流式细胞术课件

FlowCytometryBriefintroductionofflowcytomitryBasicprinciples;Whyisituseful—whatcanflowcytometrydo?BasicPrinciples概述；FCM的結構；數據的存儲、顯示和分析。標本的製備；怎樣看圖：BasicPrinciples概述

FCM：集鐳射、電子、光電測量、電腦、螢光化學、單抗的高科技儀器。對處在快速直線流動狀態中的單個細胞或生物顆粒進行快速（15000/s)、多參數的、定量分析和分選的技術。分兩類：臺式機（臨床型）

大型機（cellsorting).科研用。BDFACSVantageSE

FCM的結構FCM的結構細胞流動室；光源；聚光系統；信號檢測器；電子電腦；細胞分選裝置。BasicComponentsofFlowCytometryLaserLens(typ)FlowcellCellsuspensionForwardscatterSidescatterDichroicmirrorFluorescencedetectorDigitalComputer:SignalprocessorDigitalconversionDatastorageDisplay光源細胞流動室聚光系統信號監測器數據的存儲、顯示與分析數據的存儲：listmode顯示：單參數直方圖(histgram);：常用於分析DNA等；雙參數：二維點圖等（dotplot)；等高圖：(contour)三維等高圖：3Dplot設門（gating):DataAcquisition-Listmode一定條件下，螢光強度與細胞內DNA含量成正比。只反應一個參數與細胞數量間的關係，不能顯示兩個獨立參數與細胞的關係。GreenFluorescenceIntensityNumberofCellsUnstainedcellsFITC-CD4OneParameterHistogramRedFluorescenceIntensityNumberofCellsUnstainedcellsFITC-CD3OneParameterHistogramRelationship?Pb:lymphocytegateCD4+一定為Th?SINGLECOLORHISTOGRAMS無法顯示各抗體檢測之間的關係，兩種抗體均陽性是同一群細胞or不同細胞75%+60%+RedFluorescenceIntensityGreenFluorescenceIntensityTwoParameterHistogramCD4+CD3+CD4-CD3+CD4+/CD3-CD4+/CD3+CD4-/CD3-TWOCOLORPATTERNFL1-CD3FL2-CD4

colorCD3-CD4-

blackCD3+CD4- blueCD3-CD4+ cyanCD3+CD4+ green54%6%Contourplot3Dplot:細胞多的地方，山峰高。RedFluorescenceIntensityGreenFluorescenceIntensityTwoParameterHistogramCD4+CD3+CD4+/CD3-Gating:設立條件，符合條件的細胞納入分析。可依據：細胞內部參數：FS,SS；外部參數：CD19+R1CD4R1Gating:AmorphousQuadrantLineRectilinear數據單位：單參數橫坐標：道數channel縱坐標：頻率count線性測量：常用於分析DNA、FS，適用較小變化範圍的信號對數測量：適用較大範圍變化的信號。表面抗原測定、SS雙參數：均為channel，數字大，螢光強度高FCM檢測常用參數

內部參數—散射光測定前向角散射(forwardscatterFSC):與雷射光束平行的信號。與被測細胞大小有關。側向角散射（sidescatterSSC):與雷射光束呈90°的光信號。與細胞內結構和顆粒物質多少有關。採用FSC、SSC組合，可區分淋巴細胞、單核細胞、粒細胞等群體。FCM常用參數

外部參數—螢光測量螢光素探針標記的細胞所發螢光。螢光信號強弱、多少與細胞的抗原量、內含物多少有關。常用的螢光顯色劑：FITC：

PE：藻紅蛋白PerCP；PI：碘化丙啶，測定DNA含量。臨床常用三色螢光檢測，現有四色、多色。Excitation/EmissionSpectrum

e.g.FITC400500600ExcitationEmission488520激發波長發射波長FITCPEPerCPFSSSFCM樣品製備單個細胞，新鮮標本，抗凝保存(細胞濃度為106—107/ml)血細胞等懸液狀成分：PB、BM、CSF，漿膜腔液—PBS採集。實體組織：新鮮組織2h內製備成單細胞懸液酶處理法（胰酶、膠原酶、溶菌酶等）；機械分離法；化學法（EDTA）。；石蠟包埋組織的製備切片—脫蠟—酶處理標本的保存和固定：未染色標本表面抗原測定—最好新鮮；螢光染色標本：1—4%多聚甲醛固定。測DNA的標本可較長期保存。染色：直標：敏感性稍差，非特異染色低，多種標記同時測定。間標：敏感、非特異功能染色高DIRECTMAHMAH=Mouse-Anti-HumanFluorescentSurface

