Interlaboratory Pig‐a gene mutation assay trial: Studies of 1,3‐propane sultone with immunomagnetic enrichment of mutant erythrocytes

Abstract

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (∼20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.

Human erythrocyte PIG‐A assay: An easily monitored index of gene mutation requiring low volume blood samples

“…Immunomagnetic separation provided an effective and efficient means to enrich the proportion of mutant cells and allow the enumeration of larger numbers of rare mutant phenotype cells. Thus, as with the analogous rodent blood assay, immunomagnetic depletion of wild-type cells prior to flow cytometry facilitated evaluation of magnitudes-greater numbers of cells than would have otherwise been possible [Dertinger et al, 2011b,c; 2012]. …”

“…This low baseline frequency led us to utilize immu-nomagnetic depletion of wild-type cells prior to flow cytometric analysis in order to enrich samples for mutant phenotype cells, as has been described for rat blood [Dertinger et al, 2011b,c, 2012]. In this manner, data acquisition rates and the number of cell equivalents evaluated per sample are enhanced dramatically.…”

Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa

“…Treatments were based on previous studies (11-13) with male rats that showed these dose levels are tolerated and significant increases in Pig-a mutant phenotype cells are induced.…”

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

“…In addition, these data provided the impetus for establishing a method to dramatically increase the number of cells evaluated per sample through the use of immunomagnetic separation technology [21]. Stage IV studies have been conducted with these updated methods and demonstrate improved statistical power [21,28,29], but only a fraction of the completed work has been published or available to the Workgroup in a form that could be used to assess intra-and interlaboratory reproducibility.…”