PDF herunterladen
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
› Institutsangaben 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.
› Weitere Informationen
Auch verfügbar auf
   Lizenzen und Reprints

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



Publikationsverlauf

Eingereicht: 14. Januar 2025

Angenommen: 25. Februar 2025

Artikel online veröffentlicht:
25. März 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
 

  • References

  • 1 Milling Jr TJ, Frontera J. Exploring indications for the use of direct oral anticoagulants and the associated risks of major bleeding. Am J Manag Care 2017; 23 (Suppl. 04) S67-S80
    Suche in Google Scholar
  • 2 Bielecki S, Lee D, Hamad B. The market for oral anticoagulants. Nat Rev Drug Discov 2018; 17 (09) 617-618
    Suche in Google Scholar
  • 3 Connors JM. Testing and monitoring direct oral anticoagulants. Blood 2018; 132 (19) 2009-2015
    Suche in Google Scholar
  • 4 Lee LH. DOACs - advances and limitations in real world. Thromb J 2016; 14 (Suppl. 01) 17
    Suche in Google Scholar
  • 5 Perreault S, de Denus S, White-Guay B. et al. Oral anticoagulant prescription trends, profile use, and determinants of adherence in patients with atrial fibrillation. Pharmacotherapy 2020; 40 (01) 40-54
    Suche in Google Scholar
  • 6 Ruff CT, Giugliano RP, Braunwald E. et al. Comparison of the efficacy and safety of new oral anticoagulants with warfarin in patients with atrial fibrillation: a meta-analysis of randomised trials. Lancet 2014; 383 (9921) 955-962
    Suche in Google Scholar
  • 7 Xu Y, Siegal DM. Anticoagulant-associated gastrointestinal bleeding: framework for decisions about whether, when and how to resume anticoagulants. J Thromb Haemost 2021; 19 (10) 2383-2393
    Suche in Google Scholar
  • 8 Deutsch D, Boustière C, Ferrari E, Albaladejo P, Morange PE, Benamouzig R. Direct oral anticoagulants and digestive bleeding: therapeutic management and preventive measures. Therap Adv Gastroenterol 2017; 10 (06) 495-505
    Suche in Google Scholar
  • 9 Ballestri S, Romagnoli E, Arioli D. et al. Risk and management of bleeding complications with direct oral anticoagulants in patients with atrial fibrillation and venous thromboembolism: a narrative review. Adv Ther 2023; 40 (01) 41-66
    Suche in Google Scholar
  • 10 Yasaka M, Yokota H, Suzuki M, Yamane T, Ono Y. Incidence rates of bleeding and emergency surgery due to trauma or fracture among Japanese patients with non-valvular atrial fibrillation receiving oral anticoagulation therapy. Cardiol Ther 2020; 9 (01) 189-199
    Suche in Google Scholar
  • 11 Moia M, Squizzato A. Reversal agents for oral anticoagulant-associated major or life-threatening bleeding. Intern Emerg Med 2019; 14 (08) 1233-1239
    Suche in Google Scholar
  • 12 Brown CS, Mattson AE, Cabrera D. , al . Real world utilization of Andexanet alfa in the management of oral factor Xa inhibitor-associated gastrointestinal bleeding. Am J Emerg Med 2023; 73: 1-6
    Suche in Google Scholar
  • 13 White CM, Caroti KS, Bessada Y. et al. Andexanet alfa versus PCC products for factor Xa inhibitor bleeding: a systematic review with meta-analysis. Pharm J Human Pharmacol Drug Ther 2024; 44 (05) 394-408
    Suche in Google Scholar
  • 14 Bernier M, Lancrerot SL, Parassol N. et al. Therapeutic drug monitoring of direct oral anticoagulants may increase their benefit-risk ratio. J Cardiovasc Pharmacol 2020; 76 (04) 472-477
