The cytokinesis-block micronucleus assay for cryopreserved whole blood

The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients.

Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5-2 Gy.

The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples.

To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays.