title: In Vivo Pig-a gene mutation assay: Guidance for 3Rs-friendly implementation.
abstract: The rodent Pig-a assay is an in vivo method for the detection of gene mutation, where lack of glycosylphosphatidylinositol-anchored proteins on the surface of circulating red blood cells (RBCs) serves as a reporter for Pig-a gene mutation. In the case of rats, the frequency of mutant phenotype RBCs is measured via fluorescent anti-CD59 antibodies and flow cytometry. The Pig-a assay meets the growing expectations for novel approaches in animal experimentation not only focusing on the scientific value of the assay but also on animal welfare aspects (3Rs principles), for example, amenable to integration into pivotal rodent 28-day general toxicology studies. However, as recommended in the Organisation for Economic Co-operation and Development Test Guidelines for genotoxicity testing, laboratories are expected to demonstrate their proficiency. While this has historically involved the extensive use of animals, here we describe an alternative approach based on a series of blood dilutions covering a range of mutant frequencies. The experiments described herein utilized either non-fluorescent anti-CD59 antibodies to provide elevated numbers of mutant-like cells, or a low volume blood sample from a single N-ethyl-N-nitrosourea treated animal. Results from these so-called reconstruction experiments from four independent laboratories showed good overall precision (correlation coefficients: 0.9979-0.9999) and accuracy (estimated slope: 0.71-1.09) of mutant cell scoring, which was further confirmed by Bland-Altman analysis. These data strongly support the use of reconstruction experiments for training purposes and demonstrating laboratory proficiency with very few animals, an ideal situation given the typically conflicting goals of demonstrating laboratory proficiency and reducing the use of animals. Environ. Mol. Mutagen. 57:678-686, 2016. © 2016 Wiley Periodicals, Inc.
authors: Bayer Pharma AG, Muellerstrasse 178, Berlin, 13353, Germany.
authors: Marian
authors: M
authors: Raschke
authors: Bayer Pharma AG, Muellerstrasse 178, Berlin, 13353, Germany.
authors: Bernd-W
authors: BW
authors: Igl
authors: GlaxoSmithKline, Park Road, Ware, Hertfordshire, SG12 0DP, United Kingdom.
authors: Julia
authors: J
authors: Kenny
authors: GlaxoSmithKline, Park Road, Ware, Hertfordshire, SG12 0DP, United Kingdom.
authors: Joanne
authors: J
authors: Collins
authors: Litron Laboratories, 3500 Winton Place, Rochester, New York, 14623.
authors: Stephen D
authors: SD
authors: Dertinger
authors: Litron Laboratories, 3500 Winton Place, Rochester, New York, 14623.
authors: Carson
authors: C
authors: Labash
authors: Covance Laboratories Inc, 671 S. Meridian Rd, Greenfield, Indiana, 46140.
authors: Javed A
authors: JA
authors: Bhalli
authors: Covance Laboratories Inc, 671 S. Meridian Rd, Greenfield, Indiana, 46140.
authors: Cameron C M
authors: CC
authors: Tebbe
authors: Covance Laboratories Inc, 671 S. Meridian Rd, Greenfield, Indiana, 46140.
authors: Kylie M
authors: KM
authors: McNeil
authors: Bayer Pharma AG, Muellerstrasse 178, Berlin, 13353, Germany.
authors: Andreas
authors: A
authors: Sutter
mesh_terms: D023401:Animal Use Alternatives; D000827:Animal Welfare; D000818:Animals; D018957:CD59 Antigens / Q000032:analysis; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005038:Ethylnitrosourea / Q000633:toxicity*; D005434:Flow Cytometry; D017408:Guidelines as Topic; D007753:Laboratories / Q000592:standards; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D011919:Rats, Inbred Strains; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism
chemical_list: D018957:CD59 Antigens; D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein; D005038:Ethylnitrosourea
keywords: Bland-Altman analysis; genotoxicity testing; laboratory proficiency