Age and gender-related variability of main lymphocyte subsets (T, B and NK cell 
absolute counts and percentages from Ly; T4, T8 and DN cell absolute counts and 
percentages from lymphocytes and from T cells; T4:T8 and T:B ratios) was studied 
in a large cohort of pediatric patients (2 days - 17 years) at yearly intervals. 
4128 6-color TBNK tests performed on BD FACSCanto II flow cytometer were 
assessed; patients with immune deficiencies and tumors were not included. The 
study revealed significant age and gender-related changes in all subsets. 
Absolute counts of T, B, T4 cells dropped from neonates to adolescents, decrease 
of T8 and NK cells was milder; relative count of T cells increased with age and 
that of B cells decreased; T4:T8 ratio went down and T:B ratio grew. Total T, T4 
cells and T4:T8 ratio were significantly higher in girls, while T8, NK and DN 
cells - in boys; significantly higher relative and absolute B cell counts in 
boys appeared in adolescence. We compared our results with reference values for 
healthy children (Tosato et al., 2015), there was a good concordance, except for 
DN cells. Advantages of using patient cohort instead of healthy children as 
reference, possibilities for adjusting age and gender-specific reference ranges 
and potential international data pooling are discussed. This article is 
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