blood cell counting standardization - bipm ? Â· ¾reference method for red blood cell counting...

Download Blood Cell Counting Standardization - BIPM ? Â· ¾Reference Method for Red Blood Cell Counting ... Count Platelet RBC Histogram ... blood corrected count measured 00.02001 count

Post on 04-Feb-2018

219 views

Category:

Documents

4 download

Embed Size (px)

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 periodwithin 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 Orifice80100m

    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 BiasRBC counting2.0%WBC counting4.0%

    Sampling ErrorSampling ErrorSampling ErrorPoor Mixinghemorheological effectsInaccuracy of Dilution

    Transport ErrorTransport ErrorTransport ErrorSedimentation, cell loss and/or

    RecirculationInaccuracy of the displaced volume

    Counting ErrorCounting ErrorCounting ErrorImproper discriminationInaccurate correction for coincidence lossImprecision of reference cell count

  • 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)

    20L 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

    Yaxis350001(fold) Certified Manometer volume (l)

    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 = uB2 + uD2 + uF2 + uM2 + uCC2 + uR2

    uF uCCcount

  • 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 )

    expanded

    uncertainty

    normal

    combined std.

    uncertainty

    A -

    repeatabilityof

    measuring

    B normal 2CC

    coincidence correction

    B normal 2M

    volume of manometer

    Brectangula

    r1.73

    F

    volumeof flask

    A D

    repeatabilityof

    dispensing

    B normal 2B

    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 )

    expanded

    uncertainty

    normal

    combined std.

    uncertainty

    A -

    repeatabilityof