Ocular surface staining: Current concepts and techniques : Indian Journal of Ophthalmology

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Review Article - Clinical

Ocular surface staining: Current concepts and techniques

Srinivas, Sanjeev P; Rao, Srinivas K

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Indian Journal of Ophthalmology 71(4):p 1080-1089, April 2023. | DOI: 10.4103/ijo.IJO_2137_22
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The health of the ocular surface is vital for clear vision and comfort. Various factors can adversely influence the ocular surface and tear film homeostasis, and these include procedures like cataract and corneal refractive surgery. It is, therefore, important to assess the integrity of the ocular surface in a rapid, predictable, and consistent manner in the clinic. Various tests and devices have been described, and while these are useful, this article highlights the importance of using fluorescein staining of the ocular surface in detecting changes. This is a simple, inexpensive, rapidly performed test that is available in most eye clinics. However, a proper technique of dye instillation and assessment is important to recognize the changes that can occur. Once detected, these changes can be quantified, and the location and patterns can be used to diagnose the diseases that are present; these changes can also be used to monitor treatment outcomes and disease progression. The article discusses the technique, assessment, and interpretation of fluorescein staining of the ocular surface, along with the role of the two other vital dyes – rose bengal and lissamine green.

The “ocular surface system” is defined as the ocular surface, which includes the surface and glandular epithelia of the cornea, conjunctiva, lacrimal gland, accessory lacrimal glands, and Meibomian gland and their apical (tears) and basal (connective tissue) matrices, the eyelashes with their associated glands of Moll and Zeis, those components of the eyelids responsible for the blink, and the nasolacrimal duct.[1] There exists a complex interplay between these components, to maintain the integrity of the ocular surface. The wettability of the ocular surface, along with corneal transparency is one of the key factors which allow light to be refracted into the eye. The presence of a smooth, wet ocular surface is, therefore, essential to maintain refraction of light into the visual system. However, unlike the other epithelial surfaces of the human body, the ocular surface is directly exposed to the external environment, making it subject to desiccation, injury, and pathogens. Disruption of surface integrity and loss of the ocular surface homeostasis are linked to the pathogenesis of most ocular surface disorders (e.g., dry eye disease [DED]). The diagnosis of ocular surface disease (OSD) has become more technology dependent in recent times, with the advent of newer modalities. Yet, ocular surface staining still remains a core component in the diagnosis and treatment of various ocular surface disorders, as it is both a powerful and an inexpensive diagnostic tool which can be used to assess the integrity of the ocular surface.

Vital Dyes in Ocular Surface Staining

The term “vital stain” in casual usage denotes the ability of a dye to penetrate the living cells or tissue without inducing any immediate evident degenerative staining. In simpler terms, it refers to the ability of the dye to stain viable tissue, whereas the term “vital staining” is used to indicate when the living cells exclude the dye (staining negatively) and the dead cells take up the dye. Understanding ocular surface staining begins with being able to differentiate the etymology of these two terms.

Fluorescein sodium

Fluorescein sodium is one of the most commonly used vital dyes in clinical practice since the 19th century. Elrich first described the concept of vital staining in 1886. Pfluger first demonstrated the staining of rabbit corneas with fluorescein in 1882. Straub and colleagues, in 1888, demonstrated the clinical uses of fluorescein staining of the cornea and conjunctiva.[2] Fluorescein penetrates poorly into the lipid layer of the corneal epithelium, and therefore, it does not stain normal cornea. Instead, the surface is stained whenever there is disruption of the cell-to-cell junctions. Although fluorescein is a very effective stain for the diseased cornea, it is more difficult to detect fluorescein staining of the conjunctiva because of the poor scleral contrast. Fluorescein absorbs light in the blue spectrum (490 nm) and emits yellow-green light of a higher wavelength (530 nm), therefore staining can be more readily viewed if a cobalt blue Fig. 1a or yellow (blue-free) filter is used.

Figure 1:
(a) Punctate corneal staining demonstrated using fluorescein dye and illumination under cobalt blue filter; (b) borders of ocular surface neoplasia highlighted by rose bengal staining; (c) conjunctival surface staining with lissamine green

Rose bengal

Rose bengal is a derivative of fluorescein and is used to detect ocular surface damage. It was initially theorized that rose bengal stains dead and devitalized tissue, but it is now thought to stain any part of the ocular surface which is not protected by an overlying tear film, specifically in areas lacking membrane-associated mucins.[3] Schirmer first introduced it in 1903 for ocular use.[4] Sjogren first demonstrated mucin-staining properties of rose bengal dye in 1933. Surface irritation and discomfort are common side effects associated with the use of this agent. Surface cytotoxicity is also one of the factors behind its limited use in routine practice.

