Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens

M. Bergeron, J. K.A. Nicholson, S. Phaneuf, T. Ding, N. Soucy, A. D. Badley, N. C.Hawley Foss, Francis Mandy

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

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 isothtocyanate (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. Conclusion: By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days.

Original languageEnglish (US)
Pages (from-to)53-61
Number of pages9
JournalClinical Cytometry
Volume50
Issue number2
DOIs
StatePublished - 2002

Keywords

  • CD45 gating
  • Double anchor gate
  • Four-color immunophenotyping
  • Homogeneous and heterogeneous gating parameters
  • Integrated primary gate
  • Intrinsic and extrinsic parameters
  • Light scatter gate
  • Sequential mixed gate
  • Single-platform absolute counting
  • T gating

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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