One-stage assay of heparin

A simple and rapid one-stage plasma heparin assay based on the heparin-dependent neutralization of activated factor X (Xa) is described. Factor Xa is prepared in a concentration adjusted to produce a clotting time of 18 to 20 seconds when heparin-free plasma is tested in the system. The assay incubation mixture contains "standard human plasma", the heparinized test plasma, cephalin, and factor Xa. Clotting times are measured automatically after incugation and calcium addition. A linear relationship was found between the log of the clotting time and the heparin concentration for the standard curve from which the heparin level is determined. The lower limit of sensitivity is about 0.05 unit heparin per milliliter. Dilution of test plasma is required to bring the heparin level to between 0.05 and 0.2 unit per milliliter. The standard curve is reproducible with a coefficient of variation of 2 to 4 per cent. Ten different, nonheparinized samples had clotting times of 18.9 plus or minus 1.0 (plus or minus 2 S.D.) seconds. Theoretically calculated and experimentally determined heparin levels after pulse injection of heparin agreed well in 5 volunteer subjects with r values between 0.93 and 0.99. This assay is not affected by normal variation in the plasma levels of fibrinogen, prothrombin, and factor V or by the plasma defects induced by coumadin.