Immunostaining-DirectINDIRECTMAHSAMMAH=Mouse-Anti-HumanSAM=Sheep-Anti-MouseFluorescentSurface

Immunostaining-Indirect怎樣設計方案：雙標、三標、四標比單標好，但經費增高；Thelp:CD4—CD4/CD3—CD4/CD3/CD45價格FITC<PE<PerCPorPC5;

CD4-FITCCD4-PE

CD4-FITC/CD3-PECD4-PE/CD3PerCP細胞表面標誌較胞內標誌測定容易，穩定；先胞膜後胞內。某些共表達有特定診斷意義。CD19/CD5,CD4/CD8CD19/CD10注意事項：FCM測定值為相對值，不是絕對值(ExceptCD34count).對照設置：陰性對照：如IgG1.陽性對照：細胞數量要夠：1X106/ml太少：變異係數

太多：Ig<<Ag,假陰性雙、三標，各抗體螢光染料必須不同。選擇何種抗體據專業知識而定。細胞活力對表面抗原檢測影響大。細胞活性檢測：1）流式檢測：活細胞將拒染螢光染料PI、7-AAD或EMA（ethidiummonoazide）。優勢:同時分析表面標誌和活性通過設門即可得到活細胞的染色特性。2）手工檢測：使用Trypanblue或其他細胞活性染料。ANTIBODIESBINDNON-SPECIFICALLYTODEADCELLSFL-KAPPAPE-LAMBDAdeadcellsALLCELLSVIABLECELLSAB細胞表面抗原表達檢測，活力>75%;通過設門取出部分死細胞。FlowCytometry-AdvantagesMeasuressinglecellMeasureslargenumberofcellsIdentifiessmallsub-populationsCellscanbephysicallysortedFlowCytometry-DisadvantagesNeedssingleparticles(cells,nuclei,chromosomes)TissuearchitectureislostLittleinformationonintra-cellulardistributionsABCDEFAb1Ab1Ab1Ab1Ab1Ab1Ab2Ab2Ab2Ab2Ab2Ab2TypesofAntigenExpressionuniformlyheterogeneousTHREECOLORPATTERNCD3CD4CD3CD4CD8CD8CD3CD3CD3CD56CD56CD8FOURCOLORPATTERNCD4CD8CD56CD8CD4CD4Applicationofflowcytometry在細胞生物學及分子生物學研究上的應用；在血液學中的應用;在臨床免疫學中的應用;在臨床腫瘤學中的應用；FCM在細胞生物學及分子生物學研究上的應用DNA倍體分析細胞增值週期分析；細胞週期蛋白細胞凋亡研究；其他多藥耐藥研究。細胞內pH及Ca²+測定。核型分析、精子和精細胞分析。微生物研究。DNAProbesKeyfeatureofDNAprobesisthattheyareSTOICHIOMETRIC-thusnumberofmoleculesofprobeboundisequivalenttonumberofmoleculesofDNAPI(碘化丙啶)andEB(溴化乙啶):作用：1嵌入雙鏈DNA和RNA，無堿基特意性（須用RNA酶處理）。2可檢驗死活細胞（不能進入完整的細胞膜）。AO（AO：Acridineorange,吖啶橙；作用：用於細胞內DNA和RNA定量測定嵌入DNA雙鏈與單鏈RNA發生靜電相互作用。Definitions&TermsPloidyrelatedtothenumberofchromosomesinacellHaploid:Numberofchromosomesinagamete(germcell)iscalledtheHAPLOIDnumberforthatparticularspeciesDiploid:ThenumberofcellsinasomaticcellforaparticularspeciesHyperdiploid:greaterthanthenormal2nnumberofchromosomesHypodiploid:Lessthanthenormal2nnumberofchromosomesDNATetraploidy:ContainingdoublethenumberofchromosomesDefinitions&TermsDNAIndex（DI）:以DI表示DNA含量不正常的程度，DI=樣品G0/G1期DNA量平均數標準二倍體DNA量平均數

