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Published on: February 8, 2016
Validation of an eight parameter immunophenotyping panel in adult canines for assessment of immunotoxicity
Brandon M Zeigler1, Yvonne Boyle-Holmes, Deanna Falzone
1Department of Cell and Molecular Biology, MPI Research, 54943 North Main Street, Mattawan, MI 49009, USA. Brandon.Zeigler@mpiresearch.com
Insights
A new six-color flow cytometry method precisely analyzes canine leukocyte populations for immunotoxicity testing. This validated assay improves efficiency and allows for sample storage without impacting results in pre-clinical toxicology studies.
Area of Science:
- Immunotoxicology
- Pre-clinical Toxicology
- Flow Cytometry
Background:
- Peripheral blood leukocyte analysis is crucial for assessing immunotoxicity in pre-clinical toxicology studies.
- Current methods may lack the precision or efficiency required for comprehensive assessment.
- Standardized and validated assays are needed for reliable immunotoxicity evaluations.
Purpose of the Study:
- To validate a single, six-color flow cytometry panel for enumerating canine peripheral blood leukocyte populations.
- To assess the assay's precision, including intra-assay and inter-assay variance.
- To determine the stability of samples before and after fixation for flow cytometric analysis.
Main Methods:
- Utilized a single, six-color flow cytometry panel to simultaneously enumerate absolute cell counts and relative percentages of leukocytes.
- Performed Good Laboratory Practice (GLP) validation to assess intra- and inter-assay variance and inter-instrument variance.
- Evaluated pre- and post-fixation stability of target leukocyte populations.
Main Results:
- All assay validation parameters met acceptance criteria, with coefficient of variation (CV) values below 25%.
- Intra- and inter-assay data confirmed acceptable precision for the single-panel method.
- Stability assessments showed no statistically significant difference between immediately processed samples and stored whole blood or processed samples.
Conclusions:
- The validated six-color flow cytometry assay provides a precise and efficient tool for canine immunotoxicity assessment.
- The assay's robustness allows for flexibility in sample handling and storage, enhancing workflow efficiency.
- This method will aid researchers in evaluating the immunotoxic effects of test articles during pre-clinical drug development.
Abstract:
Analysis of peripheral blood leukocyte populations by flow cytometry in adult beagles is a critical component of immunotoxicity assessment in regulated pre-clinical toxicology studies. In this study, data is presented utilizing a single panel, six-color method to simultaneously enumerate absolute cell counts and determine the relative percentage of leukocytes. A GLP validation was performed to determine intra- and inter-assay variance, inter-instrument variance, and pre- and post-fixation stability for the target populations. The results demonstrated all samples met acceptance criteria, CV values less than 25%, for all precision and stability intervals assessed. The intra and inter-assay data demonstrated the single panel method generated acceptable precision. Furthermore, stability results indicated whole blood samples and processed samples may be stored without a statistically significant difference in the data compared to samples immediately processed and analyzed after blood collection. This assay will provide researchers a more precise and efficient tool to evaluate the immunotoxic effects of a test article on canine peripheral blood leukocytes during pre-clinical drug testing.
