Hirudin Determination in Plasma Can Be Strongly Influenced by the Prothrombin Level☆

Abstract

Recombinant hirudin is increasingly used for therapeutic and prophylactic anticoagulation. Several laboratory methods are available to measure r-hirudin, including clot-based and amidolytic methods. The snake venom ecarin converts prothrombin to meizothrombin. Hirudin inhibits meizothrombin, causing a prolongation of the ecarin clotting time (ECT). Because the ECT depends on prothrombin levels in plasma, it was compared with a chromogenic substrate assay (CSA) for the determination of r-hirudin levels in prothrombin deficient plasma samples. R-hirudin (0.0–2.0 μg/mL) was added to plasma samples with decreasing prothrombin concentrations (100–0%). Using the ECT, false high r-hirudin levels were observed even in r-hirudin-free plasma, when prothrombin levels were below 50%. This effect was more pronounced with increasing r-hirudin levels. Additionally, r-hirudin (0.5 μg/mL) was added to plasma of patients with acquired prothrombin deficiency due to oral anticoagulation n=33 . Hirudin levels were also overestimated in these plasma samples using ECT. In plasma samples of patients n=12 treated with r-hirudin, because of suspected heparin-induced thrombocytopenia (HIT), hirudin levels were already measured falsely high, when the prothrombin levels were below 70%. The chromogenic substrate assay (CSA) determined correct values in all prothrombin-deficient plasma samples. Therefore, the CSA should be used for hirudin level determination, if overestimation due to prothrombin deficiency should be avoided.