Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens.
BACKGROUND
In the past decade, human immunodeficiency virus (HIV) lymphocyte immunophenotyping has evolved significantly. New fluorochromes, new multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using the single-platform technique have all contributed to the reliability of T-cell subset measurements. In this study, four gating protocols were evaluated to select the most robust method for T-cell subset enumeration.


METHODS
Peripheral blood specimens from 21 HIV(+) and 20 HIV(-) individuals were monitored up to 96 h. Aliquots of specimens were stored at room temperature and analyzed at 6 (baseline), 48, 72, and 96 h. Aliquots were stained with CD45-fluorescein isothiocyanate (FITC)/CD3PC5/CD4RD1/CD8ECD. Data analysis was performed with all four gating protocols.


RESULTS
Only with fresh blood did all protocols provide similar results. From samples that were 48 h old, the choice of gating strategy had a dramatic impact on immunophenotyping results. The largest deviations from baseline values occurred at 96 h and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. The gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 h.


CONCLUSIONS
By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days.
National HIV Immunology Laboratory, Bureau of HIV/AIDS, TB and STD, CIDPC, PPHB, Health Canada, LCDC 0603B1, Tunney's Pasture, Ottawa, Ontario K1A 0L2, Canada.
M
M
Bergeron
J K A
JK
Nicholson
S
S
Phaneuf
T
T
Ding
N
N
Soucy
A D
AD
Badley
N C
NC
Hawley Foss
F
F
Mandy
D000163:Acquired Immunodeficiency Syndrome / Q000097:blood*; D015704:CD4 Antigens / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D018023:HIV Seronegativity; D006679:HIV Seropositivity; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000097:blood; D008027:Light; D008214:Lymphocytes / Q000166:cytology*; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D013048:Specimen Handling / Q000379:methods; D016176:T-Lymphocyte Subsets / Q000166:cytology*; D013997:Time Factors
D015704:CD4 Antigens; D016827:CD8 Antigens; D017493:Leukocyte Common Antigens