Quantification of the TREC content by quantitative PCR. The threshold cycle (Ct) is the number of amplification cycles needed to detect the TRECs and is a relative measure, inversely related with the concentration of TRECs. Control for DNA input was done by performing a quantitative PCR (qPCR) for albumin and the difference between the Ct for TRECs and the Ct for albumin was calculated (ΔCt). The ΔCt, indicative for TREC content, was determined of HC (straight line) and ESRD patients not on RRT (dotted line) and lines were compared using a linear regression analysis (p < 0.05 for the difference between lines for ESRD patients not on RRT and HC). The lines in (A) mark the discrepancy between the calendar age (i.e. 40 years) of a non-RRT patient and the immunological age, by extrapolation of the value for the deltaCT (TREC content) to the HC regression line. In addition, Spearmans Rho correlation coefficients (Rs) were calculated for HC and ESRD patients not on RRT to determine the strength of the association between TREC content (calculated as ΔCt) and age. Age is depicted on the X-axis whereas on the Y-axis the ΔCt value for TREC content is displayed. Next, the effect of RRT on the TREC content (B) was analyzed by dissecting the ESRD patients into a young (<50 years) and old (≥50 years) group and either not (white bars) or receiving (black bars) RRT. Bars represent means + SEM.