Impairment of collagen-induced thrombus formation in microfluidic assay correlates with bleeding complications better than cytofluorometric parameters

Objectives Microfluidic assays offer a promising solution for accessing the state of the hemostasis system, testing drugs and adjusting anticoagulant dosages. However, standardisation of such assays is still missing. We aimed to design a robust and readily accessible microfluidic assay, which follows recent SSC guidelines and is sensitive to hemostatic abnormalities. Methods We optimized key parameters of 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 minute of perfusion at a shear rate of 1000 s-1 over the millimetre-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. Conclusions The developed robust microfluidic assay represents an accessible tool for the assessment of primary haemostasis in patients and is promising for clinical use.