Standardization of incubation conditions for hemolysis testing of biomaterials

Abstract

Hemolysis testing is the most common method to determine the hemocompatibility properties of biomaterials. There is however no consensus on the procedures of hemolysis testing due to insufficient comparative studies on the quality of the red blood cells used and the experimental conditions of testing. In this study we determined the effects of a number of incubation variables on the sensitivity and reproducibility of the hemolysis test using positively as well as negatively responding biomaterials and compared these results to those obtained according to the American Society for Testing and Materials (ASTM) standard. The ASTM standard method recommends hemolysis testing with highly diluted rabbit blood that is static incubated for 3 h. In this study we found that 24 h incubation of a biomaterial sample at 37 °C in slightly diluted human blood or with washed red blood cells was the most sensitive hemolysis test. Moreover usage of cryopreserved human RBC in the hemolysis test seemed to be a good alternative for fresh RBC since cryopreserved and fresh human RBC gave similar results in the hemolysis test. Hemolysis testing by exposing diluted rabbit erythrocytes to biomaterials as according to the ASTM method or by exposing biomaterials extract in saline to washed human red blood cells gave a different outcome and appeared not to be representative for clinical applications.