


Plasma

Figure 9-1
The role of folates in DNA synthesis and in formation of (Reprinted from Hoffbrand AV, et al [eds]: Postgraduate
S-adenosylmethionine (SAM), which is involved in numer- Haematology, 5th ed, Oxford, UK, Blackwell Publishing,
ous methylation reactions. ATP, adenosine triphosphate; 2005; with permission.)

to megaloblastic changes have in common a disparity in is misincorporation of uracil into DNA because of a
the rate of synthesis or availability of the four immedi- buildup of deoxyuridine triphosphate (dUTP) at the
ate precursors of DNA: the deoxyribonucleoside triphos- DNA replication fork as a consequence of the block in
phates (dNTPs)dA(adenine)TP and dG(guanine)TP conversion of dUMP to dTMP.
(purines), dT(thymine)TP and dC(cytosine)TP (pyrimi-
dines). In deficiencies of either folate or cobalamin,
Cobalaminfolate relations
there is failure to convert deoxyuridine monophosphate
(dUMP) to deoxythymidine monophosphate (dTMP), Folate is required for many reactions in mammalian
the precursor of dTTP (Fig. 9-1). This is the case because tissues. Only two reactions in the body are known to
folate is needed as the coenzyme 5,10-methylene-THF require cobalamin. Methylmalonyl CoA isomerization
polyglutamate for conversion of dUMP to dTMP; the requires adocobalamin, and the methylation of homo-
availability of 5,10-methylene-THF is reduced in either cysteine to methionine requires both methylcobalamin
cobalamin or folate deficiency. An alternative theory for and 5-MTHF (Fig. 9-1). This reaction is the first step
megaloblastic anemia in cobalamin or folate deficiency in the pathway by which 5-MTHF, which enters bone
98 marrow and other cells from plasma, is converted into Neural tube defects
all the intracellular folate coenzymes. The coenzymes Folic acid supplements at the time of conception and
are all polyglutamated (the larger size aiding retention in in the first 12 weeks of pregnancy reduce by 70% the
the cell), but the enzyme folate polyglutamate synthase incidence of NTDs (anencephaly, meningomyelocele,
can use only THF, not MTHF, as substrate. In cobal- encephalocele, and spina bifida) in fetuses. Most of this
amin deficiency, MTHF accumulates in plasma, and protective effect can be achieved by taking folic acid,
intracellular folate concentrations fall due to failure of 0.4 mg daily at the time of conception.
formation of THF, the substrate on which folate poly- The incidence of cleft palate and harelip also can be
glutamates are built. This has been termed THF starva- reduced by prophylactic folic acid. There is no clear
tion, or the methylfolate trap. simple relationship between maternal folate status and
This theory explains the abnormalities of folate these fetal abnormalities, although overall the lower the
metabolism that occur in cobalamin deficiency (high maternal folate, the greater the risk to the fetus. NTDs
serum folate, low cell folate, positive purine precursor also can be caused by antifolate and antiepileptic drugs.
aminoimidazole carboxamide ribonucleotide [AICAR] An underlying maternal folate metabolic abnor-
excretion; Table 9-2) and also why the anemia of cobal- mality has also been postulated. One abnormality has
amin deficiency responds to folic acid in large doses. been identified: reduced activity of the enzyme 5,10-
methylene-THF reductase (MTHFR) (Fig. 9-1) caused
Section Iii

by a common C677T polymorphism in the MTHFR


gene. In one study, the prevalence of this polymor-
Clinical Features phism was found to be higher in the parents of NTD
fetuses and in the fetuses themselves: homozygos-
Many symptomless patients are detected through the ity for the TT mutation was found in 13% compared
finding of a raised mean corpuscular volume (MCV) with 5% in control subjects. The polymorphism codes
on a routine blood count. The main clinical features in
Anemias

for a thermolabile form of MTHFR. The homozygous


more severe cases are those of anemia. Anorexia is usu- state results in a lower mean serum and red cell folate
ally marked, and there may be weight loss, diarrhea, or level compared with control subjects, as well as signifi-
constipation. Glossitis, angular cheilosis, a mild fever in cantly higher serum homocysteine levels. Test results
more severely anemic patients, jaundice (unconjugated), for mutations in other enzymes possibly associated with
and reversible melanin skin hyperpigmentation also may NTDs, e.g., methionine synthase and serineglycine
occur with a deficiency of either folate or cobalamin. hydroxymethylase, have been negative. Autoantibod-
Thrombocytopenia sometimes leads to bruising, and ies to folate receptors were suggested to be more com-
this may be aggravated by vitamin C deficiency or alco- mon in mothers of babies with NTDs, but this has been
hol in malnourished patients. The anemia and low leu- disproved.
kocyte count may predispose to infections, particularly
of the respiratory and urinary tracts. Cobalamin defi- Cardiovascular disease
ciency has also been associated with impaired bacteri- Children with severe homocystinuria (blood levels
cidal function of phagocytes. 100 mol/L) due to deficiency of one of three
enzymes, methionine synthase, MTHFR, or cysta-
thionine synthase (Fig. 9-1), have vascular disease, e.g.,
General tissue effects of cobalamin and folate ischemic heart disease, cerebrovascular disease, or pul-
deficiencies monary embolus, as teenagers or in young adulthood.
Epithelial surfaces Lesser degrees of raised serum homocysteine and low
After the marrow, the next most frequently affected tis- levels of serum folate and homozygous inherited muta-
sues are the epithelial cell surfaces of the mouth, stom- tions of MTHFR have been found to be associated
ach, and small intestine and the respiratory, urinary, and with cerebrovascular, peripheral vascular, and coronary
female genital tracts. The cells show macrocytosis, with heart disease and with deep vein thrombosis. Prospec-
increased numbers of multinucleate and dying cells. The tive randomized trials of lowering homocysteine levels
deficiencies may cause cervical smear abnormalities. with supplements of folic acid, vitamin B12, and vitamin
B6 against placebo over a 5-year period in patients with
Complications of pregnancy vascular disease or diabetes have not, however, shown a
The gonads are also affected, and infertility is common reduction of major cardiovascular events, nor have these
in both men and women with either deficiency. Mater- supplements reduced the risk of recurrent cardiovascu-
nal folate deficiency has been implicated as a cause of lar disease after an acute myocardial infarct. It is pos-
prematurity, and both folate deficiency and cobalamin sible that these trials were not sufficiently powered to
deficiency have been implicated in recurrent fetal loss detect a small (e.g., 10%) benefit or that another under-
and neural tube defects (NTDs), as discussed later. lying factor is responsible for both the vascular damage
and the raised homocysteine. Alternatively, the ben- examination of the blood film, tests for serum gastrin 99
eficial effects of lowering homocysteine were offset in level and for antibodies to IF or parietal cells, along
these trials by the vitamins stimulating endothelial cell with serum methylmalonic acid (MMA) measure-
proliferation. Meta-analysis has suggested that folic acid ment if available. A trial of cobalamin therapy for at
supplementation reduces the risk of stroke by 18%. The least 3 months will usually also be needed to determine
results of longer and larger trials are needed to resolve whether the symptoms improve.
these uncertainties. The biochemical basis for cobalamin neuropa-
thy remains obscure. Its occurrence in the absence of
Malignancy methylmalonic aciduria in TC II deficiency suggests
Prophylactic folic acid in pregnancy has been found in that the neuropathy is related to the defect in homo-
some but not all studies to reduce the subsequent inci- cysteinemethionine conversion. Accumulation of S-
dence of acute lymphoblastic leukemia (ALL) in child- adenosylhomocysteine in the brain, resulting in inhibi-
hood. A significant negative association has also been tion of transmethylation reactions, has been suggested.
found with the MTHFR C677T polymorphism and Psychiatric disturbance is common in both folate
leukemias with mixed lineage leukemia (MLL) trans- and cobalamin deficiencies. This, like the neuropathy,
locations but a positive association with hyperdip- has been attributed to a failure of the synthesis of SAM,
loidy in infants with ALL or acute myeloid leukemia which is needed in methylation of biogenic amines

CHAPTER 9
or with childhood ALL. A second polymorphism in (e.g., dopamine) as well as that of proteins, phospholip-
the MTHFR gene, A1298C, is also strongly associated ids, and neurotransmitters in the brain (Fig. 9-1). Asso-
with hyperdiploid leukemia. There are various posi- ciations between lower serum folate or cobalamin levels
tive and negative associations between polymorphisms and higher homocysteine levels and the development of
in folate-dependent enzymes and the incidence of adult decreased cognitive function and dementia in Alzheim-
ALL. The C677T polymorphism is thought to lead to ers disease have been reported. A 2-year double-blind
increased thymidine pools and better quality of DNA

Megaloblastic Anemias
placebo-controlled randomized clinical trial involving
synthesis by shunting 1-carbon groups toward thymi- healthy subjects >65 years old given folate, cobalamin,
dine and purine synthesis. This may explain its reported and vitamin B6 supplements showed no benefit for cog-
association with a lower risk for colorectal cancer. Most nitive performance, whereas a 3-year (FACIT) study
but not all studies suggest that prophylactic folic acid did show benefit.
also protects against colon adenomas. Other tumors that
have been associated with folate polymorphisms or sta-
tus include follicular lymphoma, breast cancer, and gas-
tric cancer. Because folic acid may feed tumors, it Hematologic Findings
probably should be avoided in those with established
tumors unless there is severe megaloblastic anemia due Peripheral blood
to folate deficiency. Oval macrocytes, usually with considerable anisocyto-
sis and poikilocytosis, are the main feature (Fig. 9-2A).
Neurologic manifestations The MCV is usually >100 fL unless a cause of micro-
Cobalamin deficiency may cause a bilateral peripheral cytosis (e.g., iron deficiency or thalassemia trait) is pres-
neuropathy or degeneration (demyelination) of the pos- ent. Some of the neutrophils are hypersegmented (more
terior and pyramidal tracts of the spinal cord and, less than five nuclear lobes). There may be leukopenia due
frequently, optic atrophy or cerebral symptoms. to a reduction in granulocytes and lymphocytes, but
The patient, more frequently male, presents with this is usually >1.5 109/L; the platelet count may be
paresthesias, muscle weakness, or difficulty in walk- moderately reduced, rarely to <40 109/L. The sever-
ing and sometimes dementia, psychotic disturbances, ity of all these changes parallels the degree of anemia. In
or visual impairment. Long-term nutritional cobala- a nonanemic patient, the presence of a few macrocytes
min deficiency in infancy leads to poor brain develop- and hypersegmented neutrophils in the peripheral blood
ment and impaired intellectual development. Folate may be the only indication of the underlying disorder.
deficiency has been suggested to cause organic nervous
disease, but this is uncertain, although methotrexate
injected into the cerebrospinal fluid may cause brain or Bone marrow
spinal cord damage. In a severely anemic patient, the marrow is hypercellular
An important clinical problem is the nonanemic with an accumulation of primitive cells due to selective
patient with neurologic or psychiatric abnormali- death by apoptosis of more mature forms. The eryth-
ties and a low or borderline serum cobalamin level. In roblast nucleus maintains a primitive appearance despite
such patients, it is necessary to try to establish whether maturation and hemoglobinization of the cytoplasm.
there is significant cobalamin deficiency, e.g., by careful The cells are larger than normoblasts, and an increased
100
Section Iii

