Whole Blood for Hematology Quality Control Material

- Standardization and External Quality Assessment using Anti-

coagulated Fresh Blood for Automated Hematology Analyzers -

Hiroshi KONDO, PhD

Department of Clinical Laboratory Science,

Faculty of Health Sciences,
Kansai University of Health Sciences
Nikkoh in Japan
 Introduction
Hematology testing plays an important role in routine medical
care, and in particular a Complete Blood Count (CBC) and
differential white blood count are essential to almost all types
of clinical diagnosis.
For this reason, a high level of accuracy is demanded.
This presentation gives an outline of traceability in blood
count testing, trends in international standardization, and
external quality assurance (EQA) using fresh blood samples
as a surveillance material.
1. Current status for international standardization for
automated hematology analyzers
2. External quality assessment for automated hematology
analyzers using anti-coagulated fresh blood in Japan
1) EQA conducted by the National Federation of Industrial
Health Organizations (NFIHO)
2) EQA conducted by the Japanese Society of Laboratory
Hematology (JSLH)
 Example of complete blood cell count traceability diagram
Material Calibration (), certification () Procedure Executed by

International conventional reference measurement

procedure by international scientific organization / WHO

Anti-coagulated
fresh human blood Manufactures internal Manufacturere
reference measurement
procedure

Manufactures
calibrator End users routine End user
measurement
procedure

Routine sample

Measurement result

Hiroshi Kondo: Actual state of international standardization and external quality assurance for blood cell counts. Readout English Edition No.44 June 2015
Standards and guidelines for hematologic laboratory testing

The reference analysis methods of the Inter national Council for

Standardization in Hematology (ICSH), World Health Organization
(WHO), and Clinical and Laboratory Standards Institute (CLSI) are
recommended as international conventional reference measurement
procedures.
The ICSH and the CLSI have recently developed and published
guidelines and standards for validation and verification of
hematology analyzer (HA).

Sue Ellen Verbrugge and Albert Huisman: Verification and standardization of blood cell counters for routine clinical laboratory tests. Clin Lab Med 35 (2015) 183-196
CBC parameters
determined by
hematology analyzer

Sue Ellen Verbrugge and Albert Huisman: Verification and standardization of blood cell counters for routine clinical laboratory tests. Clin Lab Med 35 (2015) 183-196
1. Current status for international standardization for
automated hematology analyzers
2. External quality assessment for automated hematology
analyzers using anti-coagulated fresh blood in Japan
1) EQA conducted by the National Federation of Industrial
Health Organizations (NFIHO)
2) EQA conducted by the Japanese Society of Laboratory
Hematology (JSLH)
Nationwide EQA for automated blood cell count parameters

The Japanese Society of Laboratory Hematology (JSLH) has been implementing the JSLH 6-
company EQA program using anti-coagulated fresh blood once every 2 to 4 years since 2001,
with the cooperation of six companies.
There are additional EQA programs that are implemented on a local basis under the joint or
independent sponsorship of medical associations and medical technologist associations, and
most of these use fresh blood.
Hiroshi Kondo: Actual state of international standardization and external quality assurance for blood cell counts. Readout English Edition No.44 June 2015
EQA programs implemented by local medical
technologist associations
The kind of anti-coagulant
EDTA-2Na, a mixture of ACD-A and
EDTA-2K, a mixture of CPD-A and
EDTA-2Na, and others N=415

A mixture of CPD
and EDTAK
EDTA-2K N=1244
N=830

CPD-A
N=311
Processed blood and fresh blood as EQA samples
Processed blood and fresh blood are used as test samples in these
EQA programs.
Chemicals are added to fix and stabilize processed blood in the
same way as the control blood used in internal quality control, so
storage stability is excellent and it is easy to prepare a large number
of samples.
Although matrix effects that accompany the fixing process and added
chemicals sometimes cause deviations between instruments, using
fresh blood similar to routine samples eliminates this concern.
1. Current status for international standardization for
automated hematology analyzers
2. External quality assessment for automated hematology
analyzers using anti-coagulated fresh blood in Japan
1) EQA conducted by the National Federation of Industrial
Health Organizations (NFIHO)
2) EQA conducted by the Japanese Society of Laboratory
Hematology (JSLH)
National Federation of Industrial Health Organizations (NFIHO)

Preparation of anticoagulated fresh blood Distribution of

surveillance materials

Transfusion bag
containing CPD EDTA-2Ksolution

A B A B
Fresh Processed
blood blood

Two different concentrations of blood

and fresh blood were distributed.
 Time-course changes in CBC with fresh blood

Elapsed time after

blood drawing 24h (n=11) 48h (n=218) 72h (n=90) 96h (n=11)
Hemoglobin (g/L) 1191 1191 1191 1191
Erythrocytes (1012/L) 3.770.50 3.800.42 3.810.32 3.820.46
Leukocytes (109/L) 3.930.09 3.920.12 3.850.16 * 3.820.24 *
Hematocrit 0.3600.043 0.3630.075 0.3690.095 0.3670.112
Platelets (109/L) 2004 2026 2056 2026
Data presented are meanstandard deviation.
*p<0.05
 Mean leukocyte count

