March 4, 2019 – Several members of the iuvo BioScience toxicology team co-authored a paper entitled Suitability of Long-Term Frozen Rat Blood Samples for the Interrogation of Pig-a Gene Mutation by Flow Cytometry.
The paper was co-authored by Dr. Mary Richardson, Matt Barragato, Brett Schneider and John Prattico of iuvo, along with Avlasevich, et al, representing Litron Laboratories, Covance Laboratories and Exponent and was published in the January 2019 issue of the journal Environmental and Molecular Mutagenesis.
These studies have provided an effective procedure that enables the storage and subsequent labeling and analysis of rat blood samples In the Pig-a gene mutation assay for at least seven months. This ability to use long term frozen samples not only provides data supporting the reliability of data from shipped/stored samples but also allows tremendous flexibility when deciding on a toxicological testing strategy.
The abstract is available here, as well as the ability to order the full article.
The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection. In the current report, we describe the performance of freeze-thaw reagents that enable storage and subsequent labeling and analysis of rat blood samples for at least seven months. Data generated from three laboratories are presented that demonstrate rat erythrocyte recoveries in the range of 80-90%. Despite some loss of erythrocytes, Pearson coefficients and Bland-Altman analyses based on fresh blood vs. frozen/thawed matched pairs indicate that mutant cell and reticulocyte frequencies are not significantly affected, as the measurements are highly correlated and exhibit low bias. Collectively, these data support the effectiveness and suitability of a freeze-thaw procedure that endows the assay with several new advantageous characteristics that include: flexibility in scheduling personnel/instrumentation; reliability when shipping samples from in-life facilities to analytical sites; 3Rs-friendly, as blood from positive control animals can be stored frozen to serve as analytical controls; and ability to defer a decision to generate Pig-a data until more toxicological information becomes available on a test substance. Environ. Mol. Mutagen. 60:47-55, 2019.