Evaluation of the premature chromosome condensation scoring protocol after proton and X-ray irradiation of human peripheral blood lymphocytes at high doses range

Purpose of the study: One of the main difficulties in radiation dose assessment is cells inability to reach mitosis after exposure to acute radiation. Premature chromosome condensation (PCC) has become an important method used in biological dosimetry in case of exposure to high doses. Various ways to induce PCC including mitotic cells fusion, chemical stimulation with calyculin A or okadaic acid give wide spectrum of application. The main goal of this study was to evaluate the utility of drug-induced PCC scoring procedure by testing 2 experimental modes where 150 and 75 G2/M-PCC phase cells were analyzed after exposure to high dose proton and X-ray radiation. Another aim is to determine the differences in cellular response induced by proton and photon radiation using a HPBL in vitro model as a further extension of our previous studies involving doses up to 4.0 Gy. Materials and methods: Total body exposure was simulated by irradiating whole blood collected from a healthy donor. Whole blood samples were exposed to two radiation types: 60 MeV protons and 250 kVp X-rays in the dose range of 5.0-20.0 Gy, the dose rate for protons was 0.075 and 0.15 Gy/s for X-rays. Post 48 h of human peripheral blood lymphocytes (HPBL) culture, calyculin A was added. After Giemsa staining, chromosome spreads were photographed and manually analyzed by scorers in the G2/M-PCC phase. In order to check the consistency of obtained results all scorers followed identical scoring criteria. Additionally, PCC index kinetics was evaluated for first 500 cells scored. Conclusions: Here we provide a different method of results analysis. Presented dose-response curves were obtained by calculating the value of counted excess chromosome fragments. The results indicated that obtained dose estimates as adequate in the high dose range till 18.0 Gy for both studied radiation types, giving an opportunity to further improve PCC assay procedure and shorten the analysis time i.e. in case of partial-body exposure. Moreover, the study presents preliminary results of HPBL cellular response after proton irradiation at high doses range showing differences of PCC index kinetics for different cell classes and cell distribution.