流式细胞仪基本原理及应用课件

by Dr Baiqing LI免疫学教研室免疫学教研室 流式细胞仪在临床检测和流式细胞仪在临床检测和临床科研中的应用临床科研中的应用李李 柏柏 青青蚌埠医学院免疫学教研室蚌埠医学院免疫学教研室流式细胞仪在临床检测和临床科研中的应用李 柏 青All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-2-=流式细胞仪和流式细胞术概述流式细胞仪和流式细胞术概述=流式细胞仪的工作原理和测量参数流式细胞仪的工作原理和测量参数=流式细胞仪在临床检测和科研中的应用流式细胞仪在临床检测和科研中的应用=免疫学免疫学检测和科研和科研应用用=血液病血液病检测和科研和科研应用用=肿瘤学瘤学检测和科研和科研应用用流式流式细胞胞仪的基本工作原理以及在基的基本工作原理以及在基础和和临床科研中的床科研中的应用用-2-流式细胞仪和流式细胞术概述流式细胞仪的基本工作原理以All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-3-一、流式一、流式细胞胞仪和流式和流式细胞胞术的基本概念的基本概念=流式细胞仪流式细胞仪(Flow Cytometer，FCM):是集合光是集合光电子物理，光子物理，光电测量技量技术，液流技液流技术，计算机技算机技术等等现代高科技代高科技技技术为一体的一体的细胞胞测量和分析量和分析仪器。器。-3-一、流式细胞仪和流式细胞术的基本概念流式细胞仪(FloAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-4-一、流式一、流式细胞胞仪和流式和流式细胞胞术的基本概念的基本概念=流式细胞术流式细胞术(Flow Cytometry，FCM):是是应用流式用流式细胞胞仪，对处于快速流于快速流动液液体中的体中的细胞或生物胞或生物颗粒粒进行多参数、行多参数、快速快速测量和定量分析，以及分量和定量分析，以及分选的技的技术。-4-一、流式细胞仪和流式细胞术的基本概念流式细胞术(FloAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-5-一、流式一、流式细胞胞仪和流式和流式细胞胞术特点特点=多参数测量和分析（散射光和荧光）多参数测量和分析（散射光和荧光）；=快速测量和分析快速测量和分析=每秒可达每秒可达1000-100001000-10000个个细胞胞=分分选纯度可达度可达99%99%以上。以上。=获取的数据分析结果更具有统计学意义；获取的数据分析结果更具有统计学意义；=通常测量的对象如各种细胞细胞必须处在单通常测量的对象如各种细胞细胞必须处在单个细胞悬液中。此外，也可测量微生物等生物个细胞悬液中。此外，也可测量微生物等生物颗粒，或者工合成微球非生物颗粒，颗粒，或者工合成微球非生物颗粒，-5-一、流式细胞仪和流式细胞术特点多参数测量和分析（散射All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-6-台式机台式机FACSCaliburFACSCalibur=双激光双激光 488nm 635nm=四荧光参数四荧光参数=分选、浓缩系统分选、浓缩系统-6-台式机FACSCalibur双激光77All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-8-大型机大型机FACSVantage SEFACSVantage SE=科研型科研型=八荧光参数八荧光参数=高速分选高速分选=多路分选多路分选-8-大型机FACSVantage SEAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-9-流式细胞仪与荧光显微镜比较流式细胞仪与荧光显微镜比较 流式细胞仪流式细胞仪 荧光显微镜荧光显微镜 流动的细胞流动的细胞 静止的细胞样本静止的细胞样本 数量大数量大 数量少数量少 高速分选高速分选 样本难以再利用样本难以再利用 细胞群体特征量分布细胞群体特征量分布 可揭示细胞内部结构信息可揭示细胞内部结构信息-9-流式细胞仪与荧光显微镜比较 流式细胞仪All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-10-二、流式二、流式细胞胞仪的工作原理和的工作原理和测量参数量参数l光路系统光路系统：激光光源，光路系激光光源，光路系统（透（透镜、滤光片）光片）l电子系统：电子系统：光电倍增管（光电倍增管（PMT），补偿电路），补偿电路l液流系统液流系统：鞘液系统，样品流动系统鞘液系统，样品流动系统l数据系统：数据系统：计算机，数据测量和分析软件计算机，数据测量和分析软件。