正常二倍體細胞的DI應為1（1.00.15)。診斷為不正常DNA倍體時必須見到兩個或以上分離的G0/G1峰。FluorescenceIntensity#ofEventsDNA定量分析：G1峰有早S期細胞。G2期有晚S期細胞。目前採用DNA擬合分析法。DNA直方圖定量分析（PI）：計算細胞週期—電腦擬合分析各時相的%含量（PI）G1G0%G2M%S%DoubletDiscrimination-theoryPeakIntegral2n4n2ndoubletDoubletDiscriminationIntegralFluorescenceIntegralFluorescencePeakFluorescencePeakFluorescence8x125mmlaserbeamshape16x64mmlaserbeamshapeClumps1SSCFSC2IntegratedRedPeakRedDoubletsIntegratedRedG0G1SG2-MDNAAnalysis-gatingstrategyDNAfromParaffinTissueBlocksNormaldiploidnucleiAneuploidnucleiBreastCarcinomaForwardScatterSideScatterDNA倍體變化檢測DetectionMethodsforApoptotisDetectionMethodsforApoptotisPhosphatidylserine,canbedetetectedbyincubatingthecellswithfluorescein-labelledAnnexinVBystainingwiththedye,Hoechst33342(UV)BystainingwiththedyePI(visible)BystainingwiththedyeYOPRO-1(visible)FlowCytometryofApoptoticCellsPI-Fluorescence#EventsApoptoticcellsNormalG0/G1cells用FasmAB處理細胞，誘導凋亡。Activecaspase-3是凋亡早期指標Antiactivecaspase3AbBcl-2在很多細胞中可以阻斷凋亡。PBbcl-2測定結腸腺癌P53檢測IsotypecontrolAnti-p53-PEMOLT4leukemiacellsRb檢測對照Anti-Rb-PEAb非特異antiRb封閉特異性antiRb封閉AnalysisofNuclearAntigensPCNA,isaproteininvolvedinDNAreplicationandinnucleotideexcisionrepair.Itcanbefoundintwoforms:oneistightlyboundtothechromatinandisSphasespecifictheotheriscytoplasmicandispresentthroughoutthecellcyclePCNAexpressioninintactcellsisnotcellcyclespecific.PCNAexpressioninthenucleiisSphaserelated.OtherNuclearAntigensKi-67-proliferationrelatedantigenKi-S1-proliferationrelatedantigenCyclinA:expressionbeginsinlateG1/earlySphaseandincreasesascellstraverseSphase,reachingamaximuminG2.CyclinAisnotexpressedinmitoticcellsCyclinB1:accumulatesinlateSphasebutismaximallyexpressedinG2andmitosis.細胞內因子檢測Γ-IFN/CD4Γ-IFN/CD4線粒體膜電位測定

ImmunophenotypingindiagnosisandprognosisofleukemiaandlymphomaFCManalysisofacuteleukemiaDiagnosis:Acutemyeloidleukemia(FABM1)

AntigenProfile:StronglypositiveforHLADR,CD13,CD34,CD38;dimlypositiveforCD33,CD71;partlypositiveforCD7,CD11bDiagnosis:Chroniclymphocyticleukemia

