Evaluation of γ-H2AX foci distribution among different peripheral blood mononucleated cell subtypes

The detection of γ-H2AX foci in peripheral blood mononucleated cells (PBMCs) has been incorporated as an early assay for biological dosimetry. However, overdispersion in the γ-H2AX foci distribution is generally reported. In a previous study from our group, it was suggested that overdispersion could be caused by the fact that when evaluating PBMCs, different cell subtypes are analyzed, and that these could differ in their radiosensitivity. This would cause a mixture of different frequencies that would result in the overdispersion observed.

The objective of this study was to evaluate both the possible differences in the radiosensitivities of the different cell subtypes present in the PBMCs and to evaluate the distribution of γ-H2AX foci in each cell subtype.

Peripheral blood samples from three healthy donors were obtained and total PBMCs, and CD3⁺, CD4⁺, CD8⁺, CD19⁺, and CD56⁺ cells were separated. Cells were irradiated with 1 and 2 Gy and incubated at 37 °C for 1, 2, 4, and 24 h. Sham-irradiated cells were also analyzed. γ-H2AX foci were detected after immunofluorescence staining and analyzed automatically using a Metafer Scanning System. For each condition, 250 nuclei were considered.

When the results from each donor were compared, no observable significant differences between donors were observed. When the different cell subtypes were compared, CD8⁺ cells showed the highest mean of γ-H2AX foci in all post-irradiation time points. The cell type that showed the lowest γ-H2AX foci frequency was CD56⁺. The frequencies observed in CD4⁺ and CD19⁺ cells fluctuated between CD8⁺ and CD56⁺ without any clear pattern. For all cell types evaluated, and at all post-irradiation times, overdispersion in γ-H2AX foci distribution was significant. Independent of the cell type evaluated the value of the variance was four times greater than that of the mean.

Although different PBMC subsets studied showed different radiation sensitivity, these differences did not explain the overdispersion observed in the γ-H2AX foci distribution after exposure to IR.