Copyright(c) 2005 SYSMEX CORPORATION, All Rights Reserved.

Joint Committee for Traceability in Laboratory Medicine (JCTLM) Meeting

Blood Cell Counting Blood Cell Counting StandardizationStandardization

November 15, 2005November 15, 2005

Keiji FUJIMOTOVice President, Dept. of Scientific Affairs Vice President, Dept. of Scientific Affairs

SysmexSysmex Corporation Corporation Member of ICSH Expert Panel on Member of ICSH Expert Panel on CytometryCytometry

ContentsContents

Traceability of Blood Cell CountingTraceability of Blood Cell CountingICSH Reference Methods ICSH Reference Methods

Reference Method for Red Blood Cell Counting Reference Method for Red Blood Cell Counting Measurement PrincipleMeasurement PrincipleTraceability Chain Traceability Chain Estimation of Uncertainty in MeasurementEstimation of Uncertainty in Measurement

Present Situation of StandardizationPresent Situation of Standardization6 manufacturers6 manufacturers’’ comparison study in Japancomparison study in Japan

SummarySummary

3

List of Reference Methods(1/4)

RBC & WBCReference method for the enumeration of erythrocytesand leucocytesInternational Council for Standardization in Haematology; Prepared by the Expert Panel on Cytometry- Clinical Laboratory Haematology, 16, 131-138 (1994)

4

List of Reference Methods(2/4)

PlateletPlatelet Counting by the RBC/Platelet Ratio MethodA Reference Method （CD41/61)International Council for Standardization in Haematology Expert Panel on Cytometry and International Society of Laboratory Hematology Task Force on Platelet Counting - American Journal of Clinical Pathology 115:460-464 (2001)

RBC

PLT

RBC/PLT ratio

5

List of Reference Methods(3/4)

HemoglobinRecommendations for reference method for haemoglobinmetry in human blood (ICSH standard 1995)and specifications for international haemiglobincyanidestandard (4th edition)International Council for Standardization in Haematology: Expert Panel on Haemoglobinometry- Clinical Laboratory Haematology, 16, 131-138 (1994)

Reference and Selected Procedures for the QuantitativeDetermination of Hemoglobin in Blood ; Approved Standard – Third EditionNCCLS H15-A3 (HiCN method)

>> registered in “List-I, Reference Procedures of JCTLM”

6

List of Reference Methods(4/4)

PCV (Hematcrit) Recommendations for Reference Method for the PackedCell Volume (ICSH Standard 2001)International Council for Standardization in Haematology: Expert Panel on

Cytometry- Laboratory Haematology 7, 148 -170 (2001)

Procedure for Determining Packed Cell Volume by the Microhematocrit Method ;Approved Standard – Third EditionNCCLS H7-A3

ContentsContents

Traceability for Blood Cell CountingTraceability for Blood Cell CountingICSH Reference Methods ICSH Reference Methods

Reference Method for Red Cell Counting Reference Method for Red Cell Counting Principles Principles Traceability ChainTraceability ChainEstimation of Uncertainty in MeasurementEstimation of Uncertainty in Measurement

Present Situation for standardizationPresent Situation for standardization6 manufacturers6 manufacturers’’ study in Japanstudy in Japan

SummarySummary

8

RBC & WBCReference method for the enumeration of erythrocytesand leucocytesInternational Council for Standardization in Haematology; Prepared by the Expert Panel on Cytometry- Clinical Laboratory Haematology, 16, 131-138 (1994)

9

Instrument Specifications by ICSHMeasurement SystemMeasurement SystemMeasurement System

The probability of “re-circulation” of particles through the sensing zone must be low.⇒semi-automated single channel aperture-impedance particle counter

Volume displaced during the counting periodVolume displaced during the counting periodVolume displaced during the counting period“within an accuracy of 1% traceable” to a national or

international metrological standard⇒ only achieved by “Mercury column”

thermal effects should not be greater than 0.1% per℃

Diameter of OrificeDiameter of OrificeDiameter of Orifice80～100μm

Threshold settingThreshold settingThreshold settingsignal to noise ratio is greater than 100:1

10

Specifications recommended by ICSH

GlasswarePositive displacement Positive displacement pipettspipetts

calibrated to an accuracy of ±0.5%, validated by aprocess traceable to a primary metrological standardVolumetric Flasks Volumetric Flasks ：： Grade AGrade A

borosilicate glass, each with a stated volume which has been tested by an appropriate national standard bodyCounting Vials

a minimum volume of 10ml, sufficient height that the entry orifice of the electronic cell counter is at approx. half the depth of the fluid before counting commences. no adherence of cellsDiluentsterile, non-toxic, buffered salt solution,

tonicity:280 ±15mOsms, must not cause cell lysis nor alter cell volume by > 2fl over a 20 min period

11

Analysis of Error (by ICSH protocol)

Maximum Permissible BiasMaximum Permissible BiasMaximum Permissible Bias－RBC counting；2.0%－WBC counting；4.0%

