Flow cytometric quantitation of immunofluorescence intensity Problems and perspectives.docx

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1、 1998 Wiley-LissI,nc.Cytometry 33:166178(1998)Flow Cytometric Quantitation of ImmunofluorescenceIntensity:Problems and PerspectivesJan W. Gratama, *Jean-Luc Dhautcourt,Frank Mandy, Gregor Rothe, David Barnett,51234George Janossy, Stefano Papa, Gerd Schmitz, and Rodica Lenkei8674for the European Work

2、ing Group on ClinicalCell Analysis1Department of Clinicaalnd Tumor Immunology, Danielden Hoed Kliniek,Rotterdam,The Netherlands2Ho pitalde Warquignies,Boussu,Belgium3NationalLaboratoryforAnalyticaClytology,HealthProtectionBranch,HealthCanada, Ottawa, Canada4Instituftur KlinischeChemie und Laboratori

3、umsmedizin,Klinikum der UniversittaRegensburg,Regensburg,Germany5Department of Haematology, Royal HallamshireHospital,SheffieldE,ngland6Department of ClinicaIlmmunology, Royal Free HospitalSchool of Medicine,London, England7IstitutdoiScienzeMorfologiche,UniversitadiUrbino,Urbino,Italy8CALAB Research

4、,S:t.GoransSjukhus,Stockholm,SwedenReceived8 June 1998;Accepted 10 June 1998Quantitation of immunofluorescenceintensityintensityinto units of antibodies bound per cell.Theuse of antibody-binding capacity as a surrogatemarker for quantification of Ag expression wasaddressed more directly by the devel

5、opment ofantibody-binding standards. The quantitativeindi-serves to estimate the number of defined moleculesexpressed on or in cells.Clinicalapplications of thisdiagnostic tool are increasing,e.g.,aberrant expres-sion of various antigens (Ag) by leukemic blastsorlymphomacells,intensity of CD38 expre

6、ssion byrect immunofluorescenceassay is based on beadsCD8 T-lymphocytes to monitor activation status,labeled with various amounts of CD5 mAb thatcalibratethe binding of the secondary antibody inunits of antibody-binding capacity. Alternatively,goat anti-mouselabeledcalibrationbeads have beendevelope

7、d. Published resultsobtained with the lattercalibratorsshowed an unexpected inaccuracy. Thedifferentways in which calibratorsand cellsunderstudy bind mAb (i.e.F,ab mediated versus Fc medi-ated) may have contributed to this variation.Re-cently,the use of stabilizedcellpopulations express-ing Ag in a

8、specified range of concentrations hasbeen proposed as an Ag-specificcalibrationsystemof mAb binding. We identifyseveral issues on thelevel of instrumentation, reagents, and cellsunderstudy that should be solved to allow standardizationof quantitativeassessments of immunofluorescenceintensity. Cytome

9、try 33: 166178,1998.and intensityof CD62P to detect plateletactivation.In this report we discuss the quality-controlmea-sures required forquantitationof fluorescence inten-sity,and we review seven concepts that have beendeveloped to quantify fluorescence intensityduringthe past 15 years.Initialwork

10、addressed the conver-sion of logarithmic channel numbers into units ofrelativefluorescence. The design and use of calibra-tion beads labeled with predefined amounts of dyeallowed instrument-independent expression of fluo-rescence intensityin units of molecules of equiva-lentsoluble fluorochrome (MES

11、F). This method wasrefined by the combined use of such standards withmonoclonal antibodies (mAb) conjugated 1:1 withphycoerythrin (PE),allowing translationof fluores-cence intensityinto numbers of antibodies boundper cell.Alternatively,the use of 1:1 PE-conjugatedmAb under the assumption that CD4 ly

12、mphocytesreproducibly bind 50,000 CD4 mAb molecules wasproposed to convert units of relativefluorescence 1998 Wiley-Liss,Inc.Key terms: flow cytometry; immunofluorescence;quantitation; instrumentsetup; calibrationThe European Working Group on ClinicaClellAnalysis(EWGCCA) isacollaborativeinitiativoef

