Assessing Immunophenotyping In Immunotoxicology Studies With Standardized Flow Cytometry Tools
Immunotoxicology evaluations in animal models are mandatory pre-clinical studies of the drug discovery process, as they determine potential adverse health effects that a new drug may have on the immune system of patients. For this reason, the testing strategies need to be reliable and reproducible to accurately assess safety.
Among Different Immunotoxicology tests recommended by the ICH Harmonized Tripartite Guideline, Flow Cytometry-based Lymphocytes phenotyping is a useful assay.
To address this, we have developed standardized and automated tools to determine rapidly lymphocyte phenotype in a rat model.
By using a cocktail of pre-calibrated labeled antibodies that give minimum emission spectral overlap (FITC/PC7/APC), we enable the addition of a wide variety of PE-labeled
antibodies as well as the use of the dead cell exclusion 7-AAD marker. In addition to this flexible 5-color Lymphocyte Immunophenotyping assay, a no wash Red Blood Cell lysing system was optimized for enhanced accuracy. Furthermore, immunostaining and lysing steps can be processed on the Biomek Laboratory Automation Workstation and analysed on the FC 500 cytometer, with auto-setup panels and gating protocols provided.
Various examples are shown to demonstrate the usefulness of our methodology. For example, the described technique and analysis were applied for the detection of activated T cells following mitogenic activation with Concanavalin A, or for the exclusion of dead cells in Bone Marrow samples. We have established and compared the results obtained on Rat Peripheral Blood samples processed both manually and on the automation platform, and shown no significant difference. In addition, the sensitivity and specificity of our method to known immuno-suppressive drugs and/or immunogenic agents has been evaluated.