TFOS DEWS II - Conclusions and new frontiers

A two layered tear film model of the tear film's structure is preferred over the traditional three layered model, with a mucoaqueous gel layer lying beneath, but at least partly integrated with, an overlying lipid layer. The mucoaqueous layer makes up the bulk of the thin (2–6 μm) tear film, but most data on thickness is from the precorneal tear film and there is virtually no data on the pre-conjunctival and other compartment tear films. The lipid layer is very thin, at approximately 42 nm.

Tears are distributed over the ocular surface by the combined actions of the lids, capillary movement and upward drift of the lipid layer. Tear film thinning and breakup occurs mainly as a result of evaporation of water from the tear film, and breakup times are usually twice as rapid as normal, for people with DED. The development of lateral shearing interferometry or double pass methods may improve discrimination of TBUT between normal and dry eye subjects.

It is important that future studies be clear about how, and from which compartment, tears are collected, and the type of tears (reflex, basal, or after sleep). Most studies that have measured tear film osmolarity have used tears from the lower tear film meniscus and have shown increases in tear osmolarity with dry eye. However, it is likely that the osmolarity of the lower meniscus does not accurately reflect the osmolarity over the rest of the ocular surface. Development of techniques to measure tear film osmolarity over the cornea, ideally before and after the tear film ruptures, may provide important insight into diagnosis, improve discrimination of the type of DED present, assist with development of new treatments, and provide more accurate tests for monitoring treatments for DED. Tear evaporation rates increase with dry eye by approximately two-fold. However, the lack of commercially available, reliable evaporimeters to measure this in the clinic limits its use to specialized facilities. The development of evaporimeters that can be used in clinical settings and take into account variations in ambient humidity and temperature would be extremely valuable.

Mucins help to stabilize and spread tears by binding, via their high levels of glycosylation, to water. The lipid layer reduces the surface tension of tears, allowing the thin film to avoid collapse. The lipid layer is probably responsible for reducing evaporation of tears, but how this occurs and the role of proteins and mucins in this process requires further investigation. Also, further investigation is needed to understand how proteins and mucins form the mucoaqueous gel.

The traditional belief that the tear film lipid layer can reduce water evaporation from the mucoaqueous layer has been recently questioned, by experiments in vitro that have shown that meibum or artificial lipid mixtures can only reduce evaporation of underlying water by ≤ 8%. It may be that intercalating proteins and mucins in the tear film lipid layer are needed to reduce evaporation. However, if this is the case, then results showing the importance of the ability of the tear film lipid layer to reduce the surface tension of tears and that the presence of proteins in this layer disrupts this system are difficult to reconcile. Also, proteins in meibum impair its spreading and worsen its viscoelasticity. Clearly, more studies in these areas are needed.

The tear lipidome has been explored in great detail since the publication of the first TFOS DEWS report in 2007. At least five, probably more, lipid classes are present, with each class having many different types due to the large number of possible fatty acids than can be esterified within each class. The majority of lipids in meibum and the tear film lipidome are wax and cholesterol esters. Debates regarding the presence of phospholipids in meibum have largely been settled and experiments show very low levels of this lipid class in meibum. However, the presence of relatively high levels of phospholipids in the tear film lipidome then begs the questions of where these originate from, and indeed if they are really in the tear film lipid layer or are extracted from lipid-binding proteins in tears such as lipocalin (and thereby sequestered away from the tear film lipid layer). Studies have shown that dry eye may be related to changes in the tear film lipidome, especially to changes in the concentration of OAHFAs, but more work is required to confirm these findings. Multi-laboratory and multi-national testing of common pooled meibum and tear lipid extracts is required to provide baseline lipidome data that can then be used as a gold standard to examine the effects of dry eye and its treatments.

There are various mucins found in tears, with the major soluble mucin being MUC5AC and the transmembrane mucins being MUC1, MUC4 and MUC16. However, mRNA for other mucins has been found in ocular surface epithelial cells, and so may be present in tears, although perhaps in low concentrations. There are changes to the concentration of MUC5AC, MUC1, MUC4 and MUC16 in tears of dry eye subjects, but not all studies agree on the magnitude or direction of the changes. However, decreased MUC5AC expression and alteration in mucin glycosylation in dry eye is a consistent finding among most studies. Further studies using the latest glycomic, proteomic and genomic techniques will be beneficial in helping to determine the role of changes to mucin in DED.

There has been an explosion in the number of proteins known to be present in the tear film proteome, with approximately 1800 proteins being present. The use of non-biased MS methods to evaluate changes to proteins, along with more traditional immuno-assays, has helped to show that several proteins change from normal levels in dry eye tears. These proteins can be used as biomarkers, which might help to predict, diagnose and even, in some cases, be therapeutic for dry eye. However, MS is not appropriate for converting into point-of-care analysis, whereas immunological techniques may be. To date, many candidate biomarkers found by MS have yet to be validated by immunological techniques. Furthermore, validating the biomarkers in separate populations is often lacking, resulting in a lack of data on sensitivity and specificity. Data that have been generated on specificity and sensitivity of protein biomarkers show that panels of at least four proteins are superior to individual proteins. More information is needed on biomarker dynamic range, cut-off definition, specificity, sensitivity, positive predictive value and negative predictive value, and linkage of biomarker detection to treatment. Also, testing application of biomarkers in animal models to demonstrate whether they can alleviate dry eye signs is lacking for many.

Investigations into the tear film metabolome have been small and sporadic, but much could be learned from more research in this area. The metabolome is a measure of endogenous metabolites in tears. Due to the fact that these metabolites can be many and varied, sophisticated laboratory techniques are needed during the discovery phase, and so development of assays appropriate for point-of-care use will need to be made for any metabolites that can be used as biomarkers. Changes to amino acids or their derivatives in the tear film of dry eye subjects have been found to be possible markers of the disease.

This subcommittee also examined the effect of non-pathophysiological factors impacting the tear film. Epidemiological evidence pointing to the high risk of having dry eye if the person has been a contact lens wearer has focused attention in this area, as had the publication of the TFOS report of Contact Lens Discomfort in 2013 [54]. Recent data does not support an underlying inflammatory response being associated with contact lens discomfort, but there are clearly changes to the tear lipid layer and mucins during contact lens wear. Whether these changes are transient or more permanent has not been investigated. Furthermore, it is unclear whether those people who have increased discomfort or dryness symptoms during contact lens wear were predisposed to becoming dry eye patients even if they did not wear lenses, or whether lens wear accelerated their path toward DED. With our increasing use of electronic means to communicate, the associated dry eye symptoms that can occur and how to manage or treat these is an area worthy of research in the future.

The tear film is clearly required for a healthy ocular surface. There are several changes to the tear film that occur in dry eye. How biochemical changes relate to biophysical changes, and how these relate to clinical manifestations of tear film stability and volume are not currently known, although progress is being made in many of these areas. The development of a holistic model of tear film structure and function, and changes that occur during dry eye will likely be forthcoming in response to current and future research.