Automation of the in vitro Micronucleus Assay

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The in vitro nucleus test is increasingly being used as an alternative to the more costly and time consuming in vitro chromosome aberration assay as a screen for detecting a chemical's genotoxic potential (aneugenic or clastogenic activity which are associated with disorders such as Down's Syndrome, cancers, etc.) Micronucleus measurements are made using the time-consuming and tedious process of microscopic inspection. No definitive and widely-accepted automated approach has come into use. During Phase I, we will investigate the use of flow cytometry to enumerate micronuclei in mouse lymphoma L5178 cells. This cell line is used in a commonly performed forward mutation assays (i.e., thymidine kinase locus) required by regulatory agencies world-wide. These cells offer the potential to develop an assay where the cells are treated and subsequently split between micronucleus and mutation endpoints, thereby minimizing plasticware, test article, and time requirements. Studies will focus on identifying gating and fluorescent staining procedures to discriminate bodies and mitotic chromosomes from true micronucleus events. Long range, the automated scoring procedures developed can be extended to the analysis of micronuclei in human lymphocytes. Some of these applications may include: 1) assessing the genotoxicity of pharmaceuticals. 2) identifying the individuals hypersensitive in particular genotoxin, 3) evaluating compounds or diets protective against genetic damage and 4) measurement of DNA damage in a population following a radiation and/or chemical accident. PROPOSED COMMERCIAL APPLICATION: The successful development of this methodology will enable Litron Laboratories to become an expert testing facility, capable of high throughput screening which identifies agents that cause cytogenetic damage. Furthermore, the extension of this methodology to the analysis of human blood lymphocytes and into kit format represents other significant areas of commercial opportunity.