Establishment of a healthy human range for the whole blood "OX40" assay for the detection of antigen-specific CD4+ T cells by flow cytometry.
BACKGROUND
Clinical investigation of antigen-specific T cells in potentially immunodeficient patients is an important and often challenging aspect of patient diagnostic work up. Methods for detection of microbial exposure to the T-cell compartment exist but are laborious and time consuming. Recently, a whole blood technique involving flow cytometry and detection of CD25 and OX40 (CD134) expression on the surface of activated CD4+ T cells was shown to be accurate and concordant when compared with more traditional methods of antigen-specific T-cell detection.


METHODS
Whole heparinized blood was collected from healthy donors and set up using the "OX40" assay to detect antigen-specific CD4+ T-cell responses to Varicella Zoster Virus, Epstein-Barr Virus (EBV), Cytomegalovirus, Candida albicans, and Streptococcus pneumoniae.


RESULTS
The "OX40" assay technique was clinically validated for routine use in an NHS clinical immunology laboratory by analysis of incubation length (40-50 h), sample transport time (up to 24 h at room temperature), concordance with serology testing, proliferation and interferon-gamma production. In addition, 63 healthy controls (age range 21-78) were tested for responses to generate a healthy control reference range.


CONCLUSIONS
The OX40 assay, as presented in this report, represents an economical, rapid, robust whole blood technique to detect antigen-specific T cells, which is suitable for clinical immunology diagnostic laboratory use.
Department of Immunology, Churchill Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.
Ross
R
Sadler
Elizabeth A L
EA
Bateman
Victoria
V
Heath
Smita Y
SY
Patel
Phillip P
PP
Schwingshackl
Alice C
AC
Cullinane
Lisa
L
Ayers
Berne L
BL
Ferry
D000328:Adult; D000368:Aged; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002176:Candida albicans / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D014645:Herpesvirus 3, Human / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D053262:Receptors, OX40 / Q000032:analysis*; D012016:Reference Values; D013296:Streptococcus pneumoniae / Q000276:immunology; D055815:Young Adult
C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D053262:Receptors, OX40; C505716:TNFRSF4 protein, human; D007371:Interferon-gamma
CD4+ T cells; costimulation/costimulatory; diagnostics; flow cytometry