Performance Characteristics and Clinical Application of a Modified Thrombin Generation Assay.
Authors: Zhukov Olga , Popov Joanna , Sahud Mervyn , Sferruzza Anthony 
Published: April 2020

Abstract
Background: The thrombin generation assay (TGA) allows evaluation of the overall coagulation capacity of patients. The original TGA protocol, which required sub-sampling and plasma defibrination procedures, was labor intensive and suffered from technical difficulties. The use of a fluorogenic substrate simplified the assay and provided an opportunity for clinical laboratory use. The results, received via kinetic reading mode and calculations of the derivative, allow construction of a thrombin generation curve characterized by 4 parameters: lag time (min); peak time (min); maximum thrombin (nM); and area under the curve (AUC; nM x min), which is analogous to the endogenous thrombin potential. However, complex calculations are required to analyze results. We developed a modified TGA that further simplifies the assay and analysis of results. This report describes the performance of the modified TGA and its clinical usefulness for evaluating hyper- and hypo-coagulability conditions.
Material and Methods: TGA was performed as described by Hemker et al (Pathophysiol Haemost Thromb2003;33:4–15), with the following modifications: A) human alpha-thrombin from Haematologic Technologies (Essex Junction, Vermont) was used to construct the thrombin standard curve; B) Innovin® (Dade Behring, Marburg, Germany) was used as a “trigger”; C) thrombin generation was measured in a 96-well plate on a GEMINI XPS fluorescent reader (Molecular Devices Corp, Sunnyvale, CA); D) results were calculated with a MS Excel template developed in-house; and E) AUC was expressed as % of normal (pooled normal plasma) without correction for alpha2-microglobulin-thrombin complex activity. Thrombin generation was measured at 30-sec intervals for 1 h; each sample was tested with its respective calibrator. Citrate plasma samples were run on the modified TGA: 25 from normal subjects, 6 from individuals receiving warfarin, and 17 from individuals with activated protein C resistance (APCR). For simplicity, we used only the AUC of total thrombin in further calculations, since preliminary results indicated that the AUC of free thrombin is not significantly different from those of total thrombin when both values are expressed as % of normal.
Results: The inter- and intra-assay coefficients of variation of the modified TGA were &lt;30% for lag and peak time and &lt;20% for maximum thrombin and AUC. The reportable ranges were 10–800 nM for maximum thrombin and from 7% of normal to the endpoint for AUC. The thrombin from Haematologic Technologies yielded 101–106% recovery relative to thrombin from Enzyme Research Laboratories (South Bend, IN), indicating that the source of thrombin standard only slightly affected assay performance. Patients on warfarin had lower maximum thrombin (&lt;10–140 nM) and AUC (&lt;7–60% of normal) results and APCR patients had higher maximum thrombin (206–&gt;800 nM) and AUC results (98–&gt;200% of normal) than normal subjects (maximum thrombin 92–325 nM, AUC 59–121 % of normal). Normal samples showed a dose-dependent response when spiked with commercial antithrombotic drugs.
Conclusions: The difference in results among normal individuals, patients with APCR, and warfarin-treated patients demonstrates that the assay helps distinguish between hyper- and hypo-coagulability conditions. The dose-dependent response to antithrombotics indicates that the assay can be used for drug-treatment monitoring and to support the development of new drugs. Simplification of the TGA with the modifications described above make this assay suitable for use in a clinical laboratory.