Purpose. The proposed technology allows to determine the risk group for the development of lymphocyte genomic instability among the contingents exposed to ionizing radiation as a result of the Chernobyl accident, military operations during the war, as well as during medical procedures for diagnostic and therapeutic purposes. Monitoring the risk group for the development of lymphocyte genomic instability will reduce the intensity of the effect of genotoxic factors on the patient’s body due to preventive measures.
Technical properties. The percentage of -H2AX+ (damaged), live, apoptotic and necrotic lymphocytes (markers FVS537 and Caspase-3) is determined by flow cytometry. Fluorescence intensity (MFI) serves as an indicator of the expression level of proteins: γ-H2AX, LC3B, Caspase-3. Part of the peripheral blood sample is incubated with chloroquine, a substance that blocks the process of autophagosome-lysosome fusion, leading to the accumulation of LC3B+ autophagosomes in the cell. The difference between the LC3B MFI during incubation with and without chloroquine, divided by the LC3B MFI value during incubation with chloroquine, is the autophagy activity index. The efficiency of DNA damage elimination is determined by comparing the obtained indices during incubation of a blood sample in a CO2 incubator without additional genotoxic load and with bleomycin, the effect of which on cells is similar to ionizing radiation. The total assessment of the studied indices, taking into account the external irradiation dose, allows us to determine the risk of genomic instability.
Scope. Healthcare. Healthcare institutions, medical research institutions, enterprises and institutions related to nuclear technologies in Ukraine.
Advantages. The technology is highly informative, relatively simple and fast to implement and reduces the need for molecular genetic studies using PCR or Western blot. This technology allows you to identify a group of people with a risk of developing genomic instability in lymphocytes, which is a factor in somatic and oncological pathology. In addition, this technology can be effectively used to select and monitor personnel working in contact with sources of ionizing radiation.
Technical and economic effect. Determining the effectiveness of eliminating DNA damage in lymphocytes based on markers of double-strand DNA breaks, autophagy and apoptosis in the presence of a radiation factor or other damaging factors will allow identifying patients who belong to high-risk groups for developing genomic instability in lymphocytes. or radiotherapy. And will also contribute to the development of preventive measures aimed at preventing the occurrence and reducing the negative effects of genotoxic factors on the human body.
Description. Autophagy impairment or deficiency contributes to increased DNA damage, impaired reparation mechanisms, and decreased cell viability after exposure to ionizing radiation. To assess lymphocyte DNA damage, flow cytometry is used to determine the relative amount (%) of γ-H2AX+, live, necrotic, and apoptotic cells, and the expression level of intracellular proteins: γ-H2AX, a marker of double-stranded DNA breaks, LC3B, an autophagy marker, and a marker based on the fluorescence intensity index (MFI). The autophagy activity index is calculated using the formula. The studied indices are determined with and without the addition of bleomycin, which is a radiomimetic. Based on the total assessment of the obtained indices and the external irradiation dose, the efficiency of DNA damage elimination is determined, as well as the risk of lymphocyte genomic instability with additional genotoxic load, including repeated irradiation, in particular during medical procedures.