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Use of fluorescein in optometric practice.

Optometrists and contact lens fitters in routine practice frequently use sodium fluorescein for clinical assessment. Recent changes to the availability of this diagnostic dye have led to some controversy over its use in a clinical context. This article discusses the diagnostic value of fluorescein and clarifies the current position regarding its use in the UK.


Sodium fluorescein, usually termed in a clinical setting as simply 'fluorescein', has a variety of ophthalmic applications, including assessment of retinal vascular function using fluorescein angiography, Goldmann applanation tonometry (GAT), and rigid gas permeable contact lens fitting. Whereas fluorescein angiography is performed in hospital settings with highly specialised equipment beyond the remit of most optometrists, this article focuses on the properties of fluorescein, its typical applications and the specialised tests used in optometric practice.

Fluorescein application

Fluorescein is a synthetic pH and concentration-dependent fluorescent organic compound, with peak absorption at 495nm (blue light) and emission at 520nm (green light) in water. (1,2) The fluorescein molecules, known as fluorophores, absorb electromagnetic energy, exciting them to a temporary higher energy state. As they return to their original energy level, the fluorophores emit light at a longer wavelength--a phenomenon known as fluorescence. (3) It is the most common ophthalmic dye used in clinical practice, available in solution (1% and 2% unit dose eye drops) or impregnated in salt form on to sterile filter paper strips. (1,4) In a survey on the scope of clinical therapeutic practice in the UK, 96% (n=1,093) of optometrists reported frequently using it as part of their normal clinical practice. (4)

Following instillation in the eye, a slit lamp combined with a blue filter to the illumination system is used to observe the fluorescence, visible as an intense green colour on the ocular surface. (5) The blue filter allows only light of wavelengths close to the excitatory spectrum of fluorescein to reach the eye. (3,5) The contrast of the fluorescence may be further enhanced using a yellow barrier filter, such as the Kodak Wratten 12, which blocks the blue light reflected from the ocular surface so that the fluorescence is better observed. (1,5) The visualisation of the tear film with fluorescein enables procedures such as contact lens fitting (relationship between the back surface of contact lens and cornea) and Goldmann applanation tonometry (to visualise the mires) to be performed. However, the majority of slit lamps currently available do not emit optimised blue light or utilise yellow barrier filters to optimise fluorescence--the blue light of 10 different slit lamps has been shown to have an average peak at 460nm, with only 8.3%-50.6% of the blue light optimised for >80% fluorescein excitation. (1) Viewing of the fluorescence is affected by the method of instillation, owing to the concentration-dependent nature of fluorescein. (1) At concentrations greater than 0.000005%, fluorescein begins to quench (fluorescence intensity decreases), possibly due to the dissipation of the absorbed energy following collision between more prevalent neighbouring fluorescein molecules. (2,6) Instillation to the eye using fluorescein impregnated filter paper strips or minims demonstrate some degree of quenching, but studies have shown that a moistened strip and 1% minim reached a clinically useful level of fluorescence after approximately 20 seconds, lasting on for average of 160 seconds, whereas a saturated strip and 2% minim reached this level after approximately 50 seconds. (1) On the ocular surface, the intensity of fluorescence is also affected by the thickness of the tear film; thicker regions of tear film, such as the tear meniscus, appear brighter than the thinner tear film overlying the cornea and bulbar conjunctiva, although brighter areas may be observed along conjunctival folds where the fluorescein has pooled. (7) Indeed, the overall fluorescence will, therefore, vary with the quality and quantity of the tear film.

Ocular surface staining

The integrity of the ocular surface has long been assessed using ophthalmic dyes such as fluorescein, observed as 'staining', owing to its fluorescent properties at low concentrations and relatively rare and benign potential side effects upon topical application. (6,8) The assessment of ocular surface staining is typically used in contact lens fitting, aftercare, and evaluation of dry eye, ocular surface disease, and corneal abnormalities. However, the exact mechanisms that cause ocular surface staining are not well understood. (3) Traditionally, fluorescein is believed to enter only damaged cells on the ocular surface, but not healthy cells, and appears as fluorescent staining which can help to identify sight-threatening conditions such as corneal ulcers and foreign bodies. (8) However, fluorescein uptake has also been observed in healthy corneal epithelium, possibly due to normal physiologic desquamation. (9,10) As very little research has been conducted to examine the nature of ocular surface staining, interpretation of these observations is based upon assumption and clinical intuition. (3) However, studies have shown that fluorescein staining is due to uptake of fluorescein by individual corneal epithelial cells following microscopic examination of excised rabbit eyes, and not by fluorescein pooling in areas of epithelial cell drop out or filling of intracellular spaces. (9) More recent evidence has shown no significant difference in fluorescein staining in human eyes and excised rabbit eyes after three successive ocular surface rinses with saline and no significant difference between eyes that had been rinsed compared to those which had not, suggesting that pooling of fluorescein within intracellular spaces does not contribute to fluorescein staining. (11) However, low levels of fluorescein may enter healthy epithelial cells through tight junctions on the cell surface, suggesting they are naturally permeable to fluorescein. (8) Although corneal permeability increases in soft, extended contact lens wear, any corresponding increase in staining has not been observed. (12)

