Suggestions for global coagulation assays for the assessment of COVID-19 associated hypercoagulability

Abstract

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.

Serial EXTEM, FIBTEM, and tPA Rotational Thromboelastometry Observations in the Maastricht Intensive Care COVID Cohort—Persistence of Hypercoagulability and Hypofibrinolysis Despite Anticoagulation

“…Sensitivity analyses excluding patients receiving ECMO during ICU admission did not alter any of the conclusions. Additionally, heparins execute limited effects on the ROTEM parameters; EXTEM and FIBTEM contain a heparin neutralizing agent (polybrene) and no clear fibrinolytic effect of heparins can be observed in tPA ROTEM (28). Taken together, the individualized treatment regimens, though illustrating the heterogeneous ICU population, do not in itself affect ROTEM results.…”

Viscoelastometric Testing to Assess Hemostasis of COVID-19: A Systematic Review

“…Of note, thrombin generation assays (TGAs) are highly sensitive to preanalytical aspects [119]. So far, some in vitro studies have reported that there was a heparin concentration dependent decrease in thrombin generation [120,121]. Studies showed that COVID-19 patients had a higher endogenous thrombin potential [31,39,48,66,[122][123][124][125][126][127]] than manufacturer's reference range, healthy controls or patients with sepsis, sometimes despite UFH or LMWH anticoagulation at a minimum of prophylactic dosing.…”

Hemostasis and fibrinolysis in COVID‐19 survivors 6 months after intensive care unit discharge

“…Unpublished data of patients using rivaroxaban and dabigatran show a concentrationdependent effect of these DOACs on tPA ROTEM CT, whereas tPA MCF, LOT, and LT were unaffected by DOAC concentration (data not shown). Similarly, heparins had no clear effect on tPA ROTEM parameters besides CT prolongation 16. Taken together, sis laboratory parameters and tPA ROTEM after COVID-19 ICU discharge and observed that, with the exception of tPA CT, values generally normalized over time.…”

“…At 6-month follow-up, 32% of patients received anticoagulants, predominantly the direct oral anticoagulant (DOAC) apixaban (Table2). In vitro spiking studies demonstrated that LMWH, UFH, and DOACs prolong ROTEM CT 16,17. Unpublished data of patients using rivaroxaban and dabigatran show a concentrationdependent effect of these DOACs on tPA ROTEM CT, whereas tPA MCF, LOT, and LT were unaffected by DOAC concentration (data not shown).…”