Comparison of the anticoagulant response of a novel fluorogenic anti-FXa assay with two commercial anti-FXa chromogenic assays

Introduction: Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the 'gold standard' assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity. Materials and methods: Commercial plasmas were spiked with pharmacologically relevant concentrations (0-1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7 μM fluorogenic substrate, in addition to 6 μl of 100 mM CaCl ₂ and 44 μl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed. Results: The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0-1 U/ml with CVs of < 5% for UFH and tinzaparin and CVs < 9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits. Conclusions: This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.