应用DHPLC技术进行诊断性分析的质量保证体系

应用DHPLC技术进行诊断性分析的质量保证体系张泽云美国环球基因公司中国代表处诊断性分析的要求临床分子遗传学分析的复杂性临床分子检测结果的一致性和精确性变性高效液相色谱(DHPLC)作为一种高效和敏感的基因突变检测技术DHPLC技术质量控制AMERICANCOLLEGEOFMEDICALGENETICSStandardsandGuidelinesforClinicalGeneticsLaboratories2005EditionG:CLINICALMOLECULARGENETICSTheseStandardsandGuidelinesspecificallyrefertotheuseofmoleculartechniquestoexamineheritableorsomaticchangesinthehumangenome.G18DenaturingHighPerformanceLiquidChromatography(dHPLC)(SectionAddedNovember2003)CMGSBestPracticeGuidelinesUseoftheWAVESysteminDiagnosticServicePreparedandeditedbyJohnHarvey,NationalGeneticsReferenceLaboratory(Wessex),Salisbury,UKandElsSchollen,CentreforHumanGenetics,Leuven,Belgiumlastupdate:12March2004•Introduction•Laboratoryprocess•DHPLCsystem•Dataquality•Checking&reportingguidelines•ReferencesDHPLCSOPsInstrumentormaintenanceSOPTechniqueGeneralDHPLCSOP–WAVE3500,3500HTMethodDisease-specificSOPs–Rett,BRCA,HNPCCMarfan,…Applicationcompany+usersgeneralusers+companyspecificusersSupplementaryAppendix1STANDARDOPERATINGPROCEDUREWAVE®SystemOperationandMaintenanceSOP-O&MWAVE®SystemOperationandMaintenanceForWAVE®SystemModels3500,3500Aand3500HTWAVE®SystemOperationandMaintenanceAnalysisoftheWAVE®Low&HighRangeMutationStandardsThemaintenanceprocedureDNASep®andDNASep®HTcartridgemaintenanceRoleofMutationstandards:checkingofcorrectfunctioningoftheWAVE®System,includingovencalibration,cartridgeperformance,buffercompositionandstability,toensurereproducibilityandaccuracyofthechromatographicanalysis.Mutationstandardsberunwhen:lTheroutinepre-run,lWeeklyandmonthlymaintenanceprocedure,lAfterreplacementofanycomponent,lValidationforanewbatch,lAsanassaycontrol,atthebeginningandendofeveryrun,preferablyalsoafterevery100injectionsforlongruns.AnalysisoftheWAVE®Low&HighRangeMutationStandards020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43RT-Het1RT-Hom2Δ-HetΔ-HomA020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43020903standaarden02/09Time(Minutes)2.152.12.0521.951.91.851.81.751.71.651.61.55Absorbance(mV)6543210-10.431.381.451.511.661.751.972.042.43RT-Het1RT-Hom2Δ-HetΔ-HomANormalrangesofthemutationstandardsThemaintenanceprocedure3.1Filterandflush3.2Pre-runmaintenance3.3Weeklymaintenance3.4Quarterlymaintenance3.5Othermaintenanceoperations3.6PreventativemaintenanceprocedureandsystemvalidationFilterandflushTheprincipleoffiltrationinvolvespreventingunwantedcontaminantsfromenteringthesystem.Filtrationappliestotwospecificareas:solventfiltrationandin-linefiltration.Thesystemflushingistoremovemobilephasesaltcomponentsthatcanprecipitateunderstrongsolventconditions.Pre-runmaintenance1.Buffercheck2.Injectionsystemwashing3.Pressurecheck4.Checktheabsorbanceonthedetector5.PurgethelinesWeeklymaintenanceInlinefilterreplacementCheckthesyringeQuarterlymaintenanceCheckUVlampUVlampreplacementCleaningthesystem(Isopropanolcleaning)DNASep®andDNASep®HTcartridgemaintenance1.RegularmaintenancescheduleEvery96-192injections:ExtendedhotwashEvery1000injections:ReversehotwashDNASep®wash(ifreversehotwashfailstoresolvemutationstandards)2.Short-termcartridgestorage3.Long-Termcartridgestorage4.NewcartridgeinstallationDailyMaintenanceEquilibratethecartridge–50%A50%Bfor15minutes–Run1-2blanksVerifysystemperformance(pre-analysis)–RunastandardRunSamplesVerifysystemperformance(postanalysis)WeeklyMaintenanceAnExtendedActiveCleanWashisrecommendedevery~100injections.(Usuallydoneaftereach96wellplate)OvenSetto:80°CPumpSetto:100%D15-30minutesWASHOvenSetto:56°CPumpSetto:50%A-50%BEQUILIBRATE45-90minutesRunStandardstoVerifySystemPerformance!1000InjectionMaintenanceAReverseHotWashisrecommendedevery~1000injection