Estimation of Dabigatran Plasma Concentrations in the Perioperative Setting. an Ex-Vivo Study Using Dedicated Coagulation Assays

Abstract

BACKGROUND Dabigatran etexilate has received its market authorization for various indications worldwide. It was developed to be used in fixed dose regimens without the need of regular monitoring. However, the perioperative management of dabigatran could require an assessment of the drug plasma levels to ensure a safe use of the product, especially in absence of specific antidotes. The EMA stated that dabigatran concentrations under 48 ng/mL should be reached before invasive intervention. The GIHP put the threshold at 30 ng/mL. Therefore, a specific laboratory assay, accurate in the low concentration range, easily available and performable 24h/7 is requested but, until now, all coagulation tests dedicated to the measurement of plasma dabigatran concentrations showed a lower limit of quantitation from 30 to 50 ng/mL. This limits their perioperative utility and the thrombin time (TT) is currently presented as an alternative due to its very high sensitivity to dabigatran. This interesting approach has limitations because TT is affected by several analytical and biological variables that could lead to misinterpretations and expose the patient at riskier hemostatic conditions. In this study, we propose to investigate the performance of two coagulation tests (the Hemoclot Thrombin Inhibitors LOW (HTI LOW) (Hyphen BioMed) and the Ecarin Chromogenic Assay II (ECA-II) (Diagnostica Stago)) specifically developed to measure low plasma dabigatran concentrations and to compare their results with a reference LC-MS/MS. We also assessed the performance of the standard procedure of HTI and TT. MATERIALS AND METHODS Thirty-three plasma samples of patients treated with dabigatran etexilate for stroke prevention in non-valvular atrial fibrillation, were included in the study. Plasma samples were taken randomly and included after a first screening using the HTI to select plasma concentrations <200 ng/mL. For HTI, tested plasma was diluted 1:8 in Owren-Koller® buffer. Fifty μl of tested plasma were mixed with 100 μl of normal pooled plasma and were incubated during 240 sec. One hundred μl of highly purified human thrombin pre-incubated at 37°C was then added to start the reaction. For HTI LOW, the dilution of the sample was reduced to 1:2 and specific calibrators at lower concentrations were used. For ECA-II, tested plasma was diluted 1:5 in Owren-Koller® buffer. Fifty μl of tested plasma were mixed with 140 μl of prothrombin and then 70 µl of chromogenic substrate were added and incubated during 240 sec. Seventy μl of ecarin pre-incubated at 37°C were then added to start the reaction. Thrombin time was performed using Thrombin Time® reagent 1.5NIH (Diagnostica Stago) and the limit of measurement was extended to 300 sec. All of these procedures were performed on a STA-R Evolution® coagulometer (Diagnostica Stago). RESULTS AND DISCUSSION The plasma concentrations ranged from 0 to 200 ng/mL as provided by LC-MS/MS measurements. Among these samples, 17 were between 0 and 50 ng/mL. Linear correlation, Spearman correlation and Bland-Altman analyses versus LC-MS/MS are provided in Figure 1, for assays that express results in ng/mL, i.e. HTI, HTI LOW and ECA-II. For TT, the relation is described by linear correlation. Our results show that HTI LOW performs better than HTI and ECA-II on the whole concentration range providing closer correlation and a lower systematic difference compared to LC-MS/MS. For concentrations below 50 ng/mL HTI LOW and ECA-II reveal similar systematic difference with higher 95% CI for ECA-II and perform better than HTI to estimate plasma concentrations. Thrombin time is less useful than dedicated assays to provide an estimation of the pharmacodynamics of dabigatran. For concentrations above 50 ng/mL it often exceeds the limit of measurement, i.e. 120 sec as defined by the manufacturer, and is influenced by the level of fibrinogen. However, it gives an acceptable correlation for concentration below 50 ng/mL. This may help the biologist to choose which test to use to avoid unnecessary costs and ensure the use of the more accurate dedicated assay to estimate the plasma concentrations. When TT is lower than 120 sec, HTI LOW or ECA-II should be preferred to HTI. CONCLUSION We recommend the use of specific coagulation assays to assess the dabigatran plasma concentrations before invasive procedure. Thrombin time may guide the biologist on the dedicated coagulation test to perform if he uses the HTI platform. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The addition of idarucizumab to plasma samples containing dabigatran allows the use of routine coagulation assays for the diagnosis of hemostasis disorders

“…As dabigatran is the active form of the drug administered to patients, we would not expect our observations to be different with samples from patients treated with dabigatran. This hypothesis is supported by previous observations showing that the anticoagulant effect of dabigatran in ex vivo samples [6,8,10] is similar to that in samples spiked with the drug in vitro [15][16][17][18][19][20][21]. Chromogenic assays for protein C, the chromogenic FVIII test and the free protein S assay are not based on APTT, PT, or TT, and are thus not affected by the presence of dabigatran [19].…”

“…It has been validated as an anticoagulant for thromboprophylaxis in orthopedic surgery and atrial fibrillation [1,2], and for treating venous thromboembolism and the prevention of its recurrence [3,4]. The anticoagulant effect of dabigatran can be approximated by its prolongation of most routine coagulation assays or quantified by specific assays [5][6][7][8][9][10].…”

Pharmacological reversal of the direct oral anticoagulants—A comprehensive review of the literature

“…For example, the thrombin time (TT) is very sensitive to the presence of any dabigatran and a normal TT likely excludes clinically significant levels of dabigatran. [19][20][21] The sensitivity of aPTT for detecting dabigatran is variable, but a prolonged aPTT suggests clinically significant dabigatran levels (especially if using a sensitive assay). 22 The most accurate and reliable tests for measuring dabigatran levels are the dilute thrombin time (dTT), ecarin clotting time (ECT), and ecarin chromogenic assay (ECA).…”

“…22 The most accurate and reliable tests for measuring dabigatran levels are the dilute thrombin time (dTT), ecarin clotting time (ECT), and ecarin chromogenic assay (ECA). 18,19,21 The PT can be helpful for determining the presence of rivaroxaban and edoxaban, where a prolonged PT suggests clinically significant drug levels. 23,24 However, a normal PT may not exclude clinically significant levels of rivaroxaban or edoxaban.…”

Real-world variability in dabigatran levels in patients with atrial fibrillation: reply

“…Real-world variability in dabigatran levels in patients with atrial fibrillation: reply Douxfils et al are concerned about the wide range of trough dabigatran levels among individuals reported in our cohort (< 30 ng mL À1 to 510 ng mL À1 ) [1], citing their ex vivo study investigating the test performance of various assays [2] and a population pharmacokinetic (PK) substudy of the RE-LY (Randomized Evaluation of Long-Term Anticoagulation Therapy) trial [3]. These studies differ from ours in two important ways.…”