A Chromogenic Method with High Resolution for Determination of Human FIX Activity in Purified Preparations

Abstract number: P0643

Rosén S, Andersson M

Clotting methods for determination of FIX activity often show a low resolution in their dose-response with due risk of an unsatisfactory method accuracy. A chromogenic method has been developed with the following features: (1) Activation of human FIX with FVIIa and tissue factor (Instrumentation Laboratory). (2) Activation of bovine FX by generated FIXa in the presence of human FVIII, bovine thrombin, purified phospholipids and calcium ions where human FIXa constitutes the rate limiting component. (3) Determination of FXa activity through hydrolysis of the chromogenic substrate S-2765 (Chromogenix). FIX deficiency plasma is not utilized. Sample dilution is done in buffer containing 1% protease-free BSA, carefully validated for use in hemostasis methods. A linear dose-response (r > 0.99, absorbance range 0.1–1) of FIX activity was obtained over a FIX concentration range of 0.15–3 nMol/L (roughly 1.5–30 mU/mL) in the FX activation step when using 15 min activation of FIX and 2 min activation of FX followed by 4 min hydrolysis of S-2765. By extending the FX activation and substrate hydrolysis times to 5 and 10 min respectively, a linear dose-response was obtained down to 10-fold lower FIX concentrations. If considered more convenient, FIX activation may be performed on several samples in advance and terminated by EDTA followed by FX activation in a later, separate step. A contamination with human FX of <50% on a molar basis in the FIX preparation will not interfere; at a two-fold molar excess of FX, about 10% lower FIX potency was obtained due to substrate competition in the FVIIa/TF activation step. This chromogenic method offers a high-resolution alternative to the one-stage clotting method.