Background

Investigating the blood clotting on medical devices is essential for successful development of biomaterials for implantable medical devices. Until now, no biomaterial surface has been truly able to prevent blood clotting (Sabino et al., 2019). After contact with blood, the implant surface gets an adsorbed layer of blood protein, which can further activate the coagulation cascade, leading to platelet adhesion and activation, and finally to the development of the fibrin mesh (Gorbet and Sefton, 2004). Many published methods to investigate the anti-thrombogenic properties of biomaterials focus on studying the early stages of blood clotting, such as protein adsorption, and platelet adhesion and activation. Although they are important to understand the interaction between blood and the implant surface, these studies do not provide significant information about the overall coagulation process (Damodaran et al., 2013; Simon‐Walker et al., 2018; Obstals et al., 2018).

Preventing whole blood clotting on surfaces is crucial for long term success of blood-contacting implants. The formation of a fibrin clot is one of the latest stages of thrombosis, and this fibrin mesh traps the red blood cells (Leszczak et al., 2013). In this protocol, human blood is allowed to clot on biomaterial surface for up to 45 min. When the surface is transferred to DI water, only the red blood cells that are not trapped in the fibrin mesh are dissolved in water and get lysed due to pressure change. Hemolysis is the rupture of the red blood cells, followed by the release of their components, such as hemoglobin. Thus, a higher amount of hemoglobin released indicates less blood clotting on the surface. Positive control with just blood in DI water is prepared and is considered the maximum hemoglobin release. The absorbance measured at a wavelength of 540 nm is directly proportional to the concentration of free hemoglobin in water (Sabino et al., 2019). Therefore, a higher absorbance value indicates higher hemoglobin concentration, which means less clotting on the biomaterial surface.