Lissamine green

Lissamine green, a food additive, is a synthetically produced organic acid derivative. It was first introduced by Norn in 1973. It has a high affinity for staining dead and degenerated cells, including mucous strands. The dye appears to stain areas where there are disrupted intercellular junctions. Lissamine green has peak absorption at the red end of the visible spectrum (630 nm). Use of a green (red-free) filter shows the stained areas to appear black in color. As it is better tolerated than rose bengal, it is the preferred dye for staining the bulbar conjunctiva. Surface staining is, however, dependent upon the amount of dye instilled Fig. 1c.

Anatomical and Physiological Basis of Ocular Surface Staining

Topical dyes have been used for a long time to study and characterize OSD as well as to quantify its severity. The staining pattern in most cases may help to diagnose as well as prognosticate the disease. The basis of dye uptake and staining can be explained based on the following anatomical and physiological factors.

Corneal epithelial tight junctions

The corneal epithelium is a five-layered structure possessing a basal layer of columnar cells, about 10 mm wide, intermediate layers of wing-shaped cells, and a superficial layer of large, flat, polygonal cells, about 35 mm in diameter [Fig. 2].[5] The superficial cells are connected to one another by intercellular tight junctions (zonulae occludentes). These junctions are understood to reduce the passage of ions and hydrophilic molecules above a certain size from the tears into the epithelium. These junctions help make the epithelial layer into a semi-permeable membrane, allowing transport of small hydrophilic molecules.

Figure 2:
Diagrammatic representation of the layers of the corneal epithelial surface showing epithelial tight junctions (Tj), apical glycocalyx (Glx), and intracellular gap junctions (Gj)

The tight junctions of the corneal epithelium consist of the transmembrane proteins occludin, claudin, and the junctional adhesion molecules (JAM) and the peripheral membrane proteins, ZO-1, 2, and 3 and MUPP-1.[6] The zonula occludens (ZO) proteins are members of the membrane-associated guanylate kinase (MAGUK)[7] proteins and are located at the membrane contact points of the tight junctions. These protein members form complex bridges between the tight junctions and actin cytoskeleton of the cells. Although these tight junctions provide a barrier to the passage of ions and hydrophilic molecules, the apical plasma membranes are more permeable to lipophilic molecules.

Conjunctival surface epithelium

The total surface area of the human conjunctiva is around 17.65 cm2 (±2.12) and that of the cornea is 1.04 cm2 (±0.12).[8] The bulbar conjunctiva is more loosely attached to the underlying sclera and exhibits undulating folds, which helps in unrestricted movement of the globe. The tarsal conjunctiva, on the other hand, is firmly attached to the underlying tarsal plate. The conjunctiva is highly vascular, while the cornea is avascular. Abundant goblet cells are distributed throughout the conjunctival epithelium. The tight junctions of the conjunctival epithelium are more porous than those of the corneal epithelium, possibly as a result of differences in the number and type of claudin subunits that they contain.[9]

Apical glycocalyx

The glycocalyx confers additional properties to the ocular surface, the most important of which is its intrinsic wettability.[10] When the glycocalyx is affected, wetting of the cornea becomes imperfect and tear instability ensues.

Aquaporins and gap junctions

Across the thickness of the epithelium, adjacent epithelial cells are connected to one another by water channels known as aquaporins, which are responsible for transepithelial water transport, and by gap junctions, which allow the exchange of small molecules and ions. Connexins are the major family of proteins found in these structures. These proteins are responsible for maintaining cellular polarity, thereby aiding in the transcellular transport of ions. They are also responsible for epithelial cell migration following ocular surface injury. The porosity of these gap junctions is the basis for dye diffusion, once it penetrates the superficial epithelial cell layers.

Ocular Surface Staining – Terminology

Staining of the ocular surface is dependent upon the integrity of its cellular components. This includes the presence of intact tight junctions along with the presence of a mature glycocalyx. In normal circumstances, the epithelial surface undergoes rapid turnover, wherein the epithelial cells are shed from the normal surface and are replaced by cellular division arising from the basal cells of the limbal epithelium.[11] The human corneal epithelium is replaced every week, reflecting the turnover of dividing basal cells, whose progeny migrate to the epithelial surface. The turnover rate of the human bulbar and tarsal conjunctiva is, however, not known. Any disturbance in this mechanism of homeostasis results in focal or generalized epithelial cell loss. This, in turn, forms the basis of ocular surface staining, which can be summarized under the following causes:

  • Cell death (pathological causes)
  • Loss of tight junctions
  • Loss of glycocalyx or immature glycocalyx (seen in maturing epithelial cells)
  • Mucus (free-floating mucus shows positive staining)
  • Diffusion (penetration of stain into deeper epithelial layers, wherein it spreads easily due to the presence of porous gap junctions)

Ocular surface staining patterns [Fig. 3]

