*2.5.3 Matrix metalloproteinase 9 (MMP-9)*

The matrix metalloproteinase test (MMP-9) is a useful technique to complement the diagnosis of DED recently developed. Matrix metalloproteinase 9 is an inflammatory biomarker that has been shown to be elevated in the tears of dry eye patients [26]. In tears, MMP-9 expression is normally less than 40 ng/ml and is secreted from the ocular surface epithelium [27]. MMP-9 maintains epithelial barrier function by

breaking down components of the epithelial basement membrane and tight junction proteins, such as ZO-1 and occludin [28, 29]. But in addition, MMP-9 is related to the pathogenesis of various diseases, such as sterile ulceration, ocular allergy, keratoconus, conjunctivachalasia, and DED [30–32].

In the pathology of DED, since tear hyperosmolarity begins in the early phase of DED, a vicious circle probably induces inflammatory processes and triggers the release of MMP-9 in a relatively late phase of DED [33]. Detection of elevated MMP-9 in tears could be an ideal tool for the diagnosis and treatment of tear dysfunction [34].

The technique for using the MMP-9 device (InflammaDry test) is as follows: the sampling fleece is contacted three times at each location of the lower eyelid conjunctiva (temporal, middle and nasal; from nasal to temporal direction), and it rests against the temporal conjunctiva of the lower eyelid for an additional 5 s. After the sampling fleece is assembled on a sample collector and pressed, the test strip is immersed in a solution for 20 s; 10 min after taking the sample, the result is read (**Figure 3**).

InflammaDry, this type of device has the advantages of low cost, quick analysis, and ease of device preparation. However, it has been studied that a small sample volume, the reliability of the test result can be significantly affected [35].

It has been studied that the elevation of tear MMP-9 in patients with ED appears to have a good diagnostic yield and a correlation with the clinical severity of DED [26, 27]. The reliability for this test was 85% sensitivity and 94% specificity for the diagnosis of DED [36]. But despite these very promising values, two separate reports showed moderate to low positivity in an immunoassay for MMP-9 in study subjects with DED [36–38]; reporting that there were no differences between subjective symptoms and clinical signs of DED between MMP-9 positive and negative groups [37, 38]. In general, although it is a nonspecific marker for patients with DED [39, 40], various immunoassays have reported its usefulness as an aid in the diagnosis of dry eye; however, there are factors that may influence the results of MMP-9 in tears; one of them was demonstrated in an

**Figure 3.** *InflamaDry.*

immunoassay where it was concluded that the positivity of the test can depend on the volume of the load and that therefore it can cause false negatives in diseases with aqueous deficiency such as Sjögren's syndrome or graft-versus-host disease [41]. Other useful benefits are the identification of patients with inflammation of the ocular surface and autoimmune disease and can facilitate the decision to establish an anti-inflammatory treatment in these patients [30]. In the same way, its usefulness for the preoperative management of the ocular surface was reported, reducing possible complications when there is an underlying disease. Additionally, this test can also help indicate the need for anti-inflammatory therapies, such as cyclosporine or steroids, and can also predict which patients are most likely to respond [42].

## *2.5.4 The Schirmer test*

The Schirmer test was introduced by Schirmer in 1903, despite the time and disadvantages of performing it, it is still used in most parts of the world, as part of the dry eye test battery. The Schirmer test is a diagnostic method to evaluate the production of watery tears [43, 44]. Topical anesthesia may or may not be used during the procedure, depending on the physician's preference, and may even include nasal stimulation to induce reflex tearing. The Schirmer test allows the study of total tear secretion, that is, it evaluates the sum of the basal secretion plus the reflex secretion if performed without anesthesia; but if performed under anesthesia, it measures the basal secretion produced by the accessory lacrimal glands located in the conjunctiva. The Schirmer test is a diagnostic method to evaluate the production of watery tears [44, 45].

Initially, the method described to perform the Schirmer 1 test is as follows: it should be performed a few minutes after the instillation of topical anesthetic to inhibit the reflex secretion produced by the main lacrimal gland. Whatman No. 1 filter paper 5 mm wide and 35 mm long is used. It is placed in the cul-de-sac at the level of the outer third of the lower eyelid, in a low-light environment, with the patient with his eyes open, and the length of the paper that has absorbed moisture is measured at 5 min [46].

