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International Pig-a gene mutation assay trial: Evaluation of transferability across fourteen laboratories

Dertinger, Stephen D., Phonethepswath, Souk, Weller, Pamela, Nicolette, John, Murray, Joel, Sonders, Paul, Vohr, Hans-Werner, Shi, Jing, Krsmanovic, Ljubica, Gleason, Carol, Custer, Laura, Henwood, Andrew, Sweder, Kevin, Stankowski Jr, Leon F., Roberts, Daniel J., Giddings, Amanda, Kenny, Julia, Lynch, Anthony M., Defrain, Céline, Nesslany, Fabrice, van der Leede, Bas-jan M., Van Doninck, Terry, Schuermans, Ann, Tanaka, Kentaro, Hiwata, Yoshie, Tajima, Osamu, Eleanor, Wilde, Elhajouji, Azeddine, Gunther, William C., Thiffeault, Catherine J., Shutsky, Thomas J., Fiedler, Ronald D., Kimoto, Takafumi, Bhalli, Javed A., Heflich, Robert H. and MacGregor, James T. (2011) International Pig-a gene mutation assay trial: Evaluation of transferability across fourteen laboratories. Environmental and Molecular Mutagenesis, 52 (9). pp. 690-698. ISSN 0893-6692


Experiments described herein were designed 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 and SYTO 13 dye were used to label leukocyte-depleted blood samples, and the frequency of CD59-negative erythrocytes (RBCCD59-) and CD59-negative reticulocytes (RETCD59-) were determined via flow cytometric analysis. To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to the prototypical mutagen 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: pre-dosing and on Days 15 and 30. Blood samples were processed according to standardized sample processing and data acquisition protocols, and three endpoints were measured: %reticulocytes, frequency of RETCD59-, and frequency of RBCCD59-. As illustrated by the analysis of technical replicates, the methodology was found to be highly reproducible, as experimental coefficients of variation approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the responses that were observed among different laboratories. Dose-related increases in the frequency of RETCD59- and RBCCD59- were consistently observed on Day 15. Whereas maximal RETCD59- responses tended to occur by Day 15, peak RBCCD59- responses occurred at approximately Day 45. Elevated mutant phenotype cell frequencies were maintained through the latest time-point studied (Day 90). High concordance correlation coefficients show a remarkable level of agreement between the reference site and the test sites. 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.

Item Type: Article
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Additional Information: author can archive post-print (ie final draft post-refereeing); On personal web site or secure external website at authors institution; Publisher's version/PDF cannot be used
Keywords: Pig-a; mutation; flow cytometry; CD59; interlaboratory trial; genotoxicity
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Date Deposited: 13 Oct 2015 13:15
Last Modified: 13 Oct 2015 13:15


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