Fluorescence of the ocular surface may also occur where it has been indented, typically observed in the presence of mucin balls following contact lens wear, which leave depressions in the corneal surface as deep as the basal lamina, without any clear evidence of epithelial damage in the surrounding tissue. (13,14) The shearing frictional forces between the posterior contact lens surface, tear film and cornea causing the tear film mucins to form into ball-shaped structures which become trapped beneath the contact lens and indent the cornea. (14) The fluorescence occurs as the fluorescein stained tear film pools within the indentation. A similar mechanism occurs in dimple veiling, where air bubbles trapped under a rigid gas permeable lens indent the corneal surface causing the tear film to pool within the depression. (15,16) Unlike staining due to cellular uptake, fluorescein pooling in these conditions washes away following irrigation of the ocular surface.

Choice of dye

Rose bengal is another ophthalmic dye that stains the nuclei of devitalised and degenerated epithelial cells and areas of exposure, but its use is unpopular given that it causes stinging on application, and is intrinsically toxic, suppressing human conjunctival epithelial cells in vitro. (8,17,19) The ophthalmic dye lissamine green has increased in popularity recently and stains in a similar manner to rose bengal, but is far less toxic and irritating to the eye and has, therefore, superseded rose bengal for the assessment of ocular surface staining, particularly of the conjunctival region. (5,20,21) Staining of the palpebral conjunctival region of the eyelid margin in contact with the ocular surface during blinking, known as lid wiper epitheliopathy (LWE), can be observed using lissamine green. (22,23) LWE is considered to be the result of increased frictional forces between the lid wiper and the ocular surface, due to reduced level of lubrication and greater coefficient of friction caused by contact lens wear and dry eye (see Figure 1, page 45). (22-26) However, care must be taken not to confuse this observation with Marx's line, a normal anatomic feature of the eyelid margin posterior to the meibomian glands representing the oculomucocutaneous junction. (27)

The choice of dye relates to its visibility on the ocular surface. (5) Corneal staining is best observed using fluorescein (see Figure 2, page 45) given that it may be viewed over both dark and light irises, whereas lissamine green and rose bengal are difficult to view over a dark iris. (5) However, large amounts of fluorescein or thick tear films may obscure the underlying staining. (5) Although conjunctival staining may be observed with fluorescein, lissamine green and rose bengal show up well against light scattered by the white sclera and do not obscure the underlying staining pattern as they are poorly visible within the tear film. (5) Both lissamine green and rose bengal are visible for longer as they do not diffuse into the conjunctival substantia propria, whereas fluorescein diffuses rapidly into the surrounding tissue. (5) More recently, studies have shown that a mixture of 2% fluorescein and 1% lissamine green allows optimal view of staining of the cornea and conjunctiva combined with the advantage of only one application to the ocular surface. (28)

Grading scales

Staining may be graded using dedicated ocular surface damage scales such as the Oxford system, van Bijsterveld system, Collaborative Longitudinal Evaluation of Keratoconus (CLEK) study, and the National Eye Institute (NEI) system. (29,30) The Oxford, CLEK and NEI systems offer a wider range of scoring by dividing the ocular surface into different corneal and conjunctival areas, allowing for the detection of small changes in staining. However, no staining system has been demonstrated to be superior to another. (30) In clinical practice, general anterior eye grading scales such as the CCLRU and Efron scales are more likely to be encountered, but care should be taken to ensure the same grading scale is used to monitor a particular observation, as grades are not comparable between the scales. (31) More recently, digital image analysis of anterior eye features including corneal staining has shown to correlate well with grading scales, but demonstrates better sensitivity and repeatability (variation <0.5%). (32, 33) Multiple grading scale metrics are needed to describe staining (type, severity, location and depth) and hence a simple sketch may be more efficient.