Figure 3:
PATH – representative image showing annular hyperfluorescence and peripheral punctate corneal staining with fluorescein dye. PATH = preservative-associated transient hyperfluorescence

Solution-induced corneal staining/preservative-associated transient hyperfluorescence

This phenomenon is commonly associated with the use of multipurpose lens solutions used to rinse, disinfect, and store conventional contact lenses. These lens solutions vary in pH, ionic composition, and viscosity and contain surfactants, chelating agents, and preservatives such as polyhexamethyline biguanide (PHMB), polyaminopropyl biguanide, hexamethylene-bis-ethylhexyl biguanide (alexidine), polyquaternium (Polyquad [PQ-1]), and myristamidopropyl dimethylamine (Aldox). Among these, PHMB appears to be the most popular and commonly used agent owing to its widespread popularity over the past few decades. An unusual corneal fluorescein-staining phenomenon has been observed following such Maximum propylene solution (MPS) storage, particularly in care solutions containing biguanide preservatives.[12] It is observed as asymptomatic, superficial punctate, corneal hyperfluorescence, occurring within 1–4 h after contact lens insertion and resolving within 6–8 h. Corneal staining is observed in an annular pattern [Fig. 5] due to the absorption of the cationic biguanide by the corneal epithelium. Staining with anionic fluorescein at this point results in concomitant dye uptake revealing a stippled pattern of staining. These changes appear to be transient, and an overwhelming majority of cases appear to be asymptomatic.

Figure 4:
Pooling of fluorescein dye seen on a corneal surface with focal thinning and an epithelialized surface
Figure 5:
The Wratten 47 blue filter (410–500 nm) excites fluorescein (absorption spectrum: 465–490 nm) and emits brilliant green fluorescence (520–530 nm). Placement of a Wratten 12 filter deletes the emission of >510 nm (blue light which is admixed with emitted fluorescence), thereby enhancing visible fluorescence

Transient corneal stippling

Bron et al.[7] described the stippled appearance of corneas in those where there was no detectable form of OSD. It has been proposed that this phenomenon may be due to normal epithelial turnover in which there is an orderly process of programmed cell death (apoptosis). Once apoptosis is initiated, there is a signaling response initiated between the underlying cells and the senescent cell. This allows the underlying cells to take over the functions of the dying cell as it is being shed. Staining during this phase may result in transient pooling of the dye in the space vacated by the dying cell on the epithelial surface. This occurs when the underlying glycocalyx is still immature. However, the staining stops in a matter of time, as the glycocalyx matures and assumes its normal functions. Pathological cell death, on the other hand, results in loss of epithelial tight junctions, as the underlying cell is unable to take up the loss of the senescent cell (due to sudden cell death). This allows penetration and diffusion of fluorescein within the gap junctions of the deeper cells, resulting in larger areas of staining.

Dye pooling versus staining

Pooling of fluorescein dye occurs when there is a focal or localized area of thinning/loss of underlying stromal tissue with an epithelialized surface. The disparity between the depths of these surfaces when compared to the normal surface produces a localized pooling of larger amounts of dye, which, in some cases, can be misinterpreted as areas of positive staining as seen in Fig. 4.

Ocular Surface Staining Procedure

Illumination and barrier filters

The fluorescence of a dye depends upon the wavelength of light falling on it. For example, the absorption or excitation wavelength of fluorescein sodium is 465–490 nm. A cobalt blue filter, Wratten 47/47A (included with most slit-lamp biomicroscopes), is used for this purpose, but a Wratten 12 or Kodak 12 filter can be used in place of the cobalt blue filter in case the slit-lamp biomicroscope does not have one.[13] A second filter over the observation port of the slit-lamp biomicroscope (barrier or absorption filter) eliminates blue light and helps decrease scatter and improve contrast. The use of a barrier filter enables visualization of both corneal and conjunctival staining, so that an additional dye for viewing conjunctival staining is not required. Conjunctival staining with lissamine green and rose bengal dye is usually viewed with white light, although additional barrier filters can be used to render the stained areas black for enhanced viewing [as mentioned in Table 1]. Fluorescein staining warrants the use of maximum available illumination. Rose bengal and lissamine green stains appear less visible with high illumination, and hence, it is recommended to begin with lower levels and gradually scale up the illumination until the staining is most visible.[14]

