The in vitro hemolysis assay (or hemolytic potential assay) is often used as a preliminary screening tool to evaluate toxic effects of chemicals, drugs, and biomaterials before further in vivo testing. It is used to assess the hemolytic (red blood cell-lysing) activity of materials by study their effect on erythrocytes.

At a high level, red blood cells are exposed to the test substance in an in vitro setting. After exposure, the degree of hemolysis is measured by quantifying the release of hemoglobin from the ruptured cells.  This provides an indication of the test article’s compatibility with blood, and its potential to cause damage to red blood cells.  This in vitro hemolysis assay provides a sensitive and accurate method to evaluate hemoglobin release in the plasma that simulates the in vivo process.

Per the US FDA, a hemolysis assay should be considered for excipients intended for injectable use.

To perform the in vitro hemolysis assay, the test system used is whole blood in the target species.  Either EDTA or sodium citrate can be used as an anticoagulant. Concentrations for the assay cover the range of expected Cmax values for the test article during in vivo dosing.

Blood from healthy donors is collected in tubes containing anticoagulant and is evaluated for hemoglobin content. Three concentrations of the test article formulation are prepared and mixed with an appropriate volume of whole blood. Samples are then incubated, centrifuged, and the supernatant is assessed for hemoglobin concentration.

This test can be performed non-GLP or in compliance with Good Laboratory Practice Regulations.