Abstract：
BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays.
METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays.
RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation.
CONCLUSIONS: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.
METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays.
RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation.
CONCLUSIONS: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.
摘要：
背景：对细胞外囊泡（EV）的科学和临床兴趣正在增长。暴露组织因子(TF)的EV结合因子VII/VIIa并可触发凝血。在各种疾病的循环中可以检测到高促凝TF暴露的EV，比如败血症,COVID-19或癌症。已经开发了许多内部和商业上可获得的测定法来测量EV-TF活性和抗原,但是只有少数研究比较了这些测定法中的一些。ISTHSSC血管生物学小组委员会发起了一项多中心研究，以比较敏感性，这些测定的特异性和可重复性。
方法：从健康献血者的血液中制备去血小板的血浆样品。血浆样品中加入来自人乳的电动汽车，或来自TF阳性和TF阴性细胞系的EV。还从有或没有LPS刺激的人全血中制备血浆。21个实验室使用代表18个功能测定和9个抗原测定的他们自己的测定来测量制备的样品中的EV-TF活性和抗原。
结果：对于不同的EV-TF活性和抗原测定，绝对值有很大的差异。与抗原测定相比,活性测定具有更高的特异性和灵敏度。此外,有一个大的测定内和测定间的变异性。使用阻断性抗TF抗体或免疫捕获的功能测定是最特异和敏感的。与通过高速离心分离EV的测定相比,使用免疫捕获的活性测定具有较低的变异系数。
结论：基于这项多中心研究，我们建议在存在抗TF抗体的情况下使用功能测定法测量EV-TF.
方法：从健康献血者的血液中制备去血小板的血浆样品。血浆样品中加入来自人乳的电动汽车，或来自TF阳性和TF阴性细胞系的EV。还从有或没有LPS刺激的人全血中制备血浆。21个实验室使用代表18个功能测定和9个抗原测定的他们自己的测定来测量制备的样品中的EV-TF活性和抗原。
结果：对于不同的EV-TF活性和抗原测定，绝对值有很大的差异。与抗原测定相比,活性测定具有更高的特异性和灵敏度。此外,有一个大的测定内和测定间的变异性。使用阻断性抗TF抗体或免疫捕获的功能测定是最特异和敏感的。与通过高速离心分离EV的测定相比,使用免疫捕获的活性测定具有较低的变异系数。
结论：基于这项多中心研究，我们建议在存在抗TF抗体的情况下使用功能测定法测量EV-TF.
