Corneal and conjunctival vital staining (ocular surface vital staining) is a basic ophthalmic test that visualizes damage to the corneal and conjunctival epithelium on the eye surface with dyes and quantifies the distribution and severity of the damage.
The main dyes used are the following three.
The main purposes of this test are as follows:
In the 2016 Dry Eye Clinical Practice Guideline, evaluation combining tear film breakup time (BUT) measurement and fluorescein staining is recommended as a core part of dry eye diagnosis1). In the 2006 edition, corneal and conjunctival epithelial damage was essential for diagnosing dry eye, but in the 2016 edition, epithelial damage was no longer a required criterion, and diagnosis shifted to short BUT and symptoms. Even so, vital staining remains an important way to objectively record the degree and pattern of epithelial damage1).
In the initial evaluation of infectious keratitis, fluorescein staining is also a standard procedure used to understand the extent and shape of corneal epithelial defects, and it is incorporated into care based on the Infectious Keratitis Clinical Practice Guideline (3rd edition)2).
The distribution and severity of corneal and conjunctival epithelial damage are visualized. With fluorescein, punctate superficial keratopathy (SPK) and erosions or ulcers of the corneal epithelium appear as fluorescence, and with rose bengal or lissamine green, dead and degenerated cells are stained. The pattern of SPK distribution can help suggest the underlying condition (dry eye, drug toxicity, contact lens-related injury, and so on). Scoring methods can also be used to quantify the damage and track treatment effects over time.
Fluorescein is the most widely used vital dye. It is commonly used because it is easy to obtain, safe, and causes little irritation. Fluorescein is a dye that emits green fluorescence (521 nm) when excited by blue light (maximum absorption wavelength 494 nm). It can be observed with only a cobalt blue filter, but lesions are shown more clearly when a blue-free filter is added to the observation system.
Staining principle and key observation points:
Minimal staining procedure:
Rose bengal stains the ocular surface by a mechanism different from fluorescein.
Staining characteristics:
van Bijsterveld scoring (used for both rose bengal and lissamine green):
Lissamine green is an alternative dye with staining properties similar to rose bengal, but it is less irritating to patients.
Staining characteristics:
Fluorescein (fluorescein)
Absorption wavelength: 494 nm (blue light) → fluorescence 521 nm (green)
Staining target: areas where tight junctions between epithelial cells are disrupted (intercellular spaces)
Main uses: SPK detection, BUT measurement, corneal ulcer/erosion evaluation
Irritation: low (easiest to use)
Observation filter: cobalt blue + blue-blocking (barrier) filter
Rose bengal
Staining color: red
Stained targets: dead cells, degenerated cells, areas lacking mucin protection
Main use: diagnosis of Sjögren syndrome (van Bijsterveld score), dry eye evaluation
Irritation: strong (pain when instilled)
Observation filter: white light or red filter
Lissamine green
Staining color: green
Stained targets: dead cells, degenerated cells (same mechanism as rose bengal)
Main use: alternative to rose bengal. Dry eye and Sjögren syndrome evaluation
Irritation: low (less burden for patients than rose bengal)
Observation filter: clearly seen with a red filter (560 nm or above)
| Characteristic | Fluorescein | Rose bengal | Lissamine green |
|---|---|---|---|
| Staining color | Green fluorescence | Red | Green |
| Staining target | Intercellular spaces (areas where tight junctions are disrupted) | Dead cells, degenerated cells, and mucin-deficient areas | Dead cells and degenerated cells |
| Irritation | Low | High (with pain) | Low |
| Observation filter | Cobalt blue + blue-free | White light · red filter | Red filter (560 nm or higher) |
| Main uses | SPK · BUT measurement · corneal ulcer | Sjögren diagnosis · dry eye | Rose bengal alternative |
| Representative score | Oxford score / NEI score | van Bijsterveld score | van Bijsterveld score |
Both stain dead and degenerated cells, but lissamine green is less irritating and easier on patients. Rose bengal can cause pain when instilled, so in some cases topical anesthesia may be needed. Lissamine green has become more widely used in recent years as an alternative dye that solves these drawbacks. For observation, using a red filter (560 nm or higher) makes the stained areas easier to see clearly.
Several scoring systems have been established to quantify corneoconjunctival epithelial damage.
| Scoring method | Evaluation area | Score range | Total | Main use |
|---|---|---|---|---|
| van Bijsterveld score | cornea, nasal bulbar conjunctiva, temporal bulbar conjunctiva (3 areas) | 0–3 points each | 9-point maximum (abnormal at 3.5 points or higher) | Sjögren syndrome diagnostic criteria |
| Oxford scoring | cornea, bulbar conjunctiva (nasal and temporal) (3 areas) | 0–4 points each (5 levels) | 15-point maximum | dry eye severity assessment |
| NEI/Industry Workshop score | cornea (5 sections) | 0–3 points each | 15-point maximum | dry eye clinical research |
Evaluate each of the three areas—cornea, nasal bulbar conjunctiva, and temporal bulbar conjunctiva—on a 0 to 3 scale (0: no staining, 1: a few punctate stains, 2: staining tending to merge, 3: widespread staining). A total score of 3.5 or higher is considered abnormal and is internationally adopted as a diagnostic criterion for Sjögren syndrome3).