Manufacturer X Manufacturer Y

10

Leukocytes (109/L)
5

0
Processed Fresh
blood blood
 Hematocrit
Impedance Optical

60

40
Hematocrit

20

0
Processed Fresh
blood blood
1. Current status for international standardization for
automated hematology analyzers
2. External quality assessment for automated hematology
analyzers using anti-coagulated fresh blood in Japan
1) EQA conducted by the National Federation of Industrial
Health Organizations (NFIHO)
2) EQA conducted by the Japanese Society of Laboratory
Hematology (JSLH)
 The JSLH Manufacturer Survey 2013

Testing facility locations map

Phlebotomy Room
University
Manufacturer
 The JSLH Manufacturer Survey 2013
Sample preparation process

Measurement by 6 manufactures The survey samples were measured within 4 hours after drawing, on the
representative reference hematology analyzers at the six manufacturers laboratories DxH800 (Beckman-
Coulter), XN-2000 (Sysmex), ADVIA2120 (Siemens), PENTRA60 (Horiba) ,MEK7300 (Nihon Kohden) and
CELL-DYN Sapphire (Abbott).
 The JSLH Manufacturer Survey 2013
History and reference methods of the JSLH Manufacture Survey

Hematology Analyzer Reference Method

Reference Instrument
in Japan
1st 2001 CBC, Diff
2nd 2005 CBC, Diff Diff - eye
3rd 2007 CBC, Diff Diff - eye
4th 2010 CBC, Diff, Retic Diff. Retic - eye/FCM
5th 2013 CBC, Diff, Retic Diff, Plt, Retic eyeFCM
Summary longtudinal evaluation results for CBC 6 items (2001-2013)

Each plot represents the relative

compared to the mean of
6manufactures data (as 100%)
Summary longtudinal evaluation results for WBC 5-Diff and Retics (2001-2013)

Each plot represents the relative

compared to the mean of
6manufactures data (as 100%)
The hemoglobin (Hb) concentration varied markedly in 2013

Most of the variance of CBC items using automated HAs between

manufactures decreased until 2010.
However, multiple variables including the hemoglobin (Hb) concentration
varied markedly in 2013.
Although a proficient experimenter had dispensed all three samples until
2010, three samples were dispensed by another three experimenters
respectively in 2013.
It was suggested that inter-individual variations in experimenters
concerning the stirring and dispensing conditions to prepare fresh blood
samples caused the variance of CBCs.
Therefore, we studied to identify the cause of variations in the process of
dispensing to prepare fresh blood samples for EQA.
 Improvement of procedure for stirring and dispensing to prepare
anti-coagulated fresh blood samples for EQA of HA

Hb concentrations: cyanmethemoglobin method

We evaluated the following three points to identify the cause of

variations in the process of dispensing to prepare fresh blood
samples
1. Comparison between mixing using a mechanical rolling mixer
and manual mixing
2. Time-dependent change in Hb concentrations of the anti-
coagulated blood samples after mixing
3. Effects of the volume of the container for mixing the sample
1. Comparison between mixing using a mechanical rolling mixer and manual
mixing

25.000
25.000

20.000
20.000

Hb conc. (g/dL)
Hb conc. (g/dL)

15.000
15.000
20 times rolling
10.000 30 times rolling
10.000 225 times rolling
225 times rolling
5.000
5.000
Deep end Shallow end Deep end
Shallow end

Twenty-times mixing by fully Mechanical rolling mixer

inverting the tube by hand

1) There was little difference between the Hb concentrations of deep layer and surface layer
parts of a sample tube after mixing by fully inverting the tube by hand.
2) Hb concentrations of the deep layer part of the sample tubes after mixing with a mechanical
mixer 20 times showed higher values than Hb concentrations of the surface layer part.
 2. Time-dependent change in Hb concentrations of the anti-coagulated
blood samples after mixing

Time-dependent relative change in Hb

concentrations of the anticoagulated blood
samples after mixing (%)

Sample 5 sec. 15 sec. 30 sec. 60 sec. 180 sec.

