Aim: Although no regular anticoagulant monitoring is required for Dabigatran etexilate, in certain situations in may be clinical helpful to assess the anticoagulant activity in patient. The Hemoclot Thrombin Inhibitors (HTI) kit (improved thrombin time “like” clotting assay) can be used and needs to be calibrated with specific dabigatran plasma calibrators at well defined concentrations. Ecarin clotting time and a chromogenic anti-IIa assay can also be used.
Method: Preparation and validation of freeze dried plasma calibrators and controls (2 lots) supplemented with dabigatran at defined concentrations for dedicated assays. Dabigatran was added to plasma at 3 concentrations (0.05, 0.25 and 0.50 μg/ml) for calibrators, and at 2 (0.10 and 0.30 μg/ml) for controls, and 1 ml aliquots filled and lyophilized in silikon vials. These preparations were tested for stability, over time or following reconstitution, and the exact dabigatran concentration established (against dabigatran spiked in various plasma pools, or by HPLC). Basic plasma values were measured without dabigatran.
Results: Dabigatran plasma calibrators (fresh or lyophilized) yielded a linear dose response curve with HTI assay (r²>0.990, LLOQ of 0.02 μg/ml). Normals (N=31) were all below LLOQ. When measured against a fresh reference curve, lyophilized reagents were 2 to 5 % lower than frozen plasma before lyophilization (slightly higher volume after reconstitution). Tested by HPLC (N=20) the concentrations were 10 to 15 % lower. Both lots were evaluated by HPLC as the reference method. When tested with HTI assay and calibrated with lot 1 (HPLC values), concentrations measured for lot 2 matched with those obtained by HPLC. Similar results were obtained with the chromogenic assay (anti-IIa, 2 stages).
Conclusions: Lyophilized dabigatran plasmas can be proposed for calibration and quality control of dedicated assays, clotting or chromogenic, and using the values assigned by HPLC they can be used as primary standards.