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

Abstract

Introduction 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). Objectives We sought to elucidate the extent to which procoagulant conditions require correcting for fluorogenic substrate depletion and/or IFE. Methods 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. Results 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. Conclusions 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.

Effect of wavelength and filter set choices on fluorogenic thrombin generation assay: Considerations for interlaboratory differences

“…A 96‐channel pipettor (Viaflo 96, Integra Biosciences) was used to activate the plasma mixture with calcium chloride (final concentration 10 mM). In parallel, matched plasma wells with CAT thrombin calibrator (Stago) were used to generate a cubic polynomial‐fitted curve for calibration of the TG curves (also known as thrombograms) as described in Chang et al 9 The corresponding fluorescent signal was recorded on a Synergy Neo2 at 37°C simultaneously at several combinations of Ex/Em wavelengths chosen to represent the settings used in published TG assays (Table 1). A monochromator bandwidth for Ex and Em wavelengths was set up at 10 nm for all tested settings.…”

Single-Particle Plasmon Sensor to Monitor Proteolytic Activity in Real Time

“…We use thrombin as a model protease due to its central role in the coagulation cascade and its key role in clotting diseases. − Also, its involvement in the homeostasis of the gut and central nervous system has been recently suggested, − indicating its potential as a biomarker for other pathological conditions. Hence, biosensing assays to quantify thrombin’s activity are much needed in both fundamental biochemistry studies and in the future in clinical settings.…”

Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

“…Similarly, our previous investigations suggested that the IFE and substrate consumption can be of little consequence for detection of elevated TG in normal plasma spiked with increasing levels of coagulation factors I, V, VIII, IX, X, and XI [ 5 ]. On the other hand, algorithmic CAT corrections for substrate consumption were helpful in moderate cases of elevated prothrombin [ 6 ] and antithrombin deficiency [ 7 ]. Yet, in more severe conditions of substantially elevated prothrombin or deficient antithrombin, CAT algorithm failed to produce TG curve parameters due to its inability to process substantially distorted fluorescence signals.…”

“…We employed three versions of the CAT correction algorithm (Fig. 1), commercial Thrombinoscope software (denoted here as TS software) [3], our in-house app for TG analysis written in LabTalk language for Origin statistical and graphical software (Origin; denoted as OR software) [5,6,10], as well as the publicly available software, labelled SH, developed by one of us (CL) as part of a project to improve reproducibility and transparency supported by the International Society for Thrombosis and Haemostasis (ISTH) Subcommittee on Fibrinolysis [11].…”