Sampling ErrorSampling ErrorSampling Error－Poor Mixing，hemorheological effects－Inaccuracy of Dilution

Transport ErrorTransport ErrorTransport Error－Sedimentation, cell loss and/or

Recirculation－Inaccuracy of the displaced volume

Counting ErrorCounting ErrorCounting Error－Improper discrimination－Inaccurate correction for coincidence loss－Imprecision of reference cell count <=１％

Electronic Cell Counting MethodElectronic Cell Counting MethodV

tHDC

PlateletV

tHDC

Platelet

2HDC

VRBC

2HDC

VRBC

Cell Size

Lower Threshold Upper Threshold

Cel

l Cou

nt Platelet RBC

Cell Size

Lower Threshold Upper Threshold

Cel

l Cou

nt Platelet RBCHistogram

★Pulse size is proportional to Cell size.★Pulse count corresponds to Cell count.

Start electrode

Stop electrode

1 mL

Aspiration

Manometer

Mer-cury

Diluent

Hydrauliccircuit Valve

000000 386520

Count window

Detector body

Aperture

Sample (diluted blood)

Quartz Glass PartsQuartz Glass Parts

ApertureAperture

ValveValve

Flush ValveFlush Valve

Detector Unit Mercury Manometer

Manometer volume was certified by a neutral bodyManometer volume was certified by a neutral body

Volume = 1.010 ml +/- 0.004 mL

Reference Counter & MacroReference Counter & Macro--dilutiondilution

Reference Counter Preparing 50,000-fold dilution

19

RBC Reference Method - Macro-dilution-

EDTA-2K blood

1000ml Reagent(50,001-fold

dilution)

20μL blood R atio (A ) D iluent D il.R atio # of counts

(A) 1.0000 100m l 0m l 50,001 3(B ) 0.7507 75m l 25m l 66,606 4(C ) 0.5010 50m l 50m l 99,802 6(D ) 0.2507 25m l 75m l 199,446 12

Y-axis(b)；

Coincidence-corrected count

Regression line; y=ax + b

0.2507 0.5010 0.7507 1.0000

RBC True Value

Summated Count

（Yaxis／3）×50001(fold) Certified Manometer volume (μl)

A

Uncertainty of RBC count

Precision Balance

Micro-Dispenser

Flask Mano-meter

Coincidence Correction

Reference Counter

corrected

count

measured 00.02001

uB uD uM uR

uc2 = uB

2 + uD2 + uF

2 + uM2 + uCC

2 + uR2

uF uCC

count

RBC count = 4.728 +/- 0.057 ( x 1012/L ) , k = 2

codefactor of

uncertainty

value ofthe

factor

value ofthe unc.

type

probability

distribution

divisor

standardunc.

conversion

coefficient

std. unc. in

required unit

20 0.130 0.065 0.239 0.0155

mg mg mg x 1015/g x 1012/L

20.12 0.042 0.042 0.238 0.0100

uL uL uL x 1018/L2 x 1012/L

1000 0.3 0.173 0.005 0.0008

mL mL mL x 1015/L2 x 1012/L

1.010 0.004 0.002 4.735 0.0095

mL mL mL x 1015/L2 x 1012/L

110.2 0.856 0.428 0.043 0.0186 x

103countx

103countx

103countx 109/L x 1012/L

4.782 0.005 0.005 1 0.0050

x 1012/L x 1012/L x 1012/L - x 1012/L

0.0283

x 1012/L

0.057

x 1012/L

normal( k = 2 )

Ｕexpanded

uncertainty

normalｕｃ

combined std.

uncertainty

A - １ｕＲ

repeatabilityof

measuring

B normal 2ｕCC

coincidence correction

B normal 2ｕM

volume of manometer

Brectangula

r1.73 ｕ

F

volumeof flask

A － １ｕD

repeatabilityof

dispensing

B normal 2ｕB

calibrationof balance

RBC count = 4.728 +/- 0.057 ( x 1012/L ) , k = 2

codefactor of

uncertainty

value ofthe

factor

value ofthe unc.

type

probability

distribution

divisor

standardunc.

conversion

coefficient

std. unc. in

required unit

20 0.130 0.065 0.239 0.0155

mg mg mg x 1015/g x 1012/L

20.12 0.042 0.042 0.238 0.0100

uL uL uL x 1018/L2 x 1012/L

1000 0.3 0.173 0.005 0.0008

mL mL mL x 1015/L2 x 1012/L

1.010 0.004 0.002 4.735 0.0095

mL mL mL x 1015/L2 x 1012/L

110.2 0.856 0.428 0.043 0.0186 x

103countx

103countx

103countx 109/L x 1012/L

4.782 0.005 0.005 1 0.0050

x 1012/L x 1012/L x 1012/L - x 1012/L

0.0283

x 1012/L

0.057

x 1012/L

normal( k = 2 )

