blood cell counting standardization
TRANSCRIPT
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.