A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: implications for large scale monitoring of HIV-infected patients in resource-limited settings.
OBJECTIVE
To validate a simplified flow cytometry assay for CD4 and CD8 T cell counting based on monoclonal antibodies which are made resistant to high temperatures (simplified thermoresistant assay (STRA)).


METHODS
The STRA employs FITC-conjugated anti-CD4 and anti-CD8 monoclonal antibodies, predispensed into test tubes and chemically treated to be resistant to high temperatures. Five correlation studies were performed in three different laboratories on a total of 560 blood samples from HIV-1 infected patients. Each study correlated the STRA with either double or single platform assays currently available. Accelerated stability tests on the FITC-conjugated monoclonal antibodies were performed to assess the resistance of the STRA to high temperatures.


RESULTS
Comparison of STRA with both single platform and double platform assays gave correlation coefficients ranging 0.957-0.987 for CD4+ T cells and 0.946-0.968 for CD8+ T cells. In all correlation studies there was a perfect data overlapping in the low-pathological interval of CD4+ T cells (0-400 cells/ml). The FITC-conjugated CD4 and CD8 monoclonal antibodies maintained intact binding activity and fluorescence brightness after storage for 4 weeks at 45 degrees C and can be stored for up to 8 years in regular conditions (+4 degrees C).


CONCLUSIONS
The STRA correlates well with both single-platform and double-platform flow-cytometry assays currently used to assess CD4+ T cells. The test procedure is simple, rapid, and easy to perform. The reagents can be stored under unfavorable environmental conditions for long period of time. These features should facilitate access to flow cytometry testing in resource-poor settings.
Barbesti
Silva
S
BIO-D Srl, Valenzano, Italy.
Soldini
Laura
L
Carcelain
Guisline
G
Guignet
Angelique
A
Colizzi
Vittorio
V
Mantelli
Barbara
B
Corvaglia
Alessandro
A
Tran-Minh
Thun
T
Dorigatti
Fernanda
F
Autran
Brigitte
B
Lazzarin
Adriano
A
Beretta
Alberto
A
D000911:Antibodies, Monoclonal / Q000276:immunology; D016516:CD4-CD8 Ratio / Q000379:methods*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D006295:Health Resources / Q000191:economics; D006801:Humans; D007753:Laboratories; D008403:Mass Screening / Q000191:economics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D000911:Antibodies, Monoclonal; D016650:Fluorescein-5-isothiocyanate