# Age- and Sex-Adjusted Reference Intervals in Tear Cytokine Levels in Healthy Subjects
## 1 Introduction  
Cytokine profiles have been proposed as biomarkers for numerous infectious and chronic diseases due to their pro- and anti-inflammatory effects [1]. Numerous studies have also reported important alterations in tear cytokines associated with various ocular disorders, such as primary open-angle glaucoma, ocular chronic graft versus host disease, uveitis, ocular allergies, and dry eye disease (DED), among others [2, 3, 4]. In particular, ahighlight is the usefulness of these molecules as biomarkers for DED [5]. For instance, based on cytokine profiles, diagnostic, disease, severity, and therapeutic biomarkers have been established for DED by several authors [6; 7; 8]. It is widely accepted that ocular surface inflammation has a central role in the occurrence and severity of the DED condition [9; 10]. Therefore, a useful tool in evaluating the inflammatory profile of DED may be to analyze the inflammatory mediators in tears, such as tear cytokine profile. In addition, unlike other biological fluids used for cytokine profiling (such as pleural fluid, serum, or cerebrospinal fluid) tear collection is considerably less expensive and requires minimally invasive procedures. In addition, for ocular surface diseases, tear fluid is close to the disease site so it would be expected that tear cytokine profile would be an effective biomarker of them [11]. However, despite its advantages, the use of tear cytokine profiles in clinical practice is not yet widely used as a diagnostic tool. The most common way to evaluate the potential diagnostic value of cytokines has been to establish differences between two or more groups of patients and/or controls. These studies investigate the ability of cytokines to distinguish between the evaluated conditions and confirm the diagnosis. However, a more general approach would be to build reference intervals (RIs) that allow establishing \"normal\" versus \"abnormal\" cytokine levels from a sample of healthy subjects. RIs provide a quantitative diagnostic rule to classify a value as potentially pathological when this value could be considered as extreme. The value will be considered extreme if there is only a small proportion of subjects in the healthy population with larger or smaller values. Therefore, a critical issue will be collection of a representative sample from a healthy population. Unfortunately, the number of studies specifically dedicated to investigate tear cytokine profiles in healthy subjects is limited [12; 13; 14]. Commonly, a RI represents the interval between a pair of symmetrically placed extreme percentiles. Both the 95% RIs (2.5 th-97.5 th percentile) and the 90% range (5.0 th-95.0 th percentile) are in use in the literature. Consequently, the RI will be a global range and any patient outcome outside this range will be considered \"abnormal\". However, global reference values will be no useful since demographic characteristics, such as age and sex, have been shown to have an influence on biological variations of some protein and cytokine levels in normal human tears [11; 12; 15; 16]. In this research we used generalized additive models for location, scale and shape (GAMLSS) models in a large sample size of subjects without ocular pathology in order to know if age and sex are determining factors and to build age- and sex-adjusted RIs for several cytokine concentrations in tear samples. GAMLSS are very flexible semi-parametric regression models that allow modeling all the distributional parameters: location, scale and shape (skewness and kurtosis), as linear, non-linear parametric, or smooth non-parametric functions of explanatory variables [17]. Assumptions on a distribution form for the response variable are made, allowing a variety of different distribution families. This regression technique provides a platform to fit, compare and check many different models.