CONCURRENT 10: THE COMPLETE BLOOD COUNT … · concurrent 10: the complete blood count and beyond:...

CONCURRENT10:

THECOMPLETEBLOODCOUNTANDBEYOND:QUALITYISSUESANDREFERENCEMETHODS

SaturdayMay14th 20161:30– 3:00PM(13.30– 15.00hrs)

InternationalSocietyforLaboratoryHematology (ISLH)

CONCURRENT10:

THECOMPLETEBLOODCOUNTANDBEYOND:QUALITYISSUESANDREFERENCEMETHODS

CHAIRS:Dr.PieroCappelletti.SIPMEL,Pordenone, Italy

Dr.AlbertHuisman,UMCUtrecht,Utrecht,Netherlands

VerificationandQualityControlofautomatedHematologyAnalyzers

Disclosure information:

Dr AlbertHuisman:Nothing to disclose

TheUMCUtrecht,department ofClinical ChemistryandHematology hasreceived fundingfor contractresearchfrom:

Abbott DiagnosticsBeckman-CoulterDiagnostic GrifolsMechatronics

VerificationandqualitycontrolofautomatedHematologyAnalyzers– AutomatedCellAnalysis(CompleteBloodCount(CBC))

» Including5partLeukocytedifferentialcount,reticulocytecountandnewparameters(ReticulocyteHemoglobincontentReticulatedPlatelets/ImmaturePlateletFraction,……)

– Validationofanewanalyzer– Verificationofanewanalyzer

– ISO15189– CLSI,ICSH,others– Samples– Precision– Accuracyandcomparability– Sensitivityandspecificity– Referenceintervals

– Qualitycontrol– Daily/internal– ExternalQualityAssesment (EQA/Proficiencytesting)

Why bother about verification and qualitycontrolofahighly automated analyzer?

Because:

• CBCresults formthe starting pointofnumerous diagnosticschedules,treatments and interventions,for example:– Erytrocyte /Platelet transfusions– Work upofanemia (irondeficiency /thalassemia /…)– Hematological malignancies– Generalwork-upinvarious disease states– Etctetera

Because:

• TheISO15189standard[Medicallaboratories— Particularrequirementsforqualityandcompetence]requiresaverificationprocess

AdvantagesofAutomatedCellAnalysers:

• Excellentanalytical performance• Closed-tubeanalysis• Nointer-observer variability• Noslidedistribution error• Eliminate statistical variations• Potential ofreflextesting• Availabilityofextraparameterse.g.MCV,RDW,%rP,…• Moreefficient (>100analyses/hour)and cost effective than

manualmethod• ….

Automatedcellcounters:

• CompleteBloodCount(CBC):– Hemoglobin(Hb)concentration,RBCcount&RBCindices(MCV,

MCH,MCHC),WBCcount,PLTcount– WBCdifferentialcount(5‘normal’WBC’s)– RDW,MPV– Reticulocytecount– NucleatedRBCcount(NRBC’s)– …….Flagging,etc etc

– Newparameters:• AdvancedRBCparameters:%microcyticRBC’s,Reticulocyte

Hemoglobincontent,…..• ReticulatedPlatelets/ImmaturePlatelets• ……….

Furtheradvancesinautomation

• Multiple‘inline’analysers– Built-inSlide-Maker-Stainer– Auto-validationinLIS– Automatedreflex/morphologyetc.

• Includingdigitalmorphologyorflow-cytometry solutions

– AllFullbloodanalysis“inline”(ESR,HbA1c,…..)

TotalLaboratoryAutomation DigitalMorphology

Validation ofanewanalyzer:goal

• Provision ofobjective evidence that ahematologyanalyzerfulfills specified requirements (where thespecifiedrequirements areadequatefor intended use).

• Validation isprimarily amanufacturersresponsibility toensure that designgoalsaremetand performanceclaimsarestated (including safety and effectiveness).

