定量检测血小板特异性抗体的流式免疫微珠芯片技术的建立及其临床应用

Establishment of Flow Cytometric Immunobead Array Assay for Quantitation of Platelet-Specific Antibodies and Its Application

目的:建立流式免疫微珠芯片技术(FCIA)定量检测血小板特异性抗体的方法,探讨其在ITP诊治中的应用。方法:通过已知浓度的人IgG结合在包被抗体的微珠上,构建定量标准曲线,同时使用5种抗血小板GPIX(SZ1)、GPIb(SZ2)、GPIIIa(SZ21)、GPIIb(SZ22)和P-选择素(SZ51)抗体包被的微珠捕获血小板特异性抗原抗体复合物进行血小板抗体的检测。通过定量标准曲线将流式细胞仪检测的荧光信号转换成样品中血小板抗体浓度,从而建立定量检测血浆样品中血小板特异性抗体的方法(FCIA),并对该方法从性能、效率,临床应用等方面进行评价。结果:针对GPIX、GPIb、GPIIIa、GPIIb和P-选择素5种抗体的FCIA定量检测范围分别为33.29-1280、45.17-1280、42.07-1280、46.40-1280和42.48-1280 ng/ml,回收率分别为115.23%、112.58%、117.47%、107.64%和112.67%。批内精密度(CV%)分别为3.54%、3.65%、4.66%、6.43%和6.97%,批间精密度(CV%)分别为10.89%、7.57%、10.34%、6.95%和10.72%。ITP组5种血小板特异性抗体浓度值均高于NITP和健康对照组(P<0.01)。FCIA定量检测5种抗体对诊断ITP的灵敏度、特异性和准确度分别为68.29%,84.93%和78.95%,而改良间接MAIPA技术检测的相应值分别为41.46%、90.41%和72.81%。结论:建立的血小板特异性抗体FCIA定量检测方法为ITP的诊断、疗效评估、患者预后提供了一个良好工具。

Objective:To establish a flow cytometric immunobead array assay(FCIA)to quantify platelet antibodies and to explore its application in the diagnosis and treatment of ITP.Methods:The guantitative standard curve was established by binding the human IgG of known concentration on antibody-coated microbeads;at the same time,the platelet-specific antigen and antibody complex was captured and levels of platelet antibodies were detected using the microbeads coated by 5 kinds of antibodies against platelets suca as GPⅨ(SZ1),GPⅠb(SZ2),GpⅢa(SZ21),GPⅡb(SZ22)and p-selection(SZ51).The fluorescence signal detected by flow cytometry were transformed into the conentration of platelet antibodies in samples through the quantitative standard curve,thereby establishing the method for quantititive detection of platelet-specific antibodies in plasm samples(FCIA),moreover the property,efficiency and clinical application of established FCIA method were evaluated.Results:The FCIA could detect 5 kinds of antibodies against GPⅨ,GPⅠb,GpⅢa,GPⅡb andβ-selection within a broad range of 33.29-1280 ng/ml,45.17-1280 ng/ml,42.07-1280 ng/ml,46.40-1280 ng/ml,42.48-1280 ng/ml and 42.48-1280 ng/ml respectively,and their recovery rates were 115.23%,112.58%,117.47%,107.64%and 112.67%respectively.The intra-assay coefficient of variation(CV)for anti-GPⅨ,-GPⅠb,-GpⅢa,-GPⅡb and p-selection antibodies was 3.54%,3.63%,4.66%,6.43%and 6.67%respectively,and the inter-assay CV for above mentioned antibodies were 10.89%,7.57%,10.34%,6.95%and 10.72%respectively.The detection showed that the levels of 5 kinds of platelet-specific antibodies in ITP group all were higher than those in non-ITP and healthy control groups(P<0.01).The sensitivity,specificity and accuracy of quantitatively detecting 5 kinds of antibodies for diagnosis of ITP by FCIA were 68.29%,84.98%and 78.95%respectively,while the sensitivity,specificity and accuracy of detecting 5 kinds of antibodies by modified indirect MAIPA were 41.46%,90.41%and72.81%respectively.Conclusion:The established quantitative FCIA for detection of antibodies provides a powerful tool for diaghosis and evaluation of therapeutic efficacy and prognosis of ITP patients.