Each of the three areas—cornea and bulbar conjunctiva (nasal and temporal)—is scored from 0 to 4 in five grades, for a total of 15 points. Each grade is assessed semiquantitatively by comparing it with panel diagrams. It is used to assess dry eye severity and treatment response.
The cornea is divided into five zones—central, superonasal, superotemporal, inferonasal, and inferotemporal—and each zone is scored from 0 to 3, for a total of 15 points. It is widely used in clinical research and multicenter trials.
Staining criteria for corneal and conjunctival epithelial damage in the Dry Eye Clinical Practice Guidelines (2006 edition)1):
In the 2016 dry eye diagnostic criteria, epithelial damage was removed from the essential diagnostic requirements, but observing epithelial damage continues to play an important role in assessing dry eye severity and treatment response1).
Punctate superficial keratopathy (superficial punctate keratopathy: SPK) is the most common eye finding in patients who complain of a foreign body sensation. SPK is a “result” of corneal epithelial damage caused by some underlying factor, not a diagnosis of the cause. Fluorescein vital staining is essential for detecting SPK and understanding its distribution pattern, and it can reveal subtle SPK that cannot be seen with slit-lamp microscopy alone.
When SPK is detected, it is important to actively infer the underlying cause from its distribution pattern.
| Distribution pattern | Main underlying cause |
|---|---|
| Concentrated in the lower one-third of the cornea | Dry eye (aqueous-deficient type), entropion |
| Concentrated in the upper one-third of the cornea | Superior limbic keratoconjunctivitis (SLK), trachoma |
| Entire cornea (diffuse) | Drug-induced toxic keratopathy, viral keratitis |
| 3 to 9 o’clock direction (horizontal band) | Contact lens (3-9 o’clock staining) |
| Central cornea | Lagophthalmos, neuroparalytic keratitis |
Exogenous SPK:
Endogenous SPK:
In drug-induced toxic keratopathy, damage to the conjunctival epithelium is less pronounced than damage to the corneal epithelium. This finding can be clearly confirmed with fluorescein staining and is useful for distinguishing it from other causative diseases. If diffuse SPK is seen across the entire cornea, the effect of the eyedrops being used (preservatives, high-concentration medications, aminoglycoside antibiotics, etc.) should be considered2).
In infectious keratitis, fluorescein staining allows objective assessment of the shape, area, and depth (judged by the intensity of staining) of corneal epithelial defects. The extent and shape of the ulcer are used as indicators for choosing treatment and for follow-up observation2). In addition, antibacterial eyedrops (high-concentration formulations and aminoglycosides) are likely to cause corneal epithelial toxicity, so it is important to check with vital staining during treatment for any worsening of epithelial damage2).
By observing the distribution pattern of SPK, the underlying disease can be inferred. SPK concentrated in the lower cornea suggests dry eye or entropion, upper SPK suggests SLK or trachoma, and diffuse SPK throughout the cornea suggests drug toxicity or viral keratitis. SPK at the 3 to 9 o’clock position is typical of contact lens injury, and SPK in the central cornea is characteristic of lagophthalmos and neuroparalytic keratitis. SPK is only the “result” of epithelial damage, and using the distribution pattern as a clue to search for the cause is the key point in care.
Identify the cause from the distribution and severity of epithelial damage confirmed by vital staining, and choose treatment according to the cause.
Standard treatment based on the Dry Eye Clinical Practice Guidelines (2016 edition) is as follows1).
Based on the Infectious Keratitis Clinical Practice Guidelines (3rd edition), select the appropriate antimicrobial agent after identifying the causative organism2).
Fluorescein is a fluorescent dye that absorbs cobalt blue light (494 nm) and emits green fluorescence (521 nm). The principle of fluorescence is photoluminescence, in which absorbed energy is re-emitted as light.
A blue-blocking filter (barrier filter) blocks the excitation light (around 494 nm) and lets only the fluorescence wavelength (521 nm) pass. This removes background light and makes SPK fluorescein staining easier to see. When a blue-blocking filter is attached to the slit lamp microscope, the sensitivity for detecting SPK improves greatly compared with using the cobalt blue filter alone.
When the tight junctions of the corneal epithelium break down, fluorescein penetrates the intercellular spaces and fluoresces. Areas where normal tight junctions are intact do not allow fluorescein to enter and therefore are not stained.
Rose bengal selectively stains cells that are not protected by mucin. Healthy ocular surface cells are covered by a mucin layer (mainly secretory mucin MUC5AC), which prevents rose bengal staining. Dead and degenerated cells have lost this mucin protection, so they are stained. Unlike fluorescein, it stains the dead cells themselves, so it can be considered an indicator of ocular surface cell viability.
Lissamine green stains dead and degenerated cells by a mechanism similar to rose bengal. Staining can be seen most clearly under observation with a red filter (560 nm or higher). It is thought to be less irritating to the ocular surface than rose bengal because of differences in penetration into living tissue.
The blue filter is especially important for fluorescein observation. Even with cobalt blue light without a filter, lesions can be seen, but adding a blue filter:
