Comparison of ocular surface stainings.
Dry eye disease (DED) is a multifactorial disease of tears and ocular surface that results in various symptoms with the potential damage to the ocular surface. It can range from mild to severe signs and symptoms and may affect patient’s quality of life. Various techniques and methods have been developed to evaluate DED for initial examination or regular follow up. The simple evaluations that can be performed in clinic include eyelid examination, tear break-up time, and ocular surface stainings; while the advanced ones may require certain devices or laboratory equipment. Careful and thorough examinations are important to guide the clinician to assess and evaluate dry eye.
- dry eye examination
- ocular surface staining
- tear film stability
- tear volume
- laboratory test
Dry eye disease is one of the most commonly encountered problem in daily practice. It is the reason why a patient visits the eye care professional. Dry eye—as it was defined by the National Eye Institute (NEI)/Industry Workshop on Clinical Trials in Dry Eyes—is a disorder of the tear film due to tear deficiency or excessive evaporation, which causes damage to the interpalpebral ocular surface and is associated with symptoms of ocular discomfort . In 2007, the International Dry Eye Workshop updated the original definition and classified dry eye as “multi-factorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface” . In 2017, the definition was revised, which centered on the clinical effects and associated signs as “multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface” .
Based on those definitions, dry eye symptoms can change from day to day and they may vary in every patient, from mild to severe ocular discomfort and visual disturbance. Dry eye disease can affect patients’ quality of life. It is important for eye care professionals to recognize, diagnose, and treat DED; but, somehow DED can be puzzling since there is no consistent, well accepted, diagnostic test that is both readily available and reproducible . When a patient comes in due to the symptoms that may suggest DED or for a routine examination, an eye care professional should do history taking comprehensively. Various diagnostic tests may be required to determine if the patient has DED due to aqueous deficient, evaporative, or both. In daily practice, tear-film and dry eye assessment are often performed in symptomatic patients. However, it must be kept in mind that dry eye symptoms and signs may be not well associated, as reported in previous studies [5, 6, 7].
2. History taking
A careful history taking is an important thing to perform in the first place to help the eye care professional in assessing dry eye correctly, including history of previous medication, long-term contact lens wear, ocular surface surgery, or systemic condition(s). Patients with dry eye often complain of eye discomfort or irritation, gritty or foreign body sensation, burning, tearing, stinging, intermittent sharp pain, redness, or/and photophobia. Visual disturbance may occur. Dry eye patients may have all, some, or none of these symptoms.
A clinician should also understand that dry eye symptoms increase with age, menopausal status, hormonal diseases, current smoking history, certain medications, and presence of pterygium are a few factors result in dry eye [4, 5, 8, 9, 10]. Noor et al.  found that there was a likelihood of shifting from preclinical dry eye towards DED and from normal towards predisposition to dry eye in older age.
There are many questionnaires available that can be used to utilize in assessing DED, such as National Eye Institute Visual Function Questionnaire-25 (NEV-VFQ-25) , Ocular Surface Disease Index (OSDI) , Standard Patient Evaluation of Dry Eye Questionnaire (DEQ-5) , and some others more. Every clinicians have their own preferences of questionniare.
3. Staining grading system
Visual acuity assessment (including best-corrected acuity), thorough eyelid and slit-lamp of anterior segment examinations are mandatory. Patients with dry eye often complain of blurred vision which improves with blinking or instillation of artificial tear.
Whilst taking patient’s history, a clinician can examine the eyelids macroscopically. It can guide the clinician to evaluate if the patient has dry eye. Lagophthalmous, lid laxity, decreased frequency of blinking, and size of palpebral aperture. Malpositions of the eyelid have to be recognized (such as involutional or cicatrical ectropion, eversion of lacrimal punctum, dermatochalasis, full-thickness defects, inadequate lid closure due to previous eyelid surgical reconstruction) because these conditions can influence the tear turnover. Patient’s history can guide the clinician to perform identify certain ocular manifestations under careful and focused slit-lamp examination.
3.1 Slit-lamp biomicroscopy
Under the slit-lamp biomicroscopy, a clinician should evaluate anatomical structures of the lid, including the alterations of lid margins and eyelashes. The alterations of the lid margins include hyperaemia, telangiectasia, thickening, scarring, keratinization, ulceration, tear debris, abnormalities of the meibomian orifices, metaplasia, character of expressed meibomian secretions; while for the eyelashes, the alterations include misdirection (trichiasis), malposition (dystichiasis), encrustations, collarettes [14, 15, 16]. Careful evaluation of meibomian gland is important since its dysfunction is the most frequent cause of evaportive DED and is often symtomatic [17, 18, 19]. Evaluate if the meibomian gland orifices are plugged or obstructed and/or change in its secretion.