Figure 9-2
A. The peripheral blood in severe megaloblastic anemia. aematology, 5th ed, Oxford, UK, Blackwell Publishing, 2005;
H
B. The bone marrow in severe megaloblastic anemia. with permission.)
(Reprinted from AV Hoffbrand et al [eds], Postgraduate
Anemias

number of cells with eccentric lobulated nuclei or serum lactate dehydrogenase. A weakly positive direct
nuclear fragments may be present (Fig. 9-2B). Giant and antiglobulin test due to complement can lead to a false
abnormally shaped metamyelocytes and enlarged hyper- diagnosis of autoimmune hemolytic anemia.
polyploid megakaryocytes are characteristic. In less ane-
mic patients, the changes in the marrow may be difficult
to recognize. The terms intermediate, mild, and early have Causes of Cobalamin Deficiency
been used. The term megaloblastoid does not mean mildly
megaloblastic. Rather, it is used to describe cells with Cobalamin deficiency is usually due to malabsorption.
both immature-appearing nuclei and defective hemoglo- The only other cause is inadequate dietary intake.
binization and is usually seen in myelodysplasia.
Inadequate dietary intake
Chromosomes Adults
Dietary cobalamin deficiency arises in vegans who omit
Bone marrow cells, transformed lymphocytes, and other meat, fish, eggs, cheese, and other animal products from
proliferating cells in the body show a variety of changes, their diet. The largest group in the world consists of
including random breaks, reduced contraction, spread- Hindus, and it is likely that many millions of Indians are
ing of the centromere, and exaggeration of secondary at risk of deficiency of cobalamin on a nutritional basis.
chromosomal constrictions and overprominent satellites. Subnormal serum cobalamin levels are found in up to
Similar abnormalities may be produced by antimetabo- 50% of randomly selected, young, adult Indian vegans,
lite drugs (e.g., cytosine arabinoside, hydroxyurea, and but the deficiency usually does not progress to megalo-
methotrexate) that interfere with either DNA replica- blastic anemia since the diet of most vegans is not totally
tion or folate metabolism and that also cause megalo- lacking in cobalamin, and the enterohepatic circulation
blastic appearances. of cobalamin is intact. Dietary cobalamin deficiency
may also arise rarely in nonvegetarian individuals who
Ineffective hematopoiesis exist on grossly inadequate diets because of poverty or
psychiatric disturbance.
There is an accumulation of unconjugated bilirubin in
plasma due to the death of nucleated red cells in the Infants
marrow (ineffective erythropoiesis). Other evidence for Cobalamin deficiency has been described in infants born
this includes raised urine urobilinogen, reduced hap- to severely cobalamin-deficient mothers. These infants
toglobins and positive urine hemosiderin, and a raised develop megaloblastic anemia at about 36 months of
age, presumably because they are born with low stores Table 9-4 101
of cobalamin and because they are fed breast milk with Malabsorption of Cobalamin May Occur in
low cobalamin content. The babies have also shown the Following Conditions But Is Not Usually
growth retardation, impaired psychomotor develop- Sufficiently Severe and Prolonged to Cause
ment, and other neurologic sequelae. Megaloblastic Anemia
Gastric causes
Simple atrophic gastritis (food cobalamin malabsorption)
Gastric causes of cobalamin malabsorption ZollingerEllison syndrome
See Tables 9-3 and 9-4. Gastric bypass surgery
Use of proton pump inhibitors
Pernicious anemia Intestinal causes
Pernicious anemia (PA) may be defined as a severe Gluten-induced enteropathy
lack of IF due to gastric atrophy. It is a common dis- Severe pancreatitis
HIV infection
ease in north Europeans but occurs in all countries and
Radiotherapy
ethnic groups. The overall incidence is about 120 per Graft-vs.-host disease
100,000 population in the United Kingdom (UK). The Deficiencies of cobalamin, folate, protein, ?riboflavin,
ratio of incidence in men and women among whites is ?nicotinic acid
1:1.6, and the peak age of onset is 60 years, with only Therapy with colchicine, para-aminosalicylate, neomycin,

CHAPTER 9
10% of patients being <40 years of age. However, in slow-release potassium chloride, anticonvulsant drugs,
some ethnic groups, notably black individuals and Latin metformin, phenformin, cytotoxic drugs
Alcohol
Americans, the age at onset of PA is generally lower.
The disease occurs more commonly than by chance
in close relatives and in persons with other organ-
specific autoimmune diseases, e.g., thyroid diseases,

Megaloblastic Anemias
vitiligo, hypoparathyroidism, and Addisons disease. It subnormal life expectancy as a result of a higher inci-
is also associated with hypogammaglobulinemia, with dence of carcinoma of the stomach than in control sub-
premature graying or blue eyes, and persons of blood jects. Gastric output of hydrochloric acid, pepsin, and
group A. An association with human leukocyte antigen IF is severely reduced. The serum gastrin level is raised,
(HLA) 3 has been reported in some but not all series and serum pepsinogen I levels are low.
and, in those with endocrine disease, with HLA-B8,
-B12, and -BW15. Life expectancy is normal in women Gastric biopsy
once regular treatment has begun. Men have a slightly This usually shows atrophy of all layers of the body and
fundus, with loss of glandular elements, an absence of
parietal and chief cells and replacement by mucous cells,
a mixed inflammatory cell infiltrate, and perhaps intes-
tinal metaplasia. The infiltrate of plasma cells and lym-
Table 9-3 phocytes contains an excess of CD4 cells. The antral
Causes of Cobalamin Deficiency Sufficiently
mucosa is usually well preserved. Helicobacter pylori infec-
Severe to Cause Megaloblastic Anemia tion occurs infrequently in PA, but it has been sug-
gested that H. pylori gastritis occurs at an early phase
Nutritional Vegans
of atrophic gastritis and presents in younger patients as
Malabsorption Pernicious anemia iron-deficiency anemia but in older patients as PA. H.
Gastric causes Congenital absence of intrinsic pylori is suggested to stimulate an autoimmune process
factor or functional abnormality directed against parietal cells, with the H. pylori infec-
Total or partial gastrectomy tion then being gradually replaced, in some individuals,
Intestinal causes Intestinal stagnant loop by an autoimmune process.
syndrome: jejunal diverticulo-
sis, ileocolic fistula, anatomic Serum antibodies
blind loop, intestinal stricture, Two types of IF immunoglobulin G antibody may be
etc. found in the sera of patients with PA. One, the block-
Ileal resection and Crohns ing, or type I, antibody, prevents the combination of
disease
IF and cobalamin, whereas the binding, or type II,
Selective malabsorption with
proteinuria antibody prevents attachment of IF to ileal mucosa.
Tropical sprue Type I occurs in the sera of 55% of patients and type
Transcobalamin II deficiency II in 35%. IF antibodies cross the placenta and may cause
Fish tapeworm temporary IF deficiency in a newborn infant. Patients
with PA also show cell-mediated immunity to IF. Type I
102 antibody has been detected rarely in the sera of patients with crystalline cobalamin, but show malabsorption
without PA but with thyrotoxicosis, myxedema, Hashi- when a modified test using food-bound cobalamin is
motos disease, or diabetes mellitus and in relatives of PA used. The frequency of progression to severe cobalamin
patients. IF antibodies also have been detected in gastric deficiency and the reasons for this progression are not
juice in 80% of PA patients. These gastric antibodies clear.
may reduce absorption of dietary cobalamin by combin-
ing with small amounts of remaining IF. Intestinal causes of cobalamin malabsorption
Parietal cell antibody is present in the sera of almost
90% of adult patients with PA but is frequently pres- Intestinal stagnant loop syndrome
ent in other subjects. Thus, it occurs in as many as 16% Malabsorption of cobalamin occurs in a variety of intes-
of randomly selected female subjects age >60 years. tinal lesions in which there is colonization of the upper
The parietal cell antibody is directed against the and small intestine by fecal organisms. This may occur in
subunits of the gastric proton pump (H+,K+-ATPase). patients with jejunal diverticulosis, enteroanastomosis,
or an intestinal stricture or fistula or with an anatomic
blind loop due to Crohns disease, tuberculosis, or an
Juvenile pernicious anemia operative procedure.
This usually occurs in older children and resembles PA
Ileal resection
Section Iii

of adults. Gastric atrophy, achlorhydria, and serum IF


Removal of 1.2 m of terminal ileum causes malabsorp-
antibodies are all present, although parietal cell antibod-
tion of cobalamin. In some patients after ileal resection,
ies are usually absent. About one-half of these patients
particularly if the ileocecal valve is incompetent, colonic
show an associated endocrinopathy such as autoimmune
bacteria may contribute further to the onset of cobala-
thyroiditis, Addisons disease, or hypoparathyroidism; in
min deficiency.
some, mucocutaneous candidiasis occurs.
Anemias

 elective malabsorption of cobalamin


S
withproteinuria (Imerslund syndrome:
Congenital intrinsic factor deficiency or
Imerslund-Grsbeck syndrome; congenital
functional abnormality
cobalamin malabsorption; autosomal recessive
An affected child usually presents with megaloblastic megaloblastic anemia, MGA1)
anemia in the first to third year of life; a few have pre- This autosomally recessive disease is the most com-
sented as late as the second decade. The child usually mon cause of megaloblastic anemia due to cobalamin
has no demonstrable IF but has a normal gastric mucosa deficiency in infancy in Western countries. More than
and normal secretion of acid. The inheritance is autoso- 200 cases have been reported, with familial clusters in
mal recessive. Parietal cell and IF antibodies are absent. Finland, Norway, the Middle East, and North Africa.
Variants have been described in which the child is born The patients secrete normal amounts of IF and gastric
with IF that can be detected immunologically but is acid but are unable to absorb cobalamin. In Finland,
unstable or functionally inactive, unable to bind cobal- impaired synthesis, processing, or ligand binding of
min or to facilitate its uptake by ileal receptors. cubilin due to inherited mutations is found. In Nor-
way, mutation of the gene for AMN has been reported.
Other tests of intestinal absorption are normal. Over
Gastrectomy 90% of these patients show nonspecific proteinuria, but
After total gastrectomy, cobalamin deficiency is inevi- renal function is otherwise normal, and renal biopsy
table, and prophylactic cobalamin therapy should be has not shown any consistent renal defect. A few have
commenced immediately after the operation. After par- shown aminoaciduria and congenital renal abnormali-
tial gastrectomy, 1015% of patients also develop this ties, such as duplication of the renal pelvis.
deficiency. The exact incidence and time of onset are
Tropical sprue
most influenced by the size of the resection and the
Nearly all patients with acute and subacute tropical
preexisting size of cobalamin body stores.
sprue show malabsorption of cobalamin; this may persist
as the principal abnormality in the chronic form of the
Food cobalamin malabsorption disease, when the patient may present with megaloblas-
tic anemia or neuropathy due to cobalamin deficiency.
Failure of release of cobalamin from binding proteins
Absorption of cobalamin usually improves after antibi-
in food is believed to be responsible for this condition,
otic therapy and, in the early stages, folic acid therapy.
which is more common in the elderly. It is associated
with low serum cobalamin levels, with or without raised Fish tapeworm infestation
serum levels of MMA and homocysteine. Typically, these The fish tapeworm (Diphyllobothrium latum) lives in the
patients have normal cobalamin absorption, as measured small intestine of humans and accumulates cobalamin
from food, rendering the cobalamin unavailable for cobalamin and folate levels are normal, but the anemia 103
absorption. Individuals acquire the worm by eating raw responds to massive (e.g., 1 mg three times weekly)
or partly cooked fish. Infestation is common around the injections of cobalamin. Some cases show neurologic
lakes of Scandinavia, Germany, Japan, North America, complications. The protein may be present but func-
and Russia. Megaloblastic anemia or cobalamin neurop- tionally inert. Genetic abnormalities found include
athy occurs only in those with a heavy infestation. mutations of an intra-exonic cryptic splice site, exten-
sive deletion, single nucleotide deletion, nonsense
Gluten-induced enteropathy mutation, and an RNA editing defect. Malabsorption of
Malabsorption of cobalamin occurs in 30% of untreated cobalamin occurs in all cases, and serum immunoglob-
patients (presumably those in whom the disease extends ulins are usually reduced. Failure to institute adequate
to the ileum). Cobalamin deficiency is not severe in cobalamin therapy or treatment with folic acid may lead
these patients and is corrected with a gluten-free diet. to neurologic damage.
Severe chronic pancreatitis  ongenital methylmalonic acidemia and
C
In this condition, lack of trypsin is thought to cause aciduria
dietary cobalamin attached to gastric non-IF (R) binder Infants with this abnormality are ill from birth with
to be unavailable for absorption. It also has been pro- vomiting, failure to thrive, severe metabolic acido-
posed that in pancreatitis, the concentration of calcium sis, ketosis, and mental retardation. Anemia, if present,

