PB0093 - Optimized robust thrombus-on-a-chip assay sensitive to haemostatic abnormalities - a pilot study

Background: Microfluidic flow chambers have emerged as a powerful tool for studying thrombosis and hemostasis. Besides their utility in basic research, they represent a promising solution for assessing the state of the hemostasis system. However, standardization of such assays is needed, as the precise control over various parameters of the microfluidic experiments is crucial to ensure reliable results. Additionally, analysis of the assay sensitivity to haemostatic abnormalities is required.

Aims: Development, optimization and validation of the robust microfluidic clinical assay sensitive to haemostatic abnormalities.

Methods: Blood was collected from healthy volunteers or paediatric patients with Immune thrombocytopenia (ITP), X-linked agammaglobulinemia (XLA) and Wiskott–Aldrich syndrome (WAS). The study was approved by the institutional ethics committee, all subjects gave informed consent. Blood was collected on hirudin, heparin or sodium citrate. Glass slides were coated with collagen using microfluidics, PDMS flow chambers were used for the whole blood perfusion. Platelets were labeled with DIOC6 and collagen surface coverage by thrombi was visualized using an upright fluorescence microscope.

Results: The refined protocol includes anticoagulation with hirudin, 5 minute perfusion at a shear rate of 1000 s-1 over the localized collagen patch. The surface coverage by thrombi (in %) was a reproducible parameter for a given healthy donor over a time course of weeks to months. Patients with WAS, ITP and XLA showed significantly lower surface coverage compared to that of healthy individuals. In contrast to cytofluorimetric platelet function test, the microfluidic assay was able to differentiate between ITP patients with distinct Buchanan bleeding scores.

Conclusion(s): The developed robust microfluidic assay was able to differentiate between healthy donors and patients and between ITP patients with different clinical bleeding scores. We conclude that this test represents a valuable new tool for assessing the state of primary hemostasis in patients.
The study was supported by the Russian Science Foundation grant 23-44-00082.