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Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry
Published on: March 5, 2019
Canine peripheral blood lymphocyte phenotyping by 7-color multiparameter flow cytometry
Ratree Platt1, Terry Ng2, Sherry Glover3
1Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, USA.
Insights
This study fully characterized canine lymphocyte phenotypes using novel 7-color multiparameter flow cytometry. The advanced method accurately identified lymphocyte subsets and improved canine immune system analysis.
Area of Science:
- Veterinary Immunology
- Immunophenotyping
- Flow Cytometry
Background:
- Accurate characterization of canine lymphocyte phenotypes is crucial for understanding immune status.
- Previous methods had limitations in simultaneously detecting multiple lymphocyte markers.
Purpose of the Study:
- To characterize baseline canine lymphocyte phenotypes using a novel 7-color multiparameter flow cytometry.
- To identify lymphocyte subsets coexpressing multiple markers.
Main Methods:
- Analysis of peripheral blood lymphocytes from 79 healthy Beagle or Beagle-mix dogs.
- Utilized 7-color multiparameter flow cytometry for high-resolution cell analysis.
- Developed an intensive gating strategy for accurate subset identification.
Main Results:
- Successfully detected minor lymphocyte subsets coexpressing multiple markers.
- Provided averaged percentages for major lymphocyte subsets (CD3+, CD4+, CD8+, CD21+, gammadelta TCR+).
- Demonstrated the utility of combining CD21 and CD3 monoclonal antibody clones and identified a novel CD21- CD3+ CD4- CD8-gammadelta TCR- lymphocyte subset.
Conclusions:
- Canine lymphocyte phenotypes were comprehensively characterized.
- The novel multiparameter flow cytometry approach enhances the accuracy of canine lymphocyte phenotyping.
- This method provides a powerful tool for veterinary immunology research.
Objective:
To characterize baseline canine lymphocyte phenotypes including lymphocytes coexpressing multiple markers by novel 7-color multiparameter flow cytometry.
Study Design:
Fresh canine peripheral blood lymphocytes of 79 healthy 26-week-old Beagle or Beagle-mix dogs were stained and analyzed.
Results:
The high number of samples and acquired flow data (averaging 1.9 x 10(5) cells/sample) allowed the detection of minor lymphocyte subsets coexpressing multiple lymphocyte markers. The averaged percentages of major lymphocyte subsets of CD3+, CD4+, CD8+, CD21+ and gammadelta TCR+ cells from this study were 74.0, 43.6, 14.3, 9.6, and 0.2, respectively, which were comparable but uniquely different from other reports as they were simultaneously detected in the same sample. We demonstrated that the commonly used CD21 and CD3 monoclonal antibody (mAb) clones, previously recommended not to be used in the same staining, could and should be used together with the proper steps of lymphocyte gating. We found a high percentage (10.3%) of unidentified CD21- CD3+ CD4- CD8-gammadelta TCR- lymphocyte subset that has never been reported. The intensive gating strategy and the mean percentages of each lymphocyte subset to their parent subsets and to the total lymphocyte population are presented and discussed.
Conclusion:
The canine lymphocyte phenotypes were fully characterized. This novel multiparameter flow cytometry method is a powerful approach to in-crease the accuracy of lymphocyte phenotyping in dogs.
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