Dye instillation

Fluorescein was initially administered in a liquid form from an eye dropper, but this method is seldom used today because of the high risk of Pseudomonas aeruginosa to flourish in liquid fluorescein sodium, resulting in a high risk for corneal infection. This contamination risk can be overcome by using sterile single-dose units. However, the standard method of introducing fluorescein dye into the eye is via sterile, single-use fluorescein-impregnated paper strips. Single-use dye-impregnated paper strips have now become part of the standard practice protocol due to their inexpensive cost and widespread availability. The preferred technique is to introduce a small drop of sterile, unpreserved saline onto the fluorescein-impregnated tip of the paper strip and then to lightly apply the end of the strip onto the surface of the eye Fig. 6. It is preferably applied onto the lower palpebral conjuctiva (avoiding the corneal surface). This allows deposition of fluorescein on the area of “least interest”; otherwise, a very high fluorescein concentration will usually be deposited at the point of contact of the fluorescein strip (irrespective of how delicately fluorescein is applied), leaving an intense discrete region of iatrogenic “pseudo-staining.” Physical touch may not be required if a sufficient amount of fluorescein in the form of a “hanging drop” remains suspended from the moistened strip. The amount of fluorescein instilled over the ocular surface using the described technique usually maintains the right amount of fluorescein to effect surface staining without there being excess amounts (which may reduce contrast). Specially modified fluorescein strips (The “dry eye test” [DET]; Alcon Laboratories, Fort Worth, Texas, USA) developed by Korb et al.[15] with a substantially reduced area of fluorescein impregnation provide significant reduction in sensation upon application, improved single measurement reliability, and enhanced measurement precision when compared to a conventional fluorescent strip.

Figure 6:
The simple technique of fluorescein staining

The use of 1–2 μl of 2% fluorescein in 10 μl of tears produces a surface concentration of 0.2%. In most cases, this provides the ideal amount of fluorescein on the ocular surface for effective staining. Use of one drop (30–50 μl) in the inferior conjunctival cul-de-sac leads to excessive visibility of fluorescein, thereby leading to masking of subtle findings (one must keep in mind that the normal tear volume is only 7–10 μl). Ideally, drops of dye on the stain strip must be gently shaken off before application to avoid overstaining the surface.

The rationale behind the use of lower concentrations of fluorescein on the ocular surface can be explained by what is known as the “quenching phenomenon” described by Begley et al.[16] It is described as follows:

  • Low surface (<0.01%) concentration of fluorescein produces very little fluorescence.
  • Midlevels (0.1%–0.2%) of surface concentration produce optimum fluorescence.
  • High levels (>0.25%) of surface concentration result in a drop in fluorescence (tightly packed fluorescein molecules tend to reabsorb the emitted fluorescence, resulting in quenching)
  • Therefore, the use of minimal amount of dye at first gives the opportunity to stain later if the amount of dye is deemed inadequate (without the worry of fluorescein quenching).

It is always prudent to apply lissamine green or rose bengal Fig. 7 in the upper bulbar conjunctiva. This allows the dye to trickle down and spread over the ocular surface, especially in cases with reduced tear volume, where spread from the lower palpebral conjunctiva may not be possible. In all cases, overspill of the dye is to be avoided. Overspill of dye onto the cheek and around the eye may be a cosmetic problem for the patient. Although it is temporary, overspill of any of these dyes can lead to unsightly orange, blue, and pink rings around the eye and streaks down the cheeks, especially in DED patients who tend to have overspill due to lack of tear volume.

Figure 7:
Suggested order of dye application for ocular surface assessment

Diluents and anesthetics

The ideal diluent to be used for all dyes is preservative-free saline. The use of molecules like carboxymethyl cellulose and hydroxypropyl methylcellulose is to be avoided, as they tend to coat the cellular surface, leading to masking of subtle clinical findings. Mucin-like molecules are also avoided to reduce chances of false surface staining.

Proparacaine hydrochloride (0.5%) is best suited for ocular surface staining. The use of any other surface anesthetic agent is associated with loosening of the epithelial tight junctions (cytotoxicity) and is thus avoided. An alternative would be to use a 0.3%/0.4% fluorescein sodium and benoxinate hydrochloride ophthalmic solution (Paragon Bioteck, Inc. portland, Oregan, USA). It is a yellow to orange-red ophthalmic solution containing fluorescein sodium 2.6 mg/mL (0.3%) and benoxinate hydrochloride 4.4 mg/mL (0.4%).

Stain assessment – timing of observation

The time at which staining is observed is critical for both fluorescein and lissamine green dyes. The instillation of a high concentration of fluorescein dye into an eye with a relatively low tear volume can lead to concentration quenching with little or no corneal staining visible for several minutes. Another issue with fluorescein staining of the cornea is dye diffusion into the corneal stroma when the barrier function is compromised, which can occur in severe dry eye. Various authorities recommend stain pattern assessment at different times. The timing of observation ranges between immediate assessment and leaving a gap of between 1 and 4 min from the time of dye instillation.[7,14] The immediate assessment of fluorescein staining may result in an excessively thick tear film, which may produce erroneous observations. Ideal timing for assessment would be around 2 min after dye instillation. The concentration of dye on the ocular surface allows for both static and dynamic tear film assessment. The patient is asked to blink for a few times to ensure that the dye is spread evenly across the ocular surface. The upper lid is then gently lifted before examination. This technique helps avoid leakage of excess dye trapped in the upper bulbar conjuctiva under the lid over the corneal surface during examination.

