1. Abstract During theearlyphaseafterhematopoieticcell transplantation(HCT)so-calledmixedchimerismsometimesoccurs inperipheralblood.Donor/recipientchimerismanalysisusingtheHLA-FlowmethodisaveryusefultechniqueforearlydiagnosisofgraftfailureandrelapseofleukemiaandforelucidationoftheunderlyingmechanismsinvariouspathogenicstatesafterHCT.Reportedhereinaretheresultsofseveraltwelve-colorchimerismanalysesusing theSpectral flowcytometerdevelopedbySonyCorporation.Theperformancewasequivalenttoacommoncommercialinstrument.Moreover,oneoftheuniquefeaturesofthisinstrumentistheabilitytodisplayeachfluorescencespectrumforeachcell,demonstratingtheaccuracyofanalysisandguarantyingfurtheraccurateanalysisespeciallyinmulti-coloranalysis.

2. Introduction AlthoughHCThasbeenincreasinglyusedtotreathematologicalmalignancies,metabolicdisordersandcongenitalimmunodeficiencies,HCT is frequently complicated by graft failure and relapse ofprimarydiseases.Sincepersistenceor increaseof recipient-derivedhematopoieticormalignantcellshaspathogenic import,analysisofdonor/recipientchimerismshouldbeuseful tomakeearlydiagnosisofgraft failureandrelapseofprimary-diseaseandtounderstandthemechanismsofthesepathogenicconditions.Polymerasechainreaction(PCR)-based short tandem repeat analysis andX/Ychromosomeanalysisusingfluorescence in situhybridization(FISH)aftergender-mismatched transplantationare themethodsmostcommonlyusedfor chimerismanalysis.PCR-orFISH-basedmethods, however,arecomplicated, insensitive,and time-consuming. Inaddition,wecannotdirectlyanalyze lineage-specificmixedchimerism.Ifdonor-andrecipient-derivedcellshavespecificsurfacemarkerswhichcanbe stainedby fluorescence-conjugatedantibodies, it ispossible toperformchimerismanalysisbyflowcytometryinarapid,quantitative,andhighlysensitivemanner.Since thenumberofHLA-mismatched

transplantations,e.g.cordbloodtransplantationorhaplo-identicalHCT,has increased recently,wehavedevelopeda flowcytometry-basedmethodofchimerismanalysisusing fluorescence-conjugatedanti-HLAmAbsafterHLA-mismatchedtransplantation(Figure1).Sincewecananalyzelineage-specificchimerisminarapid,quantitative,andhighlysensitivemanner,HLA-Flowisaveryusefulmethodcomparedwithpresentmethods.DuetotheexpansionintheapplicationofHCT,non-remissionphaseHCThasincreased.Insuchsituations,weneedtodetect leukemiacell inadditionto lineage-specificchimerismamongnormalleukocytes.Inourlaboratorynine-coloranalysis iscommonlyusedformonitoringengraftment.Ifresidualleukemiacellsaredetectedduring theengraftmentphase,weneedmore fluorescent colors tomonitorengraftmentandminimal residualdisease simultaneously.Becauseofthelimitedavailabilityoffluorescence-conjugatedantibodiesandacomplicatedcompensationtechnique,analyseswithtenormorecolorsarestillverydifficultforcommonflowcytometryusers. In order to solve the problems,we tried to evaluate a newcytometerdevelopedbySonyCorporation (Tokyo,Japan) replacesfilter-basedopticswithaspectraldetectionsystembasedonamulti-anodespectralPMT.

3. Instrument confi guration Thespectral flowcytometer isable to increase thenumberofanalyzedcolorsandeasilyseparatessimilarfluorescencespectraviaa32channellineararrayPMT(Figure2).Itisalsoabletomeasureandsubtractvaryingautofluorescence,permittingincreasedsignaltonoiseratiosandimprovingtheresolutionofdimsignals.Toobtainadynamicrangeof fluorescence intensitydetection,Sonynewlydevelopedthe independentlyvoltage-controlled32channelPMTwithhigh-sensitivitytodetectfrom500nto800nmbyusingaseriesofprisms.Theexcitationlasersarelaserdiode(LD)with488nmwavelengthand60mWpoweranda638nmLDwith110mW.Multiplefluorochromesaremathematicallyunmixedbyusingcomponentanalysis.

