Yamaguchi Tomoko, Shinozawa Keiko, Nagatoishi Satoru, Mitsuhashi Ayano, Bingo Masato, Inaba Hiroshi, Amano Kagehiro, Tsumoto Kohei, Kinai Ei
Department of Laboratory Medicine, Tokyo Medical University, Tokyo, Japan.
The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Thromb Res. 2023 Feb;222:131-139. doi: 10.1016/j.thromres.2023.01.007. Epub 2023 Jan 13.
[Introduction] Emicizumab, a bispecific antibody mimicking activated factor VIII (FVIII), is increasingly used in prophylaxis against bleeding in hemophilia A. Human factor-based chromogenic substrate assay (hCSA) shows concentration-dependency between emicizumab and reported FVIII activity. However, the assay measurement settings have not been optimized for emicizumab, and the reported FVIII activity cannot be directly referred as surrogate FVIII activity. [Materials and Methods] For in vitro validation of hCSA-reported surrogate FVIII activity, we compared the equation curves for emicizumab concentration with surrogate FVIII activity using spiked plasma in the thrombin generation assay (TGA), hCSA, and clot waveform analysis (CWA). Then, we generated conversion equations for hCSA-reported surrogate FVIII value to that of TGA. We also assessed the additive effect of rFVIII onto 340 nM (i.e., 50 μg/mL) emicizumab using the same assays. [Results] With 1:20 diluted plasma, halving hCSA-reported surrogate FVIII activity can be approximated to that in TGA triggered by the extrinsic pathway reagent (27.3 IU/dL vs. 13.9 IU/dL) under therapeutic emicizumab concentration. Both in TGA and hCSA, the additive effect of added FVIII on therapeutic emicizumab concentration (340 nM) was maintained at low levels of FVIII but gradually decreased at higher levels. [Conclusions] Surrogate FVIII activity can be estimated simply by halving hCSA-reported FVIII value, and the additive effect of FVIII on emicizumab diminishes at high concentrations. Based on our in vitro study, a clinical study is currently being conducted to compare individual variation of surrogate FVIII activity in hCSA and TGA.
[引言] 艾美赛珠单抗是一种模拟活化凝血因子VIII（FVIII）的双特异性抗体，越来越多地用于预防A型血友病患者出血。基于人因子的发色底物法（hCSA）显示艾美赛珠单抗与报告的FVIII活性之间存在浓度依赖性。然而，该检测方法的测量设置尚未针对艾美赛珠单抗进行优化，且报告的FVIII活性不能直接作为替代FVIII活性。[材料与方法] 为了对hCSA报告的替代FVIII活性进行体外验证，我们在凝血酶生成试验（TGA）、hCSA和血凝块波形分析（CWA）中，使用加标血浆比较了艾美赛珠单抗浓度与替代FVIII活性的方程曲线。然后，我们生成了将hCSA报告的替代FVIII值转换为TGA报告值的转换方程。我们还使用相同的检测方法评估了重组FVIII对340 nM（即50 μg/mL）艾美赛珠单抗的叠加效应。[结果] 在血浆1:20稀释的情况下，在治疗性艾美赛珠单抗浓度下，将hCSA报告的替代FVIII活性减半可近似于外源性途径试剂触发的TGA中的活性（27.3 IU/dL对13.9 IU/dL）。在TGA和hCSA中，添加的FVIII对治疗性艾美赛珠单抗浓度（340 nM）的叠加效应在FVIII水平较低时保持不变，但在较高水平时逐渐降低。[结论] 替代FVIII活性可通过将hCSA报告的FVIII值减半来简单估算，且FVIII对艾美赛珠单抗的叠加效应在高浓度时减弱。基于我们的体外研究，目前正在进行一项临床研究，以比较hCSA和TGA中替代FVIII活性的个体差异。
