Age and gender leucocytes variances and references values generated using the standardized ONE-Study protocol

Cytometry A. 2016 Jun;89(6):543-64. doi: 10.1002/cyto.a.22855. Epub 2016 May 3.


Flow cytometry is now accepted as an ideal technology to reveal changes in immune cell composition and function. However, it is also an error-prone and variable technology, which makes it difficult to reproduce findings across laboratories. We have recently developed a strategy to standardize whole blood flow cytometry. The performance of our protocols was challenged here by profiling samples from healthy volunteers to reveal age- and gender-dependent differences and to establish a standardized reference cohort for use in clinical trials. Whole blood samples from two different cohorts were analyzed (first cohort: n = 52, second cohort: n = 46, both 20-84 years with equal gender distribution). The second cohort was run as a validation cohort by a different operator. The "ONE Study" panels were applied to analyze expression of >30 different surface markers to enumerate proportional and absolute numbers of >50 leucocyte subsets. Indeed, analysis of the first cohort revealed significant age-dependent changes in subsets e.g. increased activated and differentiated CD4(+) and CD8(+) T cell subsets, acquisition of a memory phenotype for Tregs as well as decreased MDC2 and Marginal Zone B cells. Males and females showed different dynamics in age-dependent T cell activation and differentiation, indicating faster immunosenescence in males. Importantly, although both cohorts consisted of a small sample size, our standardized approach enabled validation of age-dependent changes with the second cohort. Thus, we have proven the utility of our strategy and generated reproducible reference ranges accounting for age- and gender-dependent differences, which are crucial for a better patient monitoring and individualized therapy. © 2016 International Society for Advancement of Cytometry.

Keywords: adaptive immunity; aging; immunosenescence; innate immunity; standardization.

MeSH terms

  • Adult
  • Age Factors
  • Aged
  • Aged, 80 and over
  • Antigens, CD / genetics
  • Antigens, CD / immunology*
  • Cohort Studies
  • Female
  • Flow Cytometry / standards*
  • Healthy Volunteers
  • Humans
  • Immunologic Memory
  • Immunophenotyping / standards*
  • Lymphocyte Subsets / classification*
  • Lymphocyte Subsets / cytology
  • Lymphocyte Subsets / immunology
  • Male
  • Middle Aged
  • Reference Values
  • Sex Factors


  • Antigens, CD