CHAPTER 9
ions in the ileum falls below the level needed to main- is normocytic and normoblastic. The condition may
tain normal cobalamin absorption. be due to a functional defect in either mitochondrial
methylmalonyl CoA mutase or its cofactor adocobala-
HIV infection
min. Mutations in the methylmalonyl CoA mutase are
Serum cobalamin levels tend to fall in patients with
not responsive, or only poorly responsive, to treatment
HIV infection and are subnormal in 1035% of those
with cobalamin. A proportion of infants with failure of
with AIDS. Malabsorption of cobalamin not corrected

Megaloblastic Anemias
adocobalamin synthesis respond to cobalamin in large
by IF has been shown in some, but not all, patients with
doses. Some children have combined methylmalonic
subnormal serum cobalamin levels. Cobalamin defi-
aciduria and homocystinuria due to defective formation
ciency sufficiently severe to cause megaloblastic anemia
of both cobalamin coenzymes. This usually presents in
or neuropathy is rare.
the first year of life with feeding difficulties, develop-
ZollingerEllison syndrome mental delay, microcephaly, seizures, hypotonia, and
Malabsorption of cobalamin has been reported in the megaloblastic anemia.
ZollingerEllison syndrome. It is thought that there is a
 cquired abnormality of cobalamin metabolism:
A
failure to release cobalamin from R-binding protein due nitrous oxide inhalation
to inactivation of pancreatic trypsin by high acidity, as Nitrous oxide irreversibly oxidizes methylcobalamin
well as interference with IF binding of cobalamin. to an inactive precursor; this inactivates methionine
Radiotherapy synthase. Megaloblastic anemia has occurred in patients
Both total-body irradiation and local radiotherapy to undergoing prolonged N2O anesthesia (e.g., in intensive
the ileum (e.g., as a complication of radiotherapy for care units). A neuropathy resembling cobalamin neu-
carcinoma of the cervix) may cause malabsorption of ropathy has been described in dentists and anesthetists
cobalamin. who are exposed repeatedly to N2O. Methylmalonic
aciduria does not occur as adocobalamin is not inacti-
Graft-versus-host disease vated by N2O.
This commonly affects the small intestine. Malabsorp-
tion of cobalamin due to abnormal gut flora, as well as
damage to ileal mucosa, is common.
Causes of Folate Deficiency
Drugs
The drugs that have been reported to cause malabsorp- (Table 9-5)
tion of cobalamin are listed in Table 9-4. Megaloblastic
anemia due to these drugs is, however, rare. Nutritional
Dietary folate deficiency is common. Indeed, in most
Abnormalities of cobalamin metabolism patients with folate deficiency, a nutritional element
 ongenital transcobalamin II deficiency or
C is present. Certain individuals are particularly prone
abnormality to have diets containing inadequate amounts of folate
Infants with TC II deficiency usually present with meg- (Table 9-5). In the United States and other countries
aloblastic anemia within a few weeks of birth. Serum where fortification of the diet with folic acid has been
104 Table 9-5 transport into the cerebrospinal fluid, and these patients
Causes of Folate Deficiency show megaloblastic anemia, which responds to physi-
Dietarya ologic doses of folic acid given parenterally but not
Particularly in: old age, infancy, poverty, alcoholism, orally. They also show mental retardation, convulsions,
chronic disabilities, and the people with mental and other central nervous system abnormalities. Minor
illnesses; may be associated with scurvy or kwashiorkor degrees of malabsorption may also occur after jejunal
Malabsorption resection or partial gastrectomy, in Crohns disease,
Major causes of deficiency and in systemic infections, but in these conditions, if
Tropical sprue; gluten-induced enteropathy in children severe deficiency occurs, it is usually largely due to poor
and adults; and in association with dermatitis her- nutrition. Malabsorption of folate has been described
petiformis, specific malabsorption of folate, intestinal in patients receiving salazopyrine, cholestyramine, and
megaloblastosis caused by severe cobalamin or
folate deficiency
triamterene.
Minor causes of deficiency
Extensive jejunal resection, Crohns disease, partial Excess utilization or loss
gastrectomy, congestive heart failure, Whipples dis-
ease, scleroderma, amyloid, diabetic enteropathy, Pregnancy
systemic bacterial infection, lymphoma, salazopyrine Folate requirements are increased by 200300 g to
~400 g daily in a normal pregnancy, partly because of
Section Iii

Excess utilization or loss


Physiologic transfer of the vitamin to the fetus but mainly because
Pregnancy and lactation, prematurity of increased folate catabolism due to cleavage of folate
Pathologic coenzymes in rapidly proliferating tissues. Megaloblastic
Hematologic diseases: chronic hemolytic anemias, anemia due to this deficiency is prevented by prophy-
sickle cell anemia, thalassemia major, myelofibrosis lactic folic acid therapy. It occurred in 0.5% of preg-
Malignant diseases: carcinoma, lymphoma, leukemia,
nancies in the UK and other Western countries before
Anemias

myeloma
Inflammatory diseases: tuberculosis, Crohns disease, prophylaxis with folic acid, but the incidence is much
psoriasis, exfoliative dermatitis, malaria higher in countries where the general nutritional status
Metabolic disease: homocystinuria is poor.
Excess urinary loss: congestive heart failure, active
liver disease Prematurity
Hemodialysis, peritoneal dialysis A newborn infant, whether full term or premature, has
Antifolate drugsb higher serum and red cell folate concentrations than
Anticonvulsant drugs (phenytoin, primidone, barbitu- does an adult. However, a newborn infants demand for
rates), sulphasalazine folate has been estimated to be up to 10 times that of
Nitrofurantoin, tetracycline, antituberculosis (less well adults on a weight basis, and the neonatal folate level
documented) falls rapidly to the lowest values at about 6 weeks of
Mixed causes age. The falls are steepest and are liable to reach sub-
Liver diseases, alcoholism, intensive care units normal levels in premature babies, a number of whom
develop megaloblastic anemia responsive to folic acid at
a
In severely folate-deficient patients with causes other than those about 46 weeks of age. This occurs particularly in the
listed under Dietary, poor dietary intake is often present.
b
Drugs inhibiting dihydrofolate reductase are discussed in the text.
smallest babies (<1500 g birth weight) and those who
have feeding difficulties or infections or have undergone
multiple exchange transfusions. In these babies, prophy-
lactic folic acid should be given.
adopted, the prevalence of folate deficiency has dropped
dramatically and is now almost restricted to high-risk Hematologic disorders
groups with increased folate needs. Nutritional folate Folate deficiency frequently occurs in chronic hemo-
deficiency occurs in kwashiorkor and scurvy and in lytic anemia, particularly in sickle cell disease, autoim-
infants with repeated infections or those who are fed mune hemolytic anemia, and congenital spherocytosis.
solely on goats milk, which has a low folate content. In these and other conditions of increased cell turnover
(e.g., myelofibrosis, malignancies), folate deficiency
Malabsorption arises because it is not completely reutilized after per-
forming coenzyme functions.
Malabsorption of dietary folate occurs in tropical sprue
and in gluten-induced enteropathy. In the rare con- Inflammatory conditions
genital syndrome of selective malabsorption of folate Chronic inflammatory diseases such as tuberculosis,
due to mutation of the protein-coupled folate trans- rheumatoid arthritis, Crohns disease, psoriasis, exfo-
porter (PCFT), there is an associated defect of folate liative dermatitis, bacterial endocarditis, and chronic
bacterial infections cause deficiency by reducing the 105
Diagnosis of Cobalamin and
appetite and increasing the demand for folate. Systemic
Folate Deficiencies
infections also may cause malabsorption of folate. Severe
deficiency is virtually confined to the patients with the The diagnosis of cobalamin or folate deficiency has tra-
most active disease and the poorest diet. ditionally depended on the recognition of the relevant
Homocystinuria abnormalities in the peripheral blood and analysis of the
This is a rare metabolic defect in the conversion of blood levels of the vitamins.
homocysteine to cystathionine. Folate deficiency occur-
ring in most of these patients may be due to excessive Serum cobalamin
utilization because of compensatory increased conver-
This is measured by an automated enzyme-linked
sion of homocysteine to methionine.
immunosorbent assay (ELISA). Normal serum levels
Long-term dialysis range from 118148 pmol/L (160200 ng/L) to 738
As folate is only loosely bound to plasma proteins, it is pmol/L (1000 ng/L). In patients with megaloblastic
easily removed from plasma by dialysis. In patients with anemia due to cobalamin deficiency, the level is usu-
anorexia, vomiting, infections, and hemolysis, folate ally <74 pmol/L (100 ng/L). In general, the more
stores are particularly likely to become depleted. Rou- severe the deficiency, the lower the serum cobalamin

CHAPTER 9
tine folate prophylaxis is now given. level. In patients with spinal cord damage due to the
deficiency, levels are very low even in the absence of
Congestive heart failure, liver disease anemia. Values between 74 and 148 pmol/L (100 and
Excess urinary folate losses of >100 g per day may 200 ng/L) are regarded as borderline. They may occur,
occur in some of these patients. The explanation for instance, in pregnancy and in patients with mega-
appears to be release of folate from damaged liver cells. loblastic anemia due to folate deficiency. They may

Megaloblastic Anemias
also be due to heterozygous, homozygous, or com-
Antifolate drugs pound heterozygous mutations of the gene TCN1 that
A large number of people with epilepsy who are receiv- codes for HC (transcobalamin I). There is no clinical or
ing long-term therapy with phenytoin or primidone, hematologic abnormality. The serum cobalamin level is
with or without barbiturates, develop low serum and sufficiently robust, cost-effective, and most convenient
red cell folate levels. The exact mechanism is unclear. to rule out cobalamin deficiency in the vast majority of
Alcohol may also be a folate antagonist, as patients who patients suspected of having this problem.
are drinking spirits may develop megaloblastic anemia
that will respond to normal quantities of dietary folate Serum methylmalonate and homocysteine
or to physiologic doses of folic acid only if alcohol is
withdrawn. Macrocytosis of red cells is associated with In patients with cobalamin deficiency sufficient to cause
chronic alcohol intake even when folate levels are nor- anemia or neuropathy, the serum MMA level is raised.
mal. Inadequate folate intake is the major factor in the Sensitive methods for measuring MMA and homocyste-
development of deficiency in spirit-drinking alcoholics. ine in serum have been introduced and recommended
Beer is relatively folate-rich in some countries, depend- for the early diagnosis of cobalamin deficiency, even in
ing on the technique used for brewing. the absence of hematologic abnormalities or subnormal
The drugs that inhibit DHF reductase include meth- levels of serum cobalamin. Serum MMA levels fluctu-
otrexate, pyrimethamine, and trimethoprim. Metho- ate, however, in patients with renal failure. Mildly ele-
trexate has the most powerful action against the human vated serum MMA and/or homocysteine levels occur
enzyme, whereas trimethoprim is most active against in up to 30% of apparently healthy volunteers, with
the bacterial enzyme and is likely to cause megaloblas- serum cobalamin levels up to 258 pmol/L (350 ng/L)
tic anemia only when used in conjunction with sul- and normal serum folate levels; 15% of elderly subjects,
phamethoxazole in patients with preexisting folate or even with cobalamin levels >258 pmol/L (>350 ng/L),
cobalamin deficiency. The activity of pyrimethamine is have this pattern of raised metabolite levels. These find-
intermediate. The antidote to these drugs is folinic acid ings bring into question the exact cutoff points for nor-
(5-formyl-THF). mal MMA and homocysteine levels. It is also unclear
at present whether these mildly raised metabolite levels
have clinical consequences.
Congenital abnormalities of
folate metabolism Serum homocysteine is raised in both early cobala-
min and folate deficiency but may be raised in other
Some infants with congenital defects of folate enzymes conditions, e.g., chronic renal disease, alcoholism,
(e.g., cyclohydrolase or methionine synthase) have had smoking, pyridoxine deficiency, hypothyroidism, and
megaloblastic anemia. therapy with steroids, cyclosporine, and other drugs.
106 Levels are also higher in serum than in plasma, in men
c obalamin and folate assays and a bone marrow biopsy
than in premenopausal women, in women taking hor-
has been performed (if deemed necessary). Transfusion
mone replacement therapy or in oral contraceptive
is usually unnecessary and inadvisable. If it is essential,
users, and in elderly persons and patients with several
packed red cells should be given slowly, 1 or 2 units
inborn errors of metabolism affecting enzymes in trans-
only, with the usual treatment for heart failure if pres-
sulfuration pathways of homocysteine metabolism.
ent. Potassium supplements have been recommended
Thus, homocysteine levels are not used for diagnosis of
to obviate the danger of the hypokalemia but are not
cobalamin or folate deficiency.
necessary. Occasionally, an excessive rise in platelets
occurs after 12 weeks of therapy. Antiplatelet therapy,
Other tests e.g., aspirin, should be considered if the platelet count
Studies of cobalamin absorption once were widely used, rises to >800 109/L.
but difficulty in obtaining radioactive cobalamin and Cobalamin Deficiency It is usually neces-
ensuring that IF preparations are free of viruses have sary to treat patients who have developed cobalamin
made these tests obsolete. Tests to diagnose PA include deficiency with lifelong regular cobalamin injections.
serum gastrin, which is raised, and serum pepsinogen I, In the UK, the form used is hydroxocobalamin; in the
which is low in PA (9092%) but also in other condi- United States, cyanocobalamin. In a few instances, the
tions. Tests for IF and parietal cell antibodies are also
Section Iii