A 100.000 100.540 100.320 99.940 99.720

B 100.000 99.965 99.941 99.941 99.824

C 100.000 100.256 100.039 100.059 99.961

D 100.000 99.833 99.905 99.976 99.833

E 100.000 99.574 100.351 100.075 100.025

F 100.000 100.048 100.319 100.319 99.932

1) Hb concentrations of the sample were stable up to 30 seconds after mixing by fully inverting
the tube by hand; however, a declining trend was noted after 180 seconds.
2) The rate of decrease in the Hb concentration at 180 seconds compared with at 5 seconds
was less than 0.2 percent.
 3. Effects of the volume of the container for mixing the sample

15.10

15.00

Hb conc. (g/dL)
14.943 g/dL
The sample bottle contents were mixed 5 times by manual 14.90 14.890 g/dL
rolling and swinging after twenty-times mixing using a
mechanical rolling mixer. The blood was moved from the 14.80
bottom to wall of the sample bottle during manual mixing.

14.70
1st 3rd 6th 10th 14th 18th 21st

Sample tube

1 3 6 10 14 18 21

A 2-mL anti-coagulated blood sample was dispensed into Hb concentrations of the 1st, 3rd, 6th,
21 sample tubes with a size of 10 mL each within 30 10th, 14th, 18th, and 21st sample tubes
seconds after manual mixing, respectively. were from 14.890 to 14.943 g/dL.
Improvement of procedure for stirring and dispensing to prepare
anti-coagulated fresh blood samples for EQA of HA

Conclusion:
It is suggested that inter-individual variations in
experimenters performing the stirring procedure and the time
interval from stirring to dispensing caused the variance of
CBCs between manufacturers of automated HAs in 2013.
To prepare fresh blood samples for EQA, it is important that
the sample tube including anti-coagulated blood is mixed by
fully inverting the tube by hand, and that it is dispensed within
30 seconds after mixing.
 The JSLH Manufacturer Survey 2013
History and reference methods of the JSLH Manufacture Survey

Hematology Analyzer Reference Method

Reference Instrument
in Japan
1st 2001 CBC, Diff
2nd 2005 CBC, Diff Diff - eye
3rd 2007 CBC, Diff Diff - eye
4th 2010 CBC, Diff, Retic Diff. Retic - eye/FCM
5th 2013 CBC, Diff, Retic Diff, Plt, Retic eyeFCM
6th 2016 CBC, Diff, Retic Diff, Plt, Retic eye & FCM
Plt FCM ICSH/ISLH 2001 plus
plus 1: lower limit gate 1um ( Discriminator 0.5-1.0um)
plus 2: Fixation

Whole Blood

CD41 CD61
PBS Whole Blood FITC
(0.1%BSA) FITC

25L 2475L

CD41 CD61 PBS

Mixing (0.1%BSA)
FITC FITC
5L 85L 5L 5L
TruCOUNT
Tube PBS
(0.1%BSA) Mixing
50L 40L 5L 5L
Room Temp.
Dark Box
Mixing 15min. PBS
Fixation Buffer (0.1%BSA)

Room Temp. 100L 900L

Dark Box 1%
15min.
formaldehyde
TruCOUNT Tube
100L
900L

Final dilution 1: 1000 PBS

(0.1%BSA)

Mixing 200L 800L

Final dilution 1: 1000
Flowcytometer
Semi-automated, FSC discriminator : 0.5 1.0m Mixing
Semi-automated,
Single-channel, FSC lower limit : 1.0m Single-channel,
Aperture-Impedance Sample fixation : Yes
Cell Counter Plt Counting : Direct / Indirect
Aperture-Impedance Flowcytometer
Cell Counter
RBC Counting : Direct / Cell Counter

Red Cell counts Platelets/ Red Cells Ratio

Red Cell counts
Platelets/ Red Cells Ratio

Platelet counts
Platelet counts (ICSH/ISLH reference method)
(Improved ISLH/ICSH reference Method)
Retic FCM ICSH/CLSI H44-A2 Dye: Thiazole Orange
plus : RBC gate : CD235a

Reticulocyte detection by using comparison with gated events on each plot

TO Staining Control
P1-T Plot P2-T Plot P1-C Plot P2-C Plot

P1 P1

P2 P2

RBC/Retic-T Plot RBC/Retic1-C Plot

(P1-T AND P2-T) (P1-C AND P2-C)

RBC-T RBC-C
Retic-T Retic-C

TO
TO

CD235a CD235a
Retic-T Retic-C
Retic% =
RBC-T
% - RBC-C
%
Diff

Eye CLSI H26-A2, CLSI H20-A2

Identification :
Hematology
Atlas (JSLH Atlas): H20-A2
Qualified examiners :
H20-A2
Blood film, Counting :
H26-A2

FCM JSLH-Diff ( validated by

ICSH-French Method)
 Conclusion
The development of reference analysis methods and the
drafting of guidelines are being pursued in an
international framework, and a sufficient understanding
of trends in this area is desirable.
Objective evaluation using EQA has a large role to play
in the maintenance and improvement of the reliability of
test results, and in appeals made to hematology
analyzer manufacturers.
Thank you for your attention.