Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

Abstract

The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

Keywords: CD59; CD71; Flow cytometry; Glycosylphosphatidylinositol; HIS49; Immunomagnetics; In vivo gene mutation; Pig-a assay; Reticulocytes.

Fig. 1

Outline of the PIGRET assay procedure. First, stain RBCs with PE-conjugated anti-CD71 Abs and mixed with anti-PE magnet particles. Enrich CD71 positive reticulocytes using cell separation magnet(s) by a positive selection method. Next, stain the enriched RETs with APC-conjugated anti-HIS49 Ab and FITC-conjugated anti-CD59 Ab. Finally, using FCM, CD71 and HIS49 double-positive cells are analyzed for CD59 expression, and Pig-a mutant cells are detected as the FITC-negative population

Fig. 2

Plots for the PIGRET assay. Create dot-plots of FSC vs. SSC (Plot 1), PE vs. APC (Plot 3), FSC vs. FITC (Plot 4), and PE vs. FITC (Plot 5). Create one more dot-plot of FSC-H/FSC-W to eliminate doublet cells (Plot 2, option for digital FCM). Adjust or create the “Pig-a mutants” region to include 99.0% ± 0.1% of the CD59-negative cell population using the HIS49 single-stain sample

Fig. 3

Typical images of PE vs. APC plot (Plot 3). a Typical plot for a non-stained sample and a CD59 single-stain sample, b plot for a CD71 single-stain sample, and (c) plot for PIGRET samples stained with anti-CD71, anti-CD59, and anti-HIS49 Abs

Fig. 4

Gate defining and adjustment for Pig-a mutants with HIS49 single-stain samples (copy-and-paste method for FACSCantoII). Create “temporary Pig-a mutants” gate in Plot 4. Adjust the height of the “temporary Pig-a mutants” gate to include 99.0% ± 0.1% of the CD59-negative cell population using the HIS49 single-stain sample. Make sure that the “temporary Pig-a mutants” gate has reached the x-axis. If applicable, select “Bi-exponential” for the y-axis (FITC intensity scaling) in the Plot Inspector. Then, copy-paste the temporary gate so that the gate would be created as a subset of “CD71 + HIS49 + .” Rename to “Pig-a Mutants” and change Show Populations back to the “CD71 + HIS49+” population. The marker of “temporary Pig-a mutants” can be hidden if appropriate

Fig. 5

Typical images of the FSC vs. CD59-FITC plot (Plot 4). a Typical plot for a CD59 single-stain sample (Show Populations is set to “Single Cells”), b plot for a negative control sample, and (c) plot for a positive control sample treated with ENU 40 mg/kg by gavage

Fig. 6

Example of plots for %RET. Use same plots for the PIGRET assay (plots 1–3). If a gate setting, especially the quadrant gate for Plot 3, was adjusted in the PIGRET assay, make sure to use same gate setting for %RET as PIGRET assay

Fig. 7

Example of PIGRET assay results. a PIGRET assays were conducted at 0 (pretreatment), 1, 2, and 4 weeks after a single oral administration of PBS (negative control; open circles), 10 mg/kg ENU (filled squares), or 40 mg/kg ENU (open squares). b %RET analysis results at 0, 1, 2, and 4 weeks. This study was conducted at Teijin Pharma Limited, and the result of PIGRET assay was reported as the reference site response for the MMS/JEMS collaborative study (Step 1) [2]. The %RET data have not been previously published