The objective of the assay is to assess in vitro the hemolytic potential of test items in human whole blood.
Intravascular hemolysis results from the rupture or lysis of red blood cells within the circulation in vivo. When the membrane of erythrocytes is damaged, they release their hemoglobin into the plasma. For new drug candidates and the excipients intended for injectables or infusion being in direct contact with the circulating blood, an in vitro hemolysis study should be performed at least at the intended concentration for i.v. administration (bolus and/or infusion) to determine the hemolytic potential.
Although drug-induced hemolysis is relatively rare, the potential haemolytic effect should be considered prior to application. The in vitro hemolysis assay mimics the in vivo process and evaluates hemoglobin release in the plasma (as an indicator of red blood cell lysis) following human whole blood exposure to test agent(s).
In vitro test in human whole blood.
The following samples are prepared and tested in the assay:
After pre-incubation of human whole blood aliquots, the respective solutions are added to the blood and incubated. At different time points of the incubation the hemolysis is measured spectrophotometrically and the hemolysis % is calculated.
Hemolysis % of the test item and the determination of hemolytic potential of the test item.