Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring.

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations.

Nonmutagenicity of maleic acid and its sodium salts in salmonella assays.

Maleic acid (cis-butenedioic acid) and its mono- and disodium salts are shown to be non-mutagenic in the standard Ames Salmonella/mammalian microsome assay in the absence and presence of Aroclor-1254-induced rat liver S9. This lack of activity occurred despite depression of top agar pH in accordance with the degree of protonation of this polybasic acid. These results indicate that when chemical compounds which are maleate salts show activity in this assay, the effect is attributable to the base moiety rather than maleate or pH depression per se.