CD157在PNH诊断中的应用.ppt

CD157鉴定粒/单核细胞PNH克隆,唐古生副主任医师、副教授上海长海医院血液内科、全军血液病研究所,概要,PNH概念、发病机制和临床表现PNH流式诊断指标变迁及现状流式PNH诊断的临床应用建议CD157对PNH临床诊断应用的评价,WhatisPNH,AnacquiredclonaldisorderresultingfromasomaticmutationoftheX-linkedphosphatidylinositolglycancomplementationClassA(PIGA)inahematopoieticstemcellThisgeneencodesanenzymethatcatalyzesthefirststepinthebiosyntheticpathwayoftheglycosylphosphatidylinositol(GPI)anchorExpansionofthiscloneisresponsiblefortheclinicalmanifestationsofthedisease,EuropeanJournalofHaematology,2015,Eculizumab,ConsequencesofHemolysisinPNH,DefectiveRedCellsLackComplementDefenseProteins,AttackbyComplement,Anemia,IntravascularHemolysisandReleaseofCellContents,Hemoglobinemia,IronLoss,Hemoglobinuria,Nephrotoxicity,Nitricoxidesequestration,IncreasedLactateDehydrogenase,MusclespasmandabdominalpainVascularsmoothmuscleeffectPulmonaryhypertensionPlateletactivationandthrombosis,PathogenesisofPNH,Deficiencyofcomplementregulatoryproteins,isresponsiblefornearlyalltheimportantpathologicfindingsinPNHpatientsHemolyticanemiaThrombosisPainRenalfailureBonemarrowfailureappearstooccurbyadifferentmechanism,ClassificationschemeforPNH,ClassicalPNH,whichincludeshemolyticandthromboticpatients;PNHinthecontextofotherprimarydisorders,suchasaplasticanemiaormyelodysplasticsyndrome;SubclinicalPNH,inwhichpatientshavesmallPNHclonesbutnoclinicalorlaboratoryevidenceofhemolysisorthrombosis.,Blood2005;106:36993709InternationalPNHInterestGroup(I-PIG),DiagnosisofPNH,Hamtest,sucrosehemolysistestandthecomplementlysissensitivitytestAntibodiesagainstCD55andCD59onRBCswerethemostcommonflowmethodusedtodiagnosePNHuntilrecentlyFlowcytometrynowuniversallyacceptedasthebestmethodofchoiceofdiagnosingPNH,TheProblemWithCD55/CD59,Sentout18specimensto92laboratoriesusingstabilizedwholebloodmaterial10hadPNHcloneswithexpectedvaluesrangingfromabout10%to90%,8normalspecimens,ResultsofinitialEQAstudy,LabsusingonlyCD55andCD59forgranulocyteanalysisperformedpoorlyFalsenegativerateof19%andfalsepositiverateof17.6%Tenlabsfalselydetectedclonesin50%ofnormalspecimensThus,CD55andCD59arenotdesirablereagentsfordetectingPNH,Detecting1%clonesizesAdequateforpatientswithlargeclonesassociatedwithhemolytic/thromboticPNHTestingonlyredcellsmaymisspatientsPossibletotestbothmonocytesandgranulocytesasoneservesasacheckfortheotherLymphocytesnotasuitabletarget,RoutineTesting,Highsensitivityassays,Detectingclonesassmallas0.01%(orevenless)Upto40%patientswithaplasticanemiahaveidentifiablePNHpopulationsIdentificationisimportantbecauseabout20-30%ofpatientswithaplasticanemiaprogresstoPNHHighsensitivityflowcytometryisneededtodetectthesesmallclones,Prevalence,PNHclonesinAA,MDSandotherBMF,exceptclinicalPNH.PNHclones1%weredetectedin199ofall5398patients(3.7%);18.5%inAA;1.1%MDS;2.3%inotherBMF.PNHclones0.01%in167of1746patientsfromallgroups(9.6%);1.8%inMDS;39.5%inAA,and7.8%inotherBMFpatients.Upto35%ofPNHpatientsdiewithin5yearsofdiagnosis,andupto50%ofpatientsdiewithin1015yearsofdiagnosis.,AProspectiveMulticenterStudyofParoxysmalNocturnalHemoglobinuriaCellsinPatientswithBoneMarrowFailure,Highsensitivityassays,SensitivityisdeterminedbyanumberoffactorsNumberofeventscollectedBackgroundrate(frequencyofabnormalcellsinnormalpopulations)DifferencebetweennormalandabnormalpopulationNeedtocombinetwoGPI-linkedWBCmarkerstomaximizesensitivityandspecificity,Howmanycellstocollect,Collecting250,000cellsofinterest(i.e.granulocytes)andrequiring25eventstoidentifyapopulationwouldallowasensitivityof0.01%withaprecisionofabout25%;50eventsoutof500,000givesaprecisionof14%However,itispossibletoscreenfewercellsIfthereare0cellswithlossofGPIanchorsoutof50,000events,99+%probabilitythattruefrequencyis2万。,结果,纳入骨髓或外周血标本合计131，其中15份阳性标本，来自9例阳性患者（PNH克隆大小：0.19%-93.5%）Flaer/CD157组合检出全部PNH阳性患者，敏感性、特异性、准确度均达到100%。CD157/Flaer组合检出的PNH克隆与其他组合检出的克隆大小一致。本研究中常见（69.8%，88/126）单核细胞CD14弱表达而Flaer表达正常，外周血与骨髓标本类似。,PNH阴性标本,PNH阳性微小克隆,PNH阳性克隆,CD157异常证实可疑PNH克隆,1例MDS患者存在单核细胞和红细胞PNH克隆，粒细胞未见Flaer/CD24低表达克隆，该患者CD157在粒、单核细胞表面均不表达。,7例标本粒/单核细胞表面CD157表达