• Validation study (manufacturer): collectdatatosupportaregulatorysubmissionandtheregistrationofahematologyanalyzer.

Validation ofanewanalyzer:goal

• Provision ofobjective evidence that ahematologyanalyzerfulfills specified requirements (where thespecifiedrequirements areadequatefor intended use).

• Validation isprimarily amanufacturersresponsibility toensure that designgoalsaremetand performanceclaimsarestated (including safety and effectiveness).

• Validation study (manufacturer): collectdatatosupportaregulatorysubmissionandtheregistrationofahematologyanalyzer.

Validationofanewanalyzer(manufacturer)

• Objectives ofavalidation study:– Generate datato assess safety and clinical efficacy from amedical perspective– Develop performanceinformationfor labelingandmarketingpurposes– Validate appropriate operationalperformancecharacteristics ofthe

hematology analyzerinatypical end-usersetting– Develop datathat areused to supportsubmissionoftheproductfor approval

orclearanceby regulatory bodies

Source:CLSIH26A2

• Validation performancespecifications:– Limitofblank(LoB,background)– Carryover– Imprecision (reproducibility),shorttermand long-term– Analytical measuring interval(AMI)(linearity)– Lower limitofdetection (LLoD)and lower limitofquantitation (LLoQ)– Comparibility (correlation)– Interferences– Frequency and typeofdatainvalidations

Source:CLSIH26A2

• Performancespecifications:current referencemethods*:– Selective microscopy for WBCdifferential– RBCcount andWBCcount (impedance)– Selective microhematocrit (PCV)for hematocrit (HCT)– Hemoglobin by hemiglobincyanidemethod– Selective PLTmonoclonalantibody (contemporary referencemethod)– Reticulocytes by flowcytometry

Source:CLSIH26A2

*Standardized and independent ofmanufacturer

• Performancespecifications:current referencemethods:– When noreferencemethod isavailable:usually comparisonwith previous

generation instrument

Source:CLSIH26A2

• Current referencemethods,problems,need for improvement:– Current referencemethods aremoreorless outdated

• e.g.:microscopic 400cell WBCdifferential count:elaborative and imprecise

Huismanetal.Clin LabMed 2015

– Noreferencemethods available for clinically relevantparameters*:• MCV• ExtendedRBCparameters• Reticulocyte Hb content• MPV• Reticulated platelets/immatureplatelet fraction• ….

• *Clinically relevantparametersarenot standardized!– ® This may leadto differences between differentHematology analyzers:

– Differences inclinical interpretation (confusion for clinicians)– Mayprevent wider use– Invalid aggregation ofdata

– Noreferencemethods available for clinically relevantparameters:• MCV• ExtendedRBCparameters• Reticulocyte Hb content• MPV• Reticulated platelets/immatureplatelet fraction• ….

• Urgentneed for improvement,role for professionalsocieties

Verification

InstrumentVerification by the enduserlaboratory

• Theendusershould asseswhether the manufacturers claimsonperformanceofthe specific instrumentalso apply to the“intended use criteria”setby the laboratory:Verification

• Theverification process includes performanceanalysisof:– Accuracy– Precision– Reportable rangeoftestresults and reference intervals (normal ranges)– Background(limitofblank)– Carryover (sample)– Lower limits ofdetection– Quantitation– Clinically reportable intervals (CRIs)

– Accordingto ISO15189acertain levelofverification ofanynew(hematology)analyzerhasto be done (preferably)according toaprofessionalstandard.

– ISO15189also stimulates laboratories to takepatient riskfactorsinto consideration tomeetthesestandards

– There arecurrenly 2internationaldocuments available forverification ofahematology analyzer:• Consensusdocuments with recommendations (not based onstrong

evidence butrather on“expertopinion”).

– Accordingto ISO15189acertain levelofverification ofanynew(hematology)analyzerhasto be done (preferably)according toaprofessionalstandard.