AntigenProfile:PositiveforCD19,CD22,CD5,lambda,CD23,dimlypositiveforCD20;partlypositiveforCD11c紅細胞疾病PNH：錨蛋白GPI：CD59、CD55、CD14、CD16等CD59（C8—C9）；CD55（C3轉換酶抑制劑）；須同時檢測RBC及粒細胞。少數病人（5%）只有粒細胞PNH克隆。嚴重溶血期後--GPI缺乏的紅細胞可能檢測不到。病人在檢測前有多次輸血，那麼，PNH篩查可能受到輸入血的影響，導致錯誤結果。紅細胞檢測時，至少兩種GPI相關抗原（CD55、CD59、CD16、CD24或CD66）缺失，才能診斷PNH（遺傳性缺失的病人可以缺少CD55或CD59之一）。分析未輸血的PNH病人的細胞有三種類型：III型細胞（完全缺失）II型細胞（部分缺失）I型細胞（正常表達）。有時三群細胞可能不能清楚地分開。

CountCellswithParticularFunctionCellswhichhaveaparticularfunctionusuallyhaveadistinctivesubsetofmoleculesontheirsurface.Moleculeswhicharedirectlyinvolvedinthatfunction:CD3moleculesidentifyTcellsCD4moleculesidentifythehelpersubsetofTcells(CD4moleculesarenecessaryforhelperfunction)

ASSESSMENTOFPLATELETFUNCTION

TRANSLOCATIONOFPLATELETGLYCOPROTEINSANDP-SELECTINDURINGPLATELETACTIVATIONACTIVATION: -GPIbIXV:internalized -GPIIbIIIa:1)membraneexpressionincreased 2)complexoccupiedbyfibrinogen,v.WillebrandFactor... -P-selectin:translocatedtothemembraneRESTINGACTIVATEDACTIVATION

granulesP-selectinGPIVGPIIb-IIIaGPIb/IX/VP-selectinGPIIb-IIIaGPIVGPIb/IX/VFibrinogen

50,00025,000>500DetermineCellTypeandFunctionCountCellswithSpecificEnzymaticReaction:MeasureEnzymeswithinCellswithSpecificEnzymeSubstrates

Forexample-DCFH•PeroxidaseFDA•EsteraseFDA,NaF•EsteraseClAc•ClAcetateEsteraseBu•ButyrylEsterase

CountCellsofParticularTypeImmunodeficiencyCountcellsthroughrelevantmoleculesondeficientcelltypeThymicInsufficiencyCountCD3+cellsHIVinfectionCountCD3+CD4+cells

下麵討論一種CD38漿細胞設門方法，可以用於多發性骨髓瘤診斷，並判斷細胞成熟度。抗體組合：CD38/CD19/CD56 CD38/MPC-1/CD45 CD38/CD49e/CD45CD38/CD54/CD33CD38/CD13/CD3免疫分析：使用CD38/CD19/CD56判斷多發性骨髓瘤，多發性骨髓瘤細胞為CD38++/CD19-/CD56+(見圖1)。使用CD49e、CD45和MPC-1檢測多發性骨髓瘤細胞的分期：未成熟型MM中間型MM成熟型MMCD49e--+CD45--+MPC-1-++分析報告：計算CD38強陽性的細胞比例，並報告該群細胞各相關表面標誌的表現(vsCD38)。圖例說明：圖1：多發性骨髓瘤病人的CD38/CD19/CD56三色分析表現(CD38++/CD19-/CD56+)，其中R1和R2為MM細胞。

正常人，表現為CD19+/CD56-良性M蛋白血症多發性骨髓瘤

圖2：良惡性病人在做CD38/CD19/CD56三色分析時，CD38強陽性區CD19/CD56的不同表現。(A)

正常人，表現為CD19+/CD56-(B)

