Mitotic index maximization with no effect on radiation-induced dicentric chromosome frequency

Abstract

Purpose

The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500 lymphocyte metaphases or 100 Dic aberrations. Therefore, peripheral blood culture conditions able to obtain a high frequency of metaphases for efficient dose evaluation should be optimized. However, the type of blood cultures [i.e. whole blood (WB) or isolated peripheral blood mononuclear cell (PBMC)-culture] and blood volume differ between biodosimetry laboratories. The purpose of this study is to investigate the blood volume at which a high mitotic index (MI) is obtained in peripheral WB-culture and isolated PBMC-culture, and to examine the possible effect of blood volume on radiation-induced Dic frequency.

Materials and methods

Peripheral blood was collected from three healthy donors with their informed consent. The complete and differential blood counts were performed using an automated hematology analyzer. After blood count, peripheral blood was irradiated with 0 or 2 Gy X-ray. Blood was cultured with phytohemagglutinin (180 μg/ml) and demecolcine　(0.05 μg/ml) for 48 h. The MI and Dic frequency were analyzed in 5, 10, 15, 20, 25, and 30% WB-cultures and 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2 ml WB-equivalent PBMC-cultures.

Results

In WB-culture, MI showed the highest value (∼22%) in 5–15% WB-culture and then gradually decreased to ∼9% with 30% WB-culture. MI peaked at 36 and 31% in 1.8 and 2.4 ml-WB equivalent volumes for PMBC-cultures, respectively. MI progressively decreased as the amount of PBMCs increased. Although individual differences were observed in the MI values among the three subjects, all the subjects showed the same tendency and higher MI was seen in PBMC than WB-cultures. However, these factors had no significant impact on the yield of Dics. In all culture conditions, the estimated dose calculated based on the Dic frequency was equivalent to the absorbed dose of ex vivo X-ray-irradiated blood.

Conclusion

While MI was affected by the blood culture type and the volume of cultured blood, Dic yield did not differ significantly between these conditions. These results could be used by relevant laboratories to optimize MI in certain circumstances.

Keywords:

• Whole blood-culture
• PBMC-culture
• mitotic index
• Dic aberration frequency
• cytogenetic biodosimetry

Acknowledgments

The authors thank Naomi Sasaki and Yuki Sato for their technical support.

Disclosure statement

No potential conflict of interest was reported by the author(s). The opinions and assertions expressed herein are those of the author(s) and do not reflect the official policy or position of the Uniformed Services University of the Health Sciences or the U.S. Department of Defense. This research protocol was reviewed and approved by the Committee of Medical Ethics in Hirosaki University Graduate School of Health Sciences (Approval number: 2012-278)　in accordance with all applicable federal regulations governing the protection of humans in research.

Additional information

Funding

Partial funding support for WFB was provided by USUHS award AFR-11607 and AFR-B4-10971.

Notes on contributors

Kai Takebayashi

Kai Takebayashi, B.Sc. MT, is a Master’s student in the Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences.

Keito Echizenya

Keito Echizenya is an undergraduate student in the Department of Health Sciences, Hirosaki University School of Medicine.

Yuki Kameya

Yuki Kameya is an undergraduate student in the Department of Health Sciences, Hirosaki University School of Medicine.

Daichi Nakajima

Daichi Nakajima is an undergraduate student in the Department of Health Sciences, Hirosaki University School of Medicine.

Ryo Nakayama

Ryo Nakayama, M.Sc., is a Doctoral student in the Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences.

Yohei Fujishima

Yohei Fujishima, Ph.D., is an Assistant Professor in the Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine (IREM/HU), Hirosaki University.

Valerie Swee Ting Goh

Valerie Goh Swee Ting, Ph.D., is a Research Fellow in the Department of Radiobiology, Singapore Nuclear Research and Safety Initiative (SNRSI), National University of Singapore.

Yu Abe

Yu Abe, Ph.D., is an Assistant Professor in the Department of Radiation Biology and Protection, Atomic Bomb Disease Institute, Nagasaki University.

Kosuke Kasai

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

Donovan A. Anderson

Donovan A. Anderson, Ph.D., is a Specially Appointed Assistant Professor at the Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine (IREM/HU), Hirosaki University.

William F. Blakely

William F. Blakely, Ph.D. is a Senior 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 Biometrics Department, USUHS.

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.