INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection is associated with a clear prothrombotic phenotype. Although the exact pathophysiological mechanisms are not yet fully understood, thrombosis is clearly a highly important in the prognosis and outcome of COVID-19. As such, there is a need for diagnostic analysis and quantification of the coagulation potential in these patients, both at diagnosis and follow-up. Global coagulation assays like thrombin generation (TG) and rotational thromboelastometry (ROTEM) might be suitable in estimating COVID-19 associated coagulopathy and thrombosis risk. Therefore, we aimed at validating both assays for samples with high levels of fibrinogen and in the presence of anticoagulant heparins, such as commonly observed for COVID-19 ICU patients.
MATERIALS AND METHODS: Calibrated Automated Thrombography (CAT) was optimized to assess plasma thrombin generation in the presence of heparins. The final conditions with either 10 μg/mL Ellagic acid (EA) or PPP Reagent HIGH (high tissue factor; HPPH) were validated according to the EP5 protocol for within-run and between-run variability. Overall variability was well below 10%. To estimate the influences of heparins and high fibrinogen levels, CAT was performed on spiked plasma aliquots from 13 healthy volunteers. Comparable to the CAT method, tPA-ROTEM was used to validate the effect of high fibrinogen and heparins on clotting time, clot firmness and clot lysis parameters.
RESULTS: Our adjusted COVID-19 assay showed a heparin dose dependent decrease in peak height and endogenous thrombin potential (ETP) for both EA and HPPH triggered variants. High fibrinogen did not alter the inhibitory effect of either LMWH or UFH, nor did it influence the peak height or ETP in any of the conditions. The tPA-ROTEM showed a significant prolongation in clotting time with the additions of heparin, which normalized with the addition of high fibrinogen. MCF was markedly increased in all hyperfibrinogenemic conditions. A trend towards increased lysis time and, thus, decreased fibrinolysis was observed.
CONCLUSION: Thrombin generation and tPA-ROTEM protocols for measurements in the COVID-19 populations were adjusted and validated. The adjusted thrombin generation assay shows good sensitivity for measurements in heparin spiked plasma. High levels of fibrinogen did not alter the assay or the effectiveness of heparins as measured in this assay. t-PA ROTEM was effective in measurement of both high fibrinogen and heparins spiked samples and was sensitive to the expected relevant coagulant changes by these conditions. No clear fibrinolytic effect was observed in different conditions.
- Blood Coagulation Tests
- Heparin, Low-Molecular-Weight
- RNA, Viral
- Thrombin generation
- THROMBIN GENERATION