Rapid Determination of Xa Inhibitor Activity in Blood Using a Microfluidic Device that Measures Platelet Deposition and Fibrin Generation Under Flow

Abstract

Patients taking direct oral anticoagulants (DOACs) often present complicated scenarios following major bleeding, stroke, or emergency surgery. Rapid whole blood assays of DOAC levels would aid clinical decisions such as the need for DOAC reversal.We developed a single-use, storage-stable, eight-channel microfluidic device to estimate factor Xa (FXa) inhibitor (apixaban or rivaroxaban) levels in venous thromboembolism or atrial fibrillation patients. The assay simultaneously measured whole blood clotting dynamics on collagen/tissue factor (TF; wall shear rate, 200−1) under four ex vivo conditions: no-treatment control, high dose Factor Xa inhibition, low dose or high dose FXa reversal agent (andexanet alfa). Fibrin and platelet deposition dynamics were monitored via two-color epifluorescence microscopy. Plasma samples were also evaluated by LC-MS/MS for DOAC concentrations.Experiments with healthy volunteer blood spiked with DOAC verified device performance (DOAC IC50 ∼120 nM) and confirmed that andexanet alfa added to healthy donor blood had no off-target effect on platelet or fibrin signal. Patient whole blood monitored for 15 to 25 minutes (17 minutes mean runtime) allowed calculation of functional DOAC concentrations ranging from 2 to 500 nM that correlated well with LC-MS/MS determination of apixaban or rivaroxaban (R2 = 0.7 or 0.9, respectively). Platelet dysfunction was not observed in any patient on DOAC. For a threshold of 100 nM DOAC, the area under the curve (AUC) was found to be 0.881 for apixaban and 0.933 for rivaroxaban.Microfluidic testing of whole blood can provide a rapid estimate of DOAC levels over the on-therapy range.