l细胞分选系统细胞分选系统（一）（一）流式细胞仪的结构流式细胞仪的结构-10-二、流式细胞仪的工作原理和测量参数光路系统：（一）All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-11-流式细胞仪光路系统流式细胞仪光路系统=激光激光 (Laser)(Laser)是一种相干光源，它能提供是一种相干光源，它能提供单一波长、单一方向、同步的稳定光照单一波长、单一方向、同步的稳定光照=激光波长激光波长:488nm,635nm:488nm,635nm=光收集系统是由若干组透镜光收集系统是由若干组透镜,滤波片滤波片,小孔组成，将产生的光信号引导至检测器小孔组成，将产生的光信号引导至检测器-11-流式细胞仪光路系统激光(Laser)是一种相干光All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-12-光路系统光路系统-12-光路系统All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-13-Injector Tip-13-Injector All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-14-Injector TipFluorescenceFluorescencesignalssignalsFocused laserFocused laserbeambeamSheath fluid-14-Injector FluorescencesignaAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-15-流式细胞仪光学系统流式细胞仪光学系统激发光源与荧光标记物激发光源与荧光标记物-15-流式细胞仪光学系统激发光源与荧光标记物All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-16-光学检测信号光学检测信号-16-光学检测信号All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-17-流式流式细胞胞仪的光学系的光学系统 FACSCalibur-17-流式细胞仪的光学系统 FACSCaliburAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-18-流式细胞仪液流系统流式细胞仪液流系统-18-流式细胞仪液流系统All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-19-液流系统：液流系统：鞘液系鞘液系统样品流品流动系系统-19-液流系统：鞘液系统样品流动系统All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-20-流式细胞仪的电子系统流式细胞仪的电子系统 信号检测和分析信号检测和分析=当当细胞携胞携带荧光素光素,通通过激光照射区激光照射区,受到激受到激发产生不同波生不同波长的、代表的、代表细胞内不同物胞内不同物质的的荧光信号光信号=荧光信号由光光信号由光电接收器接收，接收器接收，转变为电脉冲信号，脉冲信号，=电脉冲信号脉冲信号经A/DA/D转换成数字信号成数字信号=数字信号数字信号传送到送到计算机，算机，进行行储存、作存、作图和和统计分分析析-20-流式细胞仪的电子系统 信号检测和分析All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-21-数据系统数据系统-21-数据系统All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-22-（二）（二）流式细胞仪可检测的细胞参数流式细胞仪可检测的细胞参数细胞结构细胞结构=细胞大小细胞大小=细胞颗粒性程度细胞颗粒性程度=细胞表面面积细胞表面面积=核浆比例核浆比例=DNADNA含量与细胞周期含量与细胞周期=RNARNA含量含量细胞功能细胞功能=细胞表面蛋白抗原细胞表面蛋白抗原=细胞胞浆内蛋白性细胞胞浆内蛋白性抗原抗原=细胞内细胞因子细胞内细胞因子=细胞增殖细胞增殖=细胞内钙离子浓度细胞内钙离子浓度二、流式细胞仪的工作原理和测量参数二、流式细胞仪的工作原理和测量参数-22-（二）流式细胞仪可检测的细胞参数细胞结构细胞功能二All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-23-（二）流式细胞仪可检测的细胞的参数：散射光信号散射光信号=前向角散射光（前向角散射光（FSC,Forward ScatterFSC,Forward Scatter）入射光的前向入射光的前向 (5 5)散射光信号散射光信号,可反映：可反映：细胞相对大小及其表面积细胞相对大小及其表面积 =侧向角散射光（侧向角散射光（SSC,Side ScatterSSC,Side Scatter）入射光入射光9090 角的角的散射光信号，散射光信号，可反映：可反映：细胞颗粒性程度细胞颗粒性程度 （胞内成分和构造和表面结构的复杂性）（胞内成分和构造和表面结构的复杂性）-23-（二）流式细胞仪可检测的细胞的参数：All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-24-散射光信号散射光信号=前向角散射光前向角散射光FSCFALS SensorLaser-24-散射光信号前向角散射光FSCFALS SensorAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-25-散射光信号散射光信号=侧向角散射光侧向角散射光SSCFALS Sensor90LS