流式細(xì)胞術(shù)在白血病診斷中的應(yīng)用

1、四川大學(xué)華西醫(yī)院血液科四川大學(xué)華西醫(yī)院血液科朱煥玲朱煥玲l FCM：集激光、電子、光電測(cè)量、計(jì)算機(jī)、：集激光、電子、光電測(cè)量、計(jì)算機(jī)、熒光化學(xué)、單抗的高科技儀器。熒光化學(xué)、單抗的高科技儀器。l對(duì)處在快速對(duì)處在快速直線流動(dòng)狀態(tài)直線流動(dòng)狀態(tài)中的中的單個(gè)細(xì)胞單個(gè)細(xì)胞或或生生物顆粒物顆粒進(jìn)行進(jìn)行多參數(shù)的多參數(shù)的、定量分析定量分析和和分選分選的技的技術(shù)。術(shù)。l分兩類：臺(tái)式機(jī)（臨床型）分兩類：臺(tái)式機(jī)（臨床型） 大型機(jī)（大型機(jī)（cell sorting).科研用。科研用。BD FACSVantage SE lFCM的結(jié)構(gòu)的結(jié)構(gòu)細(xì)胞流動(dòng)室；細(xì)胞流動(dòng)室；光源；光源；聚光系統(tǒng)；聚光系統(tǒng)；信號(hào)檢測(cè)器；信號(hào)檢測(cè)器；電

2、子計(jì)算機(jī)；電子計(jì)算機(jī)；細(xì)胞分選裝置。細(xì)胞分選裝置。光源細(xì)胞流動(dòng)室聚光系統(tǒng)信號(hào)監(jiān)測(cè)器l數(shù)據(jù)的存儲(chǔ)：數(shù)據(jù)的存儲(chǔ)：listmodel顯示：顯示：?jiǎn)螀?shù)直方圖單參數(shù)直方圖(histgram);：常用于分析：常用于分析DNA等；等；雙參數(shù)：二維點(diǎn)圖等（雙參數(shù)：二維點(diǎn)圖等（dot plot) ；等高圖：等高圖：(contour)三維等高圖：三維等高圖：3D plotl設(shè)門（設(shè)門（gating):一定條件下，熒光強(qiáng)度與細(xì)胞內(nèi)DNA含量成正比。只反應(yīng)一個(gè)參數(shù)與細(xì)胞數(shù)量間的關(guān)系，不能顯示兩個(gè)獨(dú)立參數(shù)與細(xì)胞的關(guān)系。 Flow Cytometry cont. Data displayedGreen Fluoresc

3、ence IntensityNumber of CellsUnstained cellsFITC-labeled cellsOne Parameter HistogramRed Fluorescence IntensityGreen Fluorescence IntensityTwo Parameter HistogramContour plot3D plot:細(xì)胞多的地方，山峰高。l前向角散射前向角散射(forward scatter FSC):與激光束平與激光束平行的信號(hào)。與行的信號(hào)。與被測(cè)細(xì)胞大小有關(guān)被測(cè)細(xì)胞大小有關(guān)。l側(cè)向角散射（側(cè)向角散射（side scatter SSC):與激光束

4、呈與激光束呈90的光信號(hào)。與的光信號(hào)。與細(xì)胞內(nèi)結(jié)構(gòu)和顆粒物質(zhì)多少細(xì)胞內(nèi)結(jié)構(gòu)和顆粒物質(zhì)多少有關(guān)。有關(guān)。l采用采用FSC、SSC組合，可區(qū)分淋巴細(xì)胞、單核組合，可區(qū)分淋巴細(xì)胞、單核細(xì)胞、粒細(xì)胞等群體。細(xì)胞、粒細(xì)胞等群體。l熒光素探針標(biāo)記的細(xì)胞所發(fā)熒光。熒光素探針標(biāo)記的細(xì)胞所發(fā)熒光。l熒光信號(hào)強(qiáng)弱、多少與細(xì)胞的抗原量、內(nèi)含物熒光信號(hào)強(qiáng)弱、多少與細(xì)胞的抗原量、內(nèi)含物多少有關(guān)。多少有關(guān)。l常用的熒光顯色劑：常用的熒光顯色劑： FITC： PE：藻紅蛋白藻紅蛋白 PerCP； PI：碘化丙啶，測(cè)定碘化丙啶，測(cè)定DNA含量。含量。l臨床常用三色熒光檢測(cè)，現(xiàn)有四色分析臨床常用三色熒光檢測(cè)，現(xiàn)有四色分析。lCD

