最新流式细胞仪在临床检测和临床科研中的应用课件.ppt

1、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=免疫学检测和科研应用免疫学检测和科研应用=血液病检测和科研应用血液病检测和科研应用=肿瘤学检测和科研应用肿瘤学检测和科研应用流式细胞仪的基本工作原理以及在基础流式细胞仪的基本工作原理以及在基础和临床科研中的应用和临床科研中的应用All ri

2、ghts reserved by Dr Baiqing LIBengbu Medical College Department of ImmunologyAll rights reserved by Dr Baiqing LIBengbu Medical College Department of ImmunologyAll rights reserved by Dr Baiqing LIBengbu Medical College Department of ImmunologyAll rights reserved by Dr Baiqing LIBengbu Medical Colleg

3、e Department of ImmunologyAll rights reserved by Dr Baiqing LIBengbu Medical College Department of ImmunologyAll 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

4、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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-10-二、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l光路系统光路系统：激光光源，光路系统（透镜、滤光片）激光光源，光路系统（透镜、滤光片）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

5、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 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,

6、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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-12-光路系统光路系统All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-13-Injector TipAll rights reserved by Dr Baiqing LIBengbu Medical College Depa

7、rtment of Immunology-14-Injector TipSheath fluidAll 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All rights reserved by Dr Ba

8、iqing LIBengbu Medical College Department of Immunology-17-流式细胞仪的光学系统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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-19-液流系统：液流系统：鞘液系统样品流

9、动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=当细胞携带荧光素当细胞携带荧光素,通过激光照射区通过激光照射区,受到激发产受到激发产生不同波长的、代表细胞内不同物质的荧光信号生不同波长的、代表细胞内不同物质的荧光信号=荧光信号由光电接收器接收，转变为电脉冲信号，荧光信号由光电接收器接收，转变为电脉冲信号，=电脉冲信号经电脉冲信号经A/DA/D转换成数字信号转换成数字信号=数

10、字信号传送到计算机，进行储存、作图和统计分数字信号传送到计算机，进行储存、作图和统计分析析All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-21-数据系统数据系统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

11、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,

12、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）All rights reserved by Dr Baiqing LIBengbu Medical College Department o

13、f 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 SensorLaserAll 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 SensorLaserAll 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

14、本信息:“形态形态”All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-27-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-28-外周全血细胞外周全血细胞（红细胞溶解后）（红细胞溶解后）以散射光表示的以散射光表示的 双参数双参数 点图点图All rights reserved by Dr Baiqing LIBengbu Medical College Dep

15、artment of Immunology-29-外周全血细胞外周全血细胞（红细胞溶解后）（红细胞溶解后）以散射光表示的以散射光表示的 双参数双参数 点图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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-30-（二）（二）流式细胞检测细胞参数流式细胞检测细胞参数:特征信息荧光（特征信息荧光（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

16、将吸收能量释放，转换为荧光素将吸收能量释放，转换为释放较入射光波长更长的光量子（发射光波长）释放较入射光波长更长的光量子（发射光波长）=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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-31-（二）（二）流式细胞检测细胞参数流式细胞检测细胞参

17、数:特征信息荧光（特征信息荧光（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）All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-

18、32-荧光信号荧光信号FreqFluorescenceFALS SensorFluorescence detector(PMT3,PMT4 etc.)All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-33-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-34-荧光信号荧光信号双色直接染色双色直接染色All rights reserved by Dr Baiqi

19、ng LIBengbu Medical College Department of Immunology-35-（三）（三）流式细胞仪分析数据常见图形：流式细胞仪分析数据常见图形：（二维）点陈图（二维）点陈图(Dot Plot)适用于：双参数分析适用于：双参数分析All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-36-（三）（三）流式细胞仪分析数据常见图形：流式细胞仪分析数据常见图形：直方图直方图(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

20、需要观察某个荧光强度的个荧光强度的变化变化All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-37-流式细胞术检测细胞的荧光流式细胞术检测细胞的荧光 标记方法标记方法单标：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

21、）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）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All rights reserved by Dr Baiqing LIBengbu Medical College Depa

22、rtment of Immunology-39-淋巴细胞亚群分析淋巴细胞亚群分析All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-40-AdhesionMetabolicAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-41-All rights reserved by Dr Baiqing LIBengbu Medical College Department

23、 of Immunology-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：T cells,T subsets,NK cells,B cellsT cells,T subsets,NK cells,B cellsAll 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

24、淋巴细胞(CD3+)名名 称称功功 能能辅助性/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/)CD45RA+CD45RO-幼稚的/静止

