Extending culture time to improve Mitotic Index for cytogenetic dosimetry

Abstract

Purpose

To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood samples with different doses of radiation.

Materials and methods

Blood samples from two healthy male volunteers were separately irradiated with three doses (3, 5, and 6 Gy) using a ⁶⁰Co gamma source (average dose rate: 1.48 kGy.h⁻¹) and cultivated in vitro for conventional (48 h) and extended (56, 68, and 72 h) amounts of time. Colcemid (0.01 µg.mL⁻¹) was added at the beginning of the culture period. Cells were fixed, stained with fluorescence plus Giemsa (FPG), and analyzed under a light microscope. The effects of prolonged culture duration on the Mitotic Index (MI), frequency of first-division cells (M1 cells), and the First-Division Mitotic Index (FDMI) were investigated. The estimation of delivered doses was conducted using a conventional 48h-culture calibration curve.

Results

Overall, cells presented higher MI (up to 12-fold) with the extension of culture, while higher radiation doses led to lower MI values (up to 80% reduction at 48 h). Cells irradiated with higher doses (5 and 6 Gy) had the most significant increase (5- to 12-fold) of MI as the cultivation was prolonged. The frequency of M₁ cells decreased with the prolongation of culture for all doses (up to 75% reduction), while irradiated cells presented higher frequencies of M₁ cells than non-irradiated ones. FDMI increased for all irradiated cultures but most markedly in those irradiated with higher doses (up to 10-fold). The conventional 48h-culture calibration curve proved adequate for assessing the delivered dose based on dicentric frequency following a 72-hour culture.

Conclusion

Compared to the conventional 48-hour protocol, extending the culture length to 72 hours significantly increased the Mitotic Index and the number of first-division metaphases of irradiated lymphocytes, providing slides with a better scorable metaphase density. Extending the culture time to 72 hours, combined with FPG staining to score exclusively first-division metaphases, improved the counting of dicentric chromosomes. The methodology presented and discussed in this study can be a powerful tool for dicentric-based biodosimetry, especially when exposure to high radiation doses is involved.

Keywords:

• Biodosimetry
• cytogenetic dosimetry
• lymphocyte culture protocol
• cultivation length
• dicentric chromosome assay
• DCA

Acknowledgment

The authors thank Dr. David C. Lloyd (Health Security Agency, UK) for his critical reading of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the International Atomic Energy Agency (IAEA-Austria - Research Contract No: 22266) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq – Brazil – Grant No: 316346/2021-7].

Notes on contributors

Luciano Rodolfo Ferreira-Lucena

Luciano Rodolfo Ferreira-Lucena, MSc, is a biologist and a Ph.D. candidate at the Laboratory of Modeling and Biological Dosimetry, Nuclear Energy Department, Federal University of Pernambuco (UFPE), Recife, Brazil.

Amanda Iumatti Santos Firmo Xavier

Amanda Iumatti Santos Firmo Xavier, MSc, is an associate biomedical researcher at the Laboratory of Modeling and Biological Dosimetry, Nuclear Energy Department, Federal University of Pernambuco (UFPE), Recife, Brazil.

André Maciel Netto

André Maciel Netto, Ph.D., is a physicist and a full professor at the Nuclear Energy Department, Federal University of Pernambuco (UFPE), Recife, Brazil.

Simey de Souza Leão Pereira Magnata

Simey de Souza Leão Pereira Magnata, PhD., is a biomedic and an associate professor at Academic Center of Vitoria, Federal University of Pernambuco (UFPE), Vitória de Santo Antão, Brazil.

Giovanna Siqueira Lima

Giovanna Siqueira Lima is a biologist and MSc candidate at the Laboratory of Modeling and Biological Dosimetry, Nuclear Energy Department, Federal University of Pernambuco (UFPE), Recife, Brazil.

Ademir Amaral

Ademir Amaral, Ph.D., is a full professor and head of the Research Group on Radioprotection and Radioecology at the Nuclear Energy Department, Federal University of Pernambuco (UFPE), Recife, Brazil.