Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society

Satsuki Chikura,Satoru Itoh,Shigeharu Muto,Hisakazu Sanada,Katsuyoshi Horibata,Takafumi Kimoto

doi:10.1186/s41021-019-0121-z

Abstract

The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs—the PIGRET assay—has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail.

Highlights

The Pig-a assay is an in vivo gene mutation assay that uses the Pig-a gene as an endogenous reporter
We describe the procedure for the rat red blood cell (RBC) Pig-a assay that was validated in the MMS/ JEMS collaborative study
When each facility establishes and validates RBC Pig-a assay techniques on-site, it is desirable to conduct an assay on rats treated with a single dose of 40 mg/kg ENU and confirm that the Pig-a mutant frequency (MF) increases significantly at 2 or 4 weeks after administration, as shown Fig. 6

Summary

Introduction

The Pig-a assay is an in vivo gene mutation assay that uses the Pig-a gene as an endogenous reporter. In the MMS/JEMS collaborative study, the RBC Pig-a assays were conducted prior to and 1, 2, and 4 weeks after a single administration. To prepare control samples for gate adjustments, an additional 10 μL blood should be collected from one of the animals in the negative or vehicle control groups.

Results

Conclusion