# Age- and Sex-Adjusted Reference Intervals in Tear Cytokine Levels in Healthy Subjects
## 4 Discussion
Although the cytokines analyzed in that work are not the same that those in this study, our results reflected the same behavior in relation to IL-7, IL-8, and IP-10 pro-inflammatory cytokines. The association between tear concentration of cytokines and DED has been widely reported (reviewed in [3,4,53,54]). However, it is often difficult to use cytokines as diagnostic tools due to the lack of diagnostic cut-offs. In this study, we have shown the utility of our RIs to diagnose DED. All of the severe DED patients in our sample showed abnormal tear levels of at least two of the evaluated cytokines when compared to the established RIs. Fractalkine, IL-1 RA, IL-8, and VEGF showed high levels in more than 70% of the DED patients. Moreover, EGF and IP-10 were found to be infra-expressed in 23.1% and 38.5% of patients respectively of our DED sample. Altered tear levels of all these molecules have already been reported in different clinical subgroups of patients with DED. So, for example, tear concentrations of Fractalkine, IL-1 RA, IL-8, and VEGF have frequently been found significantly increased in DED patients as compared to normal controls [8; 18; 55; 56; 57; 58; 59], whereas tear EGF level has been found significantly decreased in the more severe forms of DED [55; 60; 61]. IP-10 tear levels were also found decreased compared to normal in 38.5% of the DED patients' sample, in agreement with results in tears from other studies involving severe cases of DED associated to ocular graft versus host disease [62], Stevens-Johnson syndrome [63], and, as preliminary findings suggest, in primary Sjogren's syndrome subjects [64; 65]. However, several authors have also found tear IP-10 concentration to be overexpressed in tears of patients with moderate evaporative DED [21] and with Sjogren's syndrome [66]. It has been suggested that the anti-angiogenic properties of tear IP-10 may contribute to normal ocular surface immune privilege [65] and the low levels in Stevens-Johnson syndrome have been suggested to lead to a severe smoldering inflammation and progressive subconjunctival fibrosis in these patients [63]. Further studies with a bigger DED sample are warranted to evaluate the role of IP-10 in DED. A critical issue of establishing RIs is to recruit a valid group of reference individuals. Two fundamental aspects are important in this regard: the definition of \"reference individual\" and the sample size. In this case, a reference individual should be a subject without eye pathologies. However, the characterization of this type of subject is not always easy, so collecting medium/large samples from the reference population can be quite expensive. Certainly, although above 150 individuals, our sample could be considered not sufficiently large to properly represent the reference population. Nevertheless, it is very important to highlight that a single ophthalmologist evaluated all subjects included in our sample, so the eye health criterion was homogeneous. Moreover, sample-handling factors such as tear collection, storage, or processing protocol may be a strong influence on the value of cytokine level and they could help to invalidate the sample as a reference sample. In this study, the tear samples were collected and processed by a single person, so this source of variability was also controlled. It also should be considered that in this study environmental conditions during subjects' clinical evaluation and sample collection were not controlled; it is well known the fact that ocular surface parameters and tear mediator concentration are altered under adverse environmental conditions, particularly low humidity and/or high temperatures. Nevertheless, all subjects were evaluated and samples were obtained in the same air-conditioned rooms (in a normal range of temperature and humidity), assuring that all of them were evaluated under the same conditions.