Ophthalmic Drugs Aston Lecture Course Notes Diagnostic Dyes 1

DIAGNOSTIC DYES

S1 The use of ophthalmic diagnostic dyes is the first example of clinical ‘Utilisation’ of
ophthalmic drugs in the framework AFLUC. They are invaluable in clinical assessment when
superficial ocular surface damage or disease is present or if tear function is deficient. In
these situations the corneal and conjunctival epithelial surfaces and/or extracellular
junctions are damaged, altered or compromised in some way and the properties of
ophthalmic dyes can be used to indicate the extent and nature of the condition.
Diagnostic dyes can also be utilized in contact lens fitting – (consult your Anterior Eye
module for further information on specific applications) – and to facilitate a range of further
investigative techniques. Generally soft contact lenses should be removed when using
diagnostic dyes as they can cause lens discolouration and reinserted following irrigation with
saline if necessary (see however use of Fluorexon below). Fluorescein is also used in
ophthalmology to assess retinal vascular function using fluorescein angiography.

S2 The three principal dyes relevant to general optometric practice are described below. The
legal class of paper strips impregnated with fluorescein and Rose Bengal is that of a
Medical Device (Class 1) and carry a ‘CE’ marking. Fluorescein and Rose Bengal are legally
classified as P drugs in aqueous form. The legal status of Lissamine is presently unclear.

S3 Fluorescein sodium
Fluorescein (NaFl i.e. sodium fluorescein but commonly abbreviated to fluorescein) is a
xanthene dye that has the specific property of fluorescence when exposed to short
wavelength light. It is the most widely used diagnostic dye by optometrists and as mentioned
previously it has legal classification P in aqueous form and ‘Medical Device’ when in paper
strip form. Note the correct spelling if fluorescein (i.e. does not end with an ‘e’!

Uses:
1) Assess corneal integrity: it is used as a diagnostic dye for detecting damage to the
corneal epithelium (indicated by a bright green ‘stain’) or conjunctiva. Corneal and
conjunctival integrity can be affected by infection (e.g. microbial keratitis), inflammation (e.g.
UV keratitis), trauma, dry eye, toxicity (e.g. to ophthalmic preservatives or systemic
medication