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.

The cytokinesis-block micronucleus assay for cryopreserved whole blood

“…The absence of such an effect is required when cryopreserved blood samples are used in radiosensitivity assessment. Our results are in agreement with the observations of Sioen et al and Zijno et al In both studies, the authors demonstrated that the length of the cryopreservation period had also no influence on MN yields in cryopreserved isolated PBMCs (Zijno et al 2007;Sioen et al 2020). Taken together, our first results with the MN assay suggest that the radiation response of lymphocytes, cryopreserved in whole blood samples, is similar to that observed in fresh whole blood samples.…”

“…An earlier study of our group showed that X-rays induced MN in frozen isolated PBMCs were significantly higher than in fresh isolated PBMCs. However, no difference seemed to be observed between MN yields of frozen isolated PBMCs and fresh whole blood cultures (Sioen et al 2020).…”

“…Both the MN assay on fresh and cryopreserved whole blood required 4 ml of whole blood. MN results of fresh blood cultures from 10 healthy donors, recently published by our group, were reused for this study on cryopreserved blood (Sioen et al 2020).…”

“…Fresh whole blood A standard protocol from our laboratory for the MN assay was used, according to ISO 17099:2014 (Radiobiological protection -Performance criteria for laboratories using the cytokinesis block micronucleus assay (CBMN) in peripheral blood lymphocytes for biological dosimetry) and as described by Sioen et al (Sioen et al 2020). Fresh blood cultures were prepared in 25 cm 2 culture flasks by adding 0.5 ml blood to 4.5 ml of complete culture medium (RPMI-1640 medium (Gibco) supplemented with 10% FCS, 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco)).…”

“…Cryopreservation of isolated peripheral blood mononuclear cells (PBMCs) offers similar advantages and the feasibility of performing the MN assay on PBMCs has previously been demonstrated (Miszczyk et al 2015;Pajic et al 2015;Vozilova & Akhmadullina 2019;Sioen et al 2020). However, whole blood cryopreservation requires less processing steps and is thus a simple and time efficient alternative compared to PBMC isolation (Koppen et al 2018).…”