The intrinsic coagulation activity assay

Abstract

A new assay for the contact-phase-mediated generation of thrombin activity has been developed - the intrinsic coagulation activity assay (INCA). Citrated plasma (50 microl) is incubated with 5 microl SiO2, 250 mmol/l CaCl2 in polystyrole flat-bottom wells. After exactly 4 and 5 min (37 degrees C) coagulation reaction times (INCA-4 and INCA-5), 100 microl of 2.5 mol/l arginine, pH 8.6, is added to inhibit hemostasis activation in the important ascending part of the thrombin generation curve and to depolymerize fibrin. After 20 min, 50 microl of 1 mmol/l (final concentration 0.24 mmol/l) chromogenic thrombin substrate CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, is added. The increase in absorbance is determined at 405 nm using a microtiterplate photometer. The assay is calibrated against 1 IU/ml thrombin. The normal thrombin activity range of INCA-4 (main value) or INCA-5 (control value) is 100 +/- 30% of normal (mean value +/- 1 SD; 100% = 0.5 IU/ml for INCA-4 and 1.9 IU/ml for INCA-5). With the INCA the normal range of intrinsic hemostasis is reflected, low-molecular-weight heparins can be monitored, the plasma matrix is not changed significantly, and the assay results are a percentage of normal generated thrombin activity and not coagulation seconds.