underlying cause of cobalamin deficiency can be per-


used as well as tests for individual intestinal diseases. manently corrected, e.g., fish tapeworm, tropical sprue,
or an intestinal stagnant loop that is amenable to sur-
Serum folate gery. The indications for starting cobalamin therapy
This is also measured by an ELISA technique. In most are a well-documented megaloblastic anemia or other
laboratories, the normal range is from 11 nmol/L hematologic abnormalities and neuropathy due to the
Anemias

(2 g/L) to 82 nmol/L (15 g/L). The serum folate deficiency. Patients with borderline serum cobalamin
level is low in all folate-deficient patients. It also reflects levels but no hematologic or other abnormality may be
recent diet. Because of this, serum folate may be low followed to make sure that the cobalamin deficiency
before there is hematologic or biochemical evidence of does not progress (discussed later). If malabsorption
deficiency. Serum folate rises in severe cobalamin defi- of cobalamin or rises in serum MMA levels have been
ciency because of the block in conversion of MTHF to demonstrated, however, these patients also should be
THF inside cells; raised levels have also been reported given regular maintenance cobalamin therapy. Cobala-
in the intestinal stagnant loop syndrome due to absorp- min should be given routinely to all patients who have
tion of bacterially synthesized folate. had a total gastrectomy or ileal resection. Patients who
have undergone gastric reduction for control of obesity
or who are receiving long-term treatment with proton
Red cell folate pump inhibitors should be screened and, if necessary,
The red cell folate assay is a valuable test of body folate given cobalamin replacement.
stores. It is less affected than the serum assay by recent Replenishment of body stores should be complete
diet and traces of hemolysis. In normal adults, concen- with six 1000-g IM injections of hydroxocobalamin
trations range from 8803520 mol/L (160640 g/L) given at 3- to 7-day intervals. More frequent doses are
of packed red cells. Subnormal levels occur in patients usually used in patients with cobalamin neuropathy,
with megaloblastic anemia due to folate deficiency but there is no evidence that they produce a better
but also in nearly two-thirds of patients with severe response. Allergic reactions are rare and may require
cobalamin deficiency. False-normal results may occur desensitization or antihistamine or glucocorticoid cover.
if a folate-deficient patient has received a recent blood For maintenance therapy, 1000 g hydroxocobalamin
transfusion or if a patient has a raised reticulocyte count. IM once every 3 months is satisfactory. Because of the
poorer retention of cyanocobalamin, protocols gener-
ally use higher and more frequent doses, e.g., 1000 g
IM monthly for maintenance treatment.
Treatment Megaloblastic Anemia
Because a small fraction of cobalamin can be absorbed
It is usually possible to establish which of the two defi- passively through mucous membranes even when there
ciencies, folate or cobalamin, is the cause of the ane- is complete failure of physiologic IF-dependent absorp-
mia and to treat only with the appropriate vitamin. tion, large daily oral doses (10002000 g) of cyano-
In patients who enter the hospital severely ill, how- cobalamin have been used in PA for replacement and
ever, it may be necessary to treat with both vitamins in maintenance of normal cobalamin status in, e.g., food
large doses once blood samples have been taken for malabsorption of cobalamin. Sublingual therapy has
also been proposed for those in whom injections are dif- prevent neural tube defects. It is also used in chronic 107
ficult because of a bleeding tendency and who may not dialysis patients and in parenteral feeds. Prophylactic
tolerate oral therapy. If oral therapy is used, it is impor- folic acid has been used to reduce homocysteine levels
tant to monitor compliance, particularly with elderly, to prevent cardiovascular disease, but further data are
forgetful patients. needed to assess the benefit for this and for cognitive
For treatment of patients with subnormal serum B12 function in the elderly.
levels with a normal MCV and no hypersegmentation
Pregnancy Folic acid, 400 g daily, should be given
of neutrophils, a negative IF antibody test result in the
as a supplement before and throughout pregnancy. In
absence of tests of B12 absorption is problematic. Some
women who have had a previous fetus with an NTD,
(perhaps 15%) cases may be due to TC I (HC) deficiency.
5 mg daily is recommended when pregnancy is con-
Homocysteine and/or MMA measurements may help,
templated and throughout the subsequent pregnancy.
but in the absence of these tests and with otherwise
normal gastrointestinal function, repeat serum B12 assay Infancy and Childhood The incidence of folate
after 612 months may help one decide whether to deficiency is so high in the smallest premature babies
start cobalamin therapy. during the first 6 weeks of life that folic acid (e.g.,
1 mg daily) should be given routinely to those weighing
Folate Deficiency Oral doses of 515 mg folic
<1500 g at birth and to larger premature babies who

CHAPTER 9
acid daily are satisfactory, as sufficient folate is absorbed
require exchange transfusions or develop feeding diffi-
from these extremely large doses even in patients with
culties, infections, or vomiting and diarrhea.
severe malabsorption. The length of time therapy must
The World Health Organization currently recom-
be continued depends on the underlying disease. It
mends routine supplementation with iron and folic acid
is customary to continue therapy for about 4 months,
in children in countries where iron deficiency is common
when all folate-deficient red cells will have been elimi-
and child mortality, largely due to infectious diseases,

Megaloblastic Anemias
nated and replaced by new folate-replete populations.
is high. However, some studies suggest that in areas
Before large doses of folic acid are given, cobalamin
where malaria rates are high, this approach may increase
deficiency must be excluded and, if present, corrected;
the incidence of severe illness and death. Even where
otherwise cobalamin neuropathy may develop despite
malaria is rare, there appears to be no survival benefit.
a response of the anemia of cobalamin deficiency to
folate therapy. Studies in the United States, however,
suggest that there is no increase in the proportion of
individuals with low serum cobalamin levels and no
anemia since food fortification with folic acid, but it
Megaloblastic Anemia Not Due to
is unknown if there has been a change in incidence of
cobalamin neuropathy.
Cobalamin or Folate Deficiency
Long-term folic acid therapy is required when the
or Altered Metabolism
underlying cause of the deficiency cannot be corrected This may occur with many antimetabolic drugs (e.g.,
and the deficiency is likely to recur, e.g., in chronic dialy- hydroxyurea, cytosine arabinoside, 6-mercaptopurine)
sis or hemolytic anemias. It may also be necessary in that inhibit DNA replication. Antiviral nucleoside ana-
gluten-induced enteropathy that does not respond to logues used in treatment of HIV infection may also
a gluten-free diet. When mild but chronic folate defi- cause macrocytosis and megaloblastic marrow changes.
ciency occurs, it is preferable to encourage improve- In the rare disease orotic aciduria, two consecutive
ment in the diet after correcting the deficiency with enzymes in purine synthesis are defective. The condi-
a short course of folic acid. In any patient receiving tion responds to therapy with uridine, which bypasses
long-term folic acid therapy, it is important to measure the block. In thiamine-responsive megaloblastic ane-
the serum cobalamin level at regular (e.g., once-yearly) mia, there is a genetic defect in the high-affinity thia-
intervals to exclude the coincidental development of mine transport (SLC19A2) gene. This causes defec-
cobalamin deficiency. tive RNA ribose synthesis through impaired activity
Folinic Acid (5-Formyl-THF) This is a stable of trans-ketolase, a thiamine-dependent enzyme in the
form of fully reduced folate. It is given orally or paren- pentose cycle. This leads to reduced nucleic acid pro-
terally to overcome the toxic effects of methotrexate or duction. It may be associated with diabetes mellitus and
other DHF reductase inhibitors. deafness and the presence of many ringed sideroblasts in
the marrow. The explanation is unclear for megaloblas-
Prophylactic Folic Acid In many coun- tic changes in the marrow in some patients with acute
tries, food is fortified with folic acid (in grain or flour) to myeloid leukemia and myelodysplasia.
cHAPteR 10

HEMOLYTIC ANEMIAS AND ANEMIA DUE TO


ACUTE BLOOD LOSS

Lucio Luzzatto

DeFinitionS General CliniCal anD


laboratory FeatureS
A finite life span is a distinct characteristic of red cells.
Hence, a logical, time-honored classification of ane- The clinical presentation of a patient with anemia is
mias is in three groups: (1) decreased production of red greatly influenced in the first place by whether the
cells, (2) increased destruction of red cells, and (3) acute onset is abrupt or gradual, and HAs are no exception. A
blood loss. Decreased production is covered in Chaps. 7, patient with autoimmune HA or with favism may be a
9, and 11; increased destruction and acute blood loss are medical emergency, whereas a patient with mild heredi-
covered in this chapter. tary spherocytosis or with cold agglutinin disease may
All patients who are anemic as a result of either be diagnosed after years. This is due in large measure to
increased destruction or acute blood loss have two the remarkable ability of the body to adapt to anemia
important elements in common: the anemia results from when it is slowly progressing (Chap. 2).
overconsumption of red cells from the peripheral blood,
yet the supply of cells from the bone marrow (in the
absence of coexisting marrow disease) is usually increased,
as reflected by a reticulocytosis. On the other hand, Table 10-1
physical loss of red cells from the bloodstreamwhich in ClaSSiFiCation oF HemolytiC anemiaS*
most cases also means physical loss from the bodyis fun- intraCorpuSCular extraCorpuSCular
damentally different from destruction of red cells within DeFeCtS FaCtorS
the body. Therefore, the clinical aspects and the patho- Hereditary Hemoglobinopathies Familial (atypical)
physiology of anemia in these two groups of patients are Enzymopathies hemolytic uremic
quite different, and they will be considered separately. Membrane-cytoskeletal syndrome
defects
Acquired Paroxysmal nocturnal Mechanical destruction
HemolYtic AnemiAs hemoglobinuria (microangiopathic)
(PNH) Toxic agents
With respect to primary etiology, anemias due to Drugs
increased destruction of red cells, which we know as Infectious
Autoimmune
hemolytic anemias (HAs), may be inherited or acquired
(Table 10-1). From the clinical point of view they may *
Hereditary causes correlate with intracorpuscular defects because
be more acute or more chronic, they may vary from mild these defects are due to inherited mutations. The one exception is
to very severe, and the site of hemolysis may be pre- PNH because the defect is due to an acquired somatic mutation.
dominantly intravascular or extravascular. With respect to Similarly, acquired causes correlate with extracorpuscular factors
mechanisms, HAs may be due to intracorpuscular causes because mostly these factors are exogenous. The one exception is
familial hemolytic uremic syndrome (HUS; often referred to as atypi-
or to extracorpuscular causes. But before reviewing the cal HUS) because here an inherited abnormality allows complement
individual types of HA, it is appropriate to consider activation to be excessive, with bouts of production of membrane
what they have in common. attack complex capable of destroying normal red cells.