Tear film break-up time

Tear film instability, represented by the tear film break-up time (TBUT), is considered a causative mechanism of dry eye alongside tear film hyperosmolarity, but may occur in the absence of the latter. (34,35) If the TBUT is less than the blink interval (time between consecutive blinks), or less than 10 seconds (even though the blink interval maybe shorter), the tear film is regarded as unstable, causing local drying and subsequent hyperosmolarity at the site of tear film break-up, leading to inflammation and epithelial and goblet cell damage on the ocular surface. (34,35) Reduced mucin levels as a result of conjunctival goblet cell damage may, therefore, negatively reinforce tear film instability as tear film spreading becomes impaired. (35) Thus, TBUT provides clinically relevant information about the status of tear film in the assessment of dry eye.

The TBUT is defined as the time interval between the last complete blink and the first appearance of a dry spot or disruption in the tear film (see Figure 3, page 46). (29) The visualisation of the tear film may be enhanced by the application of fluorescein to the ocular surface. (36) However, as fluorescein is typically applied in solution or via a moistened filter paper strip, this may add volume to the tear film and again artificially lengthen the TBUT. (36,37) Measurement of the TBUT with fluorescein is, therefore, considered an invasive technique denoted as the fluorescein break-up time (FBUT). Studies have shown significant increases in FBUT when increasing fluorescein volume from 1- 2.7 [micro]L, but no further rises in FBUT when supplemented further to 7.4 [micro]L. (36) Further, the addition of fluorescein to the ocular surface may itself influence the measurement, as application may induce reflex lacrimation which artificially lengthens FBUT. (37) Without the use of a micropipette to carefully control the volume of fluorescein applied to the eye, larger uncontrolled volumes typically applied in clinical practice may yield variable results. (37)

The imaging of mires, usually Placido rings or grid patterns projected onto the ocular surface via specular reflection, allows noninvasive measurement of the tear film break up time (NITBUT) that is not influenced by reflex lacrimation or addition of fluid to the ocular surface, and, therefore, provides a more accurate measure. (38) However, heat given off from the light source may cause the tear film to evaporate and artificially reduce NTIBUT, particularly during periods of measurement where the eyes are kept open for a substantial time (39), although evidence to support this is lacking. The Tearscope provides subjective assessment whereas newer instruments such as the Keratograph provide useful objective analysis. (40-42)

Seidel test

Although not commonly encountered in optometric practice, patients with suspected corneal or scleral trauma should be assessed using the Seidel test to determine whether there is a penetrating injury. (43) Here, topical anaesthetic is applied to the eye, which is held open using the fingers. Fluorescein, usually with a moistened filter paper strip (10% concentration), is then applied on top of the site of the suspected corneal lesion while the patient is seated at the slit lamp, combined with blue illumination and barrier filter to improve contrast. (43) At this stage, the fluorescein is highly concentrated, and, combined with the quenching effect does not fluoresce, but appears dark orange. If there is a leakage, the fluid will flow or stream out across the cornea and dilute the fluorescein causing it to fluoresce, indicating a full thickness penetrating injury to the cornea (positive Seidel test). (43) Alternatively, weaker concentrations of fluorescein which are typically applied in clinical practice will cause the tear film to fluoresce, and any leakage will appear as a dark flowing stream across the cornea (see Figure 4). The Seidel test may also be used to assess leakage from post-operative corneal wounds and filtering blebs following glaucoma surgery. (43,44)

Jones dye test

Watery eye or epiphora, is a symptom associated with a range of aetiologies, including lacrimal hypersecretion and/or impaired lacrimal drainage. (45-47) Where an impairment of lacrimal drainage system is suspected from patient history and examination of facial features, eyes and surrounding tissue, the Jones dye test may be used to distinguish between patent lacrimal systems, functionally obstructed or blocked lacrimal systems, and lacrimal pump failure. (45-47) Jones dye test 1 (Primary Jones test) determines whether the tears drain through to the nose. Here, one drop of 1% or 2% fluorescein is applied to the ocular surface, and a sterile cotton bud is placed in the inferior meatus of the nose, close to the valve of Hasner, at the base of the nasolacrimal duct. (45-47) Topical anaesthetic may be applied to the tip of the cotton bud to aid patient comfort. After five minutes, the cotton bud is removed and the presence of fluorescein (positive) on the bud indicates a patent lacrimal drainage system and the epiphora may be the result of lacrimal hypersecretion, caused by conditions such as ocular allergy, dry eye, blepharitis, trichiasis, distichiasis, entropion, ectropion, foreign bodies, concretions, corneal disease or anterior uveitis. (45-47) A negative finding where no fluorescein is observed indicates an obstruction, which may be anywhere from the punctum to the valve of Hasner. If a negative finding is observed, Jones Dye Test 2 (Secondary Jones test) is performed to determine whether there is lacrimal pump failure, or complete lacrimal drainage obstruction. (45-47) Here, the ocular surface is irrigated to remove any remaining fluorescein, followed by application of topical anaesthetic with a new sterile cotton bud applied to the inferior meatus, and syringing of the lacrimal system with saline. The presence of fluorescein on the cotton bud (positive result) indicates a functional obstruction where the tear fluid has collected in the nasolacrimal sac but has not drained normally, due to partial steno sis or where the nasolacrimal duct has collapsed/narrowed. (45-47) If no staining of the cotton tip is observed (negative result) this indicates lacrimal pump failure such as stenosis of the puncta or canaliculi as no tear fluid entered the nasolacrimal sac prior to syringing. If no saline appears from the nose, there is complete obstruction somewhere within the lacrimal drainage system. (45-47)


