Impairment of Collagen-Induced Thrombus Formation in Microfluidic Assay Correlates with Bleeding Complications Better Than Cytofluorometric Parameters

Abstract

Objectives

Microfluidic assays offer a promising solution for accessing the state of the hemostasis system, testing drugs, and adjusting anticoagulant dosages. However, standardization of such assays is still missing. We aimed to design a robust and readily accessible microfluidic assay, which follows recent scientific and standardization committee (SSC) guidelines and is sensitive to hemostatic abnormalities.

Methods

We optimized key parameters of the whole blood perfusion system to produce a reliable assay suitable for rapid evaluation of primary hemostasis in patients. The optimized protocol includes anticoagulation with hirudin, 5 minutes of perfusion at a shear rate of 1,000 s⁻¹ over the millimeter-wide fibrillar collagen patch at room temperature, and evaluation of thrombus formation using the upright fluorescent microscope.

Results

Percentage of the activator area covered by thrombi was a reliable parameter demonstrating reproducible results for a given donor over a time course of months. Analysis of the whole blood from 7 patients with Wiscott–Aldrich syndrome, 34 patients with immune thrombocytopenia (ITP), and 8 patients with X-linked agammaglobulinemia showed a significant decrease in thrombus surface coverage compared to that of healthy individuals. Importantly, the microfluidic assay was able to differentiate between ITP patients with distinct clinical bleeding scores better than platelet counts and cytofluorometric parameters.

Conclusion

The developed robust microfluidic assay represents an accessible tool for the assessment of primary haemostasis in patients and is promising for clinical use.

Keywords

platelets - diagnostic tools - hemostasis - microfluidics - platelet aggregation

Data Availability Statement

The data supporting this article have been included as part of the Supplementary Material (available in the online version). Any personal information about patients cannot be made available due to ethical confidentiality requirements. Additional data are available upon request.

Ethical Approval Statement

This study was approved by the CTP PCP RAS Ethical Committee on January 17, 2023 (approval number: 1/2-23[НЭК]). The study was conducted according to the principles of the Declaration of Helsinki.

Authors' Contribution

E.A.M. contributed to formal analysis, investigation, methodology, writing—original draft, visualization. T.A.K. contributed to formal analysis, investigation, writing—original draft, visualization. L.P. contributed to investigation. A.A.I. contributed to Investigation, writing—review and editing. E.V.S. contributed to resources, writing—review and editing. A.Y.S. contributed to resources, writing—review and editing. G.A.N. contributed to resources, supervision. M.A.P. contributed to conceptualization, supervision, project administration. D.Y.N.: Methodology, conceptualization, project administration.

^* These author share co-first authorship.

Publication History

Received: 14 May 2025

Accepted: 25 August 2025

Accepted Manuscript online:
26 August 2025

Article published online:
08 September 2025

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