108
Table 10-2 diagnostic workup; if it is done, it will show erythroid 109
Some Common Features of Hemolytic hyperplasia. In practice, once a HA is suspected, specific
Disorders tests will usually be required for a definitive diagnosis of a
General examination Jaundice, pallor specific type of HA.
Other physical findings Spleen may be enlarged;
bossing of skull in severe
congenital cases General pathophysiology
Hemoglobin level From normal to severely The mature red cell is the product of a developmental
reduced
pathway that brings the phenomenon of differentiation
MCV, MCH Usually increased
Reticulocytes Increased
to an extreme. An orderly sequence of events produces
Bilirubin Increased (mostly synchronous changes whereby the gradual accumulation
unconjugated) of a huge amount of hemoglobin in the cytoplasm (to
LDH Increased (up to 10 normal a final level of 340 g/L, i.e., about 5 mM) goes hand
with intravascular hemolysis) in hand with the gradual loss of cellular organelles
Haptoglobin Reduced to absent (if hemo- and of biosynthetic abilities. In the end, the erythroid
lysis is part intravascular) cell undergoes a process that has features of apoptosis,
including nuclear pyknosis and actual loss of the

CHAPTER 10
Abbreviations: LDH, lactate dehydrogenase; MCH, mean corpuscular
hemoglobin; MCV, mean corpuscular volume.
nucleus. However, the final result is more altruistic than
suicidal; the cytoplasmic body, instead of disintegrating,
is now able to provide oxygen to all cells in the human
organism for some remaining 120 days of the red cell
life span.
What differentiates HAs from other anemias is that As a result of this unique process of differentiation

Hemolytic Anemias and Anemia Due to Acute Blood Loss


the patient has signs and symptoms arising directly from and maturation, intermediary metabolism is drastically
hemolysis (Table 10-2). At the clinical level, the main curtailed in mature red cells (Fig. 10-1); for instance,
sign is jaundice; in addition, the patient may report discol- cytochrome-mediated oxidative phosphorylation has
oration of the urine. In many cases of HA, the spleen is been lost with the loss of mitochondria (through a
enlarged because it is a preferential site of hemolysis, and process of physiologic autophagy); therefore, there is
in some cases, the liver may be enlarged as well. In all no backup to anaerobic glycolysis for the production
severe congenital forms of HA, there also may be skeletal of adenosine triphosphate (ATP). Also the capacity of
changes due to overactivity of the bone marrow (although making protein has been lost with the loss of ribosomes.
they are never as severe as they are in thalassemia). This places the cells limited metabolic apparatus at risk
The laboratory features of HA are related to hemo- because if any protein component deteriorates, it cannot
lysis per se and the erythropoietic response of the bone be replaced, as it would be in most other cells, and in
marrow. Hemolysis regularly produces in the serum an fact the activity of most enzymes gradually decreases as
increase in unconjugated bilirubin and aspartate transami- red cells age. Another consequence of the relative sim-
nase (AST); urobilinogen will be increased in both urine plicity of red cells is that they have a very limited range
and stool. If hemolysis is mainly intravascular, the telltale of ways to manifest distress under hardship: in essence,
sign is hemoglobinuria (often associated with hemosid- any sort of metabolic failure will eventually lead either
erinuria); in the serum, there is increased hemoglobin, to structural damage to the membrane or to failure of
lactate dehydrogenase (LDH) is increased, and hapto- the cation pump. In either case, the life span of the red
globin is reduced. In contrast, the bilirubin level may cell is reduced, which is the definition of a hemolytic
be normal or only mildly elevated. The main sign of disorder. If the rate of red cell destruction exceeds the
the erythropoietic response by the bone marrow is an capacity of the bone marrow to produce more red cells,
increase in reticulocytes (Table 10-2), a test all too often the hemolytic disorder will manifest as HA.
neglected in the initial workup of a patient with anemia. Thus, the essential pathophysiologic process common
Usually the increase will be reflected in both the percent- to all HAs is an increased red cell turnover. The gold
age of reticulocytes (the more commonly quoted figure) standard for proving that the life span of red cells is
and the absolute reticulocyte count (the more defini- reduced (compared with the normal value of about
tive parameter). The increased number of reticulocytes 120 days) is a red cell survival study, which can be car-
is associated with an increased mean corpuscular volume ried out by labeling the red cells with 51Cr and mea-
(MCV) in the blood count. On the blood smear, this is suring residual radioactivity over several days or weeks;
reflected in the presence of macrocytes; there is also poly- however, this classic test is now available in very few
chromasia and sometimes one sees nucleated red cells. In centers, and it is rarely necessary. If the hemolytic event
most cases, a bone marrow aspirate is not necessary in the is transient, it does not usually cause any long-term
110 Embden-Meyerhof pathway Hexose monophosphate shunt owever, with chronic intravascular hemolysis, the
h
Glutathione persistent hemoglobinuria will cause considerable iron
GSH reductase GSSG
Glucose loss, needing replacement. With chronic extravascular
ATP
Hexokinase
NADP+ NADPH
hemolysis the opposite problem, iron overload, is more
ADP
Glucose-6-phosphate G6PD 6-phosphogluconate
common, especially if the patient needs frequent blood
Glucose phosphate
transfusions. Chronic iron overload will cause secondary
isomerase hemochromatosis: this will cause damage particularly
Fructose-6-phosphate
to the liver, eventually leading to cirrhosis, and to the
ATP
ADP
Phosphofructokinase heart muscle, eventually causing heart failure.
Fructose-1, 6-diphosphate

Aldolase
Compensated hemolysis versus
Glyceraldehyde-3-phosphate hemolytic anemia
HbFe2+ NAD+ Glyceraldehyde 3-phosphate
HbFe3+ NADH
dehydrogenase
2,3-bisphosphoglycerate mutase
Red cell destruction is a potent stimulus for erythropoi-
1,3-bisphosphoglycerate esis, which is mediated by erythropoietin (EPO) pro-
ADP Phosphoglycerate
kinase 2,3-bisphosphoglycerate duced by the kidney. This mechanism is so effective
ATP
3-phosphoglycerate
2,3-bisphosphoglycerate phosphatase that in many cases the increased output of red cells from
Section Iii

3-phosphoglycerate the bone marrow can fully balance an increased destruc-


mutase
tion of red cells. In such cases, we say that hemolysis
2-phosphoglycerate
is compensated. The pathophysiology of compensated
Enolase
hemolysis is similar to what we have just described,
Phosphoenolpyruvate except there is no anemia. This notion is important
ADP
Pyruvate kinase from the diagnostic point of view because a patient
Anemias

ATP
with a hemolytic condition, even an inherited one,
Pyruvate
NADH Lactate
may present without anemia. It is also important from
NAD+
dehydrogenase the point of view of management because compensated
Lactate hemolysis may become decompensatedi.e., anemia
may suddenly appearin certain circumstances, for
Figure 10-1 instance pregnancy, folate deficiency, or renal failure,
Red blood cell metabolism. The Embden-Meyerhof path- interfering with adequate EPO production. Another
way (glycolysis) generates adenosine triphosphate (ATP) general feature of chronic HAs is seen when any inter-
for energy and membrane maintenance. The generation of current condition, for instance, an acute infection,
nicotinamide adenine dinucleotide phosphate (NADPH) main- depresses erythropoiesis. When this happens, in view of
tains hemoglobin in a reduced state. The hexose mono-
the increased rate of red cell turnover, the effect will be
phosphate shunt generates nicotinamide adenine dinucleo-
predictably much more marked than in a person who
tide phosphate (NADPH) that is used to reduce glutathione,
does not have hemolysis. The most dramatic example is
which protects the red cell against oxidant stress. Regulation
infection by parvovirus B19, which may cause a rather
of 2,3-bisphosphoglycerate levels is a critical determinant of
oxygen affinity of hemoglobin. Enzyme deficiency states in order
precipitous fall in hemoglobin, an occurrence some-
of prevalence: glucose-6-phosphate dehydrogenase (G6PD)
times referred to as aplastic crisis.
> pyruvate kinase > glucose-6-phosphate isomerase > rare defi-
ciencies of other enzymes in the pathway. The more common Inherited Hemolytic Anemias
enzyme deficiencies are encircled. ADP, adenosine diphosphate.
There are three essential components in the red cell: (1)
hemoglobin, (2) the membranecytoskeleton complex,
consequences, except for an increased requirement for and (3) the metabolic machinery necessary to keep (1)
erythropoietic factors, particularly folic acid. However, and (2) in working order. Diseases caused by abnormali-
if hemolysis is recurrent or persistent, the increased ties of hemoglobin, or hemoglobinopathies, are covered
bilirubin production favors the formation of gall- in Chap. 8. Here we will deal with diseases of the other
stones. If a considerable proportion of hemolysis takes two components.
place in the spleen, as is often the case, splenomegaly
may become increasingly a feature, and hypersplen-
ism may develop, with consequent neutropenia and/or Hemolytic anemias due to abnormalities of the
membrane-cytoskeleton complex
thrombocytopenia.
The increased red cell turnover also has meta- The detailed architecture of the red cell membrane
bolic consequences. In normal subjects, the iron from is complex, but its basic design is relatively simple
effete red cells is very efficiently recycled by the body; (Fig. 10-2). The lipid bilayer incorporates phospholipids
111
RhAG

Rh GPC

Band 3 Band 3
CD47

4.2 GPA p55


Adducin
Figure 10-2 4.1R
-Spectrin -Spectrin
Diagram of red cell membrane- Ankyrin
4.1R Dematin
cytoskeleton. GPA, glycophorin A; GPC,
glycophorin C; RhAG, Rh-associated gly-
coprotein (For explanation see text.) (From Tropomyosin Actin
N Young et al: Clinical Hematology. Copy- protofilament
Self-association Tropomodulin
right Elsevier, 2006; with permission.) site

CHAPTER 10
and cholesterol, and it is spanned by a number of pro- Hereditary spherocytosis (HS)
teins that have their hydrophobic transmembrane This is a relatively common type of HA, with an esti-
domains embedded in the membrane. Most of these mated frequency of at least 1 in 5000. Its identifica-
proteins have hydrophilic domains extending toward tion is credited to Minkowksy and Chauffard, who at
both the outside and the inside of the cell. Other pro- the end of the nineteenth century reported families