Tear film should be evaluated for mucus, debris, or meibomian foam. Decreased tear meniscus is often a sign of dry eye. Normally, a patient with normal tear production has tear meniscus height of 0.2–0.5 mm; but in patient with dry eye, it is usually less than 0.25 mm or absent [4, 14, 15, 20, 21].
Ocular manifestations in mild to moderate dry eye may conjunctival hyperaermia, with or without corneal epithelial erosions; or these signs may not present in some mild cases. In severe forms of the disease, conjunctival scarring or conjunctivochalasis and/or corneal complications may occur. Filamentary keratitis, persistent epithelial defects, ulceration, and even corneal perforation can complicate the course [14, 15, 22]. Corneal staining may be required to evaluate the severity of its defects.
There are several tests that can be performed to confirm the diagnosis of dry eye and to evaluate the severity of the disease. The tests can measure the following parameters: (1) stability of the tear film as related to its break-up time (TBUT); (2) tear production (Schirmer, fluorescein clearance, and tear osmolarity); and (3) ocular surface disease (corneal stains and impression cytology). There is no clinical test to confirm the diagnosis of evaporative dry eye [21, 23].
3.2 Staining of the ocular surface
Epithelial damage to the exposed ocular surface can be evaluated with vital stainings. Staining of the cornea occurs commonly in inferior part, often more in nasal and temporal areas. Corneal epithelial defect, erosions, filaments, or punctuates can be seen in dry eye. Staining of the bulbar conjunctiva occurs over a wedge-shaped zone nasally and temporally, and in advanced dry eye may become confluent; but in milder forms of dry eye, it may be present in the absence of corneal stain .
Fluorscein staining is a basic and standard method to evaluate ocular surface damage. Commonly, every clinician is able to perform this examination in daily practice. The orange-dyed fluorescein strip is wetted with a sterile drop of saline and then is applied to tarsal or bulbar conjunctiva. Excess fluid is shaken from the strip prior to application. The dye will distribute over ocular surface after blinking. Under slit-lamp examination using cobalt-blue filter, the orange dye will turn into fluoresces green in the damaged area. Common characteristic distribution of this test is confined to the exposed intrapalpebral area of the ocular surface, but the staining may extend to unexposed area in severe case.
In the case of perforation, aqueous from the anterior chamber will leak out of the eye and mix with the tear film. The fluorescein dye around the perforated area will be diluted by this leak and the leak will appear bright green (Seidel test).
3.2.2 Rose bengal
Rose bengal is a synthetic fluorescein derivative, also perhaps referred to as bengal rose and also known as a Chemical Index (C.I.) Acid Red 94 . It has the ability to bind to epithelial cells that are uncoated by certain proteins (mainly mucin) and presents high cell toxicity . The instillation of this dye causes stinging or pain, particularly in DED patients, and it may be disliked by some patients; thus topical anesthesia is best instilled first to limit stinging sensation. Although rose bengal had been thought to be a vital dye, staining dead or degenerating cells, it is known that rose bengal normally stains healthy cells [26, 27].
Under slit-lamp with a white light, rose bengal staining can revealed discrete or confluent punctuate in damaged area of cornea and visible bulbar conjunctiva which can be seen as red dots (Figure 1). But there are disadvantages in using rose bengal in addition to pain on instillation. Although bulbar conjunctival staining is demonstrated well against the white background provided by the sclera, the dye is difficult to see on the cornea against the background of a dark iris . Another disadvantage of rose bengal is its toxicity, including decreasing the chance to recover herpes viruses in human cell cultures .
3.2.3 Lissamine green
Lissamine green is a synthetically produced organic acid dye with two amino-phenyl group and it has been used as a substitute for rose bengal since it has similar laboratory and clinical staining properties, also it is a less toxic stain and less stinging upon instillation [25, 28].
Lissamine green is a vital dye that stains ocular surface epithelial cells that are unprotected by mucin or glycocalyx, as well as cells that have been damaged, and it does not stain healthy cells or damage them [28, 29, 30]. In patient with red eyes, lissamine green could provide better staining visualization than rose bengal.