5、45為造為造血細(xì)胞血細(xì)胞（紅系除（紅系除外）的共外）的共同抗原。同抗原。避免雜質(zhì)避免雜質(zhì)細(xì)胞的沾細(xì)胞的沾染。染。CD45: 淋巴細(xì)胞單核細(xì)胞成熟粒細(xì)胞原幼細(xì)胞l細(xì)胞的分化成熟、細(xì)胞內(nèi)部結(jié)構(gòu)及其分化抗原細(xì)胞的分化成熟、細(xì)胞內(nèi)部結(jié)構(gòu)及其分化抗原的逐漸增減是漸進(jìn)過程，非突變。故同時(shí)獲知的逐漸增減是漸進(jìn)過程，非突變。故同時(shí)獲知一個(gè)細(xì)胞的多種參數(shù)方能正確判斷細(xì)胞屬性。一個(gè)細(xì)胞的多種參數(shù)方能正確判斷細(xì)胞屬性。l當(dāng)細(xì)胞發(fā)生克隆增殖后，可出現(xiàn)抗原量變、丟當(dāng)細(xì)胞發(fā)生克隆增殖后，可出現(xiàn)抗原量變、丟失和錯(cuò)譯表達(dá)。失和錯(cuò)譯表達(dá)。l細(xì)胞抗原的陽(yáng)性率及熒光強(qiáng)度是同等重要的指細(xì)胞抗原的陽(yáng)性率及熒光強(qiáng)度是同等重要的指標(biāo)。標(biāo)。

6、l單個(gè)細(xì)胞，新鮮標(biāo)本，抗凝保存：?jiǎn)蝹€(gè)細(xì)胞，新鮮標(biāo)本，抗凝保存：PB；BM；（；（有時(shí)有時(shí)BM活檢標(biāo)本）活檢標(biāo)本）LN（包括包括FNA）；）；CSF；組織浸潤(rùn)組織浸潤(rùn)FNA 或活檢標(biāo)本；或活檢標(biāo)本；l白血病細(xì)胞的抗原表達(dá)一定程度遵循正常細(xì)胞白血病細(xì)胞的抗原表達(dá)一定程度遵循正常細(xì)胞抗原表達(dá)的規(guī)律。抗原表達(dá)的規(guī)律。l白血病細(xì)胞為腫瘤性生長(zhǎng)，可出現(xiàn)正常細(xì)胞表白血病細(xì)胞為腫瘤性生長(zhǎng)，可出現(xiàn)正常細(xì)胞表達(dá)以外的異常現(xiàn)象，如缺失、錯(cuò)譯表達(dá)等。達(dá)以外的異?，F(xiàn)象，如缺失、錯(cuò)譯表達(dá)等。l目前未發(fā)現(xiàn)白血病特異性抗原，須多參數(shù)分析。目前未發(fā)現(xiàn)白血病特異性抗原，須多參數(shù)分析。l臨床原臨床原/幼細(xì)胞群定性比定量更為重要。幼

7、細(xì)胞群定性比定量更為重要。l常用抗體選擇： 核心McAbs:確定淋系或髓系（系特異性） 髓系：CD33、CD13、CD14，cMPO B-cell: CD19、CD10、cCD22 CD20 T-cell:CD7、CD2、CD5、cCD3 紅系：GlyA 巨核細(xì)胞：CD41、CD61 非系列特異性：CD34、DR、CD45lCD7/CD117/CD45-protocallHLA-DR/CD13/CD45lCD14/CD2/CD45lCD19/CD5/CD45lCD33/CD34/CD45lCD20/CD10/CD45 CD64,l117,13,33,14,64myeloidl19,10,20B

8、 lineage 7,2,5T lineage lDR,34 immature FAB SubtypeCommon PhenotypeComments/VariationsM0DR.CD13,CD33,CD34,CD7, cMPO*Generally negative for lymphoid markersM1Similar to M0 except CD15M2DR,CD13,CD33,more CD15,lessCD34CD19(+) -t(8;21) favorable prognosisM3DR(-)*,CD13,CD15,CD33,CD34-/+,CD2(+),DR(-) in m

9、aturing AML,consider M3.Myeloid/naturalkiller cell ALCD56(+).M4/M5DR,CD15,CD14,CD33,CD13,CD4weakCD2(+),consider M4EoM6DR,CD13-/+,CD33+/-,CD34,CD45 weakMature forms express glycophorinM7DR-/+,CD33+/-,CD34,CD41,CD61Beware of platelet.adhesionDiagnosis: AML- M1Antigen Profile: positive : DR, CD13, CD34

