摘要
目的:建立流式微球技术(FCIA)检测血浆血管性血友病因子活性(vWF:GPIbR)的方法,及其在缺血性脑卒中(IS)预后评估的应用。方法:包被抗人血小板糖蛋白Ibα(GPIbα)单克隆抗体SZ151 IgG的微球与重组GPIbα孵育,加入待检血浆和瑞斯托霉素,最终与FITC标记的绵羊抗人vWF IgG多抗反应,上流式细胞仪检测。联合应用vWF抗原(vWF:Ag)、vWF:GPIbR活性和vWF胶原结合活性(vWF:CB)检测,分析脑卒中患者的v WF状态。结果:流式微球法批内差异和批间差异分别为7.7%和13.5%;其与经典的ELISA法有较好的相关性(r=0.813,P <0.001),两种方法的偏倚为9.95%。与ELISA法相比,流式微球法有更高的特异度和准确度(P<0.05)。脑卒中患者vWF:Ag、vWF:GPIbR和vWF:CB水平显著高于正常对照组(H值分别为7.8、6.4、6.2)(P<0.001)。脑卒中患者vWF:GPIbR与vWF:Ag、超敏CRP、Autar评分和住院天数呈较好的正相关。结论:流式微球技术检测血浆vWF:GPIbR简单可靠,特异度高,准确性好。v WF:GPIbR参与了脑卒中的发生,并能够用于预测脑卒中患者的血栓形成风险,评估预后。
Objective: To establish a novel flow cytometric immunobead array(FCIA) for detecting plasma von Willebrand factor activity(vWF:GPIbR) and apply it in ischemic stroke(IS). Methods: Microspheres coated with anti-human platelet glycoprotein Ibα(GPIbα) monoclonal antibody SZ151 IgG, were incubated with recombinant fragment of GPIbα, then added ristocetin and plasma, finally incubated with FITC-conjugated sheep-anti-human vWF IgG polyclonal antibody, and detected by flow cytometry. vWF antigen(vWF:Ag), vWF:GPIbR, and vWF collagen binding assay(vWF:CB) were also included for evaluating vWF levels in IS patients. Results: The intra-assay coefficient variations(CVs) and inter-assay CVs of FCIA were 7.7% and 13.5%, respectively. The slope of the linear regression was 0.9739(r=0.855, P<0.001), and the Bland-Altman bias was 9.95%, indicating a good correlation between FCIA and ELISA. The FCIA had better sensitivity, specificity and accuracy as compared with those by ELISA(P<0.05). The levels of vWF:Ag, vWF:GPIbR and vWF:CB in IS patients were significantly higher in comparison with those in healthy controls(H=7.8, 6.4, 6.2, respectively, P<0.01), the level of vWF:GPIbR in IS patients positively correlated with levels of vWF:Ag, high-sensitivity C-reactive protein, Autar score and hospitalization time. Conclusion: The FCIA for detecting plasma vWF:GPIbR is more specific and accurate than ELISA. The vWF:GPIbR is involved in the paroxysm of IS, which could be used to evaluate the risk of thrombosis in IS patients.
引文
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