The use of ocular staining mentioned above is helpful in assessing the integrity of the ocular surface epithelium. These tests are easy to perform in daily clinical practice in-office setting. The clinician can choose one of these staining to assess dry eye based on the availability of the equipment in the clinic. Fluorescein impregnated strips are preferred due to their availability and simplicity of use; while rose bengal and/or lissamine may not always be available in some eye care facilities. Table 1 helps the clinician to compare these three stainings. Significant staining of the conjunctiva with rose bengal or lissamine green is most common in severe dry eye to Sjögren’s syndrome .
|Fluorescein||Rose Bengal||Lissamine Green|
|Staining normal/healthy cells||No||Yes||No|
|Staining dead or degenerated cells||No||Yes||Yes|
|Clinical means||Disruption of cellular junctions and increased membrane permeability||Loss of insufficient protection by ocular surface mucin||Cell degeneration and death (unprotected by ocular mucin or glyco-calyx)|
|Slit-lamp filter||Cobalt-blue||White light or green barrier filter||Red barrier filter|
4. Staining grading systems
Staining grading systems remain an essential element of ocular examination and allow the clinician to record the level of ocular surface staining and evaluate the severity of dry eye. The three most common grading systems are: (1) the van Bijsterveld grading system (uses rose bengal staining of the conjunctiva and cornea); (2) the Oxford grading scheme (uses fluorescein, rose bengal, or lissamine green of the conjunctiva and cornea); and (3) and the NEI Workshop system (uses fluorescein staining to grade the cornea and rose bengal to grade the conjunctiva) .
The van Bijstervel grading system is the first proposed system to grade three areas in each eye: the nasal and temporal bulbar conjunctiva and the cornea (Figure 2). The intensity of the staining is graded on scale 0 (no staining), 1 (sparsely scattered staining), 2 (densely scattered), and 3 (confluent staining). The maximum score for each eye is 9. Staining score of 3 or higher is considered abnormal.
The Oxford grading scheme uses a chart consisting of a series of panels labeled A to E in order of increasing severity of staining (Figure 3). Staining is represented by punctate dots and increases by 1 log unit between panel A and B and by ½ log unit between each subsequent panel (B to E).
The NEI Workshop grading system divides cornea into five areas and conjunctiva into six areas for each eye (Figure 4). The scale of 0 to 3 is used for the grading, according to the intensity of fluorescein staining. The maximum staining score for the cornea is 15, and for conjunctiva, the maximum score is 18. The values above 3 is considered abnormal for cornea or conjunctiva in each eye.
4.1 Laboratory tests
Lack of stability in the tear film can be seen in aqueous deficient, evaporative, or both type of dry eye. It may also occur in the setting of a poor blink rate or epithelial irregularity . There are several numbers of tools designed to evaluate tear film stability to help clinician or researcher to assess and support the diagnosis of DED.
4.1.1 Tear break-up time (TBUT)
Tear break-up time was first introduced by Norn in 1969 and remains the most frequently used diagnostic test to evaluate tear film stability . It measures the time between a complete blink and the first appearance of a dry spot on the ocular surface using fluorscein. Right after applying the fluorescein strip on to the ocular surface, and under cobalt blue filter in slit-lamp biomicroscopy, patient is asked to blink completely and hold the eye open (avoid blinking). The clinician should observe the first dry spots appear on his/her ocular surface. Normally, dry spot(s) will appear after 10 seconds.
The TBUT less than 10 seconds is considered as a cut-off score for the diagnosis of dry eye, with values of 5–10 seconds are considered marginal and less than 5 seconds indicate the dry eye symptoms [31, 32, 33, 34]. But, the threshold for Asian patients may be set much lower as many Asians with TBUT between about 7–10 seconds do not have dry eye symptoms . Some studies suggest that healthy Asian subjects have an 11–24% shorter TBUT than non-Asians [35, 36]. It hypothesized that the tear lipid layer is not able to efficiently perform its usual expansion and compression during a blink in an eye with a small palpebral aperture size (Asian), resulting in a less stable tear film .
4.1.2 Non-invasive break-up time (NIBUT)
This technique was first introduced by Mengher et al. in 1983 and is defined as ‘the time taken in seconds between the last complete blink and the appearance of the first random disturbance of a grid’. [38, 39] The NIBUT test can be performed using several device options, such as topography, keratography, or Placido disc video-keratography. Tear break-up time is considered when the reflected mires become distorted.
Mengher et al.  reported a NIBUT value of 47.9 seconds (range of four to 214 seconds), but Mohidin et al  reported lower NIBUT value (15.8 ± 9.4 seconds, range of 4.2 to 48.6 seconds). Sharanjeet-Kaur et al.  reported that NIBUT values for normal Malays and Chinese were 7.74 ± 3.34 seconds and 7.15 ± 3.38 seconds respectively. Generally, in normal population, NIBUT is longer than TBUT, with range of four to 214 seconds (median 4–19 seconds); and in patients with DED, NIBUT and TBUT values are almost the same (the cut-off values for positive finding can be as low as 2.7 seconds for automated algorithms and up to 10 seconds for subjective observation techniques) .
