PB0534 - Rapid Measurement of DOAC Concentration and Platelet Function Using Whole Blood in a Disposable Microfluidic Device

Background: Increasing Direct Oral Anticoagulant (DOAC) use has created an unmet need to determine circulating drug levels quickly and reliably, in specific clinical scenarios. Available tests are not sufficiently rapid, or have not demonstrated accuracy for discriminating drug levels around ISTH thresholds for DOAC reversal.

Aims: We evaluated Xa inhibitor levels in whole blood from VTE/AF patients using a microfluidic assay, in comparison to Liquid Chromatography tandem Mass Spectroscopy (LC-MS/MS). Our aim was to demonstrate a POC assay capable of defining Xa inhibitor levels as above/below ISTH thresholds.

Methods: We deployed a single-use microfluidic device in clinic to estimate apixaban/rivaroxaban levels in VTE/AF patients. Blood from healthy volunteers was first used to verify that Xa inhibitor reversal agent (andexanet alfa) has no off-target effect in the absence of inhibitor. VTE patient blood was treated ex vivo with andexanet alfa and supratherapeutic DOAC. Dynamic fibrin and platelet deposition on collagen/tissue factor under flow was imaged via fluorescent fibrinogen and anti-CD61 within 5 minutes of collection. These same samples (plasma) were also evaluated by LC-MS/MS for DOAC concentration.

Results: Andexanet alfa added to healthy donor blood had no effect on platelet or fibrin signal. Patient whole blood treated with andexanet alpha and supratherapeutic DOAC allowed calculation of Xa inhibitor concentrations (2 nM-500 nM). Calculated concentrations for patients on either apixaban or rivaroxaban correlated well (R2= 0.7 or 0.9, respectively) with LC-MS/MS (Figure 1). The area under the curve (AUC) was 0.91 (apixaban) and 1.0 (rivaroxaban) for verifying DOAC concentration < 60 nM in patient blood (Figure 2).

Conclusion(s): Microfluidic testing of whole blood can provide a rapid estimate of DOAC levels over the on-therapy range. Blood levels of drug above and below ISTH recommended thresholds can be determined with high specificity and selectivity, while simultaneously providing an assessment of platelet and general hemostasis function.