Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.
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.
Division of Immunology, Department of Laboratory Medicine, Kantonsspital Aarau, Aarau, Switzerland. ingmar.heijnen@ksa.ch
Ingmar A F M
IA
Heijnen
David
D
Barnett
Maria J
MJ
Arroz
Simon M
SM
Barry
Marc
M
Bonneville
Bruno
B
Brando
Jean-Luc
JL
D'hautcourt
Florian
F
Kern
Thomas H
TH
Tötterman
Erik W A
EW
Marijt
David
D
Bossy
Frank W M B
FW
Preijers
Gregor
G
Rothe
Jan W
JW
Gratama
D018414:CD8-Positive T-Lymphocytes / Q000166:cytology*; D002452:Cell Count / Q000295:instrumentation / Q000379:methods*; D003587:Cytomegalovirus / Q000276:immunology / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000276:immunology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results
D015395:Histocompatibility Antigens Class I