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Updated: Apr 7, 2026
Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis
Published on: October 17, 2018
Feline leukocyte immunophenotyping: an optimised whole-blood flow cytometry protocol
Rafael S Lopes1,2,3, Paulo Rodrigues-Santos4, Maria Dos Anjos Pires1,3
1Department of Veterinary Sciences, University of Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados 5000-801 Vila Real, Portugal.
Insights
Researchers developed an optimized feline flow cytometry protocol to improve leukocyte immunophenotyping. This cost-effective method enhances sample quality and antibody performance for reliable feline diagnostics and research.
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.
Background:
: Flow cytometry is a powerful tool for immunophenotyping, but its application in feline samples is challenging due to species-specific blood characteristics, a paucity of standardised protocols and high reagent costs. These limitations may compromise sample quality, antibody performance, and the reproducibility of the results.
Methods:
: An optimal protocol for extracellular immunophenotyping of feline leukocytes from peripheral whole-blood was developed, adapting established human flow cytometry methods. Key adaptations included improved blood collection process, assessing sample preservation, and titrating antibodies. Detailed MIFlowCyt-compliant information on cytometer configuration, compensation procedures, gating strategy, and controls is provided in Supplementary File S2.
Results:
: The enhanced blood collection significantly improved erythrocyte lysis rendering the samples more suitable for cytometric analysis. Baseline leukocytes viability, assessed by trypan blue exclusion was 98-100%. The use of a cellular antigen stabilisation reagent preserved feline peripheral whole-blood samples without detectable loss of surface antigen expression for up to 14 days. Antibody titration showed that most monoclonal antibodies were effective at 1.5 µL, reducing usage by up to 85%, while CD5 required 3 µL.
Conclusion:
: This improved, cost-effective and reproducible protocol address major technical limitations in feline flow cytometry and provides a practical framework for the reliable leukocyte immunophenotyping in clinical diagnostics, research, and comparative immunology.