The timing of observation of lissamine green conjunctival staining is another concern. In some patients, the stain may fade rapidly after instillation, and hence, viewing is recommended between 2 and 4 min.[17] However, in patients with severe DED, lissamine green conjunctival staining can be persistent. The timing of observation may be less of a problem in these patients. The intensity of stain also depends upon the amount of lissamine green dye impregnated on the paper strip, as the concentration of dye delivered by the strips may not be high enough for proper staining.[18]

Order of dye application when using multiple dyes

For assessment of total ocular surface staining, multiple dyes may have to be used either simultaneously or sequentially to improve the efficiency of the clinical exam. The use of double staining techniques may allow examination of both the corneal and conjunctival surfaces simultaneously without the need for pausing the examination for reapplication of dyes, but this may require switching between illumination settings and barrier filters to view findings, which can be a potential cause for subtle findings being missed. If the dyes are being used individually, then there is no particular order in which they need to be applied, as there is no known chemical antagonism among the three compounds. The only dictum would be to complete ocular surface staining and assessment before undertaking any invasive tests. With this in mind, a suggested order of ocular surface staining is given below.

Uses of Ocular Staining

Ocular surface uses

  • DED
  • Tear meniscus height measurement
  • Fluorescein dye clearance test
  • Ocular burns – limbus assessment
  • Corneal infections
  • To detect bleb/wound leak
  • Contact lens complications
  • Identify areas of epithelial ingrowth
  • Negative staining (e.g., laser-assisted in situ keratomileusis [LASIK] flap folds vs. flap striae; flap striae do not show areas of negative stain)[24]
  • Ocular surface squamous neoplasia (OSSN) Fig. 4
  • To identify conjunctival/corneal foreign bodies

Nonocular surface uses

  • Applanation tonometry
  • Contact lens fitting (Terry RL et al)
  • Nasolacrimal duct patency
  • Assessment and repair of canalicular injury
  • Anterior segment angiography (to look for tumors and ischemia)

Ocular Surface Staining – Assessment and Grading

Importance of ocular surface staining

Ocular surface staining is an important tool used to diagnose and prognosticate ocular surface disorders. The rising prevalence of OSD in populations has stemmed a large-scale increase in technology- and algorithm-based diagnostic techniques. These are generally machine-dependent and technology-driven methods, which may not be accessible to all clinical practitioners and patients when considering the costs involved. Hence, ocular surface staining still remains an efficient and cost-effective tool in the diagnosis and management of ocular surface disorders. Even though it need not be performed during every routine eye exam, the situations that warrant a thorough ocular surface assessment using ocular surface staining techniques include the following:

  • Patient symptoms suggestive of OSD
  • Patients with any systemic disease known to affect the ocular surface (may be asymptomatic at the time of presentation)
  • To detect, diagnose, grade, and prognosticate OSD
  • To look for the response to treatment during patient follow-up

Grading of ocular surface staining

Grading of ocular surface staining can be done by diving the cornea and conjunctiva into zones and assessing the surface changes highlighted by surface staining. Although there exist numerous grading systems, there is no current consensus regarding the best assessment and scoring system available. The absence of a “gold standard” scale which is universally accepted has impacted the ability to diagnose and monitor ocular surface conditions such as dry eye, in which surface staining is often used to assess the severity and progression of the condition. Many ocular surface scales have been developed to assess various other noninfectious ocular states, such as contact lens complications, graft-versus-host disease, and keratoconus. These scales employ different methods to score staining using unique scoring systems, some of which include the conjunctiva and others include only the cornea. A large majority of these systems use the following parameters to grade ocular surface staining:

  • Extent (by dividing the cornea and conjunctiva into zones)
  • Stain density (based on the number of staining spots)
  • Confluence of stain (patches of stain)
  • Other miscellaneous parameters (pupillary area involvement, presence of filaments, and so on)

Some of the scales that are commonly used to assess staining are the National Eye Institute (NEI)/Industry scale, the Oxford scale, the Sjögren’s International Collaborative Clinical Alliance (SICCA) Ocular Staining Score (OSS) scale, and the Baylor scale. The NEI scale divides the cornea into five zones that are scored by the density of stained dots on a 0–3 scale. In addition, the nasal and temporal conjunctiva are subdivided into three zones (superior paralimbal, inferior paralimbal, and peripheral) giving a total of six conjunctival zones, which are also graded by staining density. The Oxford scale also grades the density of stained dots within the cornea and nasal and temporal conjunctiva but has introduced the new concept of log unit increases in the number of stained dots between grades. Drawings are used to picture the increasing density of dots with each grade, showing that increasing number of stained dots are unevenly distributed within each zone. Instead, the dots cluster and eventually coalesce (Grade IV) around the limbus across the interpalpebral zone. The SICCA OSS scale includes this feature of coalescence and gives equal weight to corneal and conjunctival changes by adding three additional points to the corneal staining based on the following features: patches of confluent staining, pupillary staining, and filaments. The Baylor scale is another grading scale that involves counting dots and adding points for confluence and filaments. However, the cornea is divided into five zones and points are added for confluence and filaments within each corneal zone.[16]