4.2 Tear volume
The aim of tear volume assessment is to measure the quantity of tear film produced by lacrimal gland and conjunctiva.
4.2.1 Schirmer’s test
The Schirmer’s test is the most common examination performed whenever there is a suspicion of inadequate tear secretion. The test was named after Schirmer who brought the test forward for the first time in 1903 . Basically, without anesthesia, the test measures total (basic and reflex) tear secretion, as with anesthesia it measures basic tear secretion devoid of reflex component [43, 44]. This test remains as the most common test used for tear quantity assessment . It can be divided into Schirmer I and Schirmer II test.
The Schirmer I test is performed using strip that is folded from one end and inserted into the lower conjunctival sac at the junction of lateral and middle thirds, avoiding touching the cornea. After five minutes, the length of wetted strip is recorded. Fifteen minutes later, after instillation of topical anesthesia, the strip is placed again over the same point in the same patient for five minutes. The Schirmer II test measure reflex secretion of lacrimal gland. The procedure of this test is as the same as Schirmer I test with topical anesthesia and nasal mucosa is irritated with a cotton-tiped applicator prior to measuring tear production. The result is recorded after 5 minutes. Normally, the length of wetted strip is around 10 mm or greater and if the length is less than 5 mm, it indicates symptomatic tear deficient; but Schirmer’s test values less or equal to 10 mm have greater diagnostic value and indicate hyposecretion [22, 46, 47, 48, 49, 50].
4.2.2 Phenol red thread test
The phenol red thread (PRT) test was invented by Hamano in 1982 and developed to overcome the disadvantage of Schirmer’s test including variable results, poor repeatibility, and low sensitivity in detecting dry eyes [44, 51, 52, 53]. The test uses a special cotton thread impregnated with phenol red (a pH-sensitive indicator). The procedure is performed in the similar manners to Schirmer test, which the thread is folded at the end and inserted into the lower conjunctival sac and it will absorb the tears that contact with it. The color of this thread will change from yellow to red over the pH range of normal tears. The result is recorded after 15 seconds, much shorter than Schirmer’s test. The PRT is almost comparable with Schirmer’s test and it has advantages including simpler and more comfortable to the patient and can also be performed in children .
4.3 Laboratory tests
Conjunctival biopsy may be one of the best methods to investigate and evaluate the ocular surface condition, which offers specimens of epithelial layers and conjunctival stroma to be examined under light or electron microscopy, cytology, and immunohistochemical analyses. These techniques may allow identification and counting of inflammatory cells and analysis of cell membrane markers, intracyto-plasmic cells, or extracellular matrix components . But, of all methods mentioned above, conjunctival biopsy is an invasive technique that may cause discomfort to the patient.
4.3.1 Impression cytology
Impression cytology (IC) refers to the application of cellulose acetate filter to the ocular surface to remove the superficial layers of the ocular surface epithelium . The analysis techniques used in this method vary, depend on the purpose and the equipment availability in eye care facility. The simplest analysis technique remains light microscopy, in which epithelial and goblet cells can be well visualized through hemtoxylin and periodic acid Schiff (PAS) staining. Other techniques include electron microscopy, immunohistochemistry, flow cytometry, and RT-PCR/PCR.
The RT-PCR was used in IC specimens as early as 1994 and identified inflammatory cytokins in conjunctival specimens from Sjögren’s syndrome eyes .
4.3.2 Tear osmolarity
Tear osmolarity is the most accurate method to diagnose DED. Tear hyper-osmolarity is considered as pathogenic factor causing ocular surface inflammation, symptoms, and tissue damage which can lead to DED. This condition can occur in many situations including insufficient tear production, meibomian gland dysfunction, and exposure. There is a commercially available objective point of care test (TearLab Osmolarity System; TearLab, San Diego, California) that can measure the osmolarity of a 50-nL tear sample and is easier to use [30, 58, 59]. A reading of 308 mOsms/L or greater indicates tear osmolarity disruption. This test must be completed quickly to avoid any evaporation of tear sample. Although reproducible, this test is difficult to perform in clinic setting.
4.3.3 Ferning test
Tear ferning test is a simple test for tear film quality. This test requires capillary tubes, spatula, or glass rods to collect tear from the lower tear meniscus (about 5–20 μl). The collected tear is applied to a glass slide and evaluated under light or digital microscope with various magnifications (40-100x). In DED, the delicate fronded pattern becomes fragmented or broken up and irregular and the appearances can be graded into type I and II (healthy tear film) and type III and IV (increasing degrees of dry eye) [14, 60].