    Suche in Google Scholar
  • 15 da Silva RMFL. Novel oral anticoagulants in non-valvular atrial fibrillation. Cardiovasc Hematol Agents Med Chem 2014; 12 (01) 3-8
    Suche in Google Scholar
  • 16 Conway SE, Hwang AY, Ponte CD, Gums JG. Laboratory and clinical monitoring of direct acting oral anticoagulants: what clinicians need to know. Pharmacotherapy 2017; 37 (02) 236-248
    Suche in Google Scholar
  • 17 Moner-Banet T, Alberio L, Bart PA. Does one dose really fit all? On the monitoring of direct oral anticoagulants: a review of the literature. Hamostaseologie 2020; 40 (02) 184-200
    Suche in Google Scholar
  • 18 Shaw JR, Kaplovitch E, Douketis J. Periprocedural management of oral anticoagulation. Med Clin North Am 2020; 104 (04) 709-726
    Suche in Google Scholar
  • 19 Ward C, Conner G, Donnan G, Gallus A, McRae S. Practical management of patients on apixaban: a consensus guide. Thromb J 2013; 11 (01) 27
    Suche in Google Scholar
  • 20 Cuker A, Siegal DM, Crowther MA, Garcia DA. Laboratory measurement of the anticoagulant activity of the non-vitamin K oral anticoagulants. J Am Coll Cardiol 2014; 64 (11) 1128-1139
    Suche in Google Scholar
  • 21 Dale BJ, Chan NC, Eikelboom JW. Laboratory measurement of the direct oral anticoagulants. Br J Haematol 2016; 172 (03) 315-336
    Suche in Google Scholar
  • 22 Perifanis V, Neokleous N, Tsakiris DA. Update on laboratory testing and hemostasis assessment in patients receiving direct oral anticoagulants (DOACs). Thromb Update 2021; 5: 100084
    Suche in Google Scholar
  • 23 Rimsans J, Douxfils J, Smythe MA, Gosselin RC. Overview and practical application of coagulation assays in managing anticoagulation with direct oral anticoagulants (DOACs). Curr Pharmacol Rep 2020; 6: 241-259
    Suche in Google Scholar
  • 24 Maji D, Opneja A, Suster MA. et al. Monitoring DOACs with a novel dielectric microsensor: a clinical study. Thromb Haemost 2021; 121 (01) 58-69
    Suche in Google Scholar
  • 25 Frydman GH, Ellett F, Van Cott EM. et al. A new test for the detection of direct oral anticoagulants (rivaroxaban and apixaban) in the emergency room setting. Crit Care Explor 2019; 1 (08) e0024
    Suche in Google Scholar
  • 26 Ansell J, Zappe S, Jiang X. et al. A novel whole blood point-of-care coagulometer to measure the effect of direct oral anticoagulants and heparins. Semin Thromb Hemost 2019; 45 (03) 259-263
    Suche in Google Scholar
  • 27 Dias JD, Lopez-Espina CG, Ippolito J. et al. Rapid point-of-care detection and classification of direct-acting oral anticoagulants with the TEG 6s: implications for trauma and acute care surgery. J Trauma Acute Care Surg 2019; 87 (02) 364-370
    Suche in Google Scholar
  • 28 Harenberg J, Martini A, Du S, Krämer S, Weiss C, Hetjens S. Performance characteristics of DOAC dipstick in determining direct oral anticoagulants in urine. Clin Appl Thromb Hemost 2021; 27: 1076029621993550
    Suche in Google Scholar
  • 29 Rossi JM, Diamond SL. Scalable manufacture of a disposable, storage-stable eight-channel microfluidic device for rapid testing of platelet, coagulation, and drug function under whole blood flow. Biomicrofluidics. AIP Publishing 2020; LLC: 14
    Suche in Google Scholar
  • 30 Piran S, Traquair H, Chan N, Bhagirath V, Schulman S. Peak plasma concentration of direct oral anticoagulants in obese patients weighing over 120 kilograms: a retrospective study. Res Pract Thromb Haemost 2018; 2 (04) 684-688