25、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

26、+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 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 All rights reserved by Dr Baiqing LIBengbu Medical College Department of I

27、mmunology-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All 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

28、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

29、.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SLEAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-49-HIV AND AIDSAll rights reserved by Dr Baiqing LIBengbu Medic

30、al College Department of Immunology-50-Th1/Th2细胞细胞All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-51-Th1/Th2细胞细胞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引起迟发型过敏反应、巨噬细胞活化、局部炎症引起迟发型过敏反应、巨噬细胞活化、局部炎症n刺激来源：病毒、某些细菌刺激来源：病毒、某些细菌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、细胞增殖、分泌多克

31、隆免疫球蛋白、n促进促进I型变态反应（过敏反应）型变态反应（过敏反应）n刺激来源：多细胞寄生虫、过敏原刺激来源：多细胞寄生虫、过敏原All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-52-Th1/Th2细胞细胞PMA+Ionomycin 刺激4-All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-53-流式细胞仪检测胞内细胞因子流式细胞仪检测胞内细胞因子 l新鲜分

32、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-检测免疫细胞表型和检测免疫细胞表型和结合检测胞内细胞因结合检测胞内细胞因子：子：操作和分析方法操作和分析方法；All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-54-四色标记胞内因子四色标记胞内因子FCM检测检测All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunolog

33、y-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All ri

34、ghts 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All

35、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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-5

36、8-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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-59-肿瘤学肿瘤学All rights reserved by Dr Baiqing LIBengbu Medical Coll

37、ege Department of Immunology-60-肿瘤发生发展肿瘤发生发展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)0 75 150 225 300DNA含量分析含量分析CountsG0/G1G2/MS异倍体All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunolog

38、y-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

39、 All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-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All rights reserved by Dr Baiqing LIB

40、engbu 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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunolo

41、gy-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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-67-CD44(CD44s/CD44v)人人 体体 肿肿 瘤瘤CD44 亚亚 型型与肿瘤的临床相关性与肿瘤的临床相关性肺癌非特异总 CD44 表达的下调与较差的预后相关胃肠道癌CD44v9胃肠道肿瘤的独立的预后指标CD44v8-v10与肝转移相关,并为结直肠癌的独立的预后指标CD44v6与肠癌的浸润相关恶性黑色素瘤CD44v5与侵润

42、性相关乳腺癌CD44v6受雌激素和孕激素调控CD44v3/v4,CD44v6肿瘤分级,但不是独立的预后指标CD44s,CD44v6肿瘤分级,但不是独立的预后指标甲状腺癌(乳头型)CD44v6与淋巴节转移相关非何杰金氏淋巴瘤CDv3,v6,v9与较差预后相关神经母细胞瘤CD44s总 CD44 表达的下调与较差的预后相关All 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

43、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 All rights reserved by Dr Baiqing LIBengbu Medical College Department

44、 of Immunology-70-Cyclin(细胞周期蛋白细胞周期蛋白)All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-71-细细 胞胞 凋凋 亡亡All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-72-细胞凋亡分析 All rights reserved by Dr Baiqing LIBengbu Medical College Department

45、of Immunology-73-DNA断裂断裂All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-74-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)All rights reserved by Dr Baiqing LIBengbu Medical Col

46、lege Department of Immunology-75-Caspases早期细胞凋亡早期细胞凋亡Annexin V/PIAll rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-76-移植免疫移植免疫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

47、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All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-78-血小板血小板All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-79-血小板活化

48、血小板活化静止血小板活化血小板All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-80-血小板活化血小板活化流式全血法流式全血法血小板聚集仪血小板聚集仪样本处理样本处理抗凝全血PRP(富血小板血浆)人为活化人为活化最低强烈(经离心,洗涤)样本量样本量低至2uL10mL灵敏度灵敏度5%不高相关病症相关病症检测指标检测指标临床意义临床意义贮存血小板CD62与输注血小板恢复负相关心肺分流术(CPB)冠状血管形成术肾透析不稳定心绞痛急性心肌梗塞中风脑血管局部缺血糖尿病产前痉挛子痫症心肺分

49、流术(CPB)急性冠状动脉综合征血小板-白细胞聚集较血小板活化敏感监测和预后生物相容性Cd62p,CD63Cd62p,CDAll 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

50、=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)All rights reserved by Dr Baiqing LIBengbu Medical College Department of Immunology-83-其其 它它All rights reserved by Dr Baiqing LIBengbu Medical College Departm