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Updated: Aug 9, 2026
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Published on: October 8, 2012
Flow cytometric analysis of T-lymphocyte subsets in cats
G A Dean1, S L Quackenbush, C D Ackley
1Department of Pathology, Colorado State University, Fort Collins 80523.
Insights
A new, fast method for feline lymphocyte immunophenotyping uses whole blood lysis and flow cytometry. This technique accurately identifies T-cell subsets (CD4+ and CD8+) in cats, crucial for feline immunology research.
Area of Science:
- Veterinary Immunology
- Flow Cytometry
- Feline Hematology
Background:
- Accurate immunophenotyping of feline lymphocytes is essential for understanding feline immune responses.
- Existing methods for lymphocyte isolation can be time-consuming and require large sample volumes.
Purpose of the Study:
- To develop and validate a rapid and reliable method for feline lymphocyte immunophenotyping.
- To establish reference ranges for T-cell subsets in healthy cats.
Main Methods:
- Whole blood lysis for white blood cell isolation.
- Flow cytometry using fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (f43, Fel 7, fCD8) and phycoerythrin (PE)-conjugated fCD4 (Fel 7).
- Two-color analysis for simultaneous identification of CD4+ and CD8+ T cells.
Main Results:
- Whole blood lysis proved faster, less variable, and required less sample than density gradient separation.
- The two-color analysis allowed non-overlapping identification of CD4+ and CD8+ T cell subpopulations.
- Mean lymphocyte subset percentages in conventional and specific-pathogen-free cats were similar: pan T lymphocytes (f43) 54.8%, CD4+ cells (Fel 7) 33.9%, and CD8+ cells (fCD8) 19.1%.
- The mean CD4/CD8 ratio in normal cats was 1.9 (range 1.2-2.6).
Conclusions:
- The developed method is rapid, reliable, and suitable for routine feline lymphocyte immunophenotyping.
- This technique provides accurate quantification of T-cell subsets and a reference CD4/CD8 ratio for healthy cats.
- The findings contribute to advancing feline immunology and clinical diagnostics.
Abstract:
We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.
