Suitability of Long‐Term Frozen Rat Blood Samples for the Interrogation of Pig‐a Gene Mutation by Flow Cytometry

Abstract

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. © 2018 Wiley Periodicals, Inc.

Human blood PIG‐A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra‐ and inter‐subject variation

“…However, this low frequency has made it challenging to interrogate adequate numbers of reticulocytes to precisely measure %MN-RET. Informed by work by Grawé et al (2005) Here, we report that methods recently developed for freezing and later thawing rodent blood samples for mutation analysis (Avlasevich et al 2019) are effective with human blood. This is an important advance that overcomes numerous logistical issues that would otherwise interfere with many, if not most, human biomonitoring studies.…”

“…Here, we report that methods recently developed for freezing and later thawing rodent blood samples for mutation analysis (Avlasevich et al . 2019) are effective with human blood. This is an important advance that overcomes numerous logistical issues that would otherwise interfere with many, if not most, human biomonitoring studies.…”

Recommendations for conducting the rodent erythrocyte Pig‐a assay: A report from the HESI GTTCPig‐a Workgroup

“…Briefly, as described in greater detail below, anti‐coagulated blood samples that are kept cold (~ 4°C), not frozen, throughout transportation are compatible with analysis. Furthermore, procedures for freezing blood samples have been described (Avlasevich et al ., 2019), and these can be used for shipping frozen samples to a separate analytical facility, provided samples remain frozen throughout transportation.…”

“…Procedures also have been described for freezing and later thawing blood samples for subsequent processing and flow cytometric analysis of Pig‐a MUT RET/RBC frequencies (Avlasevich et al ., 2019). These procedures can be useful for delaying analysis for reasons that include instrument failure, deferring the decision to acquire Pig‐a data, and storing blood from mutagen‐treated animals for use as analytical positive control samples.…”

Caffeic Acid Genotoxicity: Correlation of the Pig‐a Assay with Regulatory Genetic Toxicology In Vivo Endpoints

“…The data generated to date indicate that the assay is sensitive, specific, robust, HT, and has high reproducibility and transferability (Gollapudi et al ). The assay has also considered 3Rs (Reduce, Replace, and Refine) friendly and can be easily integrated into chronic and sub‐chronic general toxicology and safety assessment studies (Raschke et al ; Avlasevich et al ). An Organization of Economic Cooperation and Development (OECD) test guideline for the Pig‐a assay is being developed.…”