CONCURRENT 10: THE COMPLETE BLOOD COUNT 10: the complete blood count and beyond: ... the complete blood count and beyond: quality issues and reference methods ... further advances in automation

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CONCURRENT10:THECOMPLETEBLOODCOUNTANDBEYOND:QUALITYISSUESANDREFERENCEMETHODSSaturdayMay14th 20161:30 3:00PM(13.30 15.00hrs)InternationalSocietyforLaboratoryHematology (ISLH)CONCURRENT10:THECOMPLETEBLOODCOUNTANDBEYOND:QUALITYISSUESANDREFERENCEMETHODSCHAIRS:Dr.PieroCappelletti.SIPMEL,Pordenone, ItalyDr.AlbertHuisman,UMCUtrecht,Utrecht,NetherlandsInternationalSocietyforLaboratoryHematology (ISLH)VerificationandQualityControlofautomatedHematologyAnalyzersDr.AlbertHuisman,UMCUtrecht,Utrecht,NetherlandsInternationalSocietyforLaboratoryHematology (ISLH)VerificationandQualityControlofautomatedHematologyAnalyzersDr.AlbertHuisman,UMCUtrecht,Utrecht,NetherlandsInternationalSocietyforLaboratoryHematology (ISLH)Disclosure information:Dr AlbertHuisman:Nothing to discloseTheUMCUtrecht,department ofClinical ChemistryandHematology hasreceived fundingfor contractresearchfrom:Abbott DiagnosticsBeckman-CoulterDiagnostic GrifolsMechatronicsVerificationandqualitycontrolofautomatedHematologyAnalyzers 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?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 EtcteteraBecause: TheISO15189standard[Medicallaboratories Particularrequirementsforqualityandcompetence]requiresaverificationprocessAdvantagesofAutomatedCellAnalysers: 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 thanmanualmethod .Automatedcellcounters: CompleteBloodCount(CBC): Hemoglobin(Hb)concentration,RBCcount&RBCindices(MCV,MCH,MCHC),WBCcount,PLTcount WBCdifferentialcount(5normalWBCs) RDW,MPV Reticulocytecount NucleatedRBCcount(NRBCs) .Flagging,etc etc Newparameters: AdvancedRBCparameters:%microcyticRBCs,ReticulocyteHemoglobincontent,.. ReticulatedPlatelets/ImmaturePlatelets .Furtheradvancesinautomation Multipleinlineanalysers Built-inSlide-Maker-Stainer Auto-validationinLIS Automatedreflex/morphologyetc. Includingdigitalmorphologyorflow-cytometry solutions AllFullbloodanalysisinline(ESR,HbA1c,..)TotalLaboratoryAutomation DigitalMorphologyValidation 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 ofthehematology analyzerinatypical end-usersetting Develop datathat areused to supportsubmissionoftheproductfor approvalorclearanceby regulatory bodiesSource:CLSIH26A2Validationofanewanalyzer(manufacturer) 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 typeofdatainvalidationsSource:CLSIH26A2Validationofanewanalyzer(manufacturer) 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 flowcytometrySource:CLSIH26A2*Standardized and independent ofmanufacturerValidationofanewanalyzer(manufacturer) Performancespecifications:current referencemethods: When noreferencemethod isavailable:usually comparisonwith previousgeneration instrumentSource:CLSIH26A2Validationofanewanalyzer(manufacturer) Current referencemethods,problems,need for improvement: Current referencemethods aremoreorless outdated e.g.:microscopic 400cell WBCdifferential count:elaborative and impreciseHuismanetal.Clin LabMed 2015Validationofanewanalyzer(manufacturer) Current referencemethods,problems,need for improvement: Current referencemethods aremoreorless outdated e.g.:microscopic 400cell WBCdifferential count:elaborative and imprecise 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 ofdataHuismanetal.Clin LabMed 2015Validationofanewanalyzer(manufacturer) Current referencemethods,problems,need for improvement: Current referencemethods aremoreorless outdated e.g.:microscopic 400cell WBCdifferential count:elaborative and imprecise Noreferencemethods available for clinically relevantparameters: MCV