International Pig-a gene mutation assay trial: Evaluation of transferability across 14 laboratories
| Autor: | William C. Gunther, Ann Schuermans, John Nicolette, Fabrice Nesslany, Ronald D. Fiedler, Stephen D. Dertinger, Céline Defrain, Laura Custer, Thomas J. Shutsky, Catherine J. Thiffeault, Robert H. Heflich, Paul Sonders, Amanda Giddings, Ljubica Krsmanovic, Javed A. Bhalli, Eleanor C. Wilde, Anthony M. Lynch, James T. MacGregor, Kevin Sweder, Osamu Tajima, Takafumi Kimoto, Andrew Henwood, Jing Shi, Terry Van Doninck, Souk Phonethepswath, Carol Gleason, Bas-jan M. van der Leede, Hans-Werner Vohr, Daniel J. Roberts, Pamela Weller, Azeddine Elhajouji, Yoshie Hiwata, Leon F. Stankowski, Joel Murray, Julia Kenny, Kentaro Tanaka |
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| Rok vydání: | 2011 |
| Předmět: |
Erythrocytes
Reticulocytes Time Factors Endpoint Determination Epidemiology International Cooperation Health Toxicology and Mutagenesis Transferability CD59 Antigens Pig a gene Mutagen Gene mutation Biology medicine.disease_cause Risk Assessment Flow cytometry Rats Sprague-Dawley Andrology medicine Animals Rats Wistar Genetics (clinical) Genetics Reproducibility medicine.diagnostic_test Mutagenicity Tests Membrane Proteins Reproducibility of Results Reference Standards Flow Cytometry Rats Inbred F344 Rats Data Interpretation Statistical Ethylnitrosourea Calibration Mutation Mutation (genetic algorithm) Laboratories Genotoxicity Mutagens |
| Zdroj: | Environmental and Molecular Mutagenesis. 52:690-698 |
| ISSN: | 0893-6692 |
| Popis: | A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBCCD59−) and CD59-negative reticulocytes (RETCD59−). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1–3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RETCD59−, and frequency of RBCCD59−. The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87–0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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