Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, and the Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215006, China.
J Transl Med. 2017 Oct 23;15(1):214. doi: 10.1186/s12967-017-1317-2.
Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation.
Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs.
The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%.
A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.
血小板自身抗体检测对免疫性血小板减少症（ITP）的诊断和预后至关重要。因此，我们旨在建立一种用于评估 ITP 血小板自身抗体的定量流式细胞免疫珠分析（FCIA）。
捕获微珠与抗 GPIX、GPIb、GPIIb、GPIIIa 和 P-选择素抗体偶联，用于结合从 250 名 ITP 患者、163 名非 ITP 患者和 243 名健康对照者的血浆样本中产生的血小板结合自身抗体复合物，用荧光素异硫氰酸酯（FITC）缀合的二级抗体作为检测试剂，通过流式细胞术记录平均荧光强度（MFI）信号。评估定量 FCIA 测定的内和间分析变异。比较定量和定性 FCIA 或单克隆抗体固定血小板抗原（MAIPA）测定之间的特异性、敏感性和准确性。最后，我们通过定量 FCIA 在 8 例新诊断的 ITP 患者中监测治疗过程。
定量 FCIA 测定的变异系数（CV）分别为抗 GPIX、GPIb、GPIIIa、GPIIb 和 P-选择素自身抗体的 9.4%、3.8%、5.4%、5.1%和 5.8%。与非 ITP 患者或健康对照者相比，我们的定量 FCIA 在 ITP 患者中检测到血小板糖蛋白 GPIX、GPIb、GPIIIa、GPIIb 和 P-选择素自身抗体水平升高。当结合所有 5 种自身抗体时，我们的定量检测的敏感性、特异性和准确性分别为 73.13%、81.98%和 78.65%，而 MAIPA 检测的敏感性、特异性和准确性分别为 41.46%、90.41%和 72.81%。
建立了一种定量 FCIA 测定法。在 ITP 患者接受皮质类固醇治疗后，我们的定量 FCIA 可确认血小板自身抗体水平降低。我们的定量检测不仅有利于 ITP 的诊断，也有利于 ITP 的治疗监测。