ExtendedRBCparameters Reticulocyte Hb content MPV Reticulated platelets/immatureplatelet fraction . Urgentneed for improvement,role for professionalsocietiesHuismanetal.Clin LabMed 2015VerificationInstrumentVerification by the enduserlaboratory Theendusershould asseswhether the manufacturers claimsonperformanceofthe specific instrumentalso apply to theintended use criteriasetby the laboratory:VerificationHuismanetal.Clin LabMed 2015InstrumentVerification by the enduserlaboratory 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)Huismanetal.Clin LabMed 2015InstrumentVerification by the enduserlaboratory 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 onstrongevidence butrather onexpertopinion).Huismanetal.Clin LabMed 2015InstrumentVerification by the enduserlaboratory 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.Huismanetal.Clin LabMed 2015Consensusdocuments with recommendationsCLSI ICSH(publishedintheInternationalJournalofLaboratoryHematology,theofficialISLH journalInstrumentVerification by the enduserlaboratoryCLSI ICSHVerification Thelaboratory(enduser)should followtheproceduresofmanufacturervalidation,buttheverificationmaybeabbreviated;thegoalistoverifythatthemanufacturersstatedperformance iscorrectVerification isconformationoftheevaluationperformedbythemanufacturerorpublished intheliterature;theverificationmaybeabbreviated(ie,focusedtomeetspecificrequirementsatthetestsite)Precision/imprecision(within-run reproducibility,closeness ofagreementbetween testresultsShould be performed withnormal samplesand samplesatmedical decision levelsavailable inclinicallaboratories.Nospecifications for number ofmeasurements and reportingSingle runof10measurements onthe samesample(all reportedparameters),3levels(normal, abnormal low,andabnormal high,aroundclinical decision points).Reported asSDand %CVInstrumentVerification by the enduserlaboratoryCLSI ICSHVerification Thelaboratory(enduser)should followtheproceduresofmanufacturervalidation,buttheverificationmaybeabbreviated;thegoalistoverifythatthemanufacturersstatedperformance iscorrectVerification isconformationoftheevaluationperformedbythemanufacturerorpublished intheliterature;theverificationmaybeabbreviated(ie,focusedtomeetspecificrequirementsatthetestsite)Precision/imprecision(within-run reproducibility,closeness ofagreementbetween testresultsShould be performed withnormal samplesand samplesatmedical decision levelsavailable inclinicallaboratories.Nospecifications for number ofmeasurements and reportingSingle runof10measurements onthe samesample(all reportedparameters),3levels(normal, abnormal low,andabnormal high,aroundclinical decision points).Reported asSDand %CVInstrumentVerification by the enduserlaboratoryCLSI ICSHPrecision(betweenbatch,longtermShould beperformedwithnormalsamplesandsamplesatmedicaldecision levelsavailableinclinicallaboratories.NospecificationsfortimeperiodandnumberofmeasurementsSingle sample,repeateddaily,foraperiodof2030d.Threelevels(allparameters,abnormallowandabnormalhigh,aroundclinicaldecisionpoints).Fixedblood(controlmaterial)mayberequiredComparability(comparison betweenevaluation HAandcurrent HA)Should be performedbutnot specified, ifdifferentmodesareavailable,whether anextensive mode-to modecomparability should beperformedAtleast 250300samples,measured onboth HAwith sampleswith various disordersand with interferingsubstancesInstrumentVerification by the enduserlaboratoryCLSI ICSHPrecision(betweenbatch,longtermShould be performedwith normal samplesand samplesatmedicaldecision levelsavailableinclinical laboratories.Nospecifications fortimeperiod and numberofmeasurementsSingle sample,repeateddaily,for aperiod of2030d.Threelevels(allparameters,abnormallowand abnormal high,around clinical decisionpoints).Fixed