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

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

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Institute of Medical Immunology, Charité Universitätsmedizin Berlin, Germany.
Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, København, Denmark.
Department of Surgery, University of Regensburg, Germany.


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.


adaptive immunity; aging; immunosenescence; innate immunity; standardization

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