CYTOKINESIS-BLOCK MICRONUCLEUS (CBMN) ASSAY/CBMN CYTOME ASSAY IN HUMAN LYMPHOCYTES AFTER IN VITRO IRRADIATION AND ITS USE IN BIODOSIMETRY

Abstract

Summary Aim: Cytokinesis-block micronucleus (CBMN) assay and its comprehensive variant CBMN cytome assay are cytogenetic methods. CBMN is based on assessment of micronuclei in nucleated cells that have completed only one nuclear division. Besides micronuclei, CBMN cytome assay analyzes additional genotoxic (nucleoplasmic bridges and nuclear buds), cytostatic (nuclear division index), and cytotoxic (amount of necrotic and apoptotic cells) parameters. The aim of this study is to evaluate these parameters in human blood lymphocytes after in vitro irradiation and to assess its contribution to biodosimetry. Material and methods: Human blood from 6 donors was in vitro irradiated by 0, 1, 2, 3, 4, or 5 Gy and cultivated for 72 hours. Blood lymphocytes were stimulated with phytohemagglutinin and their cytokinesis was blocked by cytochalasin B. After cultivation, cultures were hypotonically treated, dropped onto glass slides and stained with Giemsa. Slides were evaluated by microscope. Results: We observed significantly increased amount of micronuclei, nucleoplasmic bridges, and nuclear buds measured in binucleated cells, significantly increased amount of micronuclei measured in mononucleated cells and significantly decreased nuclear division index after irradiation by 1, 2, 3, 4, and 5 Gy. Amount of death cells (apoptotic and necrotic) significantly increased after irradiation by 4 and 5 Gy. Conclusion: Although all parameters assessed by CBMN cytome assay have biodosimetric potential, practically feasible is only evaluation of micronuclei in binucleated cells. This parameter was used to construct in vitro linear-quadratic dose-response calibration curve which could be used as a biodosimetric tool for triage of radiation casualties.