流式细胞仪器参数设置与追踪(cst)

23-13536-00Rev.01CytometerPerformance—CS&TCytometerPerformance—CS&T23-13536-00Rev.012KnowYourCytometerCytometerCharacterizationForeachfluorescenceparameter:•DetectorEfficiency—Qr•BackgroundSignal—Br•Resolution(highsignal)—%rCVforabrightparticle•LinearRange—LinearityMinandMaxLimits•StandardDeviationofElectronicNoise—SDENCytometerPerformance—CS&T23-13536-00Rev.013DetectionEfficiency—QrQrisproportionaltoQ=#fluorochromemolecules#photoelectronsCytometer1APCdetectorphotoelectronsCytometer2APCdetectorphotoelectronsWhichcytometerhashigherQrforAPCdetection?CytometerPerformance—CS&T23-13536-00Rev.014WhyisQrImportant?•HigherQr→moresensitivity.•Whenotherfactorsareheldconstant,sensitivity(asquantifiedbystainindex)isproportionalto.LowQrHighQrQrCytometerPerformance—CS&T23-13536-00Rev.015FactorsAffectingDetectorEfficiency•Laserpowerandalignment•Opticalefficiency•Opticscleanlinessordamage•PMTredspectrumsensitivity•PoorPMTperformance(age)•Dirtyflowcell•DirtyordegradedfilterCytometerPerformance—CS&T23-13536-00Rev.016Brisameasureofopticalbackground.UnboundantibodyorfluorochromeSpectraloverlapfromanothermarkeronacellAutofluorescenceRamanscatterScatterfromtheflowcellandambientlightBackgroundSignal—BrBristhenumberoffluorochromemoleculesthatwouldproduceasignalequivalenttothebackgroundsignal.CytometerPerformance—CS&T23-13536-00Rev.017WhyisBrImportant?•HigherBr→lesssensitivity.•Whenotherfactorsareheldconstant,sensitivity(asquantifiedbystainindex)isproportionalto.LowBrHighBr1BrCytometerPerformance—CS&T23-13536-00Rev.018FactorsAffectingBr•Dirtyflowcell.•Damagedopticalcomponent.•BluelaserparametersFITCandPEexhibithighBrduetoRamanscatter.•VioletlaserparametersexhibithighBrduetoautofluorescence.CytometerPerformance—CS&T23-13536-00Rev.019QrandBr:ALookatSomeRealDataHighQr—LowBrHighQr—HighBrLowQr—HighBr•QrandBrcharacterizationsareforbothFITCandPerCP-Cy5.5parameters.•PlotsgatedonCD3+lymphocytes.CytometerPerformance—CS&T23-13536-00Rev.0110•Resolutionistheabilitytodistinguishbetweenpopulationsbasedonfluorescense.•Resolutionisakeyfactorinsensitivity.ResolutionGoodResolutionPoorResolutionFluorescenceIntensity•Apracticalmeasureofresolutionisthe%rCVofareferenceparticleinthemidtoupperendofthescale.CytometerPerformance—CS&T23-13536-00Rev.0111CytometerFactorsAffectingResolution•Uniformexcitationofparticles−Laserpower−Laserbeamshapeandalignment−Flowrate•WindowextensionCytometerPerformance—CS&T23-13536-00Rev.0112•Linearityistheproportionalityofoutputtoinputforfluorescencemeasurements.XAb2XAbs0100020003000WhatisLinearity?XAbs2XAbs=10002000CytometerPerformance—CS&T23-13536-00Rev.0113WhyisLinearityImportant?•AccuratefluorescencecompensationSmalldeviationsinlinearityinthelastdecadecanresultinsignificantcompensationerrors.•Quantitativemeasurementstudies–DNA–Antigen-antibodybindingLinearMFI142000MeasuredMFI140000Spillover20%CompensationError2000X20%=400CytometerPerformance—CS&T23-13536-00Rev.0114EffectofNon-LinearityonCompensationFITCMFIPEMFI179675688059218073000365DBAC•SamplesareBD™CompBeadparticlesstainedwithvaryinglevelsofFITC-Ab.•CompensationiscalculatedusingsamplesAandC.•Thiscytometerhasapoorlinearrange,with2%deviationfromlinearityabovetheMaxLinearityChannelat50,000.CytometerPerformance—CS&T23-13536-00Rev.0115CytometerFactorsAffectingLinearity•QualityofPMTtubeanditssocket•Preampandelectronics•LaserbeamshapeandalignmentCytometerPerformance—CS&T23-13536-00Rev.0116•SDENisthesignalvariationduetolowlevelbackgroundnoisecontributedbytheelectronicssystem.•LowSDEN→highersensitivityandlargerdynamicrange.•Mostcriticalforparameterswithlowcellularautofluorescence:−APC-H7−PE-Cy7−PerCP-Cy5.5−APCStandardDeviationofElectronicNoise—SDENtimeintensityCytometerPerformance—CS&T23-13536-00Rev.0117•PMTconnectors•PMTnoise•Cablesnearpowersources•Analogtodigitalconversionerror•WindowextensionFactorsAffectingSDENCytometerPerformance—CS&T23-13536-00Rev.0118WindowExtensionintensityprocessedeventtimeelectronicabortwindowextensionsoverlapthresholdwindowextensionswindowgatebelowthresholdCytometerPerformance—CS&T23-13536-00Rev.0119•SmallerwindowextensionscanlowertheSDENcontributiontovariationrelativetotheintegratedpulsesignal.WindowExtensionandSDENtimeintensitythresholdwindowextensions•Caution:smallerwindowextensionscanamplifyothermeasurementerrors.−Laserdelay−LinearitySensitivityExerciseCytometerPerformance—CS&T23-13536-00Rev.0120CS&TCS&Tisafullyautomatedsoftwareandresearchreagentsystem,uniquetoBDdigitalcytometers,providing:•Characterization•Setup•TrackingCytometerPerformance—CS&T23-13536-00Rev.0121CS&TBeads•Twosizesandthreeintensitiesofuniformbeads:–2μmdimfluorescenceintensity–3μmmidfluorescenceintensity–3μmbrightfluorescenceintensity•ExcitedanddetectedinallBDpreconfiguredcytometers.DimBright&Mid3µm2µmBrightMidDimFSC-AFITC-A(x1,000)CytometerPerformance—CS&T23-13536-00Rev.0122CS&TWorkflowAdministratorAccessAllUsersAccessPerforminitiallyforeachconfiguration.Rerunbaselineatothertimesincludingaftermajorserviceandwhenexistingbaselineexpires.CreateCytometerConfigurationCheckPerformanc