Development and Validation of a Chromogenic Direct Thrombin Inhibitor Assay on the ACL Top 700 for Quantifying Dabigatran Levels in Plasma

Dabigatran is a potent, competitive, and reversible direct thrombin inhibitor, that binds to the active site of thrombin, inhibiting both free and clot-bound thrombin. It is the active form of dabigatran etexilate, which is a low molecular weight prodrug metabolized to its active form after oral administration. Although patients on therapeutic doses of dabigatran do not require routine coagulation monitoring due to its dose dependent and predictable pharmacological profile, there may be situations in which it may be beneficial to be able to accurately measure the degree of anticoagulation (ie. urgent surgery, severe bleeding, thrombosis despite treatment, overdose, bridging with other anticoagulants, patients with a high risk of dabigatran accumulation or potential drug interactions). Accordingly, we developed and validated a chromogenic direct thrombin inhibitor assay on the ACL TOP 700 automated hemostasis analyzer for quantifying dabigatran levels in human plasma.

Our test principle was based on the in vitro thrombin inhibition by dabigatran, in which excess thrombin was added to the plasma sample, the thrombin in the sample was neutralized in proportion to the amount of dabigatran, and the residual thrombin hydrolyzed the chromogenic substrate releasing pNA which was measured photometrically at 405 nm on the ACL TOP 700. This assay was calibrated with purchased dabigatran calibration samples (Aniara). Each calibration curve consisted of five points, used a 3rd order polynomial curve fit, and was performed each time an assay was run. For all runs, an r² of 1.0 was observed and all calibrators demonstrated an acceptable accuracy and precision (± 10% from assigned value for samples ≥ 100 ng/mL or < 10 ng/mL of assigned values for all other samples).

Validation of this dabigatran level assay for accuracy, intra- and inter-assay precision, analytical specificity, analytical sensitivity, analyte stability, and robustness was completed and reportable result ranges established. To evaluate accuracy, three plasma samples containing dabigatran in the range of expected concentrations (high, medium, low) were compared to the value determined by mass spectrometry. Intra- and inter-assay precision was determined by analyzing these 3 samples over multiple assay runs spanning multiple days. All determined values were within 15% of the assigned values for samples ≥ 100ng/mL and within 10ng/mL of the assigned values for all other samples. Analytical specificity was confirmed by running sample pairs (plasma/serum, citrate/EDTA, hemolyzed/non-hemolyzed, icteric/non-icteric, lipemic/cleared-lipemic, 1 U/mL unfractionated heparin and 2 U/mL low molecular weight heparin-containing samples). Only the serum sample resulted in >10% difference from the standard plasma, indicating assay interference. Assay results were not affected by unfractionated heparin up to 1 U/mL and low molecular weight heparin up to 2 U/mL. Analytical sensitivity was established by assaying normal plasma containing no or low levels of dabigatran. The limit of blank was determined to be 3 ng/mL, limit of detection 10 ng/mL, and limit of quantitation (LoQ) 20 ng/mL. Analyte stability studies established samples thawed and refrozen up to 2 times were acceptable. Assay robustness was determined to be acceptable. The reportable range was determined to be 20-900 ng/mL based on the calibration curve and LoQ.

In summary, we have developed and validated an accurate, precise, sensitive and robust chromogenic assay on the ACL TOP 700 for the determination of dabigatran concentration in human plasma. This assay may prove useful in certain clinical circumstances (urgent surgery, severe bleeding, or thrombosis despite treatment) for the assessment of anticoagulation status.