血管性血友病诊断与分型方法的建立和临床应用

Development and clinical application of diagnostic tests for von Willebrand disease

目的建立系列的血管性血友病(vWD)筛查、诊断和分型的方法。方法以Ⅲ型胶原包被微孔板,以辣根过氧化物酶(HRP)-兔抗人血管性血友病因子(vWF)IgG 为检测抗体,建立 vWF胶原结合试验(vWF:CB);通过垂直式琼脂糖电泳、电转印和化学发光法对 vWF 多聚体进行分析。对可疑 vWD 患者测定血小板、出血时间、活化部分凝血活酶时间(APTT)、血小板聚集试验(RIPA)、FVⅢ:C、vWF:RCo、vWF:Ag、vWF:CB,并进行多聚体分析。结果所建 vWF:CB 线性范围广,灵敏度高,重复性好;所建多聚体琼脂糖电泳分析方法安全、敏感、解像度高。10例可疑病例血小板均正常,大部分出血时间延长、APTY 延长、RIPA 反应低下、FVⅢ:C 降低;vWF:RCo、vWF:Ag 含量和 vWF:CB均不同程度降低;部分患者 vWF:Ag/vWF:CB 比值>2,中、高分子量 vWF 显著缺如。8例患者可明确诊断为 vWD,其中1型2例、2A 型4例和3型2例。结论本研究建立的方法和推荐的组合实验适合于绝大多数 vWD 患者的诊断和分型需要。

Objective To develop a panel of screening, diagnostic and classifying tests for von Willebrand disease（vWD）. Methods An assay for von Willebrand factor （vWF）： CB was developed with type Ⅲ collagen and HRP labeled rabbit anti-vWF IgG. Multimeric analysis of vWF was performed with vertical agarose gel electrophoresis and then with blotting and chemiluminescent staining. Platelets, bleeding time, activation partial thromboplastin time（APTr）, RIPA, FVⅢ： C, vWF： nCo, vWF： Ag and vWF： CB were determined in plasma of ten patients. Results The vWF： CB assay was constructed with wide linear range,low detection limits and high reproducibility. The vWF multimeric assay had no radioactivity and showed high solution. Platelets was normal and the content of vWF： Rco, vWF： Ag and vWF： CB were decreased to some extent in 10 suspicious cases while bleeding time and APTr was increased, and RIPA and FvⅢ c were decreased in most of the cases. The vWF： Ag/vWF： CB ratios were 〉 2 in some patients with the loss of high and medium sized vWF multimers in plasma. Eight patients with vWD were identified, wherein two were characterized as type 1,4 as type 2A and 2 as type 3 respectively. Conclusion The panel of tests is suitable for diagnosis and classification of vWD.