Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity

Abstract

In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.

Keywords: antimicrobial peptides (AMPs); cytotoxicity; hemolysis assay; hemolysis ratio.

Figure 1

OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% erythrocyte solution originating from a mouse, rat, rabbit, and human, incubated for 60 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), or AMPs 1, 2 or 3 (at concentrations of 100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from unpaired t-test is indicated by asterisks for comparison of human and rabbit samples (p-values: * < 0.05 *** < 0.001 **** < 0.0001 ns: non-significant). See Table S1 for p-values from comparison of all species.

Figure 2

OD measurements at 405 nm (Y-axis) of free hemoglobin in 1% erythrocyte solutions originating from 10 different human individuals, as well as blood pooled from all individuals, incubated for 60 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), dH₂O, or AMPs 1, 2, or 3 (100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.

Figure 3

OD measurements at 405 nm (Y-axis) of free hemoglobin in 1%, 2%, and 5% erythrocyte (EC) solution of pooled blood originating from 10 different human individuals, incubated for 60 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), dH₂O, or AMPs 1, 2, or 3 (100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) and included in plots.

Figure 4

OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% human erythrocyte solution incubated for 15, 30, 60, 90 or 120 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), dH₂O or AMPs 1, 2 or 3 (100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.

Figure 5

OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% erythrocyte solution sourced from a mouse, rat, rabbit, and human treated with different concentrations of Triton X-100 (A), Tween (B), and SDS (C), as well as ACS or dH₂O (D). Erythrocytes were incubated for 60 min at 37 °C. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from unpaired t-test is indicated by asterisks (p-values: * < 0.05 ** < 0.01 *** < 0.001 **** < 0.0001 ns: non-significant).

Figure 6

OD measurements of free hemoglobin in human whole blood at 405 nm (A), 530 nm (B), or 570 nm (C). Samples were treated with PBS (negative control), AMPs 1, 2, 3, melittin, or polymyxin B (all at 100 μM), as well as with dH₂O, ACS, or different concentrations of Triton X-100, Tween, or SDS. All samples were incubated for 60 min at 37 °C. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.

Figure 7

OD measurements of free hemoglobin in 1% washed human erythrocytes at 405 nm (A), 530 nm (B), or 570 nm (C). Samples were treated with PBS (negative control), AMPs 1, 2, 3, melittin or polymyxin B (all at 100 μM), as well as with dH₂O, ACS, or different concentrations of Triton X-100, Tween, or SDS. All samples were incubated for 60 min at 37 °C. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.

Figure 8

OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% human erythrocyte solution incubated with PBS, dH₂O, ACS, Triton X-100, SDS, Tween, AMPs 1, 2, or 3 (100 μM), or polymyxin B (100 μM) for 60 min at 37 °C in polystyrene (black bars) or polypropylene (red bars) tubes. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from paired t-test is indicated by asterisks (ns: non-significant).

Figure 9

OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% human erythrocyte solution incubated with PBS, dH₂O, ACS, Triton X-100, SDS, Tween, AMPs 1, 2, or 3 (100 μM), or polymyxin B (100 μM) for 60 min at 37 °C using high (500 μL: black bars) or low (100 μL; red bars) volumes. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from paired t-test is indicated by asterisks (p-values: * < 0.05 ns: non-significant).