13、 scientistasctivein the eld of clinicalcellanalysisfrom 13 European countries.The goal of thisworking group,which is open to scientistsactive in this eld, is the evaluation,renement, and standardizatioonf new analyticatlechniquesforclinicalcellanalysis.The group is divided into subsectionsaccording

14、to thedifferentasksand types of analyticatlechniques,i.e.o,w and imagecytometryor molecularbiology.The coremembers oftheEWGCCA areG.Schmitz(Regensburg,Germany), coordinatorB;.Autran (ParisF,rance);B.Brando (Milan,Italy)J;.L.Dhautcourt(Boussu,Belgium);J.W. Gratama(Rotterdam,The Netherlands);A. Huber

15、(Aarau,Switzerland)G;. Janossy(London, England);H.E. Johnsen (Copenhagen, Denmark); J.Kappel-mayer (Debrecen, Hungary); R. Lenkei (Stockholm,Sweden); A. Orfao(Salamanca,Spain);S. Papa (Urbino, Italy);M. Papamichail (Athens,Greece);G. Valet(MartinsriedG,ermany); T. Totterman (Uppsala,Swe-den); G. Rot

16、he (Regensburg, Germany); and B. Zupanska (Warsaw,Poland).Contractgrantsponsor:Biomed-2 Programme (DirectoratGeeneral XII,European Commission, Brussels,Belgium);Contractgrantnumber: Con-certedActionContractBMH4972611.*Correspondence to:Jan W. Gratama,M.D.,Department of ClinicaalndTumor Immunology, D

17、aniel den Hoed Cancer Center,P.O. Box 5201,3008 AE Rotterdam,The Netherlands.E-mail:gratamaimmh.azr.nl 167QUANTITATIONOF IMMUNOFLUORESCENCEINTENSITYThe ultimategoal of ow cytometric quantitationofimmunouorescence intensitiysto enumerate thenumberof certainmoleculeson (orin)cellsof a givenpopulation.

18、During the past15 years,a varietyof methods have beendeveloped toapproach thisgoal,allof which arebased onthe detectionof the molecules (antigensAg)of interestusingmonoclonal antibodies(mAb) and thequanticationof mAb binding using a calibratiosnystem.The quantita-tion of severalAg has been shown to

19、provide usefulclinicalinformation.For example, neoplasticB lineagecellscan be distinguishedfrom normal precursor andmature B lymphocytes by overexpressionof CD10 or CD5,respectivelya,nd by underexpressionof CD24 and CD45in some cases (1820);the overexpressionof CD10 byacute B-lymphoblasticleukemia c

20、ellsis even helpfulforthe detectionof minimal residualdisease(8).Recently,themachine for Ag quantitation.This concept has beenintroducedby Schwartz etal.(33,34).Thisconcept willbeusefulin an exchangeable multi-instrumenetnvironment,e.g.,ata largeow cytometryfacilitayn,d/or ifa multitudeofAg have tob

21、e quantitativealnyalyzed.Thisapplicatioinsshown by Lenkei et al.elsewhere in thisvolume (22).Atypicalapplicationwould be, for instance,a generalprocedure forthe quantitativdeescriptionof the antigenicprole of leukemic blastcells.Quality Control of the Flow Cytometerfor Quantitation of Immunofluoresc

22、enceQualitycontrolof the ow cytometer forquantitatioonfimmunouorescenceshould include the assessment ofinstrumentparametersthataffectthe accuracyand preci-sionof data.In thisrespect,two groups ofprocedurescanbe distinguishedT.he rst group of procedures iscarriedout at relativellyarge intervals(e.g.,

23、twice a year) byqualied servicepersonnel and includesexamination ofthe efficiencaynd performance of the lasertube, opticallters,logarithmic(log)and linearampliers, and photo-multipliertubes (PMT). These procedures also includeoptimizationof the opticalalignment of ow cytometersequipped with a cuvett

