Abstract：
BACKGROUND: Factor (F)VIII inhibitors are measured using labor- and resource-expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titer inhibitors and show high variations in quality surveys, mainly because of manual assay procedures.
OBJECTIVE: The goal of this study was the development of a fast and fully automated FVIII inhibitor assay by using recombinant (r)FVIII as substrate and dedicated equipment for execution of the test.
METHODS: A new rapid, fully automated, FVIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII; Kovaltry, Bayer) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of von Willebrand factor. Dramatic shortening of incubation time facilitated full automation on an analyzer capable of 3 subsequent sample dilution steps and 3 reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated for 10 minutes at 37 °C, whereafter remaining FVIII activity was analyzed with a kinetic chromogenic assay, allowing inhibitor activity calculation without preceding FVIII activity calibration, using a Ceveron s100 analyzer (Technoclone).
RESULTS: Mean titer in 60 nonhemophiliacs was 0.0 BU/mL (SD, 0.1), yielding a limit of blank of 0.1 BU/mL and lower limit of quantification of 0.2 BU/mL. Analyses were performed with the new method and a Nijmegen assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type I inhibitor samples was 1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive samples. Reproducibility was tested by analyzing 7 samples in 3 laboratories for 5 days, twice daily; coefficients of variation of all samples were <15%.
CONCLUSIONS: We present development data of a sensitive and specific rapid, automated FVIII inhibitor assay generating results within 20 minutes that is less resource-intensive than standard assays with potential to improve assay variability.
OBJECTIVE: The goal of this study was the development of a fast and fully automated FVIII inhibitor assay by using recombinant (r)FVIII as substrate and dedicated equipment for execution of the test.
METHODS: A new rapid, fully automated, FVIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII; Kovaltry, Bayer) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of von Willebrand factor. Dramatic shortening of incubation time facilitated full automation on an analyzer capable of 3 subsequent sample dilution steps and 3 reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated for 10 minutes at 37 °C, whereafter remaining FVIII activity was analyzed with a kinetic chromogenic assay, allowing inhibitor activity calculation without preceding FVIII activity calibration, using a Ceveron s100 analyzer (Technoclone).
RESULTS: Mean titer in 60 nonhemophiliacs was 0.0 BU/mL (SD, 0.1), yielding a limit of blank of 0.1 BU/mL and lower limit of quantification of 0.2 BU/mL. Analyses were performed with the new method and a Nijmegen assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type I inhibitor samples was 1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive samples. Reproducibility was tested by analyzing 7 samples in 3 laboratories for 5 days, twice daily; coefficients of variation of all samples were <15%.
CONCLUSIONS: We present development data of a sensitive and specific rapid, automated FVIII inhibitor assay generating results within 20 minutes that is less resource-intensive than standard assays with potential to improve assay variability.
摘要：
背景：使用劳动力和资源昂贵的Nijmegen或Bethesda测定法测量因子VIII抑制剂，对低滴度抑制剂缺乏敏感性，并显示质量调查的高度差异，主要是因为手工化验程序。
方法：一种新的快速，全自动,提供了因子VIII抑制剂测定法，其核心是使用全长重组FVIII（rFVIII）（Kovaltry®）作为抑制剂底物代替血浆FVIII，由于不存在VWF,导致抑制剂与rFVIII的快速结合。孵育时间的显着缩短促进了分析仪的完全自动化，该分析仪能够进行三个后续的样品稀释步骤和三个试剂添加。将样品和rFVIII的等体积混合物(1.0U/mL)孵育10分钟/37°C,此后，用动力学显色测定法分析剩余的FVIII活性，允许在没有FVIII活性校准的情况下计算抑制剂活性，使用CeveronS100分析仪。
结果：60例非血友病患者的平均滴度为0.0BU/mL（SD0.1），产生0.1BU/mL的空白限和0.2BU/mL的定量下限。使用新方法和Nijmegen测定法对28个抑制剂阳性临床样品进行了分析，14含有emicizumab和14不含。不含emicizumab的I型抑制剂样品的相关系数为r=1.0。在抑制剂阳性样品的加标实验中排除了该方法的艾咪珠单抗依赖性。通过在五天内在三个实验室分析七个样本来测试重复性，每天两次；所有样品的CV<15%。
结论：我们提供敏感和特定的开发数据，快速,自动FVIII抑制剂测定在20分钟内产生结果，比标准测定法资源密集程度低，具有改善测定变异性的潜力。
方法：一种新的快速，全自动,提供了因子VIII抑制剂测定法，其核心是使用全长重组FVIII（rFVIII）（Kovaltry®）作为抑制剂底物代替血浆FVIII，由于不存在VWF,导致抑制剂与rFVIII的快速结合。孵育时间的显着缩短促进了分析仪的完全自动化，该分析仪能够进行三个后续的样品稀释步骤和三个试剂添加。将样品和rFVIII的等体积混合物(1.0U/mL)孵育10分钟/37°C,此后，用动力学显色测定法分析剩余的FVIII活性，允许在没有FVIII活性校准的情况下计算抑制剂活性，使用CeveronS100分析仪。
结果：60例非血友病患者的平均滴度为0.0BU/mL（SD0.1），产生0.1BU/mL的空白限和0.2BU/mL的定量下限。使用新方法和Nijmegen测定法对28个抑制剂阳性临床样品进行了分析，14含有emicizumab和14不含。不含emicizumab的I型抑制剂样品的相关系数为r=1.0。在抑制剂阳性样品的加标实验中排除了该方法的艾咪珠单抗依赖性。通过在五天内在三个实验室分析七个样本来测试重复性，每天两次；所有样品的CV<15%。
结论：我们提供敏感和特定的开发数据，快速,自动FVIII抑制剂测定在20分钟内产生结果，比标准测定法资源密集程度低，具有改善测定变异性的潜力。
