Development Of A Fluorescent Anti-Factor Xa Assay To Monitor Unfractionated And Low Molecular Weight Heparins

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 heparin-like anticoagulants including 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 to 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 U/ml and 0-0.6 U/ml, respectively. Assay percentage coefficients of variation were typically below 7 %