The Schirmer test remains a useful and repeatable test; although over the years several authors have made modifications to their methodology to improve the reliability of the test results; Such studies have been justified by mentioning the lack of practicality for ophthalmologists, especially when performing it routinely due to the 5-min time frame [45]. Therefore, correlations have been evaluated between the different wetting times, the size of the paper strip, or whether it is done with eyes open or closed [45, 47, 48]. The results are controversial. Some authors support the hypothesis that the shorter durations of the 5-min Schirmer test, such as the 1-min test under anesthesia, have a high correlation with the 5-min test and will make this test much more practical for patients [45]. Others affirm that the correlation in this modification was poor and consider it inappropriate to shorten the duration of the Schirmer test [49]. As this controversy has been generated, other tests have been tried with a shorter soak time, such as the Schirmer test of 2 min under anesthesia, and that the 3 mm wide paper strip could be used instead of the standard strip of 5 mm wide; finding results that correlate well with the 5-min test [47]. Then another study was conducted with a 3-min time, the results of which reveal that the 3-min Schirmer test is a reliable alternative for the diagnosis of dry eye and is more useful in daily ophthalmic practice [50]. The position of the eyes (open or closed) when performing the Schirmer test has shown important differences in its results; moisture values in the Schirmer test with eyes open have been reported to show significantly higher results

## *Current Diagnostic Tests for Dry Eye Disease in Sjögren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.103671*

compared with eyes closed [51, 52]; this may be due to the open eyes moving during the blink, stimulating tearing under the influence of the strip.

Other factors that could influence the results of the Schirmer test are head position, lighting, strip position, corneal condition, humidity, and ambient temperature, so it is important to consider these factors to achieve the standardization of this important diagnostic tool [51].

Therefore, the recommendation to perform the Schirmer test under anesthesia is to perform it with the eyes closed; this could reduce humidity variations, evaporation, and especially reflex tearing [51, 52].

The interpretation of the results of the Schirmer test is different depending on whether anesthesia is used or not. In patients with dry eye, there are clinical differences between the ADDE and EDE types, since the presence of ADDE strongly suggests the suspicion of Sjogren's syndrome; however, although they also present differences in the physiology of tears between these types, no significant differences are found in tear osmolarity, volume, or distribution [53]. In the Schirmer test without anesthesia, total secretion (basal and reflex) is evaluated, the cutoff point is between 10 and 15 mm, and in the Schirmer test with anesthesia, reflex secretion is evaluated, and the cutoff point for this test is ≤5.5 mm/5 min; sensitivity is 85% and specificity is 83% [54]. The Schirmer II test is used on special occasions, as it requires stimulation of the nasal surface with a cotton-tipped applicator and subsequent measurement of reflex tear production. The disadvantages of the Schirmer test, in all its variants, are the time required, patient discomfort, and the low reliability of the test [54, 55].

## *2.5.5 Tear breakup time (TBUT)*

The tear breakup time (TBUT) measures the stability of the tear film. With instilled fluorescein, TBUT is the time interval after a patient blinks until the first appearance of dryness in the tear film [56]. TBUT is measured by inoculating fluorescein into the cul-de-sac, asking the patient to hold the eyelids open after 1–2 blinks, and counting the seconds until a dry spot appears. The appearance of dry spots in less than 10 s is considered abnormal (**Figure 4**) [57]. Subsequently, alternative values

**Figure 4.** *TBUT.*

for BUT are determined by comparing young populations with dry eye and healthy controls, reporting that values less than 5 s occur in subjects with dry eye [58]. The TBUT presents a sensitivity of 72.2% and specificity of 61.6%. The standardization of the method to perform the tear rupture time was carried out by Johnson and Murphy in 2005 [59]. The procedure is summarized in **Table 2**.

Although this test is inexpensive, quick to perform, and uses readily available supplies, it is poorly reproducible and inaccurate [60, 61]. However, the average score of two separate TBUT measurements helps increase their repeatability [62].
