ABSTRACT
Anti-factor Xa methods have been generally accepted for the monitoring of heparin treatment mainly due to their sensitivity to LMWH and excellent performance on automated equipment. When such equipment is not available, as in small laboratories or on the night shift, there is a need for a simple manual method. In the present method, activated Factor X and a chromogenic peptide substrate are colyophilized in plastic cuvettes under conditions that avoid reaction between the two constituents. The assay is performed accordingly. Plasma (200 microliters) is diluted in 3.0 ml predispensed Tris buffer containing dextran sulfate 8000 to avoid losses of heparin due to platelet factor 4, for example. The diluted plasma (400 microliters) is added to a cuvette. The reagents are rapidly dissolved and the reactions start. The rate of Factor Xa inhibition is proportional to the heparin activity. During the incubation, the substrate is also hydrolyzed by the remaining enzyme. This combination of reactions is insensitive to changes in temperature making it possible to perform the assay at room temperature. After 10 minutes incubation, the reaction is stopped by adding 400 microliters of 5% acetic acid. The absorbance is read in a well-calibrated photometer and the results plotted on a graph available in the kit. Eleven commercially available heparins have been tested with the assay and they all fell within a narrow range, showing the relevance of using a standard plot for all heparins. The LMWH behaved differently and it may also be that they demand different therapeutic strategies.
