*3.3.8.1 Tear film osmolarity*

Interferon gamma is significantly increased in amount if the tear film on the ocular surface becomes hyperosmolar but other cytokines such as Th1, Th2 and Th17 have no significant increase in amount [111].

It has the highest correlation to disease severity of clinical DED tests [112].

Various literatures have proposed many cut-off values for DED from 305 mOsm/L to 316 mOsm/L [113], with reported sensitivities and specificities ranging from 64–91% and 78–96% respectively [113–117].

### *3.3.8.2 Tear film ferning*

When tear film is dried on a glass plate, it causes ferning. There are few pre requisites for the process such as slow crystal growth rate, low solution viscosity and low impurity levels to permit free-solute diffusion. Seven to ten minutes under normal room temperature of 20 to 26°C and room humidity of (RH up to 50%) has been recommended [118].

The crystallization begins with the formation of a nucleus, due to the supersaturation of ions with solvent evaporation at the peripheral edge of the drop. Normal crystals are formed when the sample solute is able to diffuse into areas with a lower solute concentration [118].

Electrolytes may play a role in ferning as hyperosmolarity has been found to result in deteriorated ferns [113, 119].

Tear ferning changes with contact lens wear have been found to have a moderately high sensitivity (78.4%) and specificity (78.4%) for predicting contact lens tolerance in a clinical setting [120].

Healthy tear samples produce compact, dense ferning patterns, while in dry eye samples, the pattern is fragmented or absent.

### *3.3.8.3 Biochemical analysis of the tear composition*

It includes lacrimal gland and serum protein analysis, mucin analysis and lipid analysis [36].

#### *3.3.9 To test the inflammation of the ocular surface*

Inflammation, although not specific, but is a recognized as one of the component of the pathophysiological mechanism of DED.

#### *3.3.9.1 Ocular or conjunctival redness*

This is the most common and consistent sign of ocular surface inflammation [121–123]. It can easily be detected with a pen torch or on slit lamp examination. It is not specific to DED and can occur in any disease with inflammation, for example, in response to chemical injury, infective conjunctivitis or allergic conjunctivitis.

#### *3.3.9.2 Matrix metalloproteinases*

They are secreted into the tears of a DED patients [72, 124–126]. It destroys the tight junctions of the ocular surface epithelium which in turn leads to loss of ocular surface barrier function. This assay produces a dichotomous outcome, with levels above 40 ng/ml producing a positive result, and is non-specific to the source of ocular surface inflammation.

#### *3.3.9.3 Cytokines and chemokines*

They reflect the level of epithelial disease. Elevation of Th1 and Th17 subclasses of cytokines suggest involvement of particular T lymphocyte differentiation pathways in the disease. Elevation of tear Th2 cytokines, on the other hand, may suggest a more allergic-based disease, although recent evidence suggests various aspects of T cell Th1, Th2 and Th17 exist across aqueous deficient, evaporative and mixed forms of DED, with a propensity towards Th1 type T cell responses as a more global indicator of DED [127].

#### *3.3.9.4 Ocular sursface immune markers*

The most commonly used ocular surface immune marker is HLA-DR expression, a Class-II MHC antigen, which indicates a loss of the normally immune-suppressed environment of the ocular surface.

Although the authors found increased expression of HLA-DR associated with increased clinical severity of DED [128], but in comparison with other studies the normal levels of HLA-DR expression showed high variability ranging from 5–54% and the study also suggested the weak correlation of HLA-DR and traditional clinical signs of DED [129]. Other relevant markers of apoptosis include CAM-1, CD14+, CD8+ and CD4+ cells [130, 131].

#### *3.3.9.5 In vivo confocal imaging*

Corneal sub-epithelial and stromal IVCM signs of inflammation have been hypothesized and studied in DED [132, 133].

#### **4. Clinical protocol for dry eye**

The recommended order and clinical practice procedural recommendations are as follows:

	- a.NIBUT The cut-off for a positive finding can be as low as 2.7 seconds for automated algorithms, and up to 10 seconds for subjective observation techniques.

b.FBUT - A positive finding has been reported to be a value < 10 seconds.

