*3.3.6.1 Ocular surface staining*

Vital and supra-vital stains are used to demonstrate the damaged epithelium. Staining occurs over cornea and conjunctiva in different fashion.

Cornea is stained in manner such that lower part (lower one-third) is stained more than upper part and nasal part stained more than temporal part.

Bulbar conjunctiva is stained nasally and temporally in a wedge-shaped zone [86]. It is a commonly used and cost -effective test.

The dyes used in the procedure are as follows:

• Fluorescein – 1% or 2% commercial preparation is used clinically for the ocular surface staining. It stains the surface when there is a disruption of cell junctions. It stains corneal epithelial damage better than conjunctiva. At physiologic pH, fluorescein is highly water soluble and hence poorly penetrates the lipid layer and doesn't stain normal cornea. It is orange in color and fluoresces green when excited by blue light.

Yokoi and Kinoshita in 1998 reported that conjunctival damage precedes that of the cornea and is more severe.

Method – Either it is instilled in form of a drop or impregnated strip. Excess dye is washed off if drop is instilled. Best results are obtained when viewed through a yellow barrier filter (such as Kodak Wratten 12 absorption filter) plus the standard blue exciter filter of the slit lamp [86].

In the absence of yellow filter, the conjunctival stain is seen poorly.

• Rose Bengal (RB) – It is available as 1% commercial preparation. Method – Firstly, topical anesthesia is instilled in the eye to limit stinging with the dye. The dye is then instilled in the lower conjunctival sac. Excess dye is washed off with normal saline.

The staining is dose-dependent, the more the dose of dye the more is the staining. Rose Bengal stains ocular surface epithelial cells that are unprotected by mucin or glycocalyx, as well as dead or degenerated cells [87]. However, RB staining has many disadvantages as well which limits its association with dry eye. Schein et al. in 1997 reported that it stains in asymptomatic patients as well and does not correlate with the subjective symptoms [88]. RB causes staining of the Marx's line i.e., the mucocutaneous junctions of the lid margin. And thus, does not seem to have sufficient sensitivity and specificity [89].


Most commonly 3 methods are used to grade the ocular surface staining –

• Van Bijsterveld system – This system was developed in 1969. The whole ocular surface is divided into 3 zones – cornea, nasal bulbar conjunctiva and temporal bulbar conjunctiva. Each zone is the scaled from 0 to 3 where 0 indicates no staining and 3 indicates confluent staining. The maximum possible score in 9 [91].


Recently, Miyata and coauthors described a new method for grading fluorescein staining in superficial punctate keratitis (SPK). Both the area and density of SPK were graded. The area was graded from A0 to A3 and the density was graded from D0 to D3 and then these two were combined in a single index [94].

#### *3.3.6.2 Impression cytology*

It is used to diagnose diseases like DED, limbal stem-cell deficiency, ocular surface neoplasia, and specific viral infections [95]. In patients of DED, it is used to study squamous metaplasia and goblet cell density of the conjunctiva for the diagnosis and monitoring of the disease [96].

Method - Cells from the first to third most superficial layers of the epithelium are removed by application of cellulose acetate filters or bio-pore membranes, and the cells can be subsequently analyzed by various methods including microscopy, immunocytochemistry, immunoblotting analysis, polymerase chain reaction, and flow cytometry, depending on the aim of the investigation [97].

Several squamous metaplasia grading systems by Nelson, Tseng and Blades are used to analyze the conjunctival impression cytology [98–100].

#### *3.3.6.3 Lid Parallel Conjunctival Folds (LIPCOF)*

These are the folds in bulbar conjunctiva in the lateral and lower quadrant which are parallel to the lower lid margin. They represent mild stage of conjuntivochalasis but clinically they are slightly different [101].

The tear meniscus height measurements may be underestimated due to LIPCOF [44].

#### *3.3.6.4 In-vivo confocal microscopy (IVCM)*

It is a non-invasive technique to evaluate the signs of ocular surface damage in DED patients at cellular level. The signs such as decreased corneal (apex and lower periphery), and conjunctival epithelial cell density, conjunctival squamous metaplasia (increased mean individual epithelial cell area, decreased nucleocytoplasmic ratio and goblet cell density), and corneal nerve changes (decreased sub-basal nerve density, increased tortuosity and increased number of bead-like formations) are evaluated [102–106].