良性M蛋白血症，表現為部分CD19+/CD56-(虛線箭頭指示)，部分CD19-/CD56+(實線箭頭指示)(C)MM，表現為CD19-/CD56+FCMinimmunologyDiagnosisofimmunodeficiencydiseasesAutoimmune:HLA-B27AIDS:LymphocyteimmunophenotypingandHIVinfectionCD4/CD8etc.Seepage25CD18LeukocytefunctionalassaysbyFCMNeutrophil:RollinginitialbindingactivationfirmadhesionandaggregationtransmigrationCountCellsofParticularTypeBoneMarrowTransplantationCountcellswithspecificlineagemoleculesonthesurfaceofcellsanddiscriminateliveversusdeadcellsHematopoieticStemCellsCountCD34+(ClassIIorClassIII)cellsLiveCellsCount7AADNegativecellsasLiveCount7AADPositivecellsasDeadadhesionmoleculesfamilies:Selectins;IntegrinsImmunoglobulinsuperfamilyMucin-likeglycoproteinsSelectinfamilyP-selectin:CD62P:PADGEMorGMP!40;Exp.byendotheliumplateletsTranslocatetotheendothelialsurfaceinminutesE-selectin:CD62E:ELAM-1Exp.byendotheliumWithin1to2hoursafterexposuretomediatorsL-selectin:CD62L:MEL-14,IECAM-1,LAM-1Exp.bymosttypesleukocytesIntegrins:Transmembraneheterodimerscontain:homologousbeta-chainVarietyalphachainsLFA-1(CD11a/CD18)Macrophageantigen1(Mac-1,CD11b-CD18)BindtoICAM-1CD11c/CD18Exp.OnlyonleukocytesImmunoglobulinsuperfamilyICAM1,2,3Bindtolymphocytefunction–associatedAg(LFA)Varyinteractionwithotheradhesionmolecules.PECAM-1(CD31)PlateletendothelialcelladhesionmoleculeFoundinbothleukocyteandendothelialcellsVCAM-1(vascularcelladhesionmolecule1)BindwithintegrinverylateAg4(VLA-4)initialbindingP-selectinandE-selectinareparticularlyimportantintheearlybindingofneutrophilsfirmadhesionandaggregationDependoninteractionLFA-1andMac-1withICAM-1OveralongertimeperiodImportantinthebindingofalltypesofleukocytesVLA-4/VCAM-1Importantintheadhesionofchronicinflammatorycells(lymphocyte,monocytesandeosinophils)Humanleukocyteadhesiondefects:LeukocyteadhesiondediciencyIAutosomalrecessivedisorderCD11bmarkedlyreduced(afterstimulate)RecurrentbacterialandfungalinfectionDiagnosisbymeansofflowcytometryMac-1(CD11b/CD18)andL-selectinabn.SpecificgranuledeficiencyReducedorabsentCD11b/CD18Formylpeptide-inducedspecificgranulereleaseassayDisorderofphagocytekillingClinicallyusefulnontraditionalapplicationsofFCMFCMinRBCAg-AbdetectionassaysDiseaseassociations:Alloimmunizationduringchildbirth.AIHA.Transfusion-relatedreactions;ErythrocytephenotypingBonemarrowengraftmentClassificationinMDSFetal-maternalhemorrhageSicklecelldiseaseSpecificareasofFCMforRBC:DetectionandquantitationofRBC-boundIg(IgG,A,M)(<30moleculespercell,esp.forDATnegative,fluorescentbeads)RBC-boundcomplementproteins(C3,C4,etc.)BloodgroupAg(Rh-CDE,ABO,le,Kell)Circulatinganti-Rh,ABOAb.Immunoglobulinsubclasses(G1,2,3,4)Intracellularnucleotides(nucleatedRBCsandRet.)Intracellularproteins(fetalhemoglobin)DetectionoffetalHbinmaternal-fetalhemorhageCriticalimportancein:Rh-Dincompatibilities(FMH)SicklecelldiseaseDyserythropoeisisMDSThalassemiasHemolyticanemiaFMH:FMHcanleadtolife-threateningautoimmmunesequelMotherRh(-),fetusRh(-)FMHcanbetreatedwithanti-Rhglobulin.FCM:Hb-FspecificmonoAbcandetectlowlevels(0.1%)ofallogeneicRBCS.Helptodeterminethedosageofanti-RhAb.SignificantseparateadultFcellfromfetalRBCs.FCM在臨床腫瘤學中的應用檢測癌前病變、協助腫瘤早期診斷：細胞DNA含量變化：癌前病變細胞不典型增生DN