Development and characterization of an automated assay of effective heparin activity in plasma

We describe a fully automated assay for determining effective heparin activity in plasma, based on heparin-catalyzed inhibition of Factor Xa (EC 3.4.21.6) by antithrombin III (AT III). Residual Factor Xa is determined kinetically by the Du Pont aca discrete clinical analyzer with a chromogenic substrate and is inversely related to heparin activity. Because the test plasma is the sole source of AT III, the assay result is dependent on AT III activity and reflects effective rather than total heparin activity. The assay range is 20-1200 USP units/L, and the assay shows equivalent sensitivity to standard and low-molecular-mass heparins. Within-run reproducibility (CV) is 1.6% at 390 units/L. There was no interference from common blood components or drugs. Results agreed well with those by the Coatest heparin kit (Kabi) adapted to the Cobas-Bio analyzer (r = 0.85, n = 122).