SensorLaser-25-散射光信号侧向角散射光SSCFALS SensorAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-26-流式细胞的基本信息流式细胞的基本信息:“形态形态”-26-流式细胞的基本信息:“形态”All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-27-27-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-28-外周全血外周全血细胞胞（红细胞溶解后）胞溶解后）以散射光表示的以散射光表示的 双参数双参数 点点图-28-外周全血细胞（红细胞溶解后）All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-29-外周全血外周全血细胞胞（红细胞溶解后）胞溶解后）以散射光表示的以散射光表示的 双参数双参数 点点图中性粒细胞中性粒细胞单核细胞单核细胞淋巴细胞淋巴细胞-29-外周全血细胞（红细胞溶解后）All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-30-（二）（二）流式流式细胞胞检测细胞参数胞参数:特征信息特征信息荧光（光（FluorescenceFluorescence）信号）信号=荧光素吸收激光（激发光波长）能量荧光素吸收激光（激发光波长）能量=荧光素将吸收能量释放，转换为荧光素将吸收能量释放，转换为释放放较入射光波入射光波长更更长的光量子（的光量子（发射光波射光波长）=荧光素与特异抗体结合（荧光抗体）荧光素与特异抗体结合（荧光抗体）=荧光抗体与细胞抗原结合越多，产生荧光信号越强荧光抗体与细胞抗原结合越多，产生荧光信号越强=荧光素与细胞核酸结合，荧光强度反映核酸含量荧光素与细胞核酸结合，荧光强度反映核酸含量-30-（二）流式细胞检测细胞参数:特征信息荧光（FAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-31-（二）（二）流式流式细胞胞检测细胞参数胞参数:特征信息特征信息荧光（光（FluorescenceFluorescence）信号）信号lForward scatter(FSC)lSide scatter(SSC)lFL1（第一荧光）（第一荧光）-FITC(异硫氰酸荧光素）异硫氰酸荧光素）lFL2（第二荧光）（第二荧光）-PE（藻红蛋白）（藻红蛋白）lFL3（第三荧光）（第三荧光）-PerCP,PeCy5等等lPL4（第四荧光）（第四荧光）-APC（藻青蛋白）（藻青蛋白）-31-（二）流式细胞检测细胞参数:特征信息荧光（FAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-32-荧光信号荧光信号FluorescenceFALS SensorFluorescence detector(PMT3,PMT4 etc.)-32-荧光信号FluorescenceFALS SensoAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-33-33-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-34-荧光信号光信号双色直接染色双色直接染色-34-荧光信号双色直接染色All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-35-（三）（三）流式流式细胞胞仪分析数据常分析数据常见图形：形：（二（二维）点）点陈图(Dot Plot)适用于：双参数分析适用于：双参数分析-35-（三）流式细胞仪分析数据常见图形：（二维）点陈图All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-36-（三）（三）流式流式细胞胞仪分析数据常分析数据常见图形：形：直方直方图(Histogram Plot)适用于：适用于：单参数分析单参数分析仅需要观察某仅需要观察某个荧光强度的个荧光强度的变化变化-36-（三）流式细胞仪分析数据常见图形：直方图(HiAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-37-流式流式细胞胞术检测细胞的胞的荧光光 标记方法方法单标：一种单抗一种单抗 :FL1,FSC,SSC:FL1,FSC,SSC（三参数）（三参数）双标：两种单抗：两种单抗：FL1,FL1,FSC,SSCFL1,FL1,FSC,SSC（四参数）（四参数）三标：三种单抗：三种单抗：FL1,FL2,FL3,FSC,SSCFL1,FL2,FL3,FSC,SSC（五参数）（五参数）四标四种荧光标记抗体：四种单抗：四种单抗：FL1,FL2,FL3,FL4,FSC,SSCFL1,FL2,FL3,FL4,FSC,SSC（六参数）（六参数）-37-流式细胞术检测细胞的荧光 标记方法单标：All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-38-淋巴细胞亚群分析淋巴细胞亚群分析细细 胞胞 凋凋 亡亡 肿瘤学肿瘤学移植免疫移植免疫三 流式细胞仪在临床检测和科研中的应用血小板血小板其其 