Assessment of Heparins, Heparinoids and Direct Thrombin Inhibitors Using a New FXa-and RVV-V-Based Assay.

UFH and direct thrombin inhibitors are usually monitored using the activated partial thromboplastin time (aPTT), which has many known limitations. These include non-linear-dose response-relationships as well as a high variability among different reagents. In addition the aPTT has a low sensitivity towards low molecular weight heparin (LMWH). Newer methods such as chromogenic substrate analysis, ecarin clotting time or the Heptest assay have provided improved sensitivity and specificity, but still have not reached broad application in routine or emergency laboratories. The performance of a new universal monitoring method (Pefakit® PiCT®, Pentapharm, Basel) in detecting anticoagulant effects of anti-Xa and / or arti-IIa agents was evaluated.

Methods: Measurements were performed by addition of platelet poor plasma to a reagent containing a combination of FXa, RVV-V (FV activating snake venom) and phospholipids. After 180 s the samples were recalcified and the clotting time was recorded. Plasma samples from 30 healthy blood donors and 50 patients receiving low molecular weight heparin were analyzed. To evaluate the assay precision 30 samples were analyzed in triplicate. Plasma pools from healthy volunteers and patients under oral anticoagulant treatment were spiked with unfractionated heparin, low molecular weight heparin, argatroban and pentasaccharide.

Results: PiCT® clotting times of the healthy volunteers were 20.6 ± 1.2 sec. Clotting times of the patient samples ranged 29–195 sec. Correlation of anti-Xa activity to PiCT® clotting times was good (r=0.94). The mean variation coefficient of the repeated determinations was 2.6%. Addition of LMWH, UFH or Argatroban to normal plasma showed a steep increase and almost linear dose-response of the PiCT clotting times. Addition of pentasaccharide showed a non-linear dose-response with lesser increase of clotting times at concentrations above 0.75 μg/ml plasma. The addition of LMWH or UFH to plasma of patients under warfarin therapy showed moderate additive effects when compared to normal plasma, while stronger additive effects of warfarin and heparin therapy were found for argatroban.

Conclusion: The prothrombinase induced clotting test (PiCT ®) is a new clotting assay, which sensitively assesses LMWH, UFH and direct thrombin inhibitors with almost linear dose-response-relationships. The results revealed a wide measuring range which allows for assessment of prophylactic, therapeutic and supratherapeutic doses of these compounds. As a clotting test with pipetting volumes and an incubation regimen identical with the aPTT, the test can be used in routine or emergency laboratories, thus facilitating 24-hour availability of the method with short turn-around times. Allowing the assessment of heparins, heparinoids as well as direct thrombin inhibitor therapy with one method may simplify their management in clinical practice.