Hemolytic Anemias and Anemia Due to Acute Blood Loss


teins are tethered to the membrane through a glyco- in which HS was inherited as an autosomal dominant
sylphosphatidylinositol (GPI) anchor, and they have condition (Fig 10-3A). From this seminal work, HS
only an extracellular domain. These proteins are came to be defined as an inherited form of HA associ-
arranged roughly perpendicular to or lying across the ated with the presence of spherocytes in the periph-
membrane; they include ion channels, receptors for eral blood. In addition, in vitro studies revealed that
complement components, receptors for other ligands, the red cells were abnormally susceptible to lysis in
and some of unknown function. The most abundant of hypotonic media: indeed, the presence of osmotic fragil-
these proteins are glycophorins and the so-called band ity became the main diagnostic test for HS. Today we
3, an anion transporter. The extracellular domains of know that HS, thus defined, is genetically heteroge-
many of these proteins are heavily glycosylated, and neous, i.e., it can arise from a variety of mutations in
they carry antigenic determinants that correspond to one of several genes (Table 10-3). Whereas classically
blood groups. Underneath the membrane and tangen- the inheritance of HS is autosomal dominant (with
tial to it is a network of other proteins that make up the patients being heterozygous), some severe forms
the cytoskeleton: the main cytoskeletal protein is spec- are instead autosomal recessive (with the patient being
trin, the basic unit of which is a dimer of -spectrin homozygous).
and -spectrin. The membrane is physically linked
to the cytoskeleton by a third set of proteins (includ- Clinical presentation and diagnosis
ing ankyrin and the so-called band 4.1 and band 4.2), The spectrum of clinical severity of HS is broad. Severe
which thus make these two structures intimately con- cases may present in infancy with severe anemia,
nected to each other. whereas mild cases may present in young adults or even
The membranecytoskeleton complex is indeed so later in life. In women, HS is sometimes first diagnosed
integrated that, not surprisingly, an abnormality of almost when anemia is investigated during pregnancy. The
any of its components will be disturbing or disrup- main clinical findings are jaundice, an enlarged spleen,
tive, causing structural failure, which results ultimately and often gallstones; indeed, it is often the finding of
in hemolysis. These abnormalities are almost invariably gallstones in a young person that triggers diagnostic
inherited mutations; thus, diseases of the membrane investigations.
cytoskeleton complex belong to the category of inherited The variability in clinical manifestations that is
HAs. Before the red cells lyse, they often exhibit more observed among patients with HS is largely due to the
or less specific morphologic changes that alter the normal different underlying molecular lesions (Table 10-3).
biconcave disk shape. Thus, the majority of the diseases Not only are mutations of several genes involved, but
in this group have been known for over a century as individual mutations of the same gene can also give
hereditary spherocytosis and hereditary elliptocytosis. very different clinical manifestations. In milder cases,
112 hemolysis is often compensated (discussed earlier),
and this may cause variation in time, even in the same
patient, because intercurrent conditions (e.g., infection)
cause decompensation. The anemia is usually normo-
cytic, with the characteristic morphology that gives the
disease its name. A characteristic feature is an increase in
mean corpuscular hemoglobin concentration (MCHC);
this is almost the only condition in which an increased
MCHC is seen.
When there is a family history (Fig. 10-3A), it is
usually easy to suspect the diagnosis, but there may
be no family history for at least two reasons. (1) The
patient may have a de novo mutation, i.e., a mutation
that has taken place in a germ cell of one of his or
her parents or early after zygote formation. (2) The
patient may have a recessive form of HS (Table 10-3).
In most cases, the diagnosis can be made on the basis
Section Iii

of red cell morphology and of a test for osmotic


fragility, a modified version of which is called the
pink test. In some cases, a definitive diagnosis can
be obtained only by molecular studies demonstrating
a mutation in one of the genes underlying HS. This is
usually carried out in laboratories with special exper-
Anemias

tise in this area.

Treatment Hereditary Spherocytosis

We dont have a causal treatment for HS; i.e., no way


has yet been found to correct the basic defect in the
membranecytoskeleton structure. However, it has
been apparent for a long time that the spleen plays
a special role in HS through a dual mechanism. On
one hand, like in many other HAs, the spleen itself is
a major site of destruction; on the other hand, tran-
sit through the splenic circulation makes the defec-
tive red cells more spherocytic and therefore acceler-
ates their demise, even though lysis may take place
elsewhere. For these reasons, splenectomy has long
been regarded as a prime, almost obligatory, therapeu-
tic measure in HS. Therefore, current guidelines (not
evidence-based) are as follows. (1) Avoid splenectomy in
mild cases. (2) Delay splenectomy until at least 4 years
of age, after the risk of severe sepsis has peaked.
(3) Antipneumococcal vaccination before splenectomy
is imperative, whereas penicillin prophylaxis postsple-
nectomy is controversial. (4) There is no doubt that HS
patients often may require cholecystectomy, in which
case the practice has been to also carry out a splenec-
tomy at the same time. Today the decision regarding
Figure 10-3 this combined surgery should not be regarded as auto-
Peripheral blood smear from patients with membrane- matic; cholecystectomy is usually done via the laparo-
cytoskeleton abnormalities. A. Hereditary spherocytosis. scopic approach, and splenectomy should be carried
B. Hereditary elliptocytosis, heterozygote. C. Elliptocytosis, out if clinically indicated.
with both alleles of the -spectrin gene mutated.
Table 10-3 113
Inherited Diseases of the Red Cell MembraneCytoskeleton
Disease(s) with
Chromosomal Protein Certain Mutations
Gene Location Produced (Inheritance) Comments

SPTA1 1q22-q23 -Spectrin HS (recessive) Rare


HE (dominant) Mutations of this gene account for about 65% of HE.
More severe forms may be due to coexistence of an
otherwise silent mutant allele
SPTB 14q23-q24.1 -Spectrin HS (dominant) Rare
HE (dominant) Mutations of this gene account for about 30% of HE,
including some severe forms
ANK1 8p11.2 Ankyrin HS (dominant) May account for majority of HS
SLC4A1 17q21 Band 3 (anion HS (dominant) Mutations of this gene may account for about 25%
channel) of HS
Southeast Asia Polymorphic mutation (deletion of 9 amino acids);
ovalocytosis clinically asymptomatic; protective against

CHAPTER 10
(dominant) Plasmodium falciparum
Stomatocytosis Certain specific missense mutations shift protein
function from anion exchanger to cation conduc-
tance
EPB41 1p33-p34.2 Band 4.1 HE (dominant) Mutations of this gene account for about 5% of HE:
mostly with prominent morphology but no hemolysis
in heterozygotes; severe hemolysis in homozygotes

Hemolytic Anemias and Anemia Due to Acute Blood Loss


EPB42 15q15-q21 Band 4.2 HS (recessive) Mutations of this gene account for about 3% of HS.
RHAG 6p21.1-p11 Rhesus Chronic nonsphero- Very rare; associated with total loss of all Rh antigens
antigen cytic hemolytic
anemia

Abbreviations: HE, hereditary elliptocytosis; HS, hereditary spherocytosis.

Hereditary elliptocytosis (HE) finding, in a blood test, of high serum K+ (pseudohy-


HE is at least as heterogeneous as HS, both from the perkalemia). In patients from some families, the cation
genetic point of view (Table 10-3) and from the clinical transport disturbance is associated with gain of water: as
point of view. Again, it is the shape of the red cells that a result, the red cells are overhydrated (low MCHC),
gives the name to these conditions, but there is no direct and on a blood smear, the normally round-shaped cen-
correlation between the elliptocytic morphology and clini- tral pallor is replaced by a linear-shaped central pallor,
cal severity. In fact, some mild or even asymptomatic cases which has earned this disorder the name stomatocytosis.
may have nearly 100% elliptocytes, whereas in severe cases, In patients from other families, the red cells are instead
it is all sorts of bizarre poikilocytes that predominate. Clini- dehydrated (high MCHC), and their consequent rigid-
cal features and recommended management are similar to ity has earned this disorder the name xerocytosis. In these
those outlined above for HS. Although the spleen may not disorders, one would suspect that the primary defect
have the specific role it has in HS, in severe cases, sple- may be in a cation transporter. In most cases, this has not
nectomy may be beneficial. The prevalence of HE caus- yet been demonstrated, but interestingly, certain mis-
ing clinical disease is similar to that of HS. However, an sense mutations of the SLC4A1 gene encoding band 3
asymptomatic form, referred to as Southeast Asia ovalocy- (Table 10-3) give stomatocytosis. Hemolysis can vary
tosis, has a frequency of up to 7% in certain populations, from relatively mild to quite severe. From the practical
presumably as a result of malaria selection. point of view, it is important to know that splenectomy
is contraindicated, as it has been followed in a majority
of cases by severe thromboembolic complications.
Disorders of cation transport
These rare conditions with autosomal dominant inheri-
Enzyme abnormalities
tance are characterized by increased intracellular sodium
in red cells, with concomitant loss of potassium: indeed, When there is an important defect in the membrane or
they are sometimes discovered through the incidental in the cytoskeleton, hemolysis is a direct consequence
114 of the fact that the very structure of the red cell is concentration gradient across the membrane. If this
abnormal. Instead, when one of the enzymes is defec- fails, due to a defect of any of the enzymes of the gly-
tive, the consequences will depend on the precise role colytic pathway, the result will be hemolytic disease
of that enzyme in the metabolic machinery of the red (Table 10-4).
cell, which, in first approximation, has two important
Pyruvate kinase deficiency
functions: (1) to provide energy in the form of ATP and
(2) to prevent oxidative damage to hemoglobin and to Abnormalities of the glycolytic pathway are all inher-
other proteins. ited and all rare. Among them, deficiency of pyruvate
kinase (PK) is the least rare, with an estimated preva-
Abnormalities of the glycolytic pathway lence of the order of 1:10,000. The clinical picture is
Since red cells, in the course of their differentiation, have that of an HA that often presents in a newborn with
sacrificed not only their nucleus and their ribsomes but neonatal jaundice; the jaundice persists, and it is usually
also their mitochondria, they rely exclusively on the associated with a very high reticulocytosis. The anemia
anaerobic portion of the glycolytic pathway for pro- is of variable severity; sometimes it is so severe as to
ducing energy in the form of ATP. Most of the ATP require regular blood transfusion treatment; sometimes
is required by the red cell for cation transport against a it is mild, bordering on a nearly compensated hemolytic
Section Iii

Table 10-4
Red Cell ENzyme Abnormalities Causing Hemolysis
Prevalence Clinical
Chromosomal of Enzyme Manifestations
Anemias

Enzyme (Acronym) Location Deficiency (Rank) Extra-Red Cell Comments

Glycolytic Hexokinase (HK) 10q22 Very rare Other isoenzymes


pathway known
Glucose 6-phosphate 19q31.1 Rare (4)* NM, CNS
isomerase (G6PI)
Phosphofructoki- 12q13 Very rare Myopathy
nase (PFK)
Aldolase 16q22-24 Very rare
Triose phosphate 12p13 Very rare CNS (severe), NM
isomerase (TPI)
Glyceraldehyde 12p13.31-p13.1 Very rare Myopathy
3-phosphate dehy-
drogenase (GAPD)
Diphosphoglycerate 7q31-q34 Very rare Erythrocytosis rather
mutase (DPGM) than hemolysis
Phosphoglycerate Xq13 Very rare CNS, NM May benefit from
kinase (PGK) splenectomy
Pyruvate kinase (PK) 1q21 Rare (2)* May benefit from
splenectomy
Redox Glucose 6-phosphate Xq28 Common (1)* Very rarely In almost all cases only
dehydrogenase granulocytes AHA from exogenous
(G6PD) trigger
Glutathione 20q11.2 Very rare CNS
synthase
-Glutamylcysteine 6p12 Very rare CNS
synthase
Cytochrome b5 22q13.31-qter Rare CNS Methemoglobinemia
reductase rather than hemolysis
Nucleotide Adenylate kinase 9q34.1 Very rare CNS
(AK)
Metabolism Pyrimidine 5- 3q11-q12 Rare (3)* May benefit from
nucleotidase (P5N) splenectomy

*
The numbers from (1) to (4) indicate the ranking order of these enzymopathies in terms of frequency.
Abbreviations: AHA, acquired hemolytic anemia; CNS, central nervous system: NM, neuromuscular manifestations.
disorder. As a result, the diagnosis may be delayed, and Abnormalities of redox metabolism 115
in some cases, it is made in young adults, for instance, G6PD deficiency
in a woman, during her first pregnancy, when the ane- Glucose 6-phosphate dehydrogenase (G6PD) is a
mia may get worse. In part, the delay in diagnosis is due housekeeping enzyme critical in the redox metabo-
to the fact that the anemia is remarkably well tolerated lism of all aerobic cells (Fig. 10-1). In red cells, its
because the metabolic block at the last step in glycoly- role is even more critical because it is the only source
sis causes an increase in bisphosphoglycerate (or DPG), of NADPH that directly and via glutathione (GSH)
a major effector of the hemoglobinoxygen dissocia- defends these cells against oxidative stress. G6PD defi-
tion curve; thus, the oxygen delivery to the tissues is ciency is a prime example of an HA due to interaction
enhanced. between an intracorpuscular cause and an extracorpus-
cular cause because in the majority of cases, hemolysis is
triggered by an exogenous agent. Although a decrease in
Treatment Pryuvate Kinase Deficiency G6PD activity is noted in most tissues of G6PD-deficient
subjects, the decrease is less marked than in red cells,
The management of PK deficiency is mainly support- and it does not seem to have a clinical impact.
ive. In view of the marked increase in red cell turnover,
oral folic acid supplements should be given constantly.

