Abstract
Background: The thrombin generation (TG) test is useful for characterizing global hemostasis potential, but fluorescence substrate artifacts, such as thrombin-α2macroglobulin (T-α2MG) signal, inner filter effect (IFE), substrate consumption, and calibration algorithms have been suggested as sources of intra- and inter-laboratory variance, which may limit its clinical utility. Methods: Effects of internal vs. external normalization, IFE and T-α2MG on TG curves in normal plasma supplemented with coagulation factors, thrombomodulin, and tissue factor were studied using the Calibrated Automated Thrombinography (CAT; Diagnostica Stago, Parsippany, NJ, USA) and in-house software. Results: The various calibration methods demonstrated no significant difference in producing TG curves, nor increased the robustness of the TG assay. Several TG parameters, including thrombin peak height (TPH), produced from internal linear calibration did not differ significantly from uncalibrated TG parameters. Further, TPH values from internal linear and nonlinear calibration with or without T-α2MG correction correlated well with TPH from external calibration. Higher coefficients of variation (CVs) for TPH values were observed in both platelet-free and platelet-rich plasma with added thrombomodulin. Conclusions: Our work suggests minimal differences between distinct computational approaches toward calibrating and correcting fluorescence signals into TG levels, with most samples returning similar or equivalent TPH results.
Highlights
The clinical application of the thrombin generation (TG) assay has been suggested by multiple research studies, and it is often used for bionanalytical assessment in clinical trials [1,2]
Three thrombin calibration methods were studied in this work: linear calibration (Figure 1D), non-linear (CAT-like, Figure 1G) and both external and internal Michaelis–Menten formula-based corrections for substrate depletion (Figure 1K,M, respectfully)
These thrombin peak height (TPH) values overlapped with the range observed in: 217 healthy donors by Kremers et al [33] (1 pM tissue factor (TF) triggered platelet-free plasma (PFP), TPH range: 41–371 nM), in severe-to-mild hemophilia donors described by van Veen et al [34] (TPH 9–107 nM), and in patients with at least one thrombotic event and a confirmed diagnosis of inherited thrombophilia from Luna-Záizar et al [35] (5 pM TF triggered PFP, TPH range: 21.8–499.3 nM)
Summary
The clinical application of the thrombin generation (TG) assay has been suggested by multiple research studies, and it is often used for bionanalytical assessment in clinical trials [1,2]. The apparent TG signal can be overestimated even further due to other enzymes which cleave most synthetic thrombin substrates, such as Factor (F) Xa [20] or, in fibrinolysis experiments, tissue plasminogen activator. Such artifacts are inseparable from the substrate properties used in the assay, and, arguably, they cannot be corrected by any means except by substituting for a different kind of substrate reporting approach, e.g., faster substrates [21]. The thrombin generation (TG) test is useful for characterizing global hemostasis potential, but fluorescence substrate artifacts, such as thrombin-α2macroglobulin (T-α2MG) signal, inner filter effect (IFE), substrate consumption, and calibration algorithms have been suggested as sources of intra- and inter-laboratory variance, which may limit its clinical utility.
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