Scientific Research Question: How can we utilize the Stago thrombin generation assay (TGA) in versatile experimental settings when studying human plasma, mouse plasma, human platelets, cultured malignant cells and their microparticles ?
Methodology: The (TGA) has at least three advantages compared to conventional clotting time assays. First the raw fluorescence data measured by a fluorimetric substrate cleavage are converted to a kinetic result by using a software (Thrombinoscope) and this provides more complex and meaningful information compared to simple clotting times. Second, the test monitors total thrombin formation in a period of 1 hour that may or may not correlate with clotting time results. The third advantage is that clotting times usually do not provide any information on hypercoagulability, while TGA is useful in this respect as well. We utilized TGA in 4 different experimental settings (i) tissue factor-phospholipid (ii) low tissue factor- phospholipid (iii) phospholipid only and (iv) no activators.
Findings: Reproducibility studies carried out on plasma samples measured by the classical tissue factor phospholipd activators provided CV values between 3.0 and 5.5% in different time parameters (lag time, timet to peak) and quantity values (Peak thrombin, ETP) that were just slightly higher than those of conventionally used clotting assays. Based on the analysis of human platelets and cultured malignant cells and their microparticles we drew the conclusion that the lag time and time to peak parameters are the most sensitive to detect pro- and anticoagulant changes (Hudák et al, Clin Chem Lab Med, 2017, 55: 1215-1223, Hudák et al, Biomed Res Int, 2017;2017:9795271, Tóth J et al, Thromb Res, 2017, 158:25-34). In human plasma samples, parameters reflecting the quantity of thrombin formation, e.g. peak thrombin and ETP values proved more valuable in detecting hypocoagulability in clinical samples (Hudák et al, PLoS One. 2017;12(7):e0180477) as well as in patients treated with anticoagulants.
Conclusion: The analytical performance of the TGA would make it suitable as a diagnostic test, nevertheless the lack of standardization in activating components, the variability during sample preparation in cellular assays, the changes in stored plasma samples as well as the considerable inter-individual variability in plasma TGA values hamper its utilization as a diagnostic test.
