Development of a fluorescent anti-factor Xa assay to monitor unfractionated and low molecular weight heparins

Abstract

Fluorogenic assays have many potential advantages over traditional clot-based and chromogenic assays such as the absence of interference from a range of factor deficiencies as well as offering the possibility of assays in platelet rich plasma or whole blood. A fluorogenic anti-factor Xa (anti-FXa) assay has been developed for the determination of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), namely enoxaparin and tinzaparin, and the synthetic heparinoid danaparoid, in commercial human pooled plasma. The assay was based on the complexation of heparin-spiked plasmas with exogenous FXa at a concentration of 4 nM in the presence of 0.9 μM of the fluorogenic substrate methylsulfonyl- d-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Pooled plasma samples were spiked with concentrations of anticoagulants in the range 0–1.6 U/ml. The assay was capable of the measurement of UFH and danaparoid in the range 0–1 U/ml, and enoxaparin and tinzaparin in the range 0–0.8 and 0–0.6 U/ml, respectively. Correlation coefficients generated by linear regression of the log/lin data analysis were between 0.93 and 0.96 for the anticoagulants tested. Assay percentage coefficients of variation were typically below 7%.