Haemostasis relies on the adequate function and cooperation of platelets, as well as the coagulation and fibrinolytic systems. Platelets quickly adhere and aggregate to a site of vascular injury, forming a platelet plug, or thrombus, to minimize bleeding. Activated platelets provide a negatively charged membrane surface, which promotes cell-based thrombin generation, and thus facilitates blood coagulation. Fibrin is the key end product of the coagulation cascade and, together with platelet-dependent clot contraction, stabilizes the platelet clot to arrest bleeding. On the other hand, the clot dissolves during wound healing, a process known as fibrinolysis.
As haemostasis is a physiological process critical for survival, any faults or challenges in this system can lead to thrombotic or bleeding complications. The assessment of these processes and their interactions allows for the detection of haemostatic dysfunction, resulting from either a genetic or acquired defect, and makes it possible to assess the effect of antiplatelet or anticoagulant therapy.
The whole blood flow assay determines platelet function in thrombus formation and can assess platelet-dependent coagulant potential as well as fibrinolysis under physiological conditions. The system is based on microfluidics and an imaging system that operate at 37°C and uses an arterial or venous shear rate. The (effects on these) processes can be studied in this assay during a 10 minute perfusion.
The microfluidics consist of a coverslip microspot-coated with a thrombogenic surface and a transparent parallel-plate perfusion channel. The microfluidics and blood sample (600 µL) are incubated at 37°C to achieve a stable temperature prior to and during thrombus formation or breakdown under flow. Platelet adhesion, aggregation and coagulation/lysis is assessed every 10s by brightfield imaging combined with the recording of platelet and fibrin specific added fluorescent labels. Three different applications are distinguished to study different parts of the haemostatic process:
Microscopy images are automatically captured in real time using the Cytosmart Lux3 and the corresponding Axion software. Multiple output parameters can be acquired through visual inspection of brightfield images on thrombus signatures, including thrombus morphology, height and contraction score. Additionally, fluorescence images analysed by a machine learning algorithm provide automated information on time-dependent platelet deposition, thrombus size and fibrin formation.
