The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells

Abstract

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G₀ CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

Keywords: PBMCS; biological dosimetry; genotoxicity tests; human blood; micronucleus assay; radiosensitivity.

Figure 1

Schematic overview of experiments: comparison between the whole blood and peripheral blood mononuclear cells (PBMC) cytokinesis-block micronucleus (CBMN) assays, performed on (A) healthy controls and (B) patient samples. (C) Effect of different blood volumes on CBMN assay results. (D) The effect of different cryopreservation times on CBMN assay results. (E) The PBMC CBMN assay performed in a biological dosimetry setting.

Figure 2

Comparison of the number of micronuclei (MNi) per 1000 binucleated (BN) cells for whole blood cultures (WBC), fresh, 2 weeks, and 25 weeks cryopreserved peripheral blood mononuclear cells (PBMCS) after 0, 0.5, 1 and 2 Gy exposure. The error bars represent SD of the mean.

Figure 3

Effect of cryopreservation time (2, 5, 10, 20 and 25 weeks) on the number of micronuclei (MNi) per 1000 binucleated (BN) cells in peripheral blood mononuclear cells (PBMCS). The error bars represent SD of the mean.

Figure 4

Radiosensitivity assessment of (A) fresh and (B) 25 weeks cryopreserved peripheral blood mononuclear cells (PBMCS) of 5 patients, exposed to 0, 0.5 and 1 Gy. Patients are regarded as being radiosensitive when their micronuclei (MNi) values exceed the mean +3 SD of a healthy control group, for each dose. According to clinical data, Patients 1 and 2 were considered radiosensitive while Patients 3, 4 and 5 were not radiosensitive.

Figure 5

Evaluation of the volume of blood required for a peripheral blood mononuclear cell (PBMC) cytokinesis-block micronucleus (CBMN) assay. PBMC isolation was performed on 1 mL, 3 mL and 5 mL of blood and the obtained PBMCS were subsequently used for the CBMN assay.

Figure 6

Whole blood was exposed to 0.5, 1 and 2 Gy, before peripheral blood mononuclear cell (PBMC) isolation. The micronuclei (MNi) mean ± SD of three donors is plotted against the linear quadratic fit of the mean ± SD of ten cryopreserved (2 w) PBMC controls.