Fluorogenic thrombin generation (TG) is a global hemostasis assay that provides an overall representation of hemostasis potential. However, the accurate detection of thrombin activity in plasma may be affected by artifacts inherent to the assay-associated fluorogenic substrate. The significance of the fluorogenic artifacts or their corrections has not been studied in hemophilia treatment applications. We sought to investigate TG in hemophilia plasma samples under typical and worst-case fluorogenic artifact conditions and assess the performance of artifact correction algorithms. Severe hemophilic plasma with or without added Factor VIII (FVIII) was evaluated using commercially available and in-house TG reagents, instruments, and software packages. The inner filter effect (IFE) was induced by spiking elevated amounts of fluorophore 7-amino-4-methylcoumarin (AMC) into plasma prior to the TG experiment. Substrate consumption was modeled by adding decreasing amounts of Z-Gly-Gly-Arg-AMC (ZGGR-AMC) to plasma or performing TG in antithrombin deficient plasma. All algorithms corrected the AMC-induced IFE and antithrombin-deficiency induced substrate consumption up to a certain level of either artifact (edge of failure) upon which TG results were not returned or overestimated. TG values in FVIII deficient (FVIII-DP) or supplemented plasma were affected similarly. Normalization of FVIII-DP resulted in a more accurate correction of substrate artifacts than algorithmic methods. Correction algorithms may be effective in situations of moderate fluorogenic substrate artifacts inherent to highly procoagulant samples, but correction may not be required under typical conditions for hemophilia treatment studies if TG parameters can be normalized to a reference plasma sample.

中文翻译：

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凝血酶检查的偏差来源和局限性：内部过滤效应和失败算法边缘的底物耗尽

荧光凝血酶生成 (TG) 是一种全局止血测定，可全面表征止血潜力。然而，血浆中凝血酶活性的准确检测可能会受到与测定相关的荧光底物固有的伪影的影响。荧光伪影或其校正的重要性尚未在血友病治疗应用中进行研究。我们试图在典型和最坏情况的荧光伪影条件下研究血友病血浆样本中的 TG，并评估伪影校正算法的性能。使用市售和内部 TG 试剂、仪器和软件包对添加或不添加因子 VIII (FVIII) 的严重血友病血浆进行评估。在 TG 实验之前，通过将大量荧光团 7-氨基-4-甲基香豆素 (AMC) 掺入血浆中来诱导内过滤效应 (IFE)。通过向血浆中添加逐渐减少量的 Z-Gly-Gly-Arg-AMC (ZGGR-AMC) 或在缺乏抗凝血酶的血浆中进行 TG 来模拟底物消耗。所有算法都将 AMC 引起的 IFE 和抗凝血酶缺乏引起的底物消耗校正到一定水平的任一伪影（失败边缘），此时 TG 结果未返回或被高估。FVIII 缺乏（FVIII-DP）或补充血浆中的 TG 值也受到类似的影响。FVIII-DP 的标准化可以比算法方法更准确地校正底物伪影。校正算法在高促凝样品固有的中度荧光底物伪影的情况下可能有效，但如果 TG 参数可以标准化为参考血浆样品，则在血友病治疗研究的典型条件下可能不需要校正。