Modified thromboelastometric tests provide improved sensitivity and specificity to direct oral anticoagulants compared to standard thromboelastometric tests in-vitro

The study was approved by the Ludwig-Maximilians-University ethics committee (No 17-525-2). Written informed consent was obtained from all individuals prior to study inclusion.

Blood samples of healthy volunteers, 10 in the first study part on rivaroxaban and dabigatran [7] and 20 for the second part with edoxaban and apixaban, were analyzed. Exclusion criteria were coagulation disorders, intake of anticoagulants or platelet inhibitors within 30 days prior to study inclusion. Apixaban/edoxaban were dissolved in dimethyl sulfoxide (DMSO) and aqua to provide stock solution with a concentration of 62.5 ng/µl. Citrated whole blood (S-Monovette Sarstedt, Nürnbrecht, Germany) was spiked with six different concentrations of edoxaban and apixaban (0 ng/ml; 31.25 ng/ml; 62.5 ng/ml; 125 ng/ml; 250 ng/ml; 500 ng/ml). Aqua was added to each sample to ensure that there was no dilutional effect between the samples. DMSO concentration in the blood samples was < 1% and did not affect blood cell viability (> 99%), as assessed using flow cytometry. In preliminary tests, we carried out the specific, calibrated anti-Xa tests to check for correct spiking.

Standard thromboelastometric tests (EXTEM, FIBTEM, HEPTEM, NATEM) were performed using ROTEM delta analyzers (TEM Innovations GmbH, Munich, Germany). Additionally, we used modified thromboelastometric tests as described previously by our group: first, a low-tissue-factor test (TFTEM), and second, an ecarin based test (ECATEM).

TFTEM contains about 10% of the tissue factor concentration compared to EXTEM. This makes the assay more sensitive to changes in thrombin generation.

The following parameters provided by the systems were analyzed in the study: clotting time (CT; time from initiation of clotting process to 2 mm clot amplitude), clot formation time (CFT; time from CT until a clot amplitude of 20 mm is reached), and maximum clot firmness (MCF; maximum amplitude of clot firmness).

Statistics

ROC curve analyzes were done as follows: If a test was to be examined for the detection of a specific DOAC substance with cutoff > 30 ng/ml (e.g., apixaban), all samples spiked with this substance were tested against all samples without a DOAC (control samples without spiking of a DOAC). This enabled us to investigate whether the thromboelastometric tests can distinguish between samples from healthy controls and samples with anticoagulant of the specific substance, even at low doses. To analyze the cutoff > 60 ng/ml the samples with spiked dose of 31.25 ng/ml were added to the controls. This variant makes it possible to investigate the extent to which higher plasma concentrations can be detected, in particular in differentiating between healthy subjects and samples with low DOAC concentrations. We therefore used data of both studies [7]. Optimal cutoff values were determined using the Youden index.

Furthermore, regression analyzes have been performed to calculate coefficient r2, slope and intercept.

Data of the edoxaban and apixaban dose-effect-curves are presented as median with interquartile range (Q1/Q3) unless indicated otherwise. The corresponding data on the dose-effect-curves can be found in the first published manuscript for this work [7]. Statistical analysis was performed using Graph Pad Prism 9.2 (Graphpad Software Inc., La Jolla, USA). Statistical differences between groups were analyzed using two-way-ANOVA with post-hoc correction for multiple testing using the Tukey’s multiple comparison test.

Preliminary reference intervals for TFTEM and ECATEM were calculated based on 60 blood samples from healthy individuals without DOAC medication from this and two recently published studies [7, 8].

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