流式微球技术检测糖蛋白Ⅱb/Ⅲ a自身抗体在特发性血小板减少性紫癜诊断中的应用

目的 建立流式微球技术检测血小板膜表面糖蛋白Ⅱ b/Ⅲa(GPⅡb/Ⅲa)特异性自身抗体的方法,比较该方法和改良间接单克隆抗体俘获血小板抗原技术(MAIPA)在特发性血小板减少性紫癜(ITP)与非免疫性血小板减少性紫癜鉴别诊断中的价值.方法 应用抗人血小板GPⅡb/Ⅲa单抗(CD41a)包被微球.分离血小板,将血小板裂解后与包被过的微球孵育,再加入PE标记的羊抗人IgG多克隆抗体,流式细胞术分析.分别用该方法和改良间接MAIPA检测血小板表面和血浆中的糖蛋白特异性自身抗体,并将检测结果进行比较.结果 将样本的平均荧光强度值(MFI)与正常对照MFI的均值比较,计算比率.该比率均值及范围在ITP组为3.36(0.84～22.94),非免疫性血小板减少组为1.16(0.67～5.59),正常对照组为1.08(0.72～1.76).非参数检验得ITP组荧光强度比率与非免疫性血小板减少组及正常对照组存在显著差异(P《0.01).若将正常对照组上限作为界值,比率大于1.76定为阳性,则流式微球技术诊断ITP的敏感性为71.43%,特异性为94.28%,阳性预测值为95.24%,其敏感性高于改良间接MAIPA的测定值(51.79%)(χx2=4.57，P<0.05)。由ROC曲线得流式微球法区分ITP患者与正常人的鉴别效度为0.916。结论 流式微球技术检测血小板膜表面糖蛋白特异性自身抗体，耗时短、重复性好，敏感性高于改良间接MAIPA，对提高ITP的实验诊断水平及指导临床治疗有一定的价值。

Determination of platelet-associate autoantibodies against platelet-specific receptors by cytometric bead array and its clinical application in idiopathic thrombocytopenic purpura

OBJECTIVE:To establish a method for detecting platelet-associate autoantibodies against platelet-specific receptors using cytometric bead array, and compare the clinical usefulness of this method and modified indirect monoclonal antibody immobilization of platelet antigen technique (MAIPA) in the differential diagnosis of idiopathic thrombocytopenic purpura (ITP) from non-immune thrombocytopenic purpura (non-ITP). METHODS: The microbeads were coated with monoclonal antibodies against glycoprotein IIb/IIIa (CD41a), platelets were isolated from blood samples, then platelet lysate was incubated with the coated microbeads, and R-Phycoerythrin-conjugated goat-antihuman IgG polyclonal antibodies, finally analyzed with flow cytometry. GP IIb/IIIa autoantibodies in sample plasma were measured by modified indirect MAIPA at the same time. RESULTS: The individual fluorescene level was calculated as the ratio to the three controls. The mean ratios were 3.36 (range 0.84 - 22.94) in the ITP group, 1.16 (range 0.67 - 5.59) in the non-ITP patient and 1.08 (range 0.72 - 1.76) in the healthy controls. There was a highly significant difference (P <0.01) between the ITP patients and either the non-ITP patients or the normal controls. If the up limit of healthy controls was set as cutoff value, ratio of greater than 1.76 was considered positive. Cytometric bead array had a sensitivity of 71.43%, a specificity of 94.28% and a positive predictive value of 95.24% for the diagnosis of ITP, the sensitivity being higher than that of modified indirect MAIPA' s (5179%) (chi2 = 4.57, P <0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.916. CONCLUSION: Flow cytometric bead method for detection of platelet-associate autoantibodies against platelet-specific receptors is a more rapid, better reproducibility and higher sensitivity than modified MAIPA, and has a potential value in promoting the diagnosis of ITP and guiding treatment.