Related Experiment Video
Updated: Aug 9, 2026
Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers
Published on: March 6, 2009
Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European
Ingmar A F M Heijnen1, David Barnett, Maria J Arroz
1Division of Immunology, Department of Laboratory Medicine, Kantonsspital Aarau, Aarau, Switzerland. ingmar.heijnen@ksa.ch
Insights
Standardizing tetramer flow cytometry is crucial for routine diagnostics. A new single-platform method for counting cytomegalovirus (CMV)-specific CD8(+) T cells proved reproducible across multiple labs.
Area of Science:
- Immunology
- Cellular immunology
- Flow cytometry
Background:
- Human Leukocyte Antigen (HLA) class I peptide tetramers are vital for detecting and monitoring antigen-specific CD8(+) T cells.
- Standardization of tetramer flow cytometry is essential for its routine diagnostic application.
- A multicenter study was initiated to develop and evaluate a standardized single-platform tetramer-based method using cytomegalovirus (CMV) as a model.
Purpose of the Study:
- To develop and evaluate a standardized single-platform tetramer-based method for enumerating antigen-specific CD8(+) T cells.
- To assess the reproducibility of the method across multiple clinical sites.
- To establish a reliable assay for routine diagnostic use.
Main Methods:
- A single-platform tetramer-based method was developed and evaluated.
- Absolute counts of CMV-specific CD8(+) T cells were determined by combining tetramer-binding cell percentages with absolute CD8(+) T-cell counts.
- Stabilized blood samples were distributed to multiple clinical sites for analysis on three separate occasions.
Main Results:
- Between-site coefficients of variation were <10% for absolute CD8(+) T-cell counts and ~30% for CMV-specific CD8(+) T-cell counts.
- Within-site coefficients of variation were ~5% for absolute CD8(+) T-cell counts, ~9% for the percentage of CMV-specific CD8(+) T cells, and ~17% for absolute counts.
- Lower variation in CMV-specific CD8(+) T-cell subsets correlated with higher proportions of these cells.
Conclusions:
- The evaluated single-platform MHC tetramer-based method is a reproducible assay.
- This method is suitable for the routine enumeration of antigen-specific CD8(+) T cells.
- The standardization efforts contribute to the reliable implementation of tetramer flow cytometry in diagnostics.
Background:
HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model.
Methods:
Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method.
Results:
Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets.
Conclusions:
The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.
More Related Videos
11:07Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination
Published on: April 29, 2015
08:10Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector
Published on: November 28, 2018