Hirudin determination in plasma can be strongly influenced by the prothrombin level

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 microg/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 microg/mL) was added to plasma of patients with acquired prothrombin deficiency due to oral anticoagulation. Hirudin levels were also overestimated in these plasma samples using ECT. In plasma samples of patients 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.