After external and anatomical considerations, the next step in diagnosing DES is evaluating which part of the tear film is dysfunctional. The tear film is a complex fluid made up of lipids, an aqueous layer, and mucins (Fig. 1.1). Most patients with DES have dysfunction in more than one layer although there is typically one that is most affected. For examples, patients with Sjogren’s syndrome or graft-versus-host disease (GVHD) typically have primarily aqueous tear deficiency (ATD), while patients with rosacea typically have mostly evaporative dry eye (LTD). Several tests are used to evaluate tear function as this information has implications for treatment (Table 1.2). Schirmer’s test with or without anesthesia can be used to assess aqueous production (Fig. 1.2), while tear break-up time and evaluation of meibomian gland parameters can evaluate the composition and function of the lipid layer (Fig. 1.3). Other tests, such as measurement of tear osmolarity, can give a global assessment of tear health as hyperosmolarity is believed to be a key player in the pathophysiology of disease (Fig. 1.4) (DEWS 2007b). It is important, however, to understand that none of the tests are a perfect mirror of tear function and that the results can vary substantially both due to testing methodology and to the dynamic biology of the tear film. With regard to the former, tear break-up times vary substantially by the amount of fluorescein placed in the eye. With regard to the latter, tear osmolarity values can vary substantially when measured in the same individual 3 times in a row, a finding that suggests the presence of ocular surface instability. Despite these limitations, a general “gestalt” of tear function can be obtained by measuring some or all of these parameters. Newer tests are currently being evaluated that may better assess the dynamic state of the tear film including functional visual acuity (FVA) testing (Goto et al. 2006; Ishida et al. 2005) and evaluating tear film stability with consecutive topographic images (Tear film Stability Analysis System) (Gumus et al. 2011). However, these newer tests are not in widespread use in the clinical arena.

Interferometry is another test that can be performed to evaluate the health of the lipid layer (Tomlinson and Khanal 2005). In this technique, interference fringes are produced by light reflected at the air-lipid and lipid-aqueous interfaces of the tear film. This specular reflection from the tear surface is imaged and recorded digitally. Several researchers have developed grading patterns to interpret the recorded images, most focusing on the uniformity of the fringe pattern. Overall, research has demonstrated that a thicker lipid layer is associated with greater tear film stability and that loss of uniformity indicates tear film instability (Tomlinson and Khanal 2005). LipiView® (Tear Science, Morrisville, NC) is a commercially available interferometer that comes as part of the Tear Science diagnosis and treatment system (described below).

Another adjuvant study that can be used to evaluate for the presence of ocular surface inflammation is the InflammaDry™ assay (Rapid Pathogen Screening, Sarasota, FL). This test detects elevated levels of matrix metalloproteinase-9 (MMP-9) in the tear film. MMP-9 is a nonspecific marker of inflammation and has been found to be elevated in various ocular surface disorders and corneal ulceration (Kaufman 2013). An advantage of the test is its ease and rapidity; the applicator is placed in the lower conjunctivae to sample tears, and the results are available within 10 min. A limitation, however, is that the test result is reported in binary fashion: two lines indicate an elevated MMP-9 level; one line indicates a normal level.

Newer data suggest that DES may be a manifestation of a corneal neuropathy (Rosenthal et al. 2009; Rosenthal and Borsook 2012) and evaluation of corneal sensation should be considered, especially in patients with extensive corneal staining and minimal disease symptoms. Available technologies include the more qualitative cotton-tip swab which can compare sensation between the eyes and the more quantitative Cochet-Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France). Belmonte et al. (1999) developed a noncontact gas esthesiometer that can measure corneal threshold to mechanical, thermal, and chemical stimulation. Unfortunately, this esthesiometer is not commercially available and only four modified versions of the instrument are available worldwide.

Another commercially available tool that can be used to study corneal nerves is the confocal microscope (Confoscan 4, Nidek, Fremont, California). In vivo confocal microscopy can be used to image corneal nerves in the sub-basal plexis. Several studies have found abnormal corneal nerve morphology in patients with DES including increased nerve tortuosity, fewer fibers, and higher bead density (Zhang et al. 2011; Villani et al. 2013). This testing, however, is in its infancy, and at this point it is not clear what role nerve imaging will have in the diagnosis and treatment of DES.