*4.1. Tears*

The tear film is composed by three layers: mucin, aqueous, and lipid that provides functional, nutritional, and protective characteristics for the ocular surface. These layers interact with the meibomian gland, lacrimal gland, conjunctiva, and cornea, which facilitate and regulate the normal production, distribution, and elimination of tears. The tear film creates a refractive surface on the cornea and protects it from environmental damage, each layer contents glycoproteins, amino acids, electrolytes, enzymes and proteins like lipocalin, lysozymes, lactoferrin, and albumin, as well phosphatidylcholine and phosphatidylethanolamine. Particular immunological components include sIgA e IgG, cytokines mainly Tumor necrosis factorα (TNFα), ILα, IL-1ß, IL-6, matrix metalloproteinases (MMP), and chemokines, which are immunological mediators in the ocular surface diseases (Figure 2). Although, they are expressed in other diseases, for example, in autoimmune diseases, diabetic retinopathy, dry eye syndrome, ocular allergies, neurological disorders like Parkinson's disease, and Multiple Sclerosis [74]. Obtaining the tear film requires minimally invasive procedures, making it an accessible matrix that needs to be further studied and used as a starting point for the study of a variety of eye diseases. The determination of a wide variety of cytokines, enzymes and metabolic residues in tears could be useful for the establishment of therapeutics targets, contributing to the improvement of treatments and facilitating their diagnosis.

The number of components in tear samples varies according to the technique used for their collection. Due to the small volume of tear sample (5–10 μL), two techniques are commonly used for their collection:


are used frequently to analyze inflammatory markers like interleukins and MMP-9 and they are measured by enzyme-linked immunosorbent assay (ELISA) or Luminex technology using tears that were collected with Merocel, Pro-ophta, or Weck-Cel sponges. However, comparative studies with simultaneous measurements of cytokines have shown that Merocel is useful for clinical assess for cytokines/chemokines levels but have the limitation with measures of IL-7 and IL-4 due to protein stability problems with the extraction bu ffer [75–77]. In the case of STS, it may also be used for cytokine analysis assays. For collection of tears, the strip is placed on the inferior fornix of the eye and the patient should close their eyes for 5 min. After completed the time, the patient should open their eyes for remove carefully the strip and then it is collocated into a sterile 1.5 mL tube. Immediately transfer the tube to the laboratory to process later in a bead based multiplex assay or store at −80 ◦C. With this method the sample contains higher amounts of cellular proteins, lipids, and mucous compared with MCT and the analysis with multiplex provide high sensitivity for analyzing cytokines and other proteins [78].

**Figure 2.** Main components in tear film. The wide variety of components in each layer of the tear film provides function, nutrition, and protection to the ocular surface. However, it is susceptible to change their composition due to oxidative stress and inflammatory processes that involve the eye structures, which makes it an easy access ocular matrix to identify these changes measuring levels of components as biomarkers. SOD1: superoxide dismutase-1, Ser: serine, Thr: Threonine, Pro: Proline, IL: Interleukin, sIgA: surface Immunoglobuline A, MMP-9: matrix metalloproteinase-9, TNF-α: Tumor necrosis factor-<sup>α</sup>. Image created with BioRender.com.
