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In Vitro Hemolysis Assay

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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 sensitised 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.

Contact us to learn more about Cyprotex's hemolysis assays.

‘For excipients intended for injectable use, an in vitro hemolysis study could be performed at the intended concentration for I.V. administration (bolus and/or infusion) to determine the hemolytic potential.’

FDA Guidance for Industry: Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (May 2005)

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Protocol

In vitro hemolysis assay protocol

Test System	Heparinised whole blood
Species	Human and rat (other species available)
Incubation Time	45 min at 37°C
Test Compound Concentration	10 concentrations up to 250µM or the highest concentration at which the compound is soluble; two-fold dilutions
Number of Replicates	N=2 per concentration
Quality Controls	0.5% DMSO (vehicle control) Triton X-100 (positive control)
Compound Requirements	50µL of 20mM solution
Analysis Method	Spectrophotometry at 540nm
Data Delivery	Degree of lysis at each concentration IC₅₀

Hemoglobin release into plasma can be assessed as a measure of red blood cell lysis following drug/chemical exposure.

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Data

Data for in vitro hemolysis assay assessment

Compound	Concentration (%, v/v)	% Lysis
Triton X-100	1%	70%
Triton X-100	0.1%	14%
Triton X-100	0.01%	2%
SDS	1%	82%
SDS	0.1%	18%
SDS	0.01%	7%
Saponin	1%	57%
Saponin	0.1%	19%

Table 1

Hemolysis by Control Compounds

Human erythrocytes were treated with agents know to cause hemolysis. The percent lysis increased with increasing concentrations of the agents.

Figure 1

Hemolysis by Hemolysin in Human and Rabbit Erythrocytes

Rabbit erythrocytes are much more susceptible to hemolysis by the bacterial exotoxin, hemolysin, than human erythrocytes, demonstrating the need to use human erythrocytes in hemolysis experiments.

Q&A

Questions and Answers on In Vitro Hemolysis Assay

Why is it important to investigate toxic hemolysis by drugs?

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.

Please provide an overview of the in vitro hemolysis assay.

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.

What controls are included in the in vitro hemolysis assay?

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).

How does in vitro hemolysis relate to in vivo drug-induced toxic hemolysis?

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 (540nm) can lead to false positives.

References

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

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