Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay

Abstract

Purpose

In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods.

Materials and methods

Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy – background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated.

Results

>94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures.

Conclusion

This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Keywords:

• Cytokinesis-block micronucleus assay
• human peripheral blood mononuclear cells
• Giemsa
• cytogenetic biodosimetry

Acknowledgments

The authors thank Yura Hayasaka and Kaito Yanagidate for optimizing the protocol, and other members of our laboratory for their technical support.

Disclosure statement

The authors report no conflicts of interest. The opinions, conclusions and recommendations expressed or implied do not necessarily reflect the views of the Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Department of Defense or any other department or agency of the U.S. Federal Government.

Additional information

Funding

The Armed Forces Radiobiology Research Institute supported this research under the work units RAB4AM and AFR-B4-4313 (awarded to W. F. B.).

Notes on contributors

Valerie Swee Ting Goh

Valerie Goh Swee Ting, M.Sc., is a doctoral student in Hirosaki University Graduate School of Health Sciences, and a post-graduate research scholar with the Singapore Nuclear Research and Safety Initiative (SNRSI).

Ryo Nakayama

Ryo Nakayama, B.Sc., is a Master’s student in Hirosaki University Graduate School of Health Sciences.

William F. Blakely

William F. Blakely, Ph.D., is a Scientist in the Armed Forces Radiobiology Research Institute (AFRRI), a component of the Uniformed Services University of the Health Sciences (USUHS), and an Assistant Professor in the Preventive Medicine and Biostatistics Department, USUHS.

Yu Abe

Yu Abe, Ph.D., is an Assistant Professor in the Department of Radiation Life Sciences, Fukushima Medical University School of Medicine.

Christelle En Lin Chua

Christelle Chua En Lin, Ph.D., is a Senior Research Scientist in the Department of Radiobiology, Singapore Nuclear Research and Safety Initiative.

Zi Huai Chew

Chew Zi Huai, B.Sc., is a Research Assistant in the Department of Radiobiology, Singapore Nuclear Research and Safety Initiative.

Akifumi Nakata

Akifumi Nakata, Ph.D., is an Associate Professor in the Department of Basic Pharmacy, Hokkaido Pharmaceutical University School of Pharmacy.

Yohei Fujishima

Yohei Fujishima, Ph.D., is an Assistant Professor in the Department of Radiation Biology, Tohoku University School of Medicine.

Mitsuaki A. Yoshida

Mitsuaki A. Yoshida, Ph.D., is the Director in the Institute of Chromosome Life Science, Japan, a private consulting firm involved in cytogenetic dose estimation and clinical cytogenetics.

Kosuke Kasai

Kosuke Kasai, Ph.D., is a Lecturer in the Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences.

Kentaro Ariyoshi

Kentaro Ariyoshi, Ph.D., is an Assistant Professor in the Integrated Center for Science and Humanities, Fukushima Medical University.

Tomisato Miura

Tomisato Miura, Ph.D., is a Professor at the Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine (IREM/HU), Hirosaki University.