高通量法测定血小板自身抗体的含量及临床意义

Clinical significance of high-throughput measurement of platelet autoantibody levels

建立高通量流式细胞检测法测定免疫性血小板减少症(ITP)患者血浆中的自身抗体,探索其在疾病中的临床意义。首先将鼠源性抗血小板膜糖蛋白(GP)Ⅸ、抗GPⅠb、抗GPⅢa、抗GPⅡb和抗P-选择素的单克隆抗体分别包被于自身带有5种不同强度APC-cy7荧光的聚苯乙烯微球上,用流式细胞仪检测。建立受试者工作特征曲线评价该方法的特异度、灵敏度以及批内、批间差异。5种单克隆抗体检测的批内变异系数分别为8.35%、5.65%、3.41%、3.63%和3.51%。批间变异系数分别为10.10%、9.13%、8.04%、9.59%和10.20%。敏感度、特异度和精密度:抗GPⅨ分别为71.0%、80.4%和73.5%;抗GPⅠb分别为82.1%、80.8%和81.8%;抗GPⅢa分别为71.7%、79.2%和73.7%;抗GPⅡb分别为71.0%、76.7%和72.5%;抗P-选择素分别为72.0%、79.2%和74.0%;5种抗体联合检测分别为89.0%、66.5%和82.9%。利用本方法检测了75例口服地塞米松ITP患者治疗前后的自身抗体水平高低。成功建立一种基于流式微球技术特异性检测免疫性血小板减少性紫癜患者体内针对血小板膜糖蛋白自身抗体新的诊断方法。

A high-throughput flow cytometry method was established to determine autoantibodies in the plasma of immune thrombocytopenia(ITP) patients and to explore the meaning of clinical significance. The monoclonal antibodies anti-platelet glycoprotein(GP) Ⅸ, Ⅰb, Ⅲa, Ⅱb and P-selectin were coated on five different strength APC-cy7 fluorescent microsbeads for flow cytometry detection. The receiver operating characteristic curve was established to evaluate the specificity and sensitivity of the method as well as the intra-assay CVs and inter-assay CVs differences. The intra-assay CVs were 8.35%, 5.65%, 3.41%, 3.63% and 3.51%, respectively. The inter-assay CVs were 10.10%, 9.13%, 8.04%, 9.59% and 10.20%, respectively. Sensitivity, specificity, and accuracy were 71.0%, 80.4% and 73.5% for anti-GPⅨ;82.1%, 80.8% and 81.8% for anti-GPⅠb;71.7%, 79.2% and 73.7% for anti-GPⅢa;71.0%,76.7% and 72.5% for anti-GPⅡb;72.0%, 79.2% and 74.0% for anti-P-selectin;89.0%, 66.5% and 82.9% for the combined detection of the five antibodies. This method was used to detect the level of autoantibody before and after treatment in 75 ITP patients with oral dexamethasone. A new diagnostic method for platelet membrane glycoprotein autoantibodies in patients with immune thrombocytopenic purpura is successfully established based on flow microsphere technology.