CHAPTER 10
Genetic Considerations
Blood transfusion should be used as necessary, and iron
chelation may have to be added if the blood transfu- The G6PD gene is X-linked, and this has important
sion requirement is high enough to cause iron overload. implications. First, as males have only one G6PD
In these patients, who have more severe disease, sple- gene (i.e., they are hemizygous for this gene), they
nectomy may be beneficial. There is a single case report must be either normal or G6PD-deficient. By contrast,
of curative treatment of PK deficiency by bone marrow females, having two G6PD genes, can be normal, defi-

Hemolytic Anemias and Anemia Due to Acute Blood Loss


transplantation from an HLA-identical PK-normal sib- cient (homozygous), or intermediate (heterozygous). As a
ling. This seems a viable option for severe cases when a result of the phenomenon of X-chromosome inactiva-
sibling donor is available. tion, heterozygous females are genetic mosaics, with a
highly variable ratio of G6PD-normal to G6PD-
deficient cells and an equally variable degree of clinical
Other glycolytic enzyme abnormalities expression: some heterozygotes can be just as affected as
All of these defects are rare to very rare (Table 10-4), hemizygous males. The enzymatically active form of
and all cause HA with varying degrees of severity. It is G6PD is either a dimer or a tetramer of a single protein
not unusual for the presentation to be in the guise of subunit of 514 amino acids. G6PD-deficient subjects
severe neonatal jaundice, which may require exchange have been found invariably to have mutations in the
transfusion. If the anemia is less severe, it may pres- coding region of the G6PD gene (Fig. 10-4). Almost
ent later in life, or it may even remain asymptom- all of some 150 different mutations known are single
atic and be detected incidentally when a blood count missense point mutations, entailing single amino acid
is done for unrelated reasons. The spleen is often replacements in the G6PD protein. In most cases, these
enlarged. When other systemic manifestations occur,
they involve the central nervous system, sometimes
entailing severe mental retardation (particularly in
the case of triose phosphate isomerase deficiency), the
neuromuscular system, or both. The diagnosis of HA G6P NADP GSH H 2O 2
Catalase
is usually not difficult because of the triad of normo- 6PG NADPH GSSG H2O
macrocytic anemia, reticulocytosis, and hyperbiliru- G6PD Glutathione Glutathione Oxidative
binemia. Enzymopathies should be considered in the reductase peroxidase agents

differential diagnosis of any chronic Coombs-negative Hb(Fe2+)


HA. In most cases of glycolytic enzymopathies, the MetHb(Fe3+)
morphologic abnormalities of red cells characteristically
seen in membrane disorders are conspicuous by their Figure 10-4
absence. A definitive diagnosis can be made only by Diagram of redox metabolism in the red cell. G6P,
demonstrating the deficiency of an individual enzyme glucose 6-phosphate; G6PD, glucose 6-phosphate dehy-
by quantitative assays carried out in only a few special- drogenase; GSH, reduced glutathione; GSSG, oxidized glu-
ized laboratories. If a particular molecular abnormality is tathione; Hb, hemoglobin; MetHb, methemoglobin; NADP,
already known in a family, then of course one could test nicotinamide adenine dinucleotide phosphate; NADPH,
directly for that at the DNA level, bypassing the need reduced nicotinamide adenine dinucleotide phosphate, 6PG,
for enzyme assays. 6-phosphogluconate.
116 mutations cause G6PD deficiency by decreasing the in the fact that it confers a relative resistance against this
vivo stability of the protein; thus, the physiologic highly lethal infection. Whether this protective effect is
decrease in G6PD activity that takes place with red cell exerted mainly in hemizygous males or in females het-
aging is greatly accelerated. In some cases, an amino erozygous for G6PD deficiency is still not quite clear.
acid replacement can also affect the catalytic function of Different G6PD variants underlie G6PD deficiency in
the enzyme. different parts of the world. Some of the more wide-
Among these mutations, those underlying chronic spread variants are G6PD Mediterranean on the shores
nonspherocytic hemolytic anemia (CNSHA; see Clini- of that sea, in the Middle East, and in India; G6PD A in
cal Manifestations below) are a discrete subset. This Africa and in Southern Europe; G6PD Vianchan and
much more severe clinical phenotype can be ascribed in G6PD Mahidol in Southeast Asia; G6PD Canton in
some cases to adverse qualitative changes (for instance, a China; and G6PD Union worldwide. The heterogeneity
decreased affinity for the substrate, glucose 6-phosphate) of polymorphic G6PD variants is proof of their indepen-
or simply to the fact that the enzyme deficit is more dent origin, and it supports the notion that they
extreme because of a more severe instability of the have been selected by a common environmental agent,
enzyme. For instance, a cluster of mutations map at or in keeping with the concept of convergent evolution
near the dimer interface, and clearly they compromise (Fig. 10-5).
severely the formation of the dimer.
Clinical manifestations
Section Iii

Epidemiology The vast majority of people with G6PD deficiency


G6PD deficiency is widely distributed in tropical remain clinically asymptomatic throughout their life-
and subtropical parts of the world (Africa, South- times; however, all of them have an increased risk of
ern Europe, the Middle East, Southeast Asia, and developing neonatal jaundice (NNJ) and a risk of
Oceania) (Fig. 10-5) and wherever people from those developing acute hemolytic anemia (AHA) when chal-
areas have migrated. A conservative estimate is that at lenged by a number of oxidative agents. NNJ related
Anemias

least 400 million people have a G6PD deficiency gene. to G6PD deficiency is very rarely present at birth.
In several of these areas, the frequency of a G6PD defi- The peak incidence of clinical onset is between day 2
ciency gene may be as high as 20% or more. It would be and day 3, and in most cases the anemia is not severe.
quite extraordinary for a trait that causes significant However, NNJ can be very severe in some G6PD-
pathology to spread widely and reach high frequencies in deficient babies, especially in association with pre-
many populations without conferring some biologic maturity, infection, or environmental factors (such as
advantage. Indeed, G6PD is one of the best characterized naphthalenecamphor balls used in babies bedding and
examples of genetic polymorphisms in the human spe- clothing), and the risk of severe NNJ is also increased
cies. Clinical field studies and in vitro experiments by the coexistence of a monoallelic or biallelic muta-
strongly support the view that G6PD deficiency has been tion in the uridyl transferase gene (UGT1A1; the same
selected by Plasmodium falciparum malaria, by virtue of mutations are associated with Gilbert syndrome). If

Figure 10-5
Epidemiology of G6PD deficiency throughout the world. one having a different mutation. (From L Luzzatto et al, in
The different shadings indicate increasingly high levels of C Scriver et al [eds]: The Metabolic & Molecular Bases of
prevalence, up to about 20%; the different colored sym- Inherited Disease, 8th ed. New York, McGraw-Hill, 2001.)
bols indicate individual genetic variants of G6PD, each
Table 10-5 117
Drugs That Carry Risk of Clinical Hemolysis in Persons With G6PD Deficiency
Definite Risk Possible Risk Doubtful Risk

Antimalarials Primaquine Chloroquine Quinine


Dapsone/chlorproguanil*
Sulphonamides/sulphones Sulfamethoxazole Sulfasalazine Sulfisoxazole
Others Sulfadimidine Sulfadiazine
Dapsone
Antibacterial/antibiotics Cotrimoxazole Ciprofloxacin Chloramphenicol
Nalidixic acid Norfloxacin p-Aminosalicylic acid
Nitrofurantoin
Niridazole
Antipyretic/analgesics Acetanilide Acetylsalicylic acid Acetylsalicylic acid (<3 g/d)
high dose (>3 g/d)
Phenazopyridine Acetaminophen
Phenacetin

CHAPTER 10
Other Naphthalene Vitamin K analogues Doxorubicin
Methylene blue Ascorbic acid >1 g Probenecid
Rasburicase

*
Marketed as Lapdap from 2003 to 2008.

inadequately managed, NNJ associated with G6PD A very small minority of subjects with G6PD deficiency

Hemolytic Anemias and Anemia Due to Acute Blood Loss


deficiency can produce kernicterus and permanent have chronic nonspherocytic hemolytic anemia (CNSHA) of
neurologic damage. variable severity. The patient is always a male, usually
AHA can develop as a result of three types of triggers: with a history of NNJ, who may present with anemia
(1) fava beans, (2) infections, and (3) drugs (Table 10-5).
Typically, a hemolytic attack starts with malaise, weak-
ness, and abdominal or lumbar pain. After an interval of
several hours to 23 days, the patient develops jaundice
and often dark urine, due to hemoglobinuria. The onset
can be extremely abrupt, especially with favism in chil-
dren. The anemia is from moderate to extremely severe.
It is usually normocytic and normochromic, and it is due
partly to intravascular hemolysis. Hence, it is associated
with hemoglobinemia, hemoglobinuria, high LDH, and
low or absent plasma haptoglobin. The blood film shows
anisocytosis, polychromasia, and spherocytes (Fig. 10-6).
The most typical feature is the presence of bizarre poi-
kilocytes, with red cells that appear to have unevenly
distributed hemoglobin (hemighosts) and red cells
that appear to have had parts of them bitten away (bite
cells or blister cells). A classical test, now rarely carried
out, is supravital staining with methyl violet that, if done
promptly, reveals the presence of Heinz bodies, consist-
ing of precipitates of denatured hemoglobin and regarded
as a signature of oxidative damage to red cells (except for
the rare occurrence of an unstable hemoglobin). LDH is
high and so is the unconjugated bilirubin, indicating that
there is also extravascular hemolysis. The most serious
threat from AHA in adults is the development of acute
renal failure (this is exceedingly rare in children). Once
the threat of acute anemia is over, and in the absence of Figure 10-6
comorbidity, full recovery from AHA associated with Peripheral blood smear from a 5-year-old G6PD-deficient
G6PD deficiency is the rule. boy with acute favism.
118 or unexplained jaundice or because of gallstones later
no previous kidney disease, recovery is the rule. The man-
in life. The spleen may be enlarged. The severity of
agement of NNJ associated with G6PD deficiency is no
anemia ranges in different patients from borderline to
different from that of NNJ due to other causes.
transfusion-dependent. The anemia is usually normo-
In cases with CNSHA, if the anemia is not severe,
macrocytic, with reticulocytosis. Bilirubin and LDH are
regular folic acid supplements and regular hemato-
increased. Although hemolysis is, by definition, chronic
logic surveillance will suffice. It will be important to
in these patients, they are also vulnerable to acute oxi-
avoid exposure to potentially hemolytic drugs, and
dative damage, and therefore the same agents that can
blood transfusion may be indicated when exacerbations
cause acute HA in people with the ordinary type of
occur, mostly in concomitance with intercurrent infec-
G6PD deficiency will cause severe exacerbations in
tion. In rare patients, regular blood transfusions may
people with the severe form of G6PD deficiency. In
be required, in which case appropriate iron chelation
some cases of CNSHA, the deficiency of G6PD is so
should be instituted. Unlike in hereditary spherocytosis,
severe in granulocytes that it becomes rate-limiting for
there is no evidence of selective red cell destruction in
their oxidative burst, with consequent increased suscep-
the spleen; however, in practice, splenectomy has proven
tibility to some bacterial infections.
beneficial in severe cases.
Laboratory diagnosis
The suspicion of G6PD deficiency can be confirmed by
Section Iii

