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Updated: Aug 21, 2026
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Published on: October 8, 2012
Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow
L Ginaldi1, N Farahat, E Matutes
1Academic Department of Haematology and Cytogenetics, Royal Marsden Hospital, London.
Insights
Quantitative flow cytometry reveals distinct T cell antigen expression levels in healthy individuals. These reference values for T cell antigens on lymphocyte subsets can aid in diagnosing disease states by identifying deviations from normal patterns.
Area of Science:
- Immunology
- Cell Biology
Background:
- Lymphocyte subsets play crucial roles in immune responses.
- Understanding the expression levels of T cell antigens is vital for characterizing lymphocyte subsets and their functions.
- Reference values for antigen expression are needed to identify deviations in disease states.
Purpose of the Study:
- To establish reference values for T cell antigen expression on normal lymphocyte subsets.
- To identify differences in antigen expression that may indicate specific functions or maturation stages of lymphocytes.
Main Methods:
- Peripheral blood from 15 healthy donors was analyzed using multiparametric flow cytometry with triple-color analysis.
- Monoclonal antibodies targeting CD2, CD3, CD4, CD5, CD7, and CD56 were used.
- Standard microbeads were employed to quantify antigen expression as antibody binding capacity (ABC) per cell.
Main Results:
- CD4+ T cells showed higher CD3 expression than CD8+ T cells.
- Distinct antigen expression patterns were observed between T cell subsets (CD4+CD7-, CD4+CD7+, CD8+CD7+).
- Natural killer (NK) cells exhibited different antigen profiles (higher CD7, CD56; lower CD2, CD5) compared to T cells. B cells showed lower CD5 expression than T cells.
Conclusions:
- Quantitative flow cytometry provides a reliable method for measuring antigen expression on lymphocyte subsets.
- Established reference values for antigen expression can aid in diagnosing immune-related diseases.
- This approach enhances diagnostic accuracy by comparing patient data to normal counterparts.
Aims:
To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both.
Methods:
Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC).
Results:
CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells.
Conclusions:
Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts.
