Thrombinaktivität basierter Test zur intrinsischen Gerinnung

The Intrinsic Coagulation Activity Assay (INCA) is a new general chromogenic hemostasis test for the intrinsic pathway of blood coagulation. At the present time, the most common assay to measure intrinsic coagulation is the Activated Partial Thromboplastin Time (aPTT). Meanwhile it is generally recognized that the aPTT is not capable of reflecting the full spectrum of coagulation activation in terms of giving clear account of a normal coagulatory response or detecting prothrombotic and anticoagulatory stages. While being able to monitor unfractionated heparin therapy, the aPTT is unsuitable for monitoring therapy with many other anticoagulants. New thrombin generation assays, chromogenic and fluorogenic ones, have been introduced, but are hampered by the problem of fibrin formation, interference of contact activation, or the need for special calculation software and fluorescence readers, respectively. Furthermore, these assays have all been developed with focus on reflecting the extrinsic pathway. At the same time, the need for a sensitive assay for the intrinsic pathway is emphasized by the rising evidence of the part played by the intrinsic coagulation factors in pathologic thrombus formation and prothrombotic diseases. INCA is designed for measuring thrombin activity in International Units (IU) using a chromogenic peptide substrate and a photometer. INCA involves two reactions which take place separately from each other: First the coagulation reaction is carried out by incubating 50 μl citrated plasma with 5 μl SiO2 (Pathromtin® SL) and 250 mmol/l CaCl2 in polystyrene flat-bottom wells at 37°C in a digitally controlled water bath. To obtain the most important part of a thrombin generation curve, after four or five minutes coagulation reaction time (CRT) the reaction is stopped by adding 100 μl of 2.5 mol/l arginine, pH 8.6, to inhibit hemostasis and depolymerize fibrin. After an incubation time of 20 minutes at room temperature, 50 μl of 1 mmol/l chromogenic peptide substrate CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, is added (final concentration 0.24 mmol/l), starting the detection reaction. The chromogenic substrate is cleaved by thrombin releasing free pNA. In a microtiter plate photometer the change in absorbance (ΔA) is detected at 405 nm. For calibration, a standard containing 1 IU/ml thrombin is used. INCA measured at four and five minutes CRT (INCA-4 and INCA-5) yields all necessary information for evaluating a plasma sample if INCA-5/INCA-4 ratio is greater than one, indicating that thrombin is measured in the ascending part of the curve, that is, in the phase of increasing thrombin activity. This work shows that the normal thrombin activity of INCA-4 (main value) is about 0.5 IU/ml (= 100% of normal) and of INCA-5 (control value) is about 1.9 IU/ml (= 100% of normal), the normal range being 100% ± 30% (mean value ± standard deviation), determined by measuring 39 plasmas of healthy adults. A 10-fold measurement of pooled fresh normal plasma demonstrated that the intra-assay or interassay coefficients of variation are < 10% and < 15%, respectively. The analysis of 173 patient plasmas revealed that the mean value for INCA-4 in patients with prolonged aPTT or increased International Normalized Ratio (INR) respectively was about 10% of the INCA-4 activity of patients without anticoagulation. The difference of the INCA-4 mean values of the groups „prolonged aPTT“ or „elevated INR“, respectively, compared to those patients without anticoagulation was statistically significant, which suggests that INCA reflects the therapeutic effect of heparins and coumarins. Correlations of INCA values with aPTT or INR values show a weak to moderate negative correlation. None of the correlations yield a value of |r| > 0.6, emphasizing that INCA provides unique information – beyond what can be assessed by the routine assays. INCA is very sensitive to changes in the plasma matrix as dilution of the activator increases INCA values artificially. Also, pre-analytic freezing/thawing of plasma enhances the INCA-4 value. As a result of three special features of the INCA, namely the use of arginine for hemostasis stabilization and fibrin depolymerization, the low concentration of the INCA activator (less than 5% of the contact activator amount of the aPTT), and the fast reacting chromogenic substrate, the INCA is extremely sensitive. Further research is needed to evaluate whether INCA can be used as a screening test for a dysfunctional intrinsic hemostasis system and whether INCA is helpful for more precisely monitoring anticoagulant therapies of various types. If so, INCA may become a valuable diagnostic instrument in the hemostasis laboratory, possibly capable of filling the gap in hemostasis analysis of the intrinsic pathway.