semiquantitative methods often referred to as screen- Other abnormalities of the redox system
ing tests, which are suitable for population studies and As mentioned earlier, GSH is a key player in the
can correctly classify male subjects, in the steady state, as defense against oxidative stress. Inherited defects of
G6PD-normal or G6PD- deficient. However, in clini- GSH metabolism are exceedingly rare, but each one
cal practice, a diagnostic test is usually needed when the of them can give rise to chronic HA (Table 10-4). A
patient has had a hemolytic attack. This implies that the rare, peculiar, usually self-limited severe HA of the first
Anemias

oldest, most G6PD-deficient red cells have been selec- month of life, called infantile poikilocytosis, may be associ-
tively destroyed, and young red cells, having higher ated with deficiency of glutathione peroxidase (GSHPx)
G6PD activity, are being released into the circulation. due not to an inherited abnormality but to transient
Under these conditions, only a quantitative test can give nutritional deficiency of selenium, an element essential
a definitive result. In males, this test will identify normal for the activity of GSHPx.
hemizygotes and G6PD-deficient hemizygotes; among
females, some heterozygotes will be missed, but those Pyrimidine 5-nucleotidase (P5N) deficiency
who are at most risk of hemolysis will be identified. P5N is a key enzyme in the catabolism of nucleotides
arising from the degradation of nucleic acids that takes
place in the final stages of erythroid cell maturation. How
exactly its deficiency causes HA is not well understood,
Treatment G6PD Deficiency but a highly distinctive feature of this condition is a mor-
phologic abnormality of the red cells known as basophilic
The acute HA of G6PD deficiency is largely preventable stippling. The condition is rare, but it probably ranks third
by avoiding exposure to triggering factors of previously in frequency among red cell enzyme defects (after G6PD
screened subjects. Of course, the practicability and cost- deficiency and PK deficiency). The anemia is lifelong, of
effectiveness of screening depends on the prevalence of variable severity, and may benefit from splenectomy.
G6PD deficiency in each community. Favism is entirely
preventable in G6PD-deficient subjects by not eating fava
beans. Drug-induced hemolysis can be prevented by test- Familial (atypical) hemolytic uremic
ing for G6PD deficiency before prescribing; in most cases, syndrome(aHUS)
one can use alternative drugs. When AHA develops and
This phrase is used to designate a group of rare disor-
once its cause is recognized, in most cases no specific treat-
ders, mostly affecting children, characterized by micro-
ment is needed. However, if the anemia is severe, it may
angiopathic HA with presence of fragmented erythro-
be a medical emergency, especially in children, requiring
cytes in the peripheral blood smear, thrombocytopenia
immediate action, including blood transfusion. This has
(usually mild), and acute renal failure. (The word atypi-
been the case with an antimalarial drug combination con-
cal is part of the phrase because it is the HUS caused
taining dapsone (called Lapdap, introduced as recently as
by infection with Escherichia coli producing the Shiga
2003) that has caused severe acute hemolytic episodes
toxin that is regarded as typical). The genetic basis of
in children with malaria in several African countries; after
aHUS has been elucidated only recently. Studies of
a few years, it was taken off the market. If there is acute
more than 100 families have revealed that family mem-
renal failure, hemodialysis may be necessary, but if there is
bers who have developed HUS have mutations in any
one of several genes encoding complement regulatory Toxic agents and drugs 119
proteins: complement factor H (CFH), CD46 or mem-
brane cofactor protein (MCP), complement factor I A number of chemicals with oxidative potential, whether
(CFI), complement component C3, complement factor medicinal or not, can cause hemolysis even in people
B (CFB), and thrombomodulin. Thus, whereas all other who are not G6PD-deficient (discussed earlier). Exam-
inherited HAs are due to intrinsic red cell abnormali- ples are hyperbaric oxygen (or 100% oxygen), nitrates,
ties, this group is unique in that hemolysis results from chlorates, methylene blue, dapsone, cisplatin, and numer-
an inherited defect external to red cells (Table 10-1). ous aromatic (cyclic) compounds. Other chemicals may
Because the regulation of the complement cascade has be hemolytic through a nonoxidative, largely unknown
considerable redundancy, in the steady state, any of the mechanism; examples are arsine, stibine, copper, and
above abnormalities can be tolerated. However, when lead. The HA caused by lead poisoning is characterized
an intercurrent infection or some other trigger activates by basophilic stippling. It is in fact a phenocopy of that
complement through the alternative pathway, the defi- seen in P5N deficiency (discussed earlier), suggesting it is
ciency of one of the complement regulators becomes mediated at least in part by lead inhibiting this enzyme.
critical. Endothelial cells get damaged, especially in the In these cases, hemolysis appears to be mediated by
kidney, and at the same time and partly as a result of a direct chemical action on red cells. But drugs can
this, there will be brisk hemolysis (thus, the more com- cause hemolysis through at least two other mecha-

CHAPTER 10
mon Shiga toxinrelated HUS can be regarded as a nisms. (1) A drug can behave as a hapten and induce
phenocopy of aHUS). aHUS is a severe disease with up antibody production. In rare subjects, this happens, for
to 15% mortality in the acute phase and up to 50% of instance, with penicillin. Upon a subsequent exposure,
cases progressing to end-stage renal disease. aHUS often red cells are caught, as innocent bystanders, in the reac-
undergoes spontaneous remission, and the best tested tion between penicillin and antipenicillin antibodies.
form of treatment is plasma exchange, which supplies Hemolysis will subside as soon as penicillin administra-
tion is stopped. (2) A drug can trigger, perhaps through

Hemolytic Anemias and Anemia Due to Acute Blood Loss


the deficient complement regulator. Because the basis
of aHUS is an inherited abnormality, it is not surprising mimicry, the production of an antibody against a red
that given exposure to an appropriate trigger, the syn- cell antigen. The best known example is methyldopa,
drome will tend to recur: when it does, the prognosis is an antihypertensive agent no longer in use, which in a
always serious. In some cases, kidney (and liver) trans- small fraction of patients stimulates the production of
plantation has been carried out, but the role of these the Rhesus antibody anti-e. In patients who have this
procedures is controversial. antigen, the anti-e is a true autoantibody, which would
then cause an autoimmune HA (discussed later). Usu-
ally this would gradually subside when methyldopa was
Acquired Hemolytic Anemia discontinued.
Severe intravascular hemolysis can be caused by the
Mechanical destruction of red cells venom of certain snakes (cobras and vipers), and HA
Although red cells are characterized by the remarkable can also follow spider bites.
deformability that enables them to squeeze through cap-
illaries narrower than themselves for thousands of times Infection
in their lifetimes, there are at least two situations in By far, the most frequent infectious cause of HA, in
which they succumb to shear, if not to wear and tear. endemic areas, is malaria. In other parts of the world,
The result is intravascular hemolysis, resulting in hemo- the most frequent cause is probably Shiga toxin
globinuria. One situation is acute and self-inflicted, producing Escherichia coli O157:H7, now recognized as
march hemoglobinuria. Why sometimes a marathon run- the main etiologic agent of the hemolytic-uremic syn-
ner may develop this complication, whereas on another drome, more common in children than in adults. Life-
occasion this does not happen, we do not know (per- threatening intravascular hemolysis, due to a toxin with
haps her or his footwear needs attention). A similar lecithinase activity, occurs with Clostridium perfringens
syndrome may develop after prolonged barefoot ritual sepsis, particularly following open wounds or septic
dancing. The other situation is chronic and iatrogenic abortion or as a disastrous accident due to a contami-
(it has been called microangiopathic hemolytic anemia); nated blood unit. Occasionally, HA is seen, especially
it takes place in patients with prosthetic heart valves, in children, with sepsis or endocarditis from a variety of
especially when paraprosthetic regurgitation is present. organisms.
If the hemolysis consequent to mechanical trauma to
the red cells is mild, and provided the supply of iron is
Autoimmune hemolytic anemia (AIHA)
adequate, it may be largely compensated. If more than
mild anemia develops, reintervention to correct regur- Except for countries where malaria is endemic, AIHA is
gitation may be required. the most common form of acquired hemolytic anemia. In
120 fact, not quite appropriately, the two phrases are some- produce jaundice; and sometimes the spleen is enlarged.
times used as synonymous. When this triad is present, the suspicion of AIHA must
be high. When hemolysis is (in part) intravascular, the
Pathophysiology
telltale sign will be hemoglobinuria, which the patient
AIHA is caused by an autoantibody directed against a may report or for which the physician must inquire and
red cell antigen, i.e., a molecule present on the surface of test. The diagnostic test for AIHA is the antiglobulin test
red cells. The autoantibody binds to the red cells. Once worked out in 1945 by R. R. A. Coombs and known
a red cell is coated by antibody, it will be destroyed by since by his name. The beauty of this test is that it
one or more mechanisms. In most cases the Fc portion directly detects the pathogenetic mediator of the disease,
of the antibody will be recognized by the Fc receptor of i.e., the presence of antibody on the red cells themselves.
macrophages, and this will trigger erythrophagocytosis When the test result is positive, it clinches the diagnosis,
(Fig. 10-7). Thus, destruction of red cells will take and when it is negative, the diagnosis is unlikely. How-
place wherever macrophages are abundant, i.e., in the ever, the sensitivity of the Coombs test varies depend-
spleen, liver, and bone marrow. Because of the spe- ing on the technology that is used, and in doubtful
cial anatomy of the spleen, it is particularly efficient in cases, a repeat in a specialized lab is advisable; the term
trapping antibody-coated red cells, and often this is the Coombs-negative AIHA is a last resort. In some cases,
predominant site of red cell destruction. Although in the autoantibody has a defined identity: it may be spe-
severe cases even circulating monocytes can take part in
Section Iii

cific for an antigen belonging to the Rhesus system (it


this process, most of the phagocytosis-mediated red cell is often anti-e). In many cases, it is regarded as unspe-
destruction takes place in the organs just mentioned, and cific because it reacts with virtually all types of red cells.
it is therefore called extravascular hemolysis. In some cases, As in autoimmune diseases in general, the real cause
the nature of the antibody (usually an IgM antibody) of AIHA remains obscure. However, from the clinical
is such that the antigenantibody complex on the sur- point of view, an important feature is that AIHA can
face of red cells is able to activate complement (C). As a
Anemias

appear to be isolated, or it can develop as part of a more


result, a large amount of membrane attack complex will general autoimmune disease, particularly systemic lupus
form, and the red cells may be destroyed directly; this is erythematosus, of which sometimes it may be the first
known as intravascular hemolysis. manifestation. Therefore, when AIHA is diagnosed,
Clinical features a full screen for autoimmune disease is imperative. In
The onset of AIHA is very often abrupt and can be dra- some cases, AIHA can be associated, on first presenta-
matic. The hemoglobin level can drop, within days, tion or subsequently, with autoimmune thrombocyto-
to as low as 4 g/dL; the massive red cell removal will penia (Evanss syndrome).

RBC Complement Complement activation Destroyed red cell


with formation of membrane and
membrane free hemoglobin
Reticuloendothelial attack complex
system

Mononuclear
phagocyte cell IgG1 or IgG3
(MPC) antibody molecules

Fc receptors

Figure 10-7
Mechanism of antibody-mediated immune
destruction of red cells. (From N Young
Phacocytosis Fragmentation Cytotoxicity et al: Clinical Hematology. Philadelphia,
(ADCC) Elsevier, 2006; with permission.)
causes of hemoglobinuria (Table 10-6), but the pres- 121
Treatment Autoimmune Hemolytic Anemia ence of the Donath-Landsteiner antibody will prove