Abstract
There has been a constant need to develop new and faster cytogenetic assays to measure the instability induced by genotoxic agents in the field of cytogenetic research, an example of which is the micronucleus assay. Micronuclei are fragments or complete chromosomes that remain in the cytoplasm during mitosis. With their high sensitivity and specificity detection, their presence can indicate environmental and occupational genotoxic effects. However, the prolonged periods of cell incubation this assay necessitates are costly and extensive. Hence, it is essential to develop an improved assay that can achieve standardization by being reproducible in practice. The standard protocol for the detection of micronuclei in lymphocytes uses a total assay time of 72 hours. Theoretically, it is possible to reduce the incubation period, and consequently, the total assay time, considering a lymphocyte, completes its mitosis in 24 hours. This study, after careful review of literature, proposes an experimental design to reduce the incubation period and demonstrates its usefulness in practice through the design of a collaborative trial.
Highlights
In 2011, the International Atomic Energy Agency (IAEA) recommended the use of four cytogenetic techniques to assess radiological emergencies and preparedness: dicentric analysis (DCA), micronucleus (MN) assay, fluorescent in situ hybridization (FISH), and premature condensation of chromosomes (PCC) [1]
We focused on the micronucleus assay
The frequency of lagging or incomplete DNA is called micronuclei; its presentation indicates the adaptation of cells to toxic agents such as ionizing radiation and is important according to the theory of the human genome [26]
Summary
In 2011, the International Atomic Energy Agency (IAEA) recommended the use of four cytogenetic techniques to assess radiological emergencies and preparedness: dicentric analysis (DCA), micronucleus (MN) assay, fluorescent in situ hybridization (FISH), and premature condensation of chromosomes (PCC) [1]. Micronuclei measure genotoxic damage from exposure to ionizing radiation by detecting chromosomal fragments This technique requires long incubation periods and monitoring of in vitro cultures for more than 72 hours. Cytochalasin B (Cyt-B) is added to stop cytokinesis and achieve binucleated cells (BN), which express micronuclei in lymphocytes [12], and BioMed Research International complete their division every 24 hours [13, 14]. The standard micronucleus protocol uses full ex vivo blood and requires an incubation time of 44-72 hours, which can be minimized [22] This is mentioned by a multiparametric study that concludes that due to time limitations, it is necessary to perform analysis in different laboratories, in order to have prolonged incubation times [23]. Mitogenic stimulation during early and late incubation periods can be studied in such experiments [25]
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