Dr. Andrew Macintyre
Three to six color TBNK panels assessing T-, B-lymphocytes, and NK cells (CD3, CD4, CD8, CD16, and CD20) are staples of both preclinical and clinical flow cytometry laboratories. Previously up to eight color flow cytometry was only available during the research phases of drug development. In recent years, twelve color instruments have become commonplace in the clinical laboratory. As new biological classes of pharmaceutical drugs are being developed for more complex mechanisms, a need arises to have the off the shelf subsets analyses available with drop in slots to customize the needs of an individual drug program, typically for receptor occupancy.
Currently there over 370 human cluster of differentiation (CD) markers established by the Human Leukocyte Differentiation Antigens (HLDA) committee. With approximately 330 of having commercially available antibodies against each to use during flow cytometry analysis. We are proposing a fresh paradigm for immunological assessment of drug toxicity on the immune system which allows for consistent datasets and a versatility previously not used during preclinical and clinical studies.
Briefly Immunophenotyping was performed using multi-color panel using up to 15 colors for an all-encompassing assessment of the main peripheral blood populations which are then supported by panels that detail the individual cell subsets.
The first combination assesses key mononuclear cells using the markers CD3, CD14, CD20, CD45, CD66, and CD159a. Mononuclear cell subsets are delineated using for T-lymphocytes (CD3, CD4, CD8, CD25, CD28, and CD45RA), B-lymphocytes (CD19, CD20, CD21, CD27, CD40, and IgD), Natural Killer Cells (CD3, CD25, CD107a, CD159a, CD335, and CD337), Monocytes (CD3, CD11b, CD14, CD16, CD32, and CD64), and Neutrophils (CD11b, CD14, CD18, CD35, CD64, and CD66).
With six colors used as the base mix, an additional nine colors are available for further delineation of cell populations. This approach allows for extensive data mining previously only performed during the research phase of drug development. Additionally, with the available space in the combination receptor occupancy or additional biomarkers can be easily added to an individual time point without extensive labor or re-compensation of the panel. These allow the study team to detect changes even in rare populations, providing a thorough and robust assessment of a drugu2019s effect on the immune system.