# Age- and Sex-Adjusted Reference Intervals in Tear Cytokine Levels in Healthy Subjects
## 4 Discussion  
In this work, we have established the sex- and age-adjusted RIs of eight (EGF, Fractalkine, IL-1 RA, IL-7, IL-8, IP-10, MCP-1 and VEGF) tear cytokine levels. RIs are used to describe the normal range of a parameter, and their important role in clinical medicine is beyond discussion as an initial indication for further investigation or treatment. They provide a basis for comparison of measured values of patients to a reference, and they could be very useful to diagnose the condition of a subject in epidemiological and clinical studies. The most commonly used definition of a RI is the interval of values containing the central 90% or 95% of a healthy population [28]. It is possible to estimate population percentiles from an adequate sample by computing observed percentiles. As a result, single cut-off values will be obtained. However, this result may not be very useful in the case of cytokine levels, since the distribution of cytokine concentrations, as inflammatory marker measurements, could be heavily dependent on individual characteristics, such as age and sex among others [29]. In addition, immunological data very frequently have asymmetrical distributions [30] and statistical techniques that take into account this issue should be chosen. For these reasons, in this study, GAMLSS models [17] were applied to derive age- and sex-dependent centle curves. The main advantage of these type of models is that the shape of the distribution, rather than just the mean or the variance, for the response variable can vary according to more than one explanatory variable. In our case, for example, the skewness parameter for VEGF concentration varied according to the subject age and it would be wrongly modeled by other more classical statistical methodology. Our results showed that tear levels of all studied cytokines, except Fractalkine, presented an age-dependent behavior in any of the distributional parameters. Age-associated changes in human tear film composition and proteome have already been shown in other studies [12; 15; 16] in agreement with that reported as well in other fluids, such as plasma or cerebrospinal fluid [31]. Indeed, the relationship between the aging process and immune response is not new. Even, the expression \"inflamm-aging\" has been coined to describe the progressive increase in pro-inflammatory status as age increases [32; 33]. There is a lot of literature about age, the role of inflammation markers and its link with the development of disease (reviewed in [31]). The pro- and anti-inflammatory cytokine balance changes with increasing age, and low-grade inflammation is more common among elderly population, leading to a chronic low-grade inflammatory phenotype [34]. Accordingly, age-dependent behavior of several cytokines levels has widely been reported in different biological fluids, such as plasma, cerebrospinal fluid, and also in tears, although tear studies are still scarce. Nevertheless, cytokine levels in these different biological sources have shown to have similar evolution patterns and common to our results in tears. For instance, in our study MCP-1 and IP-10 tear levels showed a sustained age-dependent increase, similar to what has already been observed in serum levels [35]. In addition, IP-10 increased linearly with age in cerebrospinal fluid [36], and plasma MCP-1 levels were significantly higher in an elderly group (70 to 92 years) compared to adults (21 to 50 years) [37]. This latest study also showed that plasma levels of EGF were significantly lower in the elderly group, which is compatible with our results in tears. In our study, IL-1 RA and IL-8 tear levels showed as well a very important increase but only in old ages (68 to 90 years). This same pattern has already been reported in tear [12] and in serum levels [38] of IL-8, and in plasma levels of IL-1 RA [39]. Even, these two cytokines have been proposed as markers linked to longevity in very old people [38; 40].