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Case Study

July 09, 2016

Hemolysis assay

Investigation of hemolytic potential of an anticancer compound using validated in vitro hemolysis assay in human whole blood

Four concentrations of test compound were tested. To determine the base level of the hemolysis (0% autohemolysis standard) PBS solution was added to one aliquot of whole blood. As positive control TritonÔ X-100 and as 100% positive control distilled water were used. After adding the test compound/controls/vehicle to the human blood, each tube was incubated in water bath. After the respective 6 incubation times, the vials were centrifuged and the absorption of the supernatants was measured in a spectrophotometer.

Each measured absorbance value was normalized with the respective value of PBS treated blood sample. The normalized absorption of the complete hemolysis caused by distilled water was graded as 100%.The dilutions of test item, vehicle and the positive control was measured and graded as a percentage of the positive 100% standard (maximal inducible hemolysis).

Results:

The results of the measurement in case of each donor can be accepted if the positive control 0.15% TritonÔ X-100 shows time dependent hemolytic effect and the hemolysis % is higher than 10% at each time point of the incubation.

On the basis of the recommendations of the regulatory guides and white papers listed in regulatory guidelines formulation with hemolysis value above 10% was considered to be at risk for hemolysis.

Conclusion: this test item does not have any hemolytic potential in the investigated concentration range applying incubation time up to 4 hrs. The vehicle at 10% in the blood also did not cause hemolysis in the 4 hour incubation time.

Contact: Aurigon

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E-Mail: info(at)aurigon.de

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