– ISO15189also stimulates laboratories to takepatient riskfactorsinto consideration tomeetthesestandards

– There arecurrenly 2internationaldocuments available forverification ofahematology analyzer:• Clinical Laboratory StandardsInstitute (CLSI)(standardH26A2)

published in2010• InternationalCouncilonStandardisation inHematology (ICSH)

guidelinepublished in2014.

Consensusdocuments with recommendations

CLSI ICSH(publishedintheInternationalJournalofLaboratoryHematology,theofficialISLH journal

CLSI ICSHVerification Thelaboratory(enduser)

should followtheproceduresofmanufacturervalidation,buttheverificationmaybeabbreviated;thegoalistoverifythatthemanufacturer’sstatedperformance iscorrect

Verification isconformationoftheevaluationperformedbythemanufacturerorpublished intheliterature;theverificationmaybeabbreviated(ie,focusedtomeetspecificrequirementsatthetestsite)

Precision/imprecision(within-run reproducibility,closeness ofagreementbetween testresults

Should be performed withnormal samplesand samplesatmedical decision levelsavailable inclinicallaboratories.Nospecifications for number ofmeasurements and reporting

Single runof10measurements onthe samesample(all reportedparameters),3levels(normal, abnormal low,andabnormal high,aroundclinical decision points).Reported asSDand %CV

CLSI ICSHVerification Thelaboratory(enduser)

should followtheproceduresofmanufacturervalidation,buttheverificationmaybeabbreviated;thegoalistoverifythatthemanufacturer’sstatedperformance iscorrect

Verification isconformationoftheevaluationperformedbythemanufacturerorpublished intheliterature;theverificationmaybeabbreviated(ie,focusedtomeetspecificrequirementsatthetestsite)

Precision/imprecision(within-run reproducibility,closeness ofagreementbetween testresults

Should be performed withnormal samplesand samplesatmedical decision levelsavailable inclinicallaboratories.Nospecifications for number ofmeasurements and reporting

Single runof10measurements onthe samesample(all reportedparameters),3levels(normal, abnormal low,andabnormal high,aroundclinical decision points).Reported asSDand %CV

CLSI ICSHPrecision(betweenbatch,longterm

Should beperformedwithnormalsamplesandsamplesatmedicaldecision levelsavailableinclinicallaboratories.Nospecificationsfortimeperiodandnumberofmeasurements

Single sample,repeateddaily,foraperiodof20–30d.Threelevels(allparameters,abnormallowandabnormalhigh,aroundclinicaldecisionpoints).Fixedblood(controlmaterial)mayberequired

Comparability(comparison betweenevaluation HAandcurrent HA)

Should be performedbutnot specified, ifdifferentmodesareavailable,whether anextensive mode-to modecomparability should beperformed

Atleast 250–300samples,measured onboth HAwith sampleswith various disordersand with interferingsubstances

CLSI ICSHPrecision(betweenbatch,longterm

Should be performedwith normal samplesand samplesatmedicaldecision levelsavailableinclinical laboratories.Nospecifications fortimeperiod and numberofmeasurements

Single sample,repeateddaily,for aperiod of20–30d.Threelevels(allparameters,abnormallowand abnormal high,around clinical decisionpoints).Fixed blood(controlmaterial)maybe required

Comparability(comparisonbetweenevaluationHAandcurrentHA)

Should beperformedbutnotspecified, ifdifferentmodesareavailable,whetheranextensivemode-tomodecomparabilityshouldbeperformed

Atleast 250–300samples,measured onboth HAwith sampleswith various disordersand with interferingsubstances

CLSI ICSHAccuracy (closeness ofagreementbetweenmeasurement and truevalue)

Should be performed;not otherwise specified

Depending onavailabilityofreferencemethod,often notapplicable;inpracticeoften compared withcurrent HA

Referenceintervals Mustbe established orverified for all reportableparameters

Should be calculated forall components oftheCBC;atleast 120samplesfrom apparentlyhealthy individuals (60male,60female)

CLSI ICSHAccuracy (closeness ofagreementbetweenmeasurement and truevalue)

Should be performed;not otherwise specified

Depending onavailabilityofreferencemethod,often notapplicable;inpracticeoften compared withcurrent HA

Referenceintervals Mustbe established orverified for all reportableparameters

Should be calculated forall components oftheCBC;atleast 120samplesfrom apparentlyhealthy individuals (60male,60female)

• Samples:– Bewareofpre-analytical variables!