它它-38-淋巴细胞亚群分析细 胞 凋 亡 肿瘤学移植免疫三 All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-39-淋巴细胞亚群分析淋巴细胞亚群分析-39-淋巴细胞亚群分析All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-40-AdhesionMetabolic-40-AdhesionMetabolicAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-41-41-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-42-42-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-43-T淋巴细胞淋巴细胞亚群检测：淋巴细胞亚群检测：Tcells,Tsubsets,NKcells,Bcells-43-T淋巴细胞淋巴细胞亚群检测：All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-44-淋巴细胞亚群淋巴细胞亚群T淋巴细胞淋巴细胞(CD3+)名名 称称功功 能能CD3+CD4+CD3+CD4+辅助性/诱导性T细胞(Th/Ti)辅助和诱导细胞免疫和体液免疫 CD3+CD4+CD29+CD3+CD4+CD29-辅助性T细胞(Th)诱导性T细胞(Ti)辅助细胞免疫和体液免疫诱导细胞免疫和体液免疫CD3+CD8+CD3+CD8+抑制性/杀伤性T细胞(Ts/Tc)抑制免疫反应/杀伤异源细胞 CD3+CD8+CD28-CD3+CD4+CD29-抑制性T细胞(Ts)杀伤性T细胞(Tc)抑制细胞和体液免疫细胞毒性T细胞CD3+CD4+CD8+活化的T细胞在T细胞恶性增生时升高 CD3+CD4-CD8-有调节功能的T细胞,其表达/T细胞受体(TCR/)CD4+/CD4+/CD8CD8+比值比值比值比值CD45RA+CD45RO-幼稚的/静止的T淋巴细胞 当免疫系统没有能力更新辅助性或抑制性/细胞毒性淋巴细胞的祖细胞时此细胞亚群较低 CD45RA-CD45RO+记忆性T淋巴细胞 病菌感染时升高,但在慢性病毒感染,如HIV患者中降低CD45RA+CD45RO+活化的T细胞,感染时升高 感染时升高-44-淋巴细胞亚群T淋巴细胞(CD3+)名 称功 能All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-45-淋巴细胞亚群淋巴细胞亚群活化活化T淋巴细胞淋巴细胞名名 称称功功 能能CD3+HLA-DR+活化T细胞CD4+HLA-DR+活化的辅助性/诱导性T细胞CD8+HLA-DR+活化的抑制性/杀伤性T细胞CD4+CD25+活化的辅助性/诱导性T细胞CD8+CD25+CD8+CD38+HLA-DR+活化的抑制性/杀伤性T细胞在病毒感染的患者中增加;其增加意味着HIV患者的预后较差B B淋巴细胞淋巴细胞淋巴细胞淋巴细胞(19+)(19+)CD19+CD23+活化的B细胞过敏患者中升高 CD19+CD5+在自身免疫性疾病中升高 CD19+CD5+CD23+慢性B细胞白血病及单克隆B淋巴细胞 NKNK细胞细胞细胞细胞CD3+CD(16+56)+CD3+CD(16+56)+自身免疫性疾病时降低,而病毒感染时升高 CD3+CD57+CMV(巨细胞病毒)感染的强烈征兆 -45-淋巴细胞亚群活化T淋巴细胞名 称功 All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-46-淋巴细胞亚群与疾病淋巴细胞亚群与疾病疾疾 病病CD3CD4CD8CD4/CD8NK自身免疫性疾病 系统性红斑狼疮 类风湿性关节炎 自身免疫性溶血性贫血 重症肌无力免疫缺陷病 爱滋病病毒感染 传染性单核细胞增多症慢性活动性肝炎活动性肝硬化肿瘤 消化道癌,肝癌,肺癌再生障碍性贫血粒细胞减少症-46-淋巴细胞亚群与疾病疾 病CD3CD4CD8All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-47-淋巴细胞亚群检测意义淋巴细胞亚群检测意义疾病机理研究 疾病辅助诊断移植后免疫检测治疗方案的确定及疗效评价CD25高于正常值5-10%,表明即将或已发生排斥CD4/CD8比值下降提示并发凶险感染 0.2时,必须停用免疫抑制剂CD2+HLA-DR+增加5-10%,且不伴有CD25的增加,提 示巨细胞病毒感染慢性疲劳,感染后综合征,纤维肌痛,类风湿性关节炎,过敏等 疾病预防-47-淋巴细胞亚群检测意义疾病机理研究 疾病辅助诊断移植后All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-48-淋巴细胞亚群检测意义淋巴细胞亚群检测意义Control（n=20)Active SLE(n=17)Stable SLE(n=13)CD370.4 5.8765.8 10.968.5 8.95CD435.0 5.7022.1 4.15*34.5 4.23CD827.9 5.7345.7 9.58*32.7 9.01CD4/CD81.27 0.140.43 0.12*1.05 