Development of a Label-free Imaging Technique for the Quantification of Thrombus Formation.

The characterization of platelet aggregation and thrombus formation typically requires the use of fluorescent labels followed by fluorescent confocal microscopy. However, fluorescent labels have been suspected to affect platelet function. We have developed a label-free imaging technique to characterize the volume and surface area coverage of platelet aggregates and thrombi formed under shear. Platelet aggregates were formed by perfusing anti-coagulated whole blood over fibrillar collagen. Thrombi were formed by perfusing recalcified whole blood over fibrillar collagen in the presence of coagulation. Platelet aggregates and thrombi volume and surface area coverage were quantified using a Hilbert transform differential interference contrast (DIC) microscopy technique (HT-DIC). Our data indicate that platelet aggregates and thrombi formed at a shear rate of 200 s-1 had similar volume and surface area coverage. At a shear rate of 1000 s-1, both the volume and surface area coverage of platelet aggregates significantly increased as compared to low shear conditions. In contrast, the volume of thrombi formed in the presence of coagulation appeared to remain the same at both low and high shear rates. Utilization of this HT-DIC imaging technique can allow for insights into the kinetics and mechanisms by which thrombi are formed under various shear conditions in a label-free manner.