Evaluation of coagulation assays versus LC-MS/MS for determination of dabigatran concentration in plasma

Department of Coagulation Research, Institute for Molecular Medicine and Surgery, Karolinska Institutet, & Department of Clinical Chemistry, Karolinska University Hospital, Stockholm, Sweden, .
European Journal of Clinical Pharmacology (Impact Factor: 2.97). 06/2013; 69(11). DOI: 10.1007/s00228-013-1550-4
Source: PubMed


Dabigatran is an oral direct thrombin inhibitor for which routine laboratory monitoring is currently not recommended. However, there are situations in which measurements of the drug and its effect are desirable. We therefore compared and validated different coagulation methods for assessments of dabigatran in clinical samples in relation to measurements of plasma dabigatran, without the purpose of establishing effective and safe concentrations of dabigatran in plasma.

Samples were obtained from 70 atrial fibrillation patients treated with dabigatran etexilate. Plasma concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were compared with coagulation methods Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as with prothrombin time-international normalized ratio (PT-INR) and activated partial thromboplastin time (aPTT).

A wide range of dabigatran concentrations was determined by LC-MS/MS (<0.5-586 ng/mL). Correlations between LC-MS/MS results and estimated concentrations were excellent for both HTI and ECA overall (r(2) = 0.97 and 0.96 respectively, p < 0.0001), but the precision and variability of these assays were not fully satisfactory in the low range of dabigatran plasma concentrations, in which ECA performed better than HTI. aPTT performed poorly, and was normal (<40 s) even with dabigatran levels of 60 ng/mL. PT-INR was normal even at supratherapeutic dabigatran concentrations.

LC-MS/MS is the gold standard for measurements of dabigatran in plasma. Alternatively, either HTI or ECA assays may be used, but neither of these assays is dependable when monitoring low levels or to infer total absence of dabigatran. The aPTT assay is relatively insensitive to dabigatran, and normal aPTT results may be observed even with therapeutic dabigatran concentrations.

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    • "e l s e v i e r . c o m / l o c a t e / t h r o m r e s Please cite this article as: Schmohl M, et al, Measurement of dabigatran plasma concentrations by calibrated thrombin clotting time in comparison to LC-MS/MS in human volunteers..., Thromb Res (2015), is a linear correlation between ECT and dabigatran plasma concentration, and the assay shows good sensitivity, its limited availability, the lack of standardisation and the lot-to-lot variability of reagents restrict its routine use in clinical practice [5] [6]. Thrombin time (TT) also provides a direct measure of thrombin inhibition in a plasma sample and exhibits a linear dose–response relationship to dabigatran concentration. "
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    ABSTRACT: Dabigatran etexilate is an oral direct thrombin inhibitor. Although routine anticoagulation monitoring with dabigatran is not usually required, a simple and precise laboratory test to measure dabigatran concentrations in patient plasma may be useful in certain clinical circumstances, such as emergency situations. The HEMOCLOT(®) Thrombin Inhibitors assay has demonstrated accurate and precise determination of dabigatran concentrations within a range of 50-500ng/ml. The objective of this study was to assess comparability of dabigatran concentrations determined by HEMOCLOT(®) and by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in plasma samples from human volunteers with end-stage renal disease (ESRD) undergoing regular haemodialysis (HD) during a Phase I study. Overall, 304 plasma samples were obtained from seven ESRD patients in dabigatran steady-state for measurement by HEMOCLOT(®) (calibrated diluted thrombin time [dTT]) and by LC-MS/MS. Agreement of dabigatran concentrations was assessed by regression analysis and difference plots. The measurements of calibration standards of the HEMOCLOT(®) assay showed excellent precision with coefficients of variation <5%. Accuracy determined by analysis of two quality control samples was 90% and 111%. HEMOCLOT(®)-derived dabigatran plasma concentrations paralleled those obtained by LC-MS/MS. The mean ratio of the LC-MS/MS and dTT-derived concentrations was 0.955 (67% limits of agreement: 0.771-1.18). The HEMOCLOT(®) Thrombin Inhibitors assay is suitable for measuring dabigatran plasma concentrations in volunteers with ESRD undergoing haemodialysis. The agreement between dabigatran concentrations determined by the HEMOCLOT(®) assay and the LC-MS/MS reference method met bioanalytical acceptance criteria. Copyright © 2014. Published by Elsevier Ltd.
    Thrombosis Research 01/2015; 135(3). DOI:10.1016/j.thromres.2014.12.021 · 2.45 Impact Factor
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    ABSTRACT: Structured summary AimsIn patients with atrial fibrillation (AF) prescribed dabigatran: to examine the correlation between plasma dabigatran concentrations and 1) the three screening coagulation assays (International Normalised Ratio, INR, activated partial thromboplastin time, aPTT, thrombin time, TT), and 2) the dilute thrombin time (dTT); and to examine the contribution of plasma fibrinogen concentrations towards the variability in TT results. Methods Plasma from patients with AF on dabigatran were analysed for clotting times, and fibrinogen and dabigatran concentrations. Correlation plots (and associated R2 values) were generated using these data. The variability in TT results explained by fibrinogen concentrations was quantified using linear regression. ResultsFifty-two patients (38-94 y) contributed 120 samples, with plasma dabigatran concentrations ranging from 9-408 μg l-1. The R2 values of INR, aPTT, TT and dTT against plasma dabigatran concentrations were 0.49, 0.54, 0.70 and 0.95, respectively. Plasma fibrinogen concentrations explained some of the residual variability in TT values after taking plasma dabigatran concentrations into account (R2 = 0.12, P = 0.02). Conclusions Of the screening coagulation assays, the TT correlated best with plasma dabigatran concentrations. Variability in fibrinogen concentrations account for some of the variability in the TT.
    British Journal of Clinical Pharmacology 03/2014; 78(3). DOI:10.1111/bcp.12366 · 3.88 Impact Factor
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