A microfluidic anti-Factor Xa assay device for point of care monitoring of anticoagulation therapy

The development of new point of care coagulation assay devices is necessary due to the increasing number of patients requiring long-term anticoagulation in addition to the desire for appropriate, targeted anticoagulant therapy and a more rapid response to optimization of treatment. The majority of point of care devices currently available for hemostasis testing rely on clot-based endpoints which are variable, unreliable and limited to measuring only certain portions of the coagulation pathway. Here we present a novel fluorescence-based anti-Factor Xa (FXa) microfluidic assay device for monitoring the effect of anticoagulant therapy at the point of care. The device is a disposable, laminated polymer microfluidic strip fabricated from a combination of hydrophobic and hydrophilic cyclic polyolefins to allow reagent deposition in addition to effective capillary fill. Zeonor was the polymer of choice resulting in low background fluorescence (208.5 AU), suitable contact angles (17.5°± 0.9°) and capillary fill times (20.3 ± 2.1 s). The device was capable of measuring unfractionated heparin and tinzaparin from 0-0.8 U ml(-1) and enoxaparin from 0-0.6 U ml(-1) with average CVs < 10%. A linear correlation was observed between the device and the fluorescent assay in the plate for plasma samples spiked with UFH, with an R(2) value of 0.99, while correlations with tinzaparin and enoxaparin resulted in sigmoidal responses (R(2) = 0.99). Plasma samples containing UFH resulted in a linear correlation between the device and a standard chromogenic assay with an R(2) value of 0.98, with both LMWHs resulting in sigmoidal relationships (R(2) = 0.99).