The mean age at baseline among subjects included in this study was 61 years (range, 55–70 years). Most participants were non-Hispanic Caucasian (N=22), and over half were male (N=17). Among all the analytes assayed, the proportion of measurements that were below the LOD ranged from 0% to 79%.
The median, 5th–95th percentile range, and percentage of measurements below the LOD at each collection time for the six analytes evaluated in this study are reported in . No statistically significant differences in cytokine levels by collection time were observed (P ≥ 0.09, Kruskal-Wallis test). Measures of variability in serum cytokine levels are reported in . The CVs estimating assay precision for the pooled QC replicate samples were fairly high for most analytes (4.6% to 29.0%); TNFα was the only analyte with a CV < 10%. However, after excluding a single pooled QC sample for which discrepancies between duplicate well measurements were observed for several analytes (duplicate well CVs ≥77%), the corresponding assay CVs were much lower for most analytes: IL-6, CV=7.1%; IL-7, CV=17.2%; IL-8, CV=6.0%; IL-10, CV=4.7%; IL-13, CV=4.3%; and TNFα, CV=5.1%. The within-subject CVs, which account not only for assay precision but also temporal variation within subjects over the five-year study period, were considerably higher, ranging from 36% to 137%. Exclusion of subjects with high outlying values resulted in only modest improvements in the within-subject CVs for most analytes (data not shown).
Descriptive statistics for serum cytokine levels (pg/mL) at study baseline, +1yr, and +5yr*
Measures of agreement for serum cytokine levels in serial samples collected over a 5-year period
Based on the estimated ICCs, we observed high reproducibility for IL-6, IL-7, IL-13, and TNFα (ρ ≥0.73), and fair to good reproducibility for IL-8 (ρ = 0.55) and IL-10 (ρ = 0.60). Analyses were repeated after restricting to paired samples from baseline/+5yr. The resulting ICCs were similar for IL-6, IL-7, IL-13, and TNFα. The ICC for IL-8 was higher in this analysis (ρ = 0.81), and the ICC for IL-10 was lower (ρ = 0.46). For each cytokine, sensitivity analyses were performed after excluding one subject with high outlying values (not the same subject for each analyte). After these exclusions, the ICC for IL-8 was higher (ρ = 0.84) but otherwise results were essentially unchanged. For the three analytes with measurements below the LOD (IL-6, IL-10, IL-13), the ICCs were generally similar after restricting to observations with detectable levels, except that for IL-10 which was somewhat lower (ρ = 0.48). After stratifying by sex, we observed similar ICCs among men and women for most analytes, with the exception of IL-7, for which the ICC was considerably higher among men than among women (ρ = 0.88 and 0.27, respectively). After stratifying by batch, for IL-8 we observed a lower ICC in the first batch relative to the second and third batches (ICCs of 0.32, 0.66, and 0.90, respectively); otherwise, no consistent patterns of differences in the ICCs across batches were observed ().
ICCs (95% CIs) for repeated measures of serum cytokine levels in samples collected over a 5-year period, stratified by batch
Spearman correlation coefficients comparing paired measurements of each cytokine at baseline vs. +5yr were also generally high (ρ ≥0.74) for most analytes, with the exception of the modest correlation for IL-10 (ρ = 0.44). For comparisons between analytes (based on measurements from baseline samples), correlations were highest between IL-13 and most other analytes (ρ = 0.31–0.85; Supplementary Table 1
). A fairly strong positive correlation (ρ = 0.61) between IL-7 and IL-8 was also observed.