Validation of the HEMOCLOT^® Direct Thrombin Inhibitor Assay for Dabigatran by Comparison with a Validated LC-MS/MS Method in Plasma Samples From Patients After Major Orthopaedic Surgery

Although therapy with dabigatran etexilate does not require routine blood coagulation monitoring, under some clinical scenarios (e.g., severe or life-threatening bleeding, overdose or emergency surgery) a simple assay may be valuable. The HEMOCLOT^® direct thrombin inhibitors assay (HYPHEN BioMed, France, CK002K), in conjunction with calibration standards and quality controls, allows the accurate and precise determination of dabigatran concentrations within a range of 50–500 ng/mL. Calibration and standardization of the assay minimizes intra- and interlaboratory variability. The present study was conducted to confirm the accuracy and reproducibility of the HEMOCLOT^® assay in samples derived from patients undergoing total hip arthroplasty taking dabigatran etexilate for the prevention of venous thromboembolism in the RE-NOVATE^® II trial.

Dabigatran concentrations in low- and high dabigatran quality control samples were determined by a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) method (providing concentrations of 122 ng/mL and 285 ng/mL, respectively) and by back-calculation from thrombin times obtained in the calibrated HEMOCLOT^® assay. Precision and accuracy of the HEMOCLOT^® assay were determined by comparison of results. A total of 100 samples collected 2 hours post-dose in citrate were analyzed by both HEMOCLOT^® and LC-MS/MS. The primary study endpoint was the comparison of total dabigatran concentrations determined by both methods.

Total assay imprecision for low dabigatran quality control samples was 2.6% coefficient of variation, with precision (repeatability) within-run 2.1%, between-run 1.4%, and between-day 0.6%. The respective values for high dabigatran samples were 2.8%, 1.8%, 2.0% and 0.9%. In the analysis of accuracy, the mean deviation of the calculated dabigatran concentration from the 122 ng/mL dabigatran sample target value was 14%, and from the 285 ng/mL dabigatran sample was –0.08%. In general, quality control sample concentrations of dabigatran were within ± 20% of the target concentration (acceptance limit). A total of 97 samples from patients provided valid data for the comparison of methods. The range of dabigatran concentrations was 68–438 ng/mL. The mean bias between the dabigatran concentrations determined by HEMOCLOT^® (test) and LC-MS/MS (reference) was –8.9% (95% confidence interval –11.9 to –6.0%), with 95% limits of agreement of –37.7% to 19.8%.

The HEMOCLOT^® direct thrombin inhibitors assay in conjunction with dabigatran standards and quality controls is suitable for the precise quantitative determination of dabigatran in citrate plasma samples. The accuracy is considered acceptable for the intended use of the assay, i.e., identifying patients with very high dabigatran concentrations.

Stangier:Boehringer Ingelheim: Employment. Eriksson:Bristol-Myers Squibb: Consultancy; Bayer: Consultancy; Astellas: Consultancy. Huo:Boehringer Ingelheim: Consultancy. Friedman:Johnson and Johnson: Consultancy. Feuring:Boehringer Ingelheim: Employment.