24、eto assurethatthe brightestandtightespteaks areproduced inallparameters.Most clinicalow cytometersareofthe stream-in-cuvettytpe.How-ever,the opticalalignment of the largerow cytometerswith sortingcapabilitiesw,hich are equipped with anozzle (stream-int-yapier),must be optimized at eachcosltdartbythe

25、 instrumentoperatorsdue to itsrelativeinstability.The second group of procedures consistsof frequent(e.g.,daily)monitoring of instrumentsetup and perfor-mance by the operatorsin order to identifyboth immedi-ate and potentialproblems. Afterthe appropriateinstru-ment settingshave been establishedt,he

26、targetchannelsfor the relevantuorescence parameters are recordedusing uorescent referencebeads,and the resultsof theday arethen compared with the accepted tolerancelimits.The instrumentpserformance for quantitativeuores-cence measurements ismonitored by computing calibra-tion plots for each uorescen

27、ce parameter from datagenerated by measurement of calibratiobneads labeledwith uorescent dyes in differentpredened intensities.Trends and variationscan be noted with the use ofLevey-Jenningscharts(23) thatallow visualinspectionoflongitudinadlata for monitoring instrumentperformanceforprecisionand tr

28、ends.intensitoyf expressionof the CD38 activatioAng by CD8lymphocytes of human immunodeciency virus(HIV)infectedindividualshas been shown to be a powerfulprognosticfactorfor the development of the acquiredimmunodeciency syndrome (AIDS) (24,25).In addition,the quantication of CD62P expression on plat

29、eletsisusefulforthe assessmentof theiractivatiosntatus(32).The European Working Group on ClinicaClellAnalysis(EWGCCA) was founded in 1996 to evaluate,optimize,and standardizenovelanalyticatlechniquesforclinicaclellanalysis(see footnote).Flow cytometricquantitationofimmunouorescence isone ofthesetech

30、niquesforwhichwe envisagean extensionof clinicaalpplicationsi,.e.f,orthe immunophenotyping of leukemias and lymphomasand forfunctionalassaysoflymphocytes and plateletsA.s abasisforour work, we review here the stateof the artandsummarize the issuesthat are to be resolved in thequantitatioonf Ag expre

31、ssionby ow cytometry.In general,two differenctoncepts can be distinguishedfor how to use a owimmunouorescence:cytometer for quantitationof1. The calibrationof a specic instrument for thequantitativaenalysisof one or more predened Ag. Thisconcept is used for clinicalchemistry or hematologyanalyzers.O

32、ptical testmaterialsfor the controlof thealignment of the instrumentare typicalloynly used in amonthly or similarintervalB.iologicalcalibratorsw,hichmay be instrument and testspecic, are used for thegenerationof an empiricalcalibratiocnurve thatdoes notrequireany mathematicalassumption.This procedur

33、e isrepeatedwhenever a new lotof mAb isused.Typically,every fourthanalysisseriesincludesan accuracy control(which againisinstrumentand assayspecic) in order toquantifythe relationto the truvealue.Precisioncon-trolst,o quantifyreproducibilitcya,n be stabilizebdiologi-cal samples and are included in e

34、very testseries.Thisconcept is particularluyseful for those applicationsinwhich a verylimitednumber ofAg isstudied(e.g.a,nalysisIn the subsequent sectionsof thisreview,we use thenomenclature for opticaltest materialsand calibrationmicrobeads as describedelsewhere in thisissue(35).Instrument SetupWin

35、dow of analysis.The multiparameterwindow ofanalysisof most clinicalapplicationsincludes forward(FSC) and sideward lightscatter(SSC),and up to fouruorescence parameters.Optimal positionof the multipa-rameter window of analysisimplicatesthat allcellularpopulationsare visibleon each parameter scale.Suc

36、hof CD38 expressionlevelson CD8 T lymphocytes usingthe stabilizecdellimmunouorescence assayseebelow).2. The genericsetup of a ow cytometer,which willallow the instrument-and assay-independentuse of the 168GRATAMAET AL.positioningisensured by usinga representativseample fora givenassay.For FSC and SS

37、C analysisl,inearamplicationis generallyused, although logarithmicamplication ismore usefulfor the simultaneousvisualizatioonf normaland aberrantcellpopulationswith high SSC signalss,uchas hairyleukemia cellsp,lasma cellso,r cellsderivedfromsolidtumors.For uorescence analysisl,ogarithmicampli-cation

38、 is generallyused in view of the wide dynamicrangeofuorescence intensitioefsmost biologicaslamples.Linearamplication serves specic analysesbetterifanarrow range of brightuorescence intensitieasrestudied(e.g.D,NA analyses).Correction for spectraloverlap. There are variablelevelsofoverlapbetween the e

39、missionspectraof routinelyused dyessuch asuorescein isothiocyanat(eFITC),phyco-erythrin(PE),and the energy-coupleddyes,includingthenaturalperidininchlorophyll(PerCP) and the synthetictandem conjugatessuch as PE-Cy5 or ECD. Hence, eachuorescence detectorwillnot only pick up signalsfromthe appropriate

40、dye, but also,to a varyingdegree,signalsfrom atleastone of the otherdyes.For example, a mixtureof two cell populations single-labelewdith FITC- andPE-conjugatedmAb willappear in a bivariatedot plotasclusteredalong axes thatare at an acute angle to eachother.These axes are rendered orthogonalby adjus

41、tingcolorcompensation.Such adjustmentscan be performedmanually or automaticallyA.utomatic correctionforspec-traloverlapcan eitherbe performed during data-acquisi-tion(themajor ow cytometersare equipped with suchsoftware)or during data analysisusing WinListsoftware(VeritySoftware,Topsham, ME). The es

42、tablishmentofappropriatecolorcompensation forany n dyes requiresncellsuspensions single-labelewdith mAb conjugated tothosedyes,plusone suspensionstainedwith alldyes.Eachcellpopulationshould span the uorescence intensitiesrangingfrom dimly positiveto a representativheigh forthatassay.The use of unsta

43、inedcellsfor adjustingcolorcompensation isdebatabledue to the possibleinterfer-ence of opticaland electronicnoise with the very weakuorescence signalsof unstainedcells.Establishing application-specifictarget channels.Stablepositioningof the multiparameterwindow ofanaly-sisallows convenient compariso

44、n of data collectedindifferentexperiments, i.e.,by visualinspectionof theshape of clustersin bivariatheistograms.FSC and SSC areprimarilyused for discriminatincgellpopulationson thebasisof theirrelativsecatterpropertiesF.or most immuno-phenotyping applicationsi,tsufficetso run a representa-tive or n

45、ormal specimen and to verifythat allmajorpopulationsare in theircharacteristipcositionson theFSC-versus-SSCdiagram.Type IIreferencemicrobeads areusefulto dene the positionof the uorescence windowof analysisA.ppropriatecolorcompensation settingshav-ing been activatedt,ype IIbeads are run, and the mea

46、nvalueof the singlebead populationin each uorescencechannel isrecorded.These are the so-calledtargetchan-nelsforthatapplicationT.he use of these targetchannelsallows,at subsequent occasions,a rapid and reliableverication of instrumentsettingsT.his strategyensuresthata constantpositioningof the windo

47、w of analysisismaintainedasa functionof time.Application-specificinstrument setup on subse-quent occasions. Generally,eitherone of two strategiesisfollowed.According to the rst one, type IIreferencebeads are placed in the application-specictargetchan-nels,and instrument settings(i.e.p,hotomultipliertubePMT voltages)arerecorded inLevey-Jenningsplots.Thisprocedure is,for example, followed by the FACSCompsoftware (Becton Dickinson Immunocytometry SystemsBDIS,San Jose,CA) and involvesfrequentminor adjust-ments ofPMT settingasnd,conseque