0.26*NK17.8 5.484.37 3.31*7.59 5.01*鉴别活动期和非活动期SLE-48-淋巴细胞亚群检测意义Control（n=20)AAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-49-HIV AND AIDS-49-HIV AND AIDSAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-50-Th1/Th2细胞细胞-50-Th1/Th2细胞All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-51-Th1/Th2细胞胞lTh1型细胞因子介导型细胞因子介导 细胞免疫反应细胞免疫反应n引起引起迟发型型过敏反敏反应、巨噬、巨噬细胞活化、局部炎症胞活化、局部炎症n刺激来源：病毒、某些刺激来源：病毒、某些细菌菌lTh2型介导体液免疫反应型介导体液免疫反应n引起引起B细胞增殖、分泌多克隆免疫球蛋白、胞增殖、分泌多克隆免疫球蛋白、n促促进I型型变态反反应（过敏反敏反应）n刺激来源：多刺激来源：多细胞寄生虫、胞寄生虫、过敏原敏原-51-Th1/Th2细胞Th1型细胞因子介导 细胞免疫反应All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-52-Th1/Th2细胞细胞PMA+Ionomycin 刺激4-52-Th1/Th2细胞PMA+Ionomycin 刺激4All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-53-流式流式细胞胞仪检测胞内胞内细胞因子胞因子 l新鲜分离新鲜分离PBMC l加加PMAIo刺激后刺激后6hl测定胞内细胞因子测定胞内细胞因子IL-4和和IFN-g g l检测免疫细胞表型和结检测免疫细胞表型和结合检测胞内细胞因子：合检测胞内细胞因子：操作和分析方法操作和分析方法；-53-流式细胞仪检测胞内细胞因子 新鲜分离PBMC 检测免All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-54-四色四色标记胞内因子胞内因子FCM检测-54-四色标记胞内因子FCM检测All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-55-Th1/Th2失衡相关疾病失衡相关疾病嗜酸性细胞增多和嗜酸性细胞增多和IgE升高相关疾病升高相关疾病 Ommens Syndrome(Ommens综合征)Hyperosinophilic Syndrome(高嗜曙红细胞综合征)Kimuras Disease(Kimuras疾病)Wells Syndrome(Wells 综合征)传染病传染病 病毒性疾病病毒性疾病 人类免疫缺陷性病毒感染和爱滋病(AIDS)麻疹 呼吸道合胞体病毒感染 慢性活动性乙型肝炎病毒感染 分枝杆菌和其它细菌感染分枝杆菌和其它细菌感染 麻疯病/肺结核 鸟结核分枝杆菌复合感染 滑膜炎反应性关节炎 淋病和梅毒 -55-Th1/Th2失衡相关疾病嗜酸性细胞增多和IgE升高All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-56-Th1/Th2失衡相关疾病失衡相关疾病寄生虫病 原寄生虫感染：皮肤和内脏利什曼病 蠕虫病：丝虫病和血吸虫病真菌性疾病 过敏性支气管肺霉菌病 念珠菌病 隐球菌病 类球孢子菌病 球孢子菌病 组织胞浆菌病恶性肿瘤 皮肤基底细胞和鳞细胞癌 Sezary Syndrome(Sezary综合征)淋巴瘤 EBV相关的B细胞和非何杰金氏淋巴瘤 何杰金氏淋巴瘤 大细胞退行性(CD30+)淋巴瘤 多发性骨髓瘤和Castlemans综合征 卡氏肉瘤-56-Th1/Th2失衡相关疾病寄生虫病www.gotofAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-57-Th1/Th2失衡相关疾病失衡相关疾病变应性疾病变应性疾病 皮肤性哮喘 变应性哮喘 春季结膜炎炎性和自身免疫性疾病炎性和自身免疫性疾病 巨细胞(暂时性的)动脉炎 系统性红斑狼疮 类风湿性关节炎 自身免疫性甲状腺疾病 类肉瘤病 银屑病(牛皮癣)IgA肾病 多发性硬化-57-Th1/Th2失衡相关疾病变应性疾病www.gotoAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-58-Th1/Th2细胞检测意义细胞检测意义疾病治疗发病机制研究使用Th1和Th2相关的细胞因子 IFN-治疗麻疯病 用抗Th1和Th2相关细胞因子或其受体的抗体 抗IL-4抗体治疗真菌和寄生虫感染 使用调节Th2/Th2细胞因子平衡的药物:萨力多胺(Thaliodomide)治疗巨细胞动脉炎疾病治疗监测-58-Th1/Th2细胞检测意义疾病治疗发病机制研究使用TAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-59-肿瘤学肿瘤学-59-肿瘤学All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-60-肿瘤发生发展肿瘤发生发展-60-肿瘤发生发展All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-61-0 