Although multiple ocular surface stain scoring systems exist, there is no concurrence among clinicians regarding the use of a single scale as the clinical standard in all research studies. More recently, newer digital scoring scales are being evaluated in the study of DED. Such a scale was described by Tan et al.[27] in 2013, which is useful in subjects who are aged between 18 and 36 years with an OSD index (OSDI) of <13. The grading scale uses software-based image analysis to determine the threshold pixel intensity for staining (set manually and automatically) as well as the percentage of fluorescent (stained) area.

Irrespective of the grading system being employed, the following remain time-tested caveats in the assessment of ocular staining patterns:

  • Nasal conjunctival stain is seen first in DED.
  • Temporal conjunctival staining in Sjögren is an important red flag. (Reduced tear clearance and natural tear production results in tear movement toward the nasal puncta with each blink. This is followed by evaporation of water, resulting in concentration of tear film debris and inflammatory products at the nasal bulbar area.)
  • Measurement of the extent of surface staining (serial diagrams or anterior segment digital photography)

Ocular surface stress test

It indicates the staining pattern observed by the examining physician after routine preliminary examination (refraction, applanation, dilatation, and so on). Punctate staining of the ocular surface indicates that the surface is stressed and is therefore unable to withstand the rigors of a normal examination. It also indicates the need for topical therapy for surface stabilization before undertaking any planned interventions (refractive surgeries – surface ablation, LASIK, intraocular lens [IOL] surgery). The findings of the ocular stress test help identify patients with unhealthy ocular surfaces, which may become prone to recurrent breakdown if not stabilized before any surgical or therapeutic intervention.

Dynamic ocular surface staining assessment for dry eye

The majority of ocular surface stain grading systems for DED depend on static assessment of the tear film and its components. A novel approach described by Yokoi and Georgiev[28] utilizes dynamic assessment of the tear film. The diagnosis of DED can be done based on the tear film dynamics and the tear film break-up pattern after the eye is opened and when the eye is kept open. Using tear film-oriented diagnosis (TFOD), not only are the dysfunctional components of the ocular surface responsible for the accelerated tear film breakup identified, but also the necessary components required to stabilize the tear film can be discovered in a layer-by-layer fashion. Therefore, on the basis of TFOD, dry eye subtypes are diagnosed as aqueous deficient dry eye (ADDE), decreased wettability dry eye (DWDE), and increased evaporation dry eye (IEDE) Bron AJ et al.[21] TFOD is based on tear film instability and breakup, which is regarded as one of the core pathophysiology of DED in Japan.[29] Abnormal breakup time (i.e., ≤5 s) and symptoms are considered to be only a part of the diagnostic criteria for dry eye. The emergence of newer modalities of medical management of dry eye, including diquafosol sodium and rebamipide, which enable tear film stabilization via targeted supplementation of deficient ocular surface components, has helped popularize the use of TFOD and targeted tear film-oriented therapy (TFOT) in countries like Japan.

TFOD in its current stage is still based on a number of subjective and objective parameters, which are used to determine the cause of tear film instability. The Dry Eye Workshop (DEWS) II[30] report, however, states that not only tear film instability, but also hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play an etiological role in the evolution of DED. TFOD, therefore, has the promise to be an ideal method for a clinician to diagnose the Dry Eye (DE) subtype[31] in its development within the field of ocular surface staining, as it focuses primarily on using fluorescein staining of the ocular surface to determine the type of DED and also determine the insufficient tear film component, which can then be supplemented with topical therapy in order to reduce tear film instability.

Interpretation of Ocular Surface Staining

The appearance of staining patterns on different parts of the ocular surface usually provides good diagnostic clues regarding the etiology of the condition. The interpretation of corneal staining is as follows:

  • Inferior one-third of the cornea – lid margin disease [Fig. 8a]
  • Upper one-third of the cornea – upper lid pathology – allergic/ vernal keratoconjunctivitis (VKC) [Fig. 8b]
  • Interpalpebral region (middle one-third of the cornea) – DED [Fig. 8c]
  • Interpalpebral region along with superior corneal involvement – preservative/medication toxicity [Fig. 8d]

Figure 8:
Composite image showing patterns of corneal fluorescein staining

Conjunctival staining patterns Ogawa et al.[25] can also be used to indicate disease etiology, based on their location, intensity of staining, and amount of dye uptake. Interpretation of conjunctival staining based on anatomical site includes the following:

  • Superior bulbar – superior limbic keratoconjunctivitis (SLKC)[23]
  • Inferior – conjunctivochalasis Fig. 9
  • Temporal – Moraxella angular conjunctivitis
  • Nasal – ocular cicatricial pemphigoid (associated ulceration), canaliculitis

Figure 9:
Surface drug toxicity (medicamentosa) inferior bulbar conjunctival staining with corneal involvement

Preservative/medication-induced ocular toxicity often manifests with conjunctival staining limited to the inferior bulbar conjuctiva. An imaginary line drawn between the two canthi demarcates the upper limit of staining. In these cases, the amount of corneal staining far exceeds that of the conjunctival staining [Fig. 10]. The presence of extensive symptoms with minimal or absent corneal and conjunctival staining is indicative of ocular surface inflammation.

Figure 10:
(a) Superior lid margin staining in lid wiper epitheliopathy; (b) rose bengal stain used to identify the line of Marx

Lid margin signs associated with ocular surface disorders include staining of the upper lid margin in cases of lid wiper epitheliopathy [Fig. 10a]. In cases of DED, there is a posterior migration of the line of Marx (which is a stained line that runs along the inner eyelid). The position of the line of Marx [Fig. 10b] can serve as an accurate indicator of Meibomian gland function. This is based on the Marx line (ML) score[32] as follows:

  • 0 – entirely on the conjuctival side of Meibomian orifices
  • 1 – part of the ML touches the Meibomian orifices
  • 2 – ML runs through all the Meibomian orifices
  • 3 – ML runs on the eyelid margin side of the Meibomian orifices

The three tenets of ocular surface assessment include

  • ocular surface staining,
  • Schirmer’s test, and
  • tear film break-up time (TBUT).

The use of all of the above surface parameters may be required to identify neuropathic pain disorder, which itself is a diagnosis of exclusion. Neuropathic pain disorders must not be omitted, as they are part of the spectrum of ocular surface disorders. Prompt identification and referral to a pain management specialist can bring significant symptomatic relief to patients, who often have chronic symptomatology that has been attributed to other causes. Neuropathic pain disorders often present similarly in another entity known as short break-up time (short BUT) DED. The two conditions can be differentiated as follows:

  • short BUT dry eye: symptomatic patient with normal Schirmer’s, absence of ocular surface staining, and reduced TBUT and
  • neuropathic pain: symptomatic patient with normal Schirmer’s, absence of ocular surface staining, and normal TBUT.


With the advent of advanced algorithm-based and technology-driven models Rodriguez et al[26] for the diagnosis of ocular conditions such as dry eye, the outcomes of management of OSD have drastically improved. Incorporating the use of artificial intelligence (AI)-based algorithms has only helped further this cause. Yet, ocular surface staining continues to be one of the fundamental principles in diagnosing and prognosticating the outcome of OSD. Future innovations in this field should be directed toward establishing a standardized system for scoring and grading OSD. A single standardized staining scale acceptable to researchers, clinicians, industry and regulatory authorities would allow better understanding and interpretation of the results of clinical trials. A widely available scale accompanied by a concise manual of procedures for use that outlines the operational conditions for its use is what is currently needed. Integration of such grading systems with technological advancements such as AI-based learning can prove to be a very promising development within the spectrum of OSD.

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Conflicts of interest

There are no conflicts of interest.


The authors state that the manuscript has been read and approved by all the authors, the requirements for authorship as stated earlier in this document have been met, and each author believes that the manuscript represents honest work, if that information is not provided in another form.


