Feline leukocyte immunophenotyping: an optimised whole-blood flow cytometry protocol

Area of Science:

• Veterinary Immunology
• Cellular Biology
• Biotechnology

Background:

• Flow cytometry application in feline samples faces challenges due to species-specific blood traits, lack of standardized protocols, and high reagent costs.
• These limitations can negatively impact sample quality, antibody efficacy, and result reproducibility in feline immunophenotyping.
• Existing challenges hinder accurate leukocyte analysis in cats, impacting both clinical diagnostics and research.

Purpose of the Study:

• To develop an optimal and cost-effective protocol for extracellular immunophenotyping of feline leukocytes from peripheral whole blood.
• To adapt established human flow cytometry methods for feline-specific applications.
• To address limitations in feline flow cytometry for improved reproducibility and reliability.

Main Methods:

• Adapted established human flow cytometry methods for feline peripheral whole blood.
• Optimized blood collection and sample preservation techniques, including the use of a cellular antigen stabilization reagent.
• Performed antibody titration to determine optimal concentrations, reducing usage significantly.

Main Results:

• Enhanced blood collection improved erythrocyte lysis, making samples more suitable for cytometric analysis.
• Leukocyte viability remained high (98-100%) and sample preservation was effective for up to 14 days without loss of surface antigen expression.
• Antibody titration reduced usage by up to 85% for most monoclonal antibodies, with CD5 requiring a slightly higher concentration.

Conclusions:

• The developed protocol is cost-effective, reproducible, and addresses key technical limitations in feline flow cytometry.
• Provides a practical framework for reliable leukocyte immunophenotyping in feline clinical diagnostics, research, and comparative immunology.
• Enhances the utility of flow cytometry for advancing feline health studies and veterinary medicine.