blood(controlmaterial)maybe requiredComparability(comparisonbetweenevaluationHAandcurrentHA)Should beperformedbutnotspecified, ifdifferentmodesareavailable,whetheranextensivemode-tomodecomparabilityshouldbeperformedAtleast 250300samples,measured onboth HAwith sampleswith various disordersand with interferingsubstancesInstrumentVerification by the enduserlaboratoryCLSI ICSHAccuracy (closeness ofagreementbetweenmeasurement and truevalue)Should be performed;not otherwise specifiedDepending onavailabilityofreferencemethod,often notapplicable;inpracticeoften compared withcurrent HAReferenceintervals Mustbe established orverified for all reportableparametersShould be calculated forall components oftheCBC;atleast 120samplesfrom apparentlyhealthy individuals (60male,60female)InstrumentVerification by the enduserlaboratoryCLSI ICSHAccuracy (closeness ofagreementbetweenmeasurement and truevalue)Should be performed;not otherwise specifiedDepending onavailabilityofreferencemethod,often notapplicable;inpracticeoften compared withcurrent HAReferenceintervals Mustbe established orverified for all reportableparametersShould be calculated forall components oftheCBC;atleast 120samplesfrom apparentlyhealthy individuals (60male,60female)InstrumentVerification by the enduserlaboratory Samples: Bewareofpre-analytical variables! Timeisone ofthe mostimportantvariablesdue to timedependentalterations ofcells (volumeand morphology) Usually anonymous surplusmaterial Broad rangeofunderlyingpathology (garantee that all cells arerecognized) 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 laboratoryInstrumentVerification by the enduserlaboratoryHuismanetalClin LabMed 2015InstrumentVerification by the enduserlaboratory:PrecisionAccuracyistheproximityofameasurementresulttothetruevalueandmainlydependentonsystematicerror(thetermbiasshouldbeavoided);precisionisthereproducibilityofthemeasurementsandmainlydependentonrandomerror.HuismanetalIJKH2016Precision Thedesired precision ofaCBCparameterisdependentonthe biological variation ofthis parameter Work ofGeorgeCembrowski etal (Clin LabMed 2015;IJLH2016).Cembrowski etalIJLH2016Precision: Inorderto generate (clinically)relevantresults the analyticalprecision (%CVreproducibility)ofaCBCresult should be lessthan halfand preferable less than ofthe biologicalvariation. Stateofthe artpossibilities ofcurrentgeneration ofHAInstrumentVerification by the enduserlaboratoryInstrumentVerification by the enduserlaboratoryDeanalytical %CVdoesaddto the total %CVInstrumentVerification by the enduserlaboratoryRoomfor improvement:ReticulocytesWBCWBCdifferentialInstrumentVerification by the enduserlaboratory Carry-over,isit important? Background,isit important?InstrumentVerification by the enduserlaboratory 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 of20x109/L(100%increase)InstrumentVerification by the enduserlaboratory Background,isit important? Abackground(for example intheWBCchannel)can leadtofalse positiveresults inCSFsamplesInstrumentVerification by the enduserlaboratoryReference:TracyGeorgeetal.QualityControl InternalQualityControl Performedonadailybasis Electronicbuiltinqualitycontrolflags 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 CheapQualityControl ExternalQualityControl/Proficiencytesting Severaltimes/year Howdoyouperformincomparisonwithotherlabsandothertypesofequipment? CalibratorAcknowledgements: JolandeVis SueEllenVerbruggeThanks!Contact:Dr AlbertHuismanUniversityMedicalCenterUtrechtDepartmentofClinicalChemistryandHaematology G.03.550Heidelberglaan 1003584CXUtrechtNetherlandsEmail:a.Huisman@umcutrecht.nlThank you for being with us!Seeyou atISLH2017inHonolulu,HawaiISLH2017Seeyou atISLH2017inHonolulu,HawaiISLH2017May4-62017 www.islh.org

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