Standardization and evaluation of the performance of the thrombin generation test under hypo- and hypercoagulability conditions

Abstract

Background In order to standardize a thrombin generation() protocol, we analyzed the analytical variables and sensitivity of this test to hypo/hypercoagulability states. Methods The effect of the tissue factor concentration and the intra- and interassay precision were analyzed. To evaluate the hypercoagulability status, the plasma of women under an oral contraceptive was tested, while plasma from hemophilia A patients at 1, 3 and 7 days after recombinant FVIII infusion, and lyophilized plasma deficient in FVII or FVIII were used for the evaluation of hypocoagulability. Results The intra-assay coefficient of variation was <10% with 1 and 5 pM of low and high TF. The oral contraceptive users showed increased thrombin generation in comparison to non-users, which was more pronounced with low TF (endogenous thrombin potential ETP) p = 0.0009; peak p = 0.0009; lagtime p = 0.0008). In relation to the FVIII-deficient plasma, a higher TG was observed as FVIII levels were increased and a better discrimination was obtained for different concentrations of FVIII with low TF (ETP p < 0.0001; peak p < 0.0001; lagtime p = 0.0004). Using low TF, plasma from hemophilia A patients showed higher TG values after 1 day of recombinant FVIII infusion vs after 3 days (ETP p < 0.0001; peak p < 0.0001; lagtime p = 0.0407), while the lowest values were observed after 7 days. With FVII-deficient plasma, thrombin generation was lower than normal plasma and a more pronounced difference was observed with high TF compared to low TF (ETP p < 0.0001; peak p < 0.0001; lagtime p < 0.0001). Conclusion Under our conditions the thrombin generation test seems to be sensitive to evaluation of hyper/hypocoagulability states. Standardization of the thrombin generation test may have an application in the evaluation of bleeding and thrombotic disorders.

ST Genesia reference values of 117 healthy donors measured with STG‐BleedScreen, STG‐DrugScreen and STG‐ThromboScreen reagents

“…The thrombin generation (TG) assay is an established research tool in the research field of thrombosis and hemostasis 1 . It mirrors a significant part of the overall function of the blood‐clotting system.…”

“…The thrombin generation (TG) assay is an established research tool in the research field of thrombosis and hemostasis. 1 It mirrors a significant part of the overall function of the blood-clotting system. The assay has been used since the early 1950s, but back then it was performed by subsampling, which was very labor intensive.…”

Validation of the Role of Thrombin Generation Potential by a Fully Automated System in the Identification of Breast Cancer Patients at High Risk of Disease Recurrence

“…According to the HYPERCAN main study proposal, we performed a series of prechemotherapy evaluation of different hemostatic biomarkers that allowed us to identify and internally validate a risk assessment model of E-DR based on ETP obtained with the CT assay. 16 However, partial automation, lack of reagent standardization, and clinical validation limit the use of CT assay in the clinical practice, 20 and this prompted us to reevaluate in this cohort patients, the role of ETP by performing the TG analysis by the fully automatized ST Genesia system. In this re-evaluation, we also included the study of the anticoagulant C pathway by the same system with dedicated reagents to further explore the hypercoagulable profile of these patients.…”

“…16 TG by the CAT assay is well reproducible within a single laboratory when standardized preanalytical and analytical conditions for measurement are applied. [18][19][20] However, partial automation, lack of reagent standardization, and clinical validation limit the use of CAT assay in the clinical practice. [21][22][23][24] Recently, a new TG analyzer (ST Genesia, Stago) has been released as the first fully automated TG system for clinical routine laboratories together with a set of reagents balanced for sensitivity to procoagulant and anticoagulant protein deficiencies.…”

Recommendations for the measurement of thrombin generation: Communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies

“…The current lack of standardization remains a major issue in the setting of TG. [21][22][23] In 2018 a working party of the Scientific and Standardization Committee for Lupus Anticoagulant/ Antiphospholipid Antibodies of the International Society on Thrombosis and Haemostasis (ISTH-SSC LA/aPL) organized a survey to investigate how laboratories measure TG in practice. 24 The ISTH SSC LA/aPL aims to contribute to standardization, as TG assays are also used in patients with antiphospholipid syndrome.…”