Effects of culturing technique on human peripheral blood lymphocytes response to proton and X-ray radiation

Miszczyk, Justyna; Rawojć, K.

doi:10.1080/09553002.2020.1704907

Cited by 6 publications

(10 citation statements)

References 30 publications

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“…No significant differences were observed between cryopreserved and fresh cultures. All obtained NDI values were also in agreement with those described in literature for fresh blood cultures (Fenech et al 2003;Miszczyk & Rawojć 2020).…”

Section: Accepted Manuscript Discussionsupporting

confidence: 90%

The cytokinesis-block micronucleus assay for cryopreserved whole blood

Beyls

Baeyens

Vral

2021

International Journal of Radiation Biology

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The cytokinesis-block micronucleus assay for cryopreserved whole blood Purpose: 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. Materials and methods: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.Results: 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. Conclusions: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.

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Section: Accepted Manuscript Discussionsupporting

confidence: 90%

The cytokinesis-block micronucleus assay for cryopreserved whole blood

Beyls

Baeyens

Vral

2021

International Journal of Radiation Biology

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“…While WBC are commonly used for cytogenetic assays, isolated PBMCS are also finding their way in chromosomal damage research [21][22][23]. The potential for cryopreservation of PBMCS makes this an exceptionally suitable cell type for cytogenetic assays, as it has several major advantages: (1) the ability to obtain a bulk of cells; (2) no need for further blood sampling; (3) the potential for batching and (4) the ability to perform the analysis within a chosen time frame.…”

Section: Discussionmentioning

confidence: 99%

“…PBMCS, including monocytes and lymphocytes, can be isolated in bulk (1–2 × 10 6 PBMCS/mL) from anticoagulated blood. One fraction can be used immediately for multiple assays while another fraction can be frozen for future tests [ 21 , 22 , 23 , 24 ]. While freshly isolated PBMCS samples are optimal for many downstream applications, the cryopreservation of PBMCS allows for batching and analysis within a chosen time frame [ 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

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

Sioen

Cloet

Vral

et al. 2020

JPM

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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.

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“…The proton and X-ray irradiation procedures were previously described in detail [8][9][10]17]. Briefly, the facility used for proton irradiation was the Proteus C-235 isochronous cyclotron, located in the IFJ PAN.…”

Section: Proton and X-ray Irradiation And Dosimetrymentioning

confidence: 99%

“…In a previous series of studies, we demonstrated that therapeutic proton irradiation and X-rays induce DNA damage and cell death in normal tissue by various modes at doses in the range of 0.3-4.0 Gy [8][9][10]. These differences influence the type, incidence, and intensity of the adverse effects of radiation therapy while allowing the development of predictive biomarkers of clinical outcomes and facilitating the selection and/or stratification of patients or patient cohorts for a given modality of radiation treatment [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Investigation of DNA Damage and Cell-Cycle Distribution in Human Peripheral Blood Lymphocytes under Exposure to High Doses of Proton Radiotherapy

Miszczyk

2021

Biology

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This study systematically investigates how a single high-dose therapeutic proton beam versus X-rays influences cell-cycle phase distribution and DNA damage in human peripheral blood lymphocytes (HPBLs). Blood samples from ten volunteers (both male and female) were irradiated with doses of 8.00, 13.64, 15.00, and 20.00 Gy of 250 kV X-rays or 60 MeV protons. The dose–effect relations were calculated and distributed by plotting the frequencies of DNA damage of excess Premature Chromosome Condensation (PCC) fragments and rings in the G2/M phase, obtained via chemical induction with calyculin A. The Papworth’s u test was used to evaluate the distribution of DNA damage. The study shows that high doses of protons induce HPBL DNA damage in the G2/M phase differently than X-rays do. The results indicate a different distribution of DNA damage following high doses of irradiation with protons versus photons between donors, types of radiation, and doses. The proliferation index confirms the impact of high doses of mitosis and the influence of radiotherapy type on the different HPBL response. The results illuminate the cellular and molecular mechanisms that underlie differences in the distribution of DNA damage and cell-cycle phases; these findings may yield an improvement in the efficacy of the radiotherapies used.

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Effects of culturing technique on human peripheral blood lymphocytes response to proton and X-ray radiation

Cited by 6 publications

References 30 publications

The cytokinesis-block micronucleus assay for cryopreserved whole blood

The cytokinesis-block micronucleus assay for cryopreserved whole blood

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

Investigation of DNA Damage and Cell-Cycle Distribution in Human Peripheral Blood Lymphocytes under Exposure to High Doses of Proton Radiotherapy

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