In Vitro Hemolysis Assay

Drug-induced hemolysis is a relatively rare but serious toxicity liability. It occurs by two mechanisms¹:

• Toxic hemolysis − direct toxicity of the drug, its metabolite, or an excipient in the formulation.
  
• Allergic hemolysis − toxicity caused by an immunological reaction in patients previously sensitized to a drug.

Cyprotex’s in vitro hemolysis testing is designed to screen for toxic hemolysis, as recommended by the US FDA.

Although the majority of normal individuals may suffer toxic hemolysis at sufficiently high concentrations of hemolytic drugs, for most drugs toxic hemolysis involves lower doses given to individuals who are genetically predisposed to hemolysis¹.

The US FDA recommends that for excipients intended for injectable use, an in vitro hemolysis study should be performed at the intended concentration for IV administration to test for hemolytic potential².

The in vitro hemolysis assay evaluates hemoglobin release in the plasma (as an indicator of red blood cell lysis) following test agent exposure.

Serial dilutions of the compound are mixed with human blood and incubated for 45 minutes at 37°C. The cells are centrifuged and the absorbance of the supernatant, which includes plasma and lysed erythrocytes, is measured. Percent lysis is calculated from a standard curve of lysed erythrocytes.

Exposure to high concentrations of drugs can cause hemolysis even in normal adults, but in individuals who are genetically predisposed to hemolysis, this can occur at lower concentrations. Neutropenia, thrombocytopenia, hemolytic anemia, aplastic anemia, and macrocytic anemia are known to occur in some patients treated with drugs³. Drugs, their metabolites, or excipients used in formulation can cause toxic hemolysis; therefore the FDA recommends testing for hemolytic potential².

Positive control is Triton X-100, a known hemolytic agent⁴, 1% v/v and 0.1% v/v. Negative control is the vehicle (typically 0.5% DMSO).

Cyprotex’s in vitro toxic hemolysis assay is a sensitive and accurate method for predicting toxic hemolysis of a drug. However, it should be noted that hemolysis sometimes occurs when blood is drawn. This should be taken into account when performing the assay. Also, coloured drugs that absorb light at the same wavelength at which the assay is measured (540 nm) can lead to false positives

_{1) Dausset J and Contu L (1967) Ann Rev Med 18; 55-70
2) FDA Guidance for Industry - Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (May 2005)
3) Lubran MM (1989) Ann Clin Lab Sci 19; 114-21
4) Deibler GE (1959) J Appl Physiol 14; 133-6}