    Suche in Google Scholar
  • 31 Grottke O, Braunschweig T, Rossaint R. et al. Transient or extended reversal of apixaban anticoagulation by andexanet alfa is equally effective in a porcine polytrauma model. Br J Anaesth 2019; 123 (02) 186-195
    Suche in Google Scholar
  • 32 Lu G, Conley PB, Leeds JM. et al. A phase 2 PK/PD study of andexanet alfa for reversal of rivaroxaban and edoxaban anticoagulation in healthy volunteers. Blood Adv 2020; 4 (04) 728-739
    Suche in Google Scholar
  • 33 Wong SL, Marshall LZ, Lawson KA. Direct oral anticoagulant prescription trends, switching patterns, and adherence in Texas Medicaid. Am J Manag Care 2018; 24 (8 Spec No.): SP309-SP314
    Suche in Google Scholar
  • 34 Gómez-Outes A, Suárez-Gea ML, Lecumberri R, Terleira-Fernández AI, Vargas-Castrillón E. Direct-acting oral anticoagulants: pharmacology, indications, management, and future perspectives. Eur J Haematol 2015; 95 (05) 389-404
    Suche in Google Scholar
  • 35 Dunois C. Laboratory monitoring of direct oral anticoagulants (Doacs). Biomedicines 2021; 9 (05) 9
    Suche in Google Scholar
  • 36 Tripodi A, Ageno W, Ciaccio M. et al. Position paper on laboratory testing for patients on direct oral anticoagulants. a consensus document from the SISET, FCSA, SIBioC and SIPMeL. Blood Transfus 2018; 16 (05) 462-470
    Suche in Google Scholar
  • 37 Gosselin RC, Adcock DM, Douxfils J. An update on laboratory assessment for direct oral anticoagulants (DOACs). Int J Lab Hematol 2019; 41 (Suppl. 01) 33-39
    Suche in Google Scholar
  • 38 Willekens G, Studt JD, Mendez A. et al. A universal anti-Xa assay for rivaroxaban, apixaban, and edoxaban measurements: method validation, diagnostic accuracy and external validation. Br J Haematol 2021; 193 (06) 1203-1212
    Suche in Google Scholar
  • 39 Derogis PBM, Sanches LR, de Aranda VF. et al. Determination of rivaroxaban in patient's plasma samples by anti-Xa chromogenic test associated to high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). PLoS One 2017; 12 (02) e0171272
    Suche in Google Scholar
  • 40 Gosselin RC, Favaloro EJ, Douxfils J. The myths behind DOAC measurement: analyses of prescribing information from different regulatory bodies and a call for harmonization. J Thromb Haemost 2022; 20 (11) 2494-2506
    Suche in Google Scholar
  • 41 Henskens YMC, Gulpen AJW, van Oerle R. et al. Detecting clinically relevant rivaroxaban or dabigatran levels by routine coagulation tests or thromboelastography in a cohort of patients with atrial fibrillation. Thromb J 2018; 16: 3
    Suche in Google Scholar
  • 42 Margetić S, Ćelap I, Huzjan AL. et al. DOAC dipstick testing can reliably exclude the presence of clinically relevant DOAC concentrations in circulation. Thromb Haemost 2022; 122 (09) 1542-1548
    Suche in Google Scholar
  • 43 Cuker A, Burnett A, Triller D. et al. Reversal of direct oral anticoagulants: guidance from the anticoagulation forum. Am J Hematol 2019; 94 (06) 697-709
    Suche in Google Scholar
  • 44 Byon W, Garonzik S, Boyd RA, Frost CE. Apixaban: a clinical pharmacokinetic and pharmacodynamic review. Clin Pharmacokinet 2019; 58 (10) 1265-1279
    Suche in Google Scholar
  • 45 Mueck W, Stampfuss J, Kubitza D, Becka M. Clinical pharmacokinetic and pharmacodynamic profile of rivaroxaban. Clin Pharmacokinet 2014; 53 (01) 1-16
    Suche in Google Scholar