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CC BY 4.0 · TH Open 2025; 09: a25475710
DOI: 10.1055/a-2547-5710
Original Article

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

Jason M. Rossi
1   Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
2   FloBio LLC, Philadelphia, Pennsylvania, United States
,
Karen A. Panckeri
3   Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
,
Soumita Ghosh
4   Institute for Translational Medicine and Therapeutics University of Pennsylvania, Philadelphia, Pennsylvania, United States
,
Tilo Grosser
4   Institute for Translational Medicine and Therapeutics University of Pennsylvania, Philadelphia, Pennsylvania, United States
5   Department of Translational Pharmacology, Bielefeld University, Bielefeld, Germany
,
Adam Cuker
3   Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
6   Department of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
,
Scott L. Diamond
1   Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
4   Institute for Translational Medicine and Therapeutics University of Pennsylvania, Philadelphia, Pennsylvania, United States
› Author Affiliations
Funding The research reported in this article was supported by an NIH grant (no. 1R43HL149480-01 [FloBio LLC]). Andexanet alfa provided by Dr. R. Camire (CHOP/Penn) and AstraZeneca (MTA with FloBio LLC. All reagents and fluidic devices were provided by FloBio LLC for subjects 11–36.
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Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

Microfluidic testing of whole blood can provide a rapid estimate of DOAC levels over the on-therapy range.

Keywords

oral anticoagulant - fibrin - thrombin - microfluidic

Authors' Contributions

All microfluidic experiments were conducted by J.M.R. LC-MS/MS procedure was designed and performed by either T.G. and S.G. or absorption systems (now Pharmaron). Subjects were recruited by K.P. and A.C. All authors contributed to either research design, acquisition of data, data analysis/interpretation, manuscript preparation, revision, or approval.


Supplementary Material



Publication History

Received: 14 January 2025

Accepted: 25 February 2025

Article published online:
25 March 2025

© 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

Bibliographical Record
Jason M. Rossi, Karen A. Panckeri, Soumita Ghosh, Tilo Grosser, Adam Cuker, Scott L. Diamond. Rapid Determination of Xa Inhibitor Activity in Blood Using a Microfluidic Device that Measures Platelet Deposition and Fibrin Generation Under Flow. TH Open 2025; 09: a25475710.
DOI: 10.1055/a-2547-5710
 

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