10、, CD38; partially positive for CD7,negative cCD3.Diagnosis: Acute myeloid leukemia with differentiation (AML M2) with features consistent with t(8;21)Antigen Profile: Positive for CD33(dim), CD34, CD13, CD15 CD11b (partial) with CD19. Negative for CD56,CD10,CD2 72 , female . A bone marrow examinatio

11、n was performed.Diagnosis: AML- M5Antigen Profile: Positive for CD33, CD38,CD34,HLADR, CD4(dim), CD64, CD13,CD14, CD11b and CD15.Diagnosis: Erythroleukemia (FAB M6)Antigen Profile: Blasts positive for CD33, CD71, CD34, CD38, CD13; Diagnosis: Acute megakaryoblastic leukemia (FAB M7)Antigen Profile: P

12、ositive for CD33, CD71, CD34, CD61SubtypeCommon PhenotypeComments/VariationsB-precursor ALLDR, CD19, CD20-/+, CD10+/-, CD34, TdT,sIg(-)Infants CD10(-) Multile myeloid Agt(9;22)Pre-B ALLDR,CD19,CD20+/-,CD10, CD34(-),cIgM(+),TdT+/-t(1;19)-CD34(-)poor prognosis.B-ALLDR,CD19,CD20,CD22,CD24, CD10+/-,CD34

13、(-),SIg L3T-ALLcCD3,CD5,CD7,CD1,CD2,dual CD4/8,CD10+/-CD34-/+Frequently lose T-cell Ag.6y. Girl, Hb: 100g/LWBC:10X10G/L,LC80%Plt. 85X10G/LDiagnosis: ALL, B-precursor type (common ALL)Antigen Profile: Positive for CD71, CD19, CD10, CD22, HLADR, CD34; dimly positive for CD33A 15 year old girl who play

14、ed for her high school soccer team noted to have difficulty breathing during her workouts. A chest x-ray revealed a large mediastinal mass, and she was found to have anemia and an elevated white blood countDiagnosis: ALL, T-cell Antigen Profile: Positive for CD7, CD5, CD3, CD34, CD10; partly positiv

15、e for CD4 and CD8Diagnosis: B-Precursor ALL (common ALL)Antigen profile: Positive for CD19, CD10, CD34, HLADR; negative for CD20, CD45, smIglAUL：僅占僅占1%典型的典型的AUL：DR+，CD34+ with no lineage-specific antigens.lBiphenotypic leukemia(mixed):僅有僅有7%能嚴(yán)格符合此型。過度診能嚴(yán)格符合此型。過度診斷是由于：斷是由于：未排除非白血病細(xì)胞；未排除非白血病細(xì)胞；非特異性結(jié)合；

16、非特異性結(jié)合；某些抗體缺乏系特異性；某些抗體缺乏系特異性；l特異性抗原為：特異性抗原為： BcCD22; TCD3; MMPO;Points B-淋巴淋巴 T-淋巴淋巴 髓系髓系 2 cCD79a cCD3 cMPO CD22 TCRab cIgM TCRrd 1 CD19 CD2 CD117 CD10 CD5 CD13 CD20 CD8 CD33 0.5 TdT TdT CD14 CD7Diagnosis: Acute mixed lineage leukemia with t(4;11)Antigen Profile: One population CD19+CD22+DR+CD20-CD

17、10-; another CD33+DR+CD64+CD14+DisorderCommon PhenotypeComments/VariationsCLLDR,CD19,CD20,CD5, CD22(-), CD23,CD10(-).clonal SIgM,FMC7(-)CD20 dimPLLDR,CD19,CD20.CD5(-),CD22, CD23(-),CD10(-),bright clonal SIgCD20 bright。CLL/PLL(30%幼淋細(xì)胞)MCLDR, CD19,CD20, CD5, CD22, CD23(-),CD10(-)FMC7(+)SIg bright Cycl

18、in D1 overexpressedFCC lymphomaDR,CD19,CD20,CD5(-),CD22, C23+/-,CD10CD10 negative20% HCLDR,CD19,CD20,CD5(-),CD22,CD23(-),CD10(-),CD11c,CD25,CD103SIgD common,very bright CD22 and CD11cPlasma cell dyscrasiasDR(-),CD19(-),CD20(-),CD22(-),CD38,CD45,clonal cIg,Bright CD38,dim CD45 73 , woman,mild splenomegaly ,WBC normal,with slight relative lymphocytosis, a PB specimen was sent for flow cytometryDiagnosis: Chronic lymphocytic leukemiaAntigen Profile: Positive for CD19, CD22, CD5, kappa, CD23, dimly positive for CD20; MCLAg profile: positive for CD19,CD5,lambda(bright),CD20（bright),FMC7.