Chang William C, Jackson Joseph W, Machlus Kellie R, Wolberg Alisa S, Ovanesov Mikhail V
Office of Tissues and Advanced Therapies Center for Biologics Evaluation and Research US Food and Drug Administration Silver Spring MD USA.
Department of Pathology and Laboratory Medicine and UNC Blood Research Center University of North Carolina at Chapel Hill Chapel Hill North Carolina USA.
Res Pract Thromb Haemost. 2021 Mar 26;5(3):447-455. doi: 10.1002/rth2.12499. eCollection 2021 Mar.
The thrombin generation (TG) test is a global hemostasis assay sensitive to procoagulant conditions. However, some TG assays may underestimate elevated TG when the thrombin fluorogenic substrate is depleted or fluorescence is attenuated by the inner filter effect (IFE).
We sought to elucidate the extent to which procoagulant conditions require correcting for fluorogenic substrate depletion and/or IFE.
We analyzed corrections for substrate depletion and IFE and their effect on TG parameters in plasma samples with elevated blood coagulation factors in the presence or absence of thrombomodulin via commercial calibrated automated thrombogram (CAT) platform and in-house software capable of internal thrombin calibration with or without CAT-like artifact correction.
Elevated thrombin peak height (TPH) and endogenous thrombin potential (ETP) were detected with 2× and 4× increases in blood coagulation factors I, V, VIII, IX, X, and XI, or prothrombin in the presence or absence of artifact correction. The effect of the CAT algorithm was evident in TG curves from both low procoagulant (thrombomodulin-supplemented) and procoagulant (factor-supplemented) plasma samples. However, in all samples, with the exception of elevated prothrombin, CAT's correction was small (<10%) and did not affect detection of procoagulant samples versus normal plasma. For elevated prothrombin samples, uncorrected TPH or ETP values were underestimated, and CAT correction produced drastically elevated TG curves.
Our data suggest that correction for substrate consumption and IFE, as offered by the CAT algorithm, is critical for detecting a subset of extremely procoagulant samples, such as elevated prothrombin, but is not necessary for all other conditions, including elevated factors XI and VIII.
凝血酶生成（TG）检测是一种对促凝条件敏感的全面止血检测方法。然而，当凝血酶荧光底物耗尽或荧光因内滤光片效应（IFE）而减弱时，一些TG检测可能会低估升高的TG。
我们试图阐明促凝条件在多大程度上需要校正荧光底物耗尽和/或IFE。
我们通过商业校准自动血栓图（CAT）平台和能够进行内部凝血酶校准（有无类似CAT伪影校正）的内部软件，分析了底物耗尽和IFE的校正及其对凝血因子升高的血浆样本中TG参数的影响，这些样本存在或不存在血栓调节蛋白。
在存在或不存在伪影校正的情况下，当凝血因子I、V、VIII、IX、X和XI或凝血酶原增加2倍和增加4倍时，检测到凝血酶峰值高度（TPH）和内源性凝血酶潜力（ETP）升高。CAT算法的效果在低促凝（补充血栓调节蛋白）和促凝（补充因子）血浆样本的TG曲线中都很明显。然而，在所有样本中，除了凝血酶原升高外，CAT的校正很小（<10%），并且不影响促凝样本与正常血浆的检测。对于凝血酶原升高的样本，未校正的TPH或ETP值被低估，而CAT校正使TG曲线大幅升高。
我们的数据表明，CAT算法提供的底物消耗和IFE校正对于检测一部分极端促凝样本（如凝血酶原升高）至关重要，但对于所有其他情况（包括因子XI和VIII升高）则不是必需的。
