流式微球法检测vWF活性及其在评估脑卒中预后中的应用  

Establishment of Flow Cytometric Immunobead Array for Detecting Plasma Von Willebrand Factor Activity and Its Clinical Application in the Prognosis of Ischemic Stroke

作　　者：闫彬 何杨[2] 陆士奇[3] 徐梦乔[2] 王琪 赵益明[2] 阮长耿[2] YAN Bin;HE Yang;LU Shi-Qi;XU Meng-Qiao;WANG Qi;ZHAO Yi-Ming;RUAN Chang-Geng(Department of Laboratorial Medicine,Nanyang City Center Hospital,Nanyang 473000,Henan Province,China;The First Affiliated Hospital of Soochow University,Jiangsu Institute of Hematology,Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou 215006,Jiangsu Province,China;Department of Emergency,The First Affiliated Hospital of Soochow University,Suzhou 215006,Jiangsu Province,China)

机构地区：[1]南阳市中心医院检验科,河南南阳473000 [2]苏州大学附属第一医院江苏省血液研究所卫生部血栓与止血重点实验室,江苏苏州215006 [3]苏州大学附属第一医院急诊科,江苏苏州215006

出　　处：《中国实验血液学杂志》2019年第1期208-214,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金面上项目(81270593);国家自然科学基金项目(81873431)

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.

关 键 词：流式微球技术 血管性血友病因子 血管性血友病 脑卒中 

分 类 号：R554.1[医药卫生—血液循环系统疾病]