1. Gipson IK. The ocular surface:The challenge to enable and protect vision. Invest Ophthalmol Vis Sci 2007;48:4390–8.
2. Norn MS. Vital staining of cornea and conjunctiva. Acta Ophthalmologica 1962;40:389–401.
3. Feenstra RP, Tseng SC. What is actually stained by rose Bengal?. Arch Ophthalmol 1992;110:984–93.
4. Savini G, Prabhawasat P, Kojima T, Grueterich M, Espana E. The challenge of dry eye diagnosis. Clin Ophthalmol 2008;2:31–55.
5. Lemp MA, Mathers WD. Corneal epithelial cell movement in humans. Eye (Lond) 1989;3:438–45.
6. Ban Y, Dota A, Cooper LJ, Fullwood NJ, Nakamura T. Tight junction-related protein expression and distribution in human corneal epithelium. Exp Eye Res 2003;76:663–9.
7. Bron AJ, Argüeso P, Irkec M, Bright FV. Clinical staining of the ocular surface. Mechanisms and interpretations. Prog Retin Eye Res 2015;44:36–61.
8. Watsky MA, Olsen TW, Edelhauser HF. Human sclera:Thickness and surface area Tasman B, Jaeger E. Cornea and Sclera. Philadelphia and New York:Lippincott-Raven;1995.
9. Yoshida Y, Ban Y, Kinoshita S. Tight junction transmembrane protein claudin subtype expression and distribution in human corneal and conjunctival epithelium. Invest Ophthalmol Vis Sci 2009;50:2103–8.
10. Cope C, Dilly PN, Kaura R, Tiffany JM. Wettability of the corneal surface:A reappraisal. Curr Eye Res 1986;5:777–85.
11. Osei-Bempong C, Figueiredo FC, Lako M. The limbal epithelium of the eye:A review of limbal stem cell biology, disease and treatment. Bioessays 2013;35:211–9.
12. Jones L, MacDougall N, Sorbara LG. Asymptomatic corneal staining associated with the use of balafilcon silicone-hydrogel contact lenses disinfected with a polyaminopropyl biguanide-preserved care regimen. Optom Vis Sci 2002;79:753–61.
13. Bron AJ, Evans VE, Smith JA. Grading of corneal and conjunctival staining in the context of other dry eye tests. Cornea 2003;22:640–50.
14. Foulks GN. Challenges and pitfalls in clinical trials of treatments for dry eye. Ocul Surf 2003;1:20–30.
15. Korb DR, Greiner JV, Herman J. Comparison of fluorescein break-up time measurement reproducibility using standard fluorescein strips versus the dry eye test (DET) method. Cornea 2001;20:811–5.
16. Begley C, Caffery B, Chalmers R, Situ P, Simpson T, Nelson JD. Review and analysis of grading scales for ocular surface staining. Ocul Surf 2019;17:208–20.
17. Hamrah P, Alipour F, Jiang S, Sohn JH, Foulks GN. Optimizing evaluation of Lissamine green parameters for ocular surface staining. Eye (Lond) 2011;25:1429–34.
18. Awisi Gyau D, Begley CG, Daniel Nelson J. A simple and cost effective method for preparing FL and LG solutions. Ocul Surf 2018;16:139–45.
19. van Bijsterveld OP. Diagnostic tests in the Sicca syndrome. Arch Ophthalmol 1969;82:10–4.
20. Terry RL, Schnider CM, Holden BA. CCLRU standards for success of daily and extended wear contact-lenses. Optom Vis Sci 1993;70:234–43.
21. Bron AJ. The Doyne lecture. Reflections on the tears. Eye (Lond) 1997;11:583–602.
22. Efron N. Clinical application of grading scales for contact lens complications. Optician 1997;213:26–34.
23. Whitcher JP, Shiboski CH, Shiboski SC, Heidenreich AM, Kitagawa K, Zhang S, et al. A simplified quantitative method for assessing keratoconjunctivitis sicca from the Sjögren's syndrome international registry. Am J Ophthalmol 2010;149:405–15.
24. De Paiva CS, Pflugfelder SC. Corneal epitheliopathy of dry eye induces hyperesthesia to mechanical air jet stimulation. Am J Ophthalmol 2004;137:109–15.
25. Ogawa Y, Kim SK, Dana R, Clayton J, Jain S, Rosenblatt MI, et al. International chronic ocular graft-vs-host-disease (GVHD) consensus group:Proposed diagnostic criteria for chronic GVHD (Part I). Sci Rep 2013;3:3419.
26. Rodriguez JD, Lane K, Ousler GW, Angjeli E, Smith L. Automated grading system for evaluation of corneal superficial punctate keratitis associated with dry eye. Invest Ophthalmol Vis Sci 2015;56:2340–7.
27. Tan B, Zhou Y, Svitova T, Lin MC. Objective quantification of fluorescence intensity on the corneal surface using a modified slit-lamp technique. Eye Contact Lens 2013;39:239–46.
28. Yokoi N, Georgiev GA. Tear-film-oriented diagnosis for dry eye. Jpn J Ophthalmol 2019;63:127–36.
29. Shimazaki J, Yokoi N, Watanabe H, Amano S, Ohashi Y. Definition and diagnosis of dry eye in Japan. Atarashii Ganka 2016;34:309–13.
30. Craig JP, Nichols KK, Akpek EK, Cafery B, Dua HS. TFOS DEWS II Definition and classification report. Ocul Surf 2017;15:276–83.
31. Yokoi N, Georgiev GA. Tear film–oriented diagnosis and tear film–oriented therapy for dry eye based on tear film dynamics. Invest Ophthalmol Vis Sci 2018;59:13–22.
32. Yamaguchi M, Kutsuna M, Uno T, Xiaodong Z, Kodama T. Marx Line:Fluorescein staining line on the inner lid as indicator of meibomian gland function. Am J Ophthalmol 2006;141:669–75.

Dry eye disease; fluorescein; interpretation; ocular surface staining; vital dyes

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