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Conditions influencing the measurement of heparin levels in the range 0 to 0.3 units/ml of plasma using the chromogenic substrate, B2-Ile-Glu-Gly-Arg-pNA HCl (S2222, KabitVitrum Ltd) have been analysed to find the most suitable incubation mixture.  Factor Xa chromogenic activity was observed to be stable between pH 5.5 and 9.0 at 37°C. An optimal pH of 7.9 was found for the action of Factor Xa upon the substrate and also for the action of plasma inhibitor upon Factor Xa. A Michaelis constant (Km) of 0.6mM was determined for the hydrolytic cleavage of the substrate by Factor Xa. At a substrate concentration of 3 times Km, Factor Xa was measureable in the range of 0.02 to 0.12 units/incubation mixture with a change in optical density at 405 mμ of 0.06/second/unit corresponding to 5.7 n Kat/unit.  Using 100 μl pooled plasma/unit of factor Xa, 55% of the Xa activity was inhibited at 3 minutes. Taking 3 minutes as a preincubation period, Xa inhibitor was measureable in the range 5 to 25 μl normal plasma per 0.25 units of Xa with a net change in optical density at 405 mμ of 0.8. The greatest difference between the percentage inhibition of Factor Xa by heparinised (0.3 units/ml) and non heparinised plasma, however, was observed to occur after 1 minute preincubation. Using these observations a heparin calibration curve was constructed using 10 μl aliquots of heparinised plasma. Heparin was measured in the range 0.06 to 0.3 units/ml plasma with a net change in optical density at 400 mμ of 0.8.

A Chromogenic Semi-Micro Method for the Determination of Factor Xa, Factor Xa Inhibitor and Mini-Dose Heparin Levels