Recently, there has been considerable debate about the use of CE marked fluorescein strips since Fluorets, the only medically licensed (pharmacy only medicinal product) fluorescein strips, were withdrawn from the global market. However, some fluorescein strips have been classed as medicinal devices and CE marked, but whether they should be regulated as a pharmaceutical, especially when used for diagnosis, has been challenged. In light of the potential consequences of not using fluorescein, such as failure or delay in detecting potentially sight threatening conditions, the Medicines and Healthcare Products Regulatory Agency (MHRA) in the UK have not stopped the sale and supply of CE marked medicinal device fluorescein strips to the UK market until the European Union position on these strips has been clarified. The Optical Confederation (September 2013) has issued a clinical consensus panel statement where optometrists and contact lens opticians may currently use CE marked fluorescein strips within their normal scope of practice in the UK until further notice. However, 0.5ml unit dose fluorescein eye drops (minims 1% and 2%) may continue to be used in the UK as they are classed as licensed medicinal products.


Fluorescein is a widely used ophthalmic dye owing to its high visibility in low concentrations and benign nature upon topical application. Although most slit lamps are not optimised for fluorescence observation, clinically useful levels can be achieved with moistened filter paper strips and 1% minim formulations. Fluorescein is most commonly used to measure intraocular pressure and assess ocular surface integrity by evaluating pooling and staining patterns, but the mechanism of the latter is not well understood. In addition, specific tests for diagnosis of dry eye, penetrating ocular trauma, and lacrimal drainage function can also be assessed with fluorescein. Lissamine green and rose bengal are useful adjuncts to assess conjunctival and lid margin damage.

Course code: C-36209 | Deadline: May 23, 2014

Learning objectives

To be able to understand the appropriate use of diagnostic drugs for examination of the tear film and anterior eye (Group 3.1.10) To be able to understand the use of diagnostic dyes when undertaking contact lens aftercare (Group 5.2.1) To be able to understand the use of diagnostic drugs to identify external pathology (Group 6.1.4)

Learning objectives

To be able to understand the use of diagnostic drugs to assess the tear film and anterior eye (Group 3.1.2)

To be able to understand the use of diagnostic dyes when undertaking contact lens aftercare (Group 5.2.2)

Learning objectives

To be able to understand the use of diagnostic drugs to assess the tear film and anterior eye (Group 3.2.4)

To be able to understand the use of diagnostic dyes when undertaking contact lens aftercare (Group 5.3.1)

Learning objectives

To be able to understand the use of diagnostic drugs to evaluate the severity of ocular r surface disease (Group 1.1.1)

To be able to understand the use of diagnostic drugs to assess the tear film and anterior eye (Group 2.1.2)


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Dr Paramdeep Bilkhu is an optometrist and post-doctoral researcher with numerous publications in peer-reviewed journals. He is the postgraduate module leader for the therapeutics and prescribing course at Aston University.

Dr Shehzad Naroo is a senior lecturer at Aston University. He is the Editor-in-Chief for the journal Contact Lens and Anterior Eye and Global President of the International Association of Contact Lens Educators. He holds Fellowships of International Association of Contact Lens Educators, the American Academy of Optometrists, and the British Contact Lens Association, European Academy of Optometry and Optics and the College of Optometrists.

Professor James Wolffsohn is deputy executive dean for life and health sciences at Aston University, teaching and researching in the field of anterior eye. He has been awarded fellowships from the International Association of Contact Lens Educators, the American Academy of Optometrists, the British Contact Lens Association, and the College of Optometrists.
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Title Annotation:1 CET POINT
Author:Bilkhu, Paramdeep; Naroo, Shehzad; Wolffsohn, James
Publication:Optometry Today
Article Type:Essay
Geographic Code:4EUUK
Date:Apr 25, 2014
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