200 400 600 8001000PI 荧光强度荧光强度(DNA含量含量)DNA含量分析含量分析2N2N4N4NCountsG0/G1G2/MS异倍体-61-0 200 400 600 8001000PI 荧光All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-62-DNA含量分析含量分析70%人类癌症中存在着异倍体或细胞周期的改变1.Ross JS.DNA Ploidy and Cell Cycle Analysis in Pathology.New York:Igaku-Shoin;1996:47-60DNA倍体和S期比例-指导治疗2 卵巢癌，乳腺癌，结肠癌，子宫内膜癌，原发部位不明的癌症 -早期 (I/II期)DNA倍体和DNA指数-预后长期和无病生存期2 卵巢癌-晚期 2.The Recommend Medicare National Coverage Policy for Flow Cytometry.HCFA Website,1999 -62-DNA含量分析70%人类癌症中存在着异倍体或细胞周期All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-63-P53/Bcl-63-P53/Bcl-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-64-P53乳腺癌 与病理学分级有关,并与乳腺癌的淋巴结转移显著相关 大肠肿瘤 早期发现大肠癌胃 癌 与预后相关肝细胞癌 与组织分级,肿瘤预后,转移及肿瘤大小密切相关-64-P53乳腺癌 与病理学分级有关,并与乳腺癌All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-65-Bcl-2Bcl-2在正常组织中的表达 造血系统细胞 淋巴,单核细胞等 受激素或生长因子调节的腺上皮 前列腺上皮,子宫内膜等 干细胞或增值细胞 皮肤的基底细胞,肠上皮 寿命长的分裂细胞 神经元 胚胎组织Bcl-2在肿瘤组织中的表达 肾脏肿瘤 肾癌,肾盂乳头瘤,Wilms瘤,肾嗜酸细胞瘤 前列腺癌 B细胞性淋巴瘤 乳腺癌(43%)甲状腺癌-65-Bcl-2Bcl-2在正常组织中的表达 造血All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-66-nm23nm23与多种肿瘤的转移及预后相关 肝细胞癌 甲状腺肿瘤 乳腺癌 结肠癌 肉瘤-66-nm23nm23与多种肿瘤的转移及预后相关 All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-67-CD44(CD44s/CD44v)-67-CD44(CD44s/CD44v)www.gotofAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-68-肿瘤多药耐药性肿瘤多药耐药性(MDR)检测检测P-gp在肿瘤组织中的表达 乳腺癌,膀胱癌,胃癌,食管癌,卵巢癌,前列腺癌等 化疗前高表达化疗后高表达 肾细胞癌,结肠癌,肝癌,肾上腺皮质癌,胰腺癌等 化疗前低表达 非何杰金氏淋巴瘤,成神经细胞瘤,急性髓白血病等 化疗不敏感化疗敏感化疗不敏感-68-肿瘤多药耐药性(MDR)检测P-gp在肿瘤组织中的表All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-69-乳腺癌乳腺癌石蜡切片脱腊ER(雌激素受体)ER抗体染色酶解流式检测固定(多聚甲醛)破膜(70%甲醇)细 胞 -69-乳腺癌石蜡切片脱腊ER(雌激素受体)ER抗体染色酶解All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-70-Cyclin(细胞周期蛋白细胞周期蛋白)-70-Cyclin(细胞周期蛋白)www.gotofcm.All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-71-细细 胞胞 凋凋 亡亡-71-细 胞 凋 亡All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-72-细胞凋亡分析-72-细胞凋亡分析 All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-73-DNA断裂断裂-73-DNA断裂All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-74-乳腺癌Fas/FasL与疾病与疾病慢性病毒性肝炎慢性病毒性肝炎恶性肿瘤细胞恶性肿瘤细胞-黑色素瘤、结肠癌、肺癌、肝癌等黑色素瘤、结肠癌、肺癌、肝癌等脑缺血后细胞凋亡脑缺血后细胞凋亡自身免疫性疾病自身免疫性疾病系统性红斑狼疮系统性红斑狼疮Fas/FasL(CD95/CD95L)-74-乳腺癌Fas/FasL与疾病慢性病毒性肝炎恶性肿瘤细All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-75-乳腺癌Caspases早期细胞凋亡早期细胞凋亡Annexin