concurrent 10: the complete blood count … · concurrent 10: the complete blood count and beyond:...
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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
InternationalSocietyforLaboratoryHematology (ISLH)
VerificationandQualityControlofautomatedHematologyAnalyzers
Dr.AlbertHuisman,UMCUtrecht,Utrecht,Netherlands
InternationalSocietyforLaboratoryHematology (ISLH)
VerificationandQualityControlofautomatedHematologyAnalyzers
Dr.AlbertHuisman,UMCUtrecht,Utrecht,Netherlands
InternationalSocietyforLaboratoryHematology (ISLH)
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?
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
Validationofanewanalyzer(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 typeofdatainvalidations
Source:CLSIH26A2
Validationofanewanalyzer(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 flowcytometry
Source:CLSIH26A2
*Standardized and independent ofmanufacturer
Validationofanewanalyzer(manufacturer)
• Performancespecifications:current referencemethods:– When noreferencemethod isavailable:usually comparisonwith previous
generation instrument
Source:CLSIH26A2
Validationofanewanalyzer(manufacturer)
• Current referencemethods,problems,need for improvement:– Current referencemethods aremoreorless outdated
• e.g.:microscopic 400cell WBCdifferential count:elaborative and imprecise
Huismanetal.Clin LabMed 2015
Validationofanewanalyzer(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 ofdata
Huismanetal.Clin LabMed 2015
Validationofanewanalyzer(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 professionalsocieties
Huismanetal.Clin LabMed 2015
Verification
InstrumentVerification by the enduserlaboratory
• Theendusershould asseswhether the manufacturers claimsonperformanceofthe specific instrumentalso apply to the“intended use criteria”setby the laboratory:Verification
Huismanetal.Clin LabMed 2015
InstrumentVerification 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 2015
InstrumentVerification 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 onstrong
evidence butrather on“expertopinion”).
Huismanetal.Clin LabMed 2015
InstrumentVerification 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 2015
Consensusdocuments with recommendations
CLSI ICSH(publishedintheInternationalJournalofLaboratoryHematology,theofficialISLH journal
InstrumentVerification by the enduserlaboratory
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
InstrumentVerification by the enduserlaboratory
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
InstrumentVerification by the enduserlaboratory
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
InstrumentVerification by the enduserlaboratory
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
InstrumentVerification by the enduserlaboratory
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)
InstrumentVerification by the enduserlaboratory
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)
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 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
InstrumentVerification by the enduserlaboratory
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
InstrumentVerification by the enduserlaboratory
InstrumentVerification by the enduserlaboratory
Deanalytical %CVdoesaddto the total %CV
InstrumentVerification by the enduserlaboratory
Roomfor improvement:ReticulocytesWBCWBCdifferential
InstrumentVerification 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 of
20x109/L(100%increase)
InstrumentVerification by the enduserlaboratory
• Background,isit important?– Abackground(for example intheWBCchannel)can leadto
“false positive”results inCSFsamples
InstrumentVerification by the enduserlaboratory
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:[email protected]
Thank you for being with us!
Seeyou atISLH2017inHonolulu,Hawai
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