The dicentric chromosome assay is a well-established biodosimetric method used to assess absorbed ionizing radiation doses by detecting dicentric chromosomal aberrations. Here, we present a detailed, reproducible protocol for applying the dicentric chromosome assay for in vitro evaluation of radioprotective agents, including novel piperazine derivatives compared with amifostine and its active metabolite WR-1065. The protocol covers all key steps-blood sample preparation, in vitro irradiation, lymphocyte culture, metaphase preparation, and scoring of dicentric chromosomes. It highlights critical stages that affect data quality and reproducibility. Integrating manual scoring with automated analysis using the Metafer system ensures accurate and efficient assessment. Thus, this protocol bridges the fields of biological dosimetry and preclinical screening of radioprotective agents, providing a reliable framework for emergency radiation dose estimation and the development of new radiation medical countermeasures.

Department of Molecular Pathology and Biology Military Faculty of Medicine University of Defence Třebešská 1575 Hradec Králové 500 01 Czech Republic

Department of Radiobiology Military Faculty of Medicine University of Defence Třebešská 1575 Hradec Králové 500 02 Czech Republic

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