Comparative Analysis of Thrombin Calibration Algorithms and Correction for Thrombin-α2macroglobulin Activity

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.

Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples

“…Similarly, Hemker and Kremers 23 previously suggested that the TG assay performance is not affected by the calculation method but rather by the preanalytical treatment of the sample, that is, removing cell material when preparing plasma, and insufficient temperature control. However, Hemker and Kremers’s conclusions about the calculation methods do not apply to severely procoagulant samples, such as those with elevated prothrombin (this work) and antithrombin deficiency 12,15 …”

“…This study is the extension of the previous computational work comparing algorithms for TG assay analysis 15 . Both studies are based on a reanalysis of the raw experimental data generated in TG experiments performed previously 16 .…”

“…TG parameters were calculated using both the CAT‐associated Thrombinoscope software version 3.0.0.29 (Thrombinoscope BV, Maastricht, the Netherlands) and an in‐house software based on Origin (OriginLab, Northampton, MA, USA; the package is available from us upon request). The in‐house software, which was previously described, 15,17 can use the internal calibration in two ways: without (ie, linear calibration) or with the nonlinear calibration (ie, CAT approach) as published 9,10,18,19 and detailed below. Note that our software is not a direct replica of CAT, as it calculates several steps slightly differently than the CAT’s software.…”

“…This study is the extension of the previous computational work comparing algorithms for TG assay analysis. 15 Both studies are based on a reanalysis of the raw experimental data generated in TG experiments performed previously. 16 Briefly, normal pooled plateletfree plasma (PFP) or single-donor platelet-rich plasma (PRP) was spiked with 100% (to make the final level of 2× the normal) or 300% (4× the normal level) of blood coagulation factors V, VIII, IX, X, XI, I (fibrinogen) and II (prothrombin) to generate a range of normal to highly procoagulant samples for TG analysis described in Machlus et al 16 Essentially, all PFP experiments used a single pool of frozen normal plasma, while PRP experiments used fresh samples from six separate healthy individuals.…”

“…Since substrate consumption and IFE correction is aimed to address underreporting of high TG activity, we investigated whether these can indeed reduce the detection of known procoagulant plasma conditions. To demonstrate that the observed results are not affected by fluorescence artifacts such as plasma fluorescence, we previously developed an internal linear calibration software 15 that retains correction for plasma optical properties via internal calibration but does not correct for substrate consumption and IFE. We therefore applied this linear software along with commercial CAT and an in‐house algorithm, similar to CAT, to analyze TG with and without correction, to a data set from procoagulant samples created by supplementing normal plasma with elevated levels of coagulation factors.…”