V/PI-75-乳腺癌Caspases早期细胞凋亡Annexin VAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-76-移植免疫移植免疫-76-移植免疫All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-77-CD25高于正常值高于正常值5-10%,表明即将或已发生排斥表明即将或已发生排斥CD4/CD8比值下降提示并发凶险感染比值下降提示并发凶险感染 0.2时时,必须停用免疫抑制剂必须停用免疫抑制剂CD2+HLA-DR+增加增加5-10%,且不伴有且不伴有CD25的增加的增加 提示巨细胞病毒感染提示巨细胞病毒感染移植后免疫监测移植后免疫监测-77-CD25高于正常值5-10%,表明即将或已发生排斥All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-78-血小板血小板-78-血小板All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-79-血小板活化血小板活化静止血小板活化血小板-79-血小板活化静止血小板活化血小板www.gotofcmAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-80-血小板活化血小板活化-80-血小板活化All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-81-网织血小板网织血小板ITP(特发性血小板减少症)化疗鉴别诊断骨髓移植-81-网织血小板ITP(特发性血小板减少症)化疗鉴别诊断骨All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-82-血小板计数新标准血小板计数新标准(间接双平台法间接双平台法)R=红细胞数/血小板数(流式细胞术)血小板数血小板数=红细胞数(血球仪)/R(国际血液学标准化委员会(ICSH)/国际实验血液学协会(ISLH)2001)灵敏度高(1-400 x109/L)，重复性好，室内/室外变异系数均很小尤其应用于血小板预输注病人的检测(阈值:20 x109/L)-82-血小板计数新标准(间接双平台法)R=红细胞数/血All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-83-其其 它它-83-其 它All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-84-HLA-B27HLA-B-84-HLA-BAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-85-正常人患 者HLA-B27HLA-B-85-正常人患 者HLA-B27www.gotofcm.cAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-86-细胞介导的细胞毒作用细胞介导的细胞毒作用活靶细胞死靶细胞替代51Cr释放实验-86-细胞介导的细胞毒作用 活All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-87-HLA-B27检测Calibration beadsHLA-B27 calibration beadsDecision markerHLAB27FITC（MFI)-87-HLA-B27检测Calibration beadsAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-88-流式临床应用发展流式临床应用发展白血病/淋巴瘤免疫分型HIV感染的CD4绝对计数CD34绝对计数/异体移植的免疫重建抗原特异性T细胞检测树突状细胞（DC）循环微转移肿瘤细胞IIIIIIIVMarti GE,Maryalice SS,et al.Introduction to Flow cytometry.Seminars in Hematology,2001;38(2):93-99-88-流式临床应用发展 白血病All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-89-流式微球分析流式微球分析 Cytometric Bead Array（CBA）-89-流式微球分析 Cytometric Bead ArAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-90-CBACBA与与ELISAELISA相比优点相比优点“多元多元”和和“同步同步”检测检测样本量少样本量少灵敏度相似灵敏度相似稳定性好稳定性好-90-CBA与ELISA相比优点All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology91 以上有不当之处，请大家给与批评指正，以上有不当之处，请大家给与批评指正，谢谢大家！谢谢大家！91