Ｕexpanded

uncertainty

normalｕｃ

combined std.

uncertainty

A - １ｕＲ

repeatabilityof

measuring

B normal 2ｕCC

coincidence correction

B normal 2ｕM

volume of manometer

Brectangula

r1.73 ｕ

F

volumeof flask

A － １ｕD

repeatabilityof

dispensing

B normal 2ｕB

calibrationof balance

Measurand :Mean of 10 times

measurements by the reference counter

Expanded uncertainty :Uc is doubled to express 95 %

confidence interval of measurand

Traceability Chain for RBC count calibrationTraceability Chain for RBC count calibration

* ISO 17511:2003 (Cases with international conventional reference measurement procedure (which is not primary) but no international conventional calibrator and without metrological traceability to SI)

Uncertainty in ＲＢＣ count on SCS-1000Coincidence

correctionPrecision balance

Micro-dispenser

Flask Mano-meter

Reference counter

Fleshblood

corrected

count

measured 00.02001

count

uc2 = u1

2 + u22 + u3

2 + ……… + un2

Fleshblood

Instability Vial to Vial

Standard analyzers SCS-1000Kobe Ono kakogawa

assaydate

exp.date

RBC

Traceability & Uncertainty for CalibratorTraceability & Uncertainty for Calibrator

Traceability Uncertainty

ContentsContents

Traceability for Blood Cell CountingTraceability for Blood Cell CountingICSH Reference Methods ICSH Reference Methods

Reference Method for Red Cell Counting Reference Method for Red Cell Counting Principles Principles Traceability ChainTraceability ChainEstimation of Uncertainty in MeasurementEstimation of Uncertainty in Measurement

Present Situation for standardizationPresent Situation for standardization6 manufacturers6 manufacturers’’ study in Japanstudy in Japan

SummarySummary

6 Manufacturers6 Manufacturers’’ Comparison Study in JapanComparison Study in Japan

Purpose: Purpose: To evaluate hematological values obtained with Standard To evaluate hematological values obtained with Standard

Automated Hematology Analyzers of 6 manufactures. Automated Hematology Analyzers of 6 manufactures.

Method:Method:Prepare fresh whole blood samples from 3 healthy Prepare fresh whole blood samples from 3 healthy donersdoners. . Measure 3 samples on standard automated analyzers of each Measure 3 samples on standard automated analyzers of each

manufacture at the same time within 6 hours after drawing.manufacture at the same time within 6 hours after drawing.Compare each data obtained by each manufacture.Compare each data obtained by each manufacture.This study was done in 1991, 1997, 2002 ,2003 and 2005 This study was done in 1991, 1997, 2002 ,2003 and 2005

(still under investigation).(still under investigation).

Difference among 6 manufactures for RBCDifference among 6 manufactures for RBC

<RBC >6 manufactures data showed well-harmonized (within +/- 1.5%) in 2003 study

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

110.0%

Sysm ex B.C oulter Bayer Abbott Horiba Kohden

1997

2002

2003

* 100% represents mean of all 6 manufacturers’ data.

Difference among 6 manufactures for WBCDifference among 6 manufactures for WBC

<WBC >6 manufactures data showed wide variation (> +/- 5%) in 2003 study.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

110.0%

Sysm ex B.C oulter Bayer Abbott Horiba Kohden

1997

2002

2003

* 100% represents mean of all 6 manufacturers’ data.

Difference among 6 manufactures for HGBDifference among 6 manufactures for HGB

<HGB >6 manufactures data showed well-harmonized (within +/- 1.5%) in 2003 study.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

110.0%

Sysm ex B.C oulter Bayer Abbott Horiba Kohden

1997

2002

2003

* 100% represents mean of all 6 manufacturers’ data.

Difference among 6 manufactures for PLT Difference among 6 manufactures for PLT

<PLT >6 manufactures data showed wide variation (> +/- 5%) in 2003 study.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

110.0%

Sysm ex B.C oulter Bayer Abbott Horiba Kohden

1997

2002

2003

* 100% represents mean of all 6 manufacturers’ data.

Difference among 6 manufactures for HCTDifference among 6 manufactures for HCT

<ＨＣＴ >6 manufactures data showed well-harmonized (within +/- 2%) in 2003 study.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

110.0%

Sysm ex B.C oulter Bayer Abbott Horiba Kohden

1997

2002

2003

* 100% represents mean of all 6 manufacturers’ data.

SummarySummary

Reference Method and Traceability has been Reference Method and Traceability has been established by using ICSH recommended protocol, established by using ICSH recommended protocol, which each manufacturers agreed.which each manufacturers agreed.Discrepancy of fresh whole blood data among major Discrepancy of fresh whole blood data among major manufacturers has been less than +/manufacturers has been less than +/-- 1% for RBC and 1% for RBC and HgbHgb. . Announcement of Uncertainty for Calibrator is ready Announcement of Uncertainty for Calibrator is ready for endfor end--users.users.