• Timeisone ofthe mostimportantvariablesdue to timedependentalterations ofcells (volumeand morphology)

– Usually anonymous surplusmaterial– Broad rangeofunderlyingpathology (garantee that all cells are

“recognized”)– Results should encompass the entire analytical range(very low

(e.g.extremetrombocytopenia)to very highlevels(e.g.extremeleucocytosis /CML).

– Ageand gender(pediatric samples?)– All typesoftubesthatmay enterthe laboratory

HuismanetalClin LabMed 2015

InstrumentVerification by the enduserlaboratory:Precision

Accuracyistheproximityofameasurementresulttothetruevalueandmainlydependentonsystematicerror(theterm‘bias’shouldbeavoided);precisionisthereproducibilityofthemeasurementsandmainlydependentonrandomerror.

HuismanetalIJKH2016

Precision

• Thedesired precision ofaCBCparameterisdependentonthe biological variation ofthis parameter– Work ofGeorgeCembrowski etal

• (Clin LabMed 2015;IJLH2016).

Cembrowski etalIJLH2016

Precision:

• Inorderto generate (clinically)relevantresults the analyticalprecision (%CVreproducibility)ofaCBCresult should be lessthan halfand preferable less than ¼ofthe biologicalvariation.

• Stateofthe artpossibilities ofcurrentgeneration ofHA

Deanalytical %CVdoesaddto the total %CV

Roomfor improvement:ReticulocytesWBCWBCdifferential

• Carry-over,isit important?• Background,isit important?

• Carry-over,isit important?– Example:carry over1%,

• Sample1:platelet count 1000x109/L• Sample2:platelet count 10x10exp9/L(subsequent sample)• Acarry-overof1%will result inafalsely increased platelet count of

20x109/L(100%increase)

• Background,isit important?– Abackground(for example intheWBCchannel)can leadto

“false positive”results inCSFsamples

Reference:TracyGeorgeetal.

QualityControl

• InternalQualityControl– Performedonadailybasis– Electronic‘builtin’qualitycontrolflags

• Aretheremechanical/electronical problems?

– Controlmaterialsoftenprovidedbymanufacturer• Usually3-controllevels(low-/medium-/high- level)

– ~Widerange• Oftenmanipulatedblood• 1versusmultipletimes/daydependingonstabilityofanalyser• Comparisonbetweendifferentanalysers possible• Expensive• 3.5SD(Cembrowski etal)

QualityControl

• InternalQualityControl– StatisticalQCbasedongeneratedresults

• MovingAverage– BuiltinsoftwareorLIS– Largeworkloadrequired– AlwaysAvailable– Cheap

QualityControl

• ExternalQualityControl/Proficiencytesting– Severaltimes/year– Howdoyouperformincomparisonwithotherlabsandother

typesofequipment?– ¹Calibrator

Acknowledgements:

• JolandeVis• SueEllenVerbrugge

Thanks!

Contact:

Dr AlbertHuismanUniversityMedicalCenterUtrechtDepartmentofClinicalChemistryandHaematology G.03.550Heidelberglaan 1003584CXUtrechtNetherlands

Email:a.Huisman@umcutrecht.nl

Thank you for being with us!

Seeyou atISLH2017inHonolulu,Hawai

ISLH2017

Seeyou atISLH2017inHonolulu,Hawai

ISLH2017May4-62017 www.islh.org

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