Objective

Genetic toxicity assessments are essential in pharmaceutical safety testing to detect drug-induced mutation or chromosomal damage. A flow cytometry-based in vivo mutagenicity assay has recently been established based on the phosphatidylinositol class A (Pig-a) gene. Pig-a forms the catalytic subunit of an enzyme, N-acetylglucosaminyltransferase, required for glycophosphatidylinositol (GPI) anchor biosynthesis. Inactivating mutations in the hemizygous Pig-a gene halt GPI-anchor synthesis, resulting in loss of cell-surface GPI-linked proteins.  Using loss of GPI-linked CD90.2 as a measure of Pig-a mutation, we have previously developed a complementary in vitro assay in L5178Y mouse lymphoma cells; 24-h exposure to known genotoxins, induced dose-dependent increases in CD90.2(-) cells. Here, we aimed to further validate the assay, focusing on mutant phenotypic expression time and rare event detection by flow cytometry. Following 24-h incubation with the mutagen ethyl-methanesulfonate (EMS;1.5-4.5mM), cells were harvested 2, 3, 4, 8, 10, 14, 16 and 24 days post-treatment and co-stained for CD90.2 and CD45, a transmembrane protein, to identify non-specific cell-surface alterations. Flow cytometric analysis indicated the optimal phenotypic expression period was 8 days.To determine the suitability of flow cytometry for the detection of rare mutational events, immunomagnetically-enriched CD90.2(-) cells (~80% purity) were serially diluted and measured by flow cytometry. The measured and expected frequencies of CD90.2(-) cells strongly correlate (R²=0.99). Using untreated cells, increasing numbers of events were recorded at different flow rates to determine the optimal acquisition parameters for analysing rare events (<0.1%). The optimum flow rate and total number of events were concluded to be medium and 100,000 events, respectively. Furthermore, CD90.2(-) immunofluorescence in cells confirmed the CD90.2(-) phenotype. In conclusion, the in vitro Pig-a assay sensitively detects rare mutational events, offering utility as a complement to the in vivo assay and potentially predicting its outcome prior to commencing 56-day animal studies.