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 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 heparinspiked 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 %.3

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs

“…of ANSN fluorophore release as a result of FXa substrate 219 cleavage and is compared to the rate of AMC fluorophore 220 release which has been published previously [16]. Opti- 1.6 U mL −1 ).…”

“…Comparing CV values between the two fluorogenic assays, Harris et al [16] in the presence and absence of two 439 LMWHs (i.e., enoxaparin and tinzaparin) and danaparoid.…”

Comparison of a fluorogenic anti-FXa assay with a central laboratory chromogenic anti-FXa assay for measuring LMWH activity in patient plasmas

“…The reaction was started by adding 50 µl of 2.7 µM Pefafluor FXa fluorogenic substrate. These assay concentrations were optimized as previously outlined [13]. All the measurements were carried out in triplicate.…”

“…A potential advantage of anti-FXa assays is that they are not affected by many of the biological variables that interfere with clot-based endpoints, thus reducing assay variability [13]. Despite improvements in the chromogenic assay mechanism, poor correlative data between commercial anti-FXa chromogenic assays has been reported.…”

“…Fluorogenic assays on the other hand, do allow for measurement in whole blood and PRP, as sample opacity is not imperative to the performance of the assay and the fluorescent signal is not hampered by fibrin formation or turbidity caused by platelets [15]. Hence assays using fluorescent labels offer significant advantages over assays using colorimetric determination, in addition to the greater sensitivity and specificity associated with fluorescence measurements [13,16,17]. In this paper, we evaluated the suitability of a fluorogenic antiFXa assay for monitoring LMWH therapy in patient samples using the standard laboratory chromogenic anti-FXa assay as the reference method.…”