5. Assessing the dry eye
The diagnosis of dry eye depends on the results of several of tests mentioned above, which ideally could be performed at a single clinic visit. It is important to keep in mind that a clinician should carry out the test in an appropriate order. Table 2 suggests a suitable order for diagnostic test, although there are various and informal data to justify a particular sequence of the tests. Some of the standard tests are specific for subgroup of the disease (Table 3). Table 4 helps the clinician to grade the severity of the DED.
|Dry Eye Severity Level|
|Discomfort, severity, and frequency||Mild and/or episodic; occurs under enviromental stress||Moderate episodic or chronic, strees or no stress||Severe frequent or constant without stress||Severe and/or disabling and constant|
|Visual symptoms||None or episodic mild fatigue||Annoying and/or activity-limiting episodic||Annoying, chronic and/or constant, limiting activity||Constant and/or possibly dissabling|
|Conjunctival injection||None to mild||None to mild||Mild or not present||Mild to moderate|
|Corneal staining (severity/location)||None to mild||Variable||Marked central||N/A|
|Cornea/tear signs||None to mild||Mild debris, decreased meniscus||Filamentary keratitis, mucus clumping, increased tear debris||Filamentary keratitis, mucus clumping, increased tear debris, ulceration|
|Lid/meibomian glands||MGD variably present||MGD variably present||MGD frequent||Trichiasis, keratinization, symblepharon|
|TBUT (seconds)||Variable||≤ 10||≤ 5||Immediate|
|Schirmer score (mm/5 minutes)||Variable||≤ 10||≤ 5||≤ 2|
Despite the wide use in clinical practice, standard tests for assessing DED and ocular surface disorders (including history taking and symptoms recording, TBUT, meibomian gland testing, ocular surface saining, and Schirmer’s testing) have shown poor repeatability and lack of efficacy [22, 57]. Moreover, it is well know that subjective symptoms often do not correlate with objective signs. Additional exploratory technique may be required to assess DED and evaluate the severity of the disease. The laboratory test may be required in patient who have subjective complaint that is not identified as dry eye (such as fluctuating vision) but do not show a lot of ocular finding such as corneal staining or any other marked conditions.
Patient with xerostomia in addition to dry eye must be investigated for the possible presence of Sjögren’s syndrome. The revised criteria of the European-American Consensus Group for the diagnosis of Sjögren’s syndrome are summarized in Table 5. The diagnosis of Sjögren’s syndrome is made if four of the six criteria are fulfilled. If SSA/SSB diagnostic testing is negative, a positive ANA (antinuclear antibody) test or positive rheumatoid factors may be indicative .
Careful and thorough examinations help the clinician to assess and evaluate dry eye disease. Various examinations are available, but the clinicians must adjust the examination to the available tools or equipments in their facilities. Ocular surface staining is the simplest test that can be performed in every clinic. If the case is complicated with or without underlying disease and needs further examniations, a clinician should refer to higher facility or dry eye specialist.
Author would like to acknowledge Susi Heryati, Angga Fajriansyah, Arief Mustaram, Patriotika Muslima, and Elfa Ali Idrus as supervisors in Infection and Immunology Division, Department of Ophthalmology, Cicendo National Eye Hospital, Bandung, Indonesia.
Conflict of interest
The author declares that there is no conflict of interest in this work.
Appendices and nomenclature
antinuclear antibody best-corrected visual acuity chemical index dry eye disease Standard Patient Evaluation for Dry Eye Questionnaire impression cytology meibomian gland dysfunction milliosmole National Eye Institute National Eye Institute Function Questionnaire-25 non-invasive break-up time Ocular Surface Disease Index periodic acid Schiff polymerase chain reaction power of hydrogen phenol red thread reverse transcription polymerase chain reaction anti-Sjögren’s syndrome type A anti-Sjögren’s syndrome type B tear break-up time uncorrected visual acuity
best-corrected visual acuity
dry eye disease
Standard Patient Evaluation for Dry Eye Questionnaire
meibomian gland dysfunction
National Eye Institute
National Eye Institute Function Questionnaire-25
non-invasive break-up time
Ocular Surface Disease Index
periodic acid Schiff
polymerase chain reaction
power of hydrogen
phenol red thread
reverse transcription polymerase chain reaction
anti-Sjögren’s syndrome type A
anti-Sjögren’s syndrome type B
tear break-up time
uncorrected visual acuity