Figure 1. Flow cytometry-based method of chimerism analysis using anti-HLA mAbs Figure 2. Next-generation spectral fl ow cytometry

488 nm 638 nm

prisms

32 ch PMT

replaceable fl ow cell

White PaperJune 2012

Twelve-color Chimerism Analysis after HLA-mismatched Hematopoietic Cell Transplantation Using Anti-HLA Antibody and Spectral Flow CytometerEri Watanabe1, Yusuke Nakauchi1, Nobukazu Watanabe1, Hiromitsu Nakauchi2

Laboratory of Diagnostic Medicine1 and Division of Stem Cell Therapy2, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Sony CorporationBio Science Business Dept.Life Science Div.Medical Business Unit

Gotenyama Tec. 5-1-12 Kitashinagawa Shinagawa-ku, Tokyo, 141-0001 JapanTel: 0120-667-010 Fax: 0120-388-060E-mail: cytometry@sony.co.jp

4. Twelve-color chimerism analysis Twelve-color chimerism analyses using artificial chimerismsamplescomposedofcordbloodandhealthyindividual’sbloodwerecarriedoutatotalof3timestoverifytheperformanceandequivalencebetweenthespectralflowcytometerandaconventionalflowcytometer.CordbloodwasprovidedfromJapaneseResearchCordBloodBank.WedeterminedthetypeofHLAusingSRL,Inc.contractservicebeforethisexperiment.HLA-A2positiveandHLA-A24negativecordbloodwasusedforexperiments.Ontheotherhand,peripheralbloodfromhealthy individualswithHLA-A2negativeandHLA-A24positivewasused.Weselectedtwelve-colorimmunostainingstodeterminethelineage-specificchimerismofhematopoieticcellsubsets:HLA-A2/HLA-A24 for chimerism,CD34/CD45 for hematopoietic stem/progenitorcellsor leukemiacells,andCD3/CD4/CD8/CD14/CD19/CD56fornormalleukocytesubsets(Figure3). Since the spectral flowcytometer is equippedwithonly twolasers,488and638nmlasers, itappears tobea littlebitharder toanalyze twelve-color samples simultaneously.However, becauseof thecombinationofprismoptics,32charrayPMT,andSpectralDeconvolution, the spectral flow cytometer could separatetwelve-color sampleswith extraordinary fluorochromesand theircombinationsuchasCy2,PE-AlexaFluor700,PE-TR/PI,andPE-Cy5/APC(Figure4). ThefrequenciesofeachcellpopulationbythenumberofCD45+

populationwerecalculatedandcomparedbetweenbothinstruments.Acquiredbythespectralflowcytometer,hematopoieticstem/progenitorcellpopulationandwhitebloodcellsubsetswereclearlydeterminedandequivalent todata from thecommoncommercial instrument.Figure4B&Cshowsspectrachartsof themeasurement resultsoftwelve-colorchimerismanalysisexcitedwith488and638nmlasers,respectively.Wecanseemany linesderived fromeachfluorescentreagent.Wecanalsoconfirmeachspectrumshapederivedfromeachcellonebyone.Thisuniqueanduseful featureenablesus tomakemoreaccuratejudgment,whichisespeciallyimportantforbothmulti-coloranalysisandrarecellanalysis.

5. Conclusion Twelve-colorchimerismanalysesusing3artificial chimerismsamples composedof cordblood andhealthy individual’s bloodwere carried out to verify the performance of the spectral flowcytometer.Thefrequenciesofeachsubpopulationofhematopoieticstem/progenitorcells,maturewhitebloodcells,andlineage-specificchimerismacquiredby the spectral flowcytometerwere clearlydeterminedandwereequivalentofdatafromacommoncommercialinstrument.Moreover,oneof theuniquefeatureof thespectralflowcytometer,displayofeachspectrumderived fromthe fluorescenceofeachcell,demonstratedtheaccuracyofanalysisandguaranteeforfurtheraccurateanalysisespeciallyinmulti-coloranalysis.

Figure 4. Result of twelve-color chimerism analysis (A), Spectra chart of sample stained with twelve-color fluorochromes excited with 488 nm laser (B), 638 nm laser (C)

Figure 3. Spectra of fluorochromes: excited with 488 nm laser (A), 638 nm laser (B)Table. Fluorochrome panel for twelve-color chimerism analysis