# Age- and Sex-Adjusted Reference Intervals in Tear Cytokine Levels in Healthy Subjects
## 3 Results  
A total of 157 subjects were included in the study. The mean age of the sample was 46.36 \(\pm\) 17.7 years (range, 18-88), and approximately half were women (n = 82; 52.2%; Cl95%: 44.1%, 60.2%). There were no significant sex differences in age (men: 48.65 \(\pm\) 19.19; women: 44.26 \(\pm\) 16.04; Student's \(t\) test \(p\)-value = 0.1202). Individual tear samples were analyzed. The minimum detectable concentrations (in pg/mL) and detection rates for the molecules analyzed are shown in Table 1.  
Eight (EGF, Fractalkine, IL-1 RA, IL-7, IL-8, IP-10, MCP-1, and VEGF) out of the 18 molecules analyzed were detected in at least the 82% of the participants. Table 2 summarizes the distribution of the levels of these eight cytokines by age and sex. The rest of the molecules analyzed (IL-1\(\beta\), IL-2, IL-6, IL-10, IL-12p70, IL-15, IL-17A, IL-23, TNF-\(\alpha\), and IFN-\(\gamma\)) were not further considered for subsequent statistical analysis because their detection rates were below 50% (Table 1). Table 3 shows the best GAMLSS models of each cytokine. Model selection involved the selection of the distribution for the corresponding cytokine and the selection of relevant explicative factors by the comparison of fitted models according to the smallest AIC. Four different distribution families were considered: Normal, Box-Cox Cole and Green, Power Exponential, and Box-Cox power exponential. For IL-8, IP-10, and VEGF, Power Exponential was chosen as the most appropriate distribution. Normal distribution was selected for Fractalkine, IL-7, and MCP-1. Finally, Box-Cox power exponential was the less selected distribution, chosen in two of the eight cytokines considered (EGF and IL-1 RA). The potential explanatory variables were age and sex. The age influence on the location parameter was common to EGF, IL-1 RA, IL-7, IL-8, IP-10, MCP-1, and VEGF, but not for Fractalkine, whose mean parameter depends exclusively on sex. In addition, age was added to model variability of IP-10 and skewness of VEGF. Sex had a relevant influence on location parameter of Fractalkine, IL-7, IL-8, and IP-10 and on variability of IP-10 (Table 3). A residual analysis of each of the final fitted models was carried out. In this diagnostic stage, none of the cytokines showed inadequacies in the final fitted model (see Residual Analysis in Appendix A). The age- and sex-related 90% reference (5%, 95%) and median (50%) curves calculated using the best-fitted GAMLSS model for each cytokine are shown in Figure 2; complete numerical values can be found in Appendix B. The levels of IL-1 RA, IL-8, IP-10, and MCP-1 showed an age-related increase; in the case of IP-10 and MCP-1 this increase was approximately linear but for IL-1 RA and IL-8, the increase was only relevant in older subjects (70 years old or more). In contrast, EGF tear concentration decreased linearly with age. Age patterns were also observed for IL-7 and VEGF tear levels. IL-7 concentration increased up to approximately 40 years, then a mild decrease was observed up to 70 years and then increased again. For VEGF, higher levels were observed in the middle age range (30 to 60 years approximately). \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Minimum Detectable Concentration** & \multicolumn{2}{c}{**Detection Rate**} \\  & **(pg/mL)** & **\%** & **95\% CI for \%** \\ \hline EGF & 3.2 & 100 & 97.02; 100 \\ Fractalkine & 3.83 & 87.9 & 81.51; 92.37 \\ IFN-y & 0.1 & 1.3 & 0.22; 5 \\ IL-10 & 0.3 & 36.3 & 28.9; 44.4 \\ IL-12p70 & 0.4 & 5.1 & 2.39; 10.13 \\ IL-15 & 0.4 & 29.9 & 23.03; 37.84 \\ IL-17A & 0.2 & 0 & 0; 2.98 \\ IL-18 & 0.4 & 19.1 & 13.45; 26.31 \\ IL-1 RA & 3.2 & 100 & 97.02; 100 \\ IL-2 & 0.3 & 0.6 & 0.03; 4.03 \\ IL-6 & 0.3 & 21 & 15.1; 28.39 \\ IL-7 & 1.08 & 87.3 & 80.78; 91.86 \\ IL-8 & 0.2 & 96.8 & 92.34; 98.82 \\ IP-10 & 3.2 & 100 & 97.02; 100 \\ MCP-1 & 3.2 & 100 & 97.02; 100 \\ TNF-\(\alpha\) & 0.1 & 1.9 & 0.49; 5.92 \\ VEGF & 3.73 & 82.2 & 75.08; 87.63 \\ IL-23 (*) & 1.01 & 10.2 & 6.02; 16.55 \\ \hline \hline \end{tabular}
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