A CHROMOGENIC METHOD FOR DETERMINATION OF FIXA IN PURIFIED PREPARATIONS
Abstract number: P-M-050
Rosén1 P., Andersson1 M., Rosén1 S.
1Bioanalytical laboratory, Rossix, Mölndal, Sweden
How-to-cite Rosén P, Andersson M, Rosén S. A CHROMOGENIC METHOD FOR DETERMINATION OF FIXA IN PURIFIED PREPARATIONS. J Thromb Haemost 2007; 5 Supplement 2: P-M-050
Introduction: FIX concentrates may contain small amounts of activated FIX (FIXa) and the measurement of FIXa is of importance as a control of risk of thrombogenicity of FIX concentrates. For this purpose, a chromogenic method has been developed.
Methods: Bovine FX is activated by FIXa in the presence of human FVIII, human thrombin, phospholipids (PL) and calcium ions. FXa activity is measured through hydrolysis of the chromogenic substrate S-2787 (Chromogenix).
The PL emulsion (Phospholipid-TGT, Rossix) is stabilized and contains synthetic/highly purified phosphatidyl choline, phosphatidyl serine and sphingomyelin.
Dilutions were made in 0.05 M Tris pH 7.3, 0.2% BSA (Rossix). The BSA was validated for use in hemostasis methods.
Two different reagents were used. Reagent A comprised FX (1 U/mL), FVIII (2.4 U/mL) and PL (12 or 43 microM) and Reagent B comprised thrombin (0.6 U/mL) and Ca2+ (9.8 mM).
50 mL FIXa, 50 mL Reagent A and 100 mL Reagent B were added to a microplate well. The mixture was incubated for 5 min at 37°C and 50 mL S-2787, 3 mM, was then added, followed by hydrolysis for 12 min. The absorbance was read at 405-490 nm.
Results: A linear dose-response (r >=0.99) of FXa activity vs FIXa was obtained over the FIXa range 1-22 ng/mL (sample concentration) at 11 mM PL. A similar linear dose-response was obtained also at 3 mM PL and with indications of a slightly higher FXa generation.
Addition of 12 and 40 mg/mL prothrombin in the sample did not influence the results.
Addition of 0.6 and 1.8 mg/mL FIX still allowed the detection of 2 ng/mL of FIXa.
Conclusions: A sensitive method for measuring trace amounts of FIXa in FIX concentrates has been developed, using a stabilized, well defined PL emulsion. The sensitivity for FIXa is 2 ng/mL in the presence of a 900-fold excess of FIX zymogen. The sensitivity can be further improved by increasing the amount of FVIII and/or increasing incubation times.
References: 1. Gray E et al. Thromb Haemost 73, 675-679 (1995)
2. Moola Z et al. Thromb Haemost 82, Suppl.,92 (1999)