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International Pig‐a gene mutation assay trial: Evaluation of transferability across 14 laboratories
Author(s) -
Dertinger Stephen D.,
Phonethepswath Souk,
Weller Pamela,
Nicolette John,
Murray Joel,
Sonders Paul,
Vohr HansWerner,
Shi Jing,
Krsmanovic Ljubica,
Gleason Carol,
Custer Laura,
Henwood Andrew,
Sweder Kevin,
Stankowski Leon F.,
Roberts Daniel J.,
Giddings Amanda,
Kenny Julia,
Lynch Anthony M.,
Defrain Céline,
Nesslany Fabrice,
van der Leede Basjan M.,
Van Doninck Terry,
Schuermans Ann,
Tanaka Kentaro,
Hiwata Yoshie,
Tajima Osamu,
Wilde Eleanor,
Elhajouji Azeddine,
Gunther William C.,
Thiffeault Catherine J.,
Shutsky Thomas J.,
Fiedler Ronald D.,
Kimoto Takafumi,
Bhalli Javed A.,
Heflich Robert H.,
MacGregor James T.
Publication year - 2011
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.20672
Subject(s) - cd59 , transferability , flow cytometry , microbiology and biotechnology , biology , enumeration , reproducibility , concordance , andrology , genetics , chemistry , chromatography , medicine , statistics , mathematics , logit , combinatorics , complement system , antibody
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 (RBC CD59− ) and CD59‐negative reticulocytes (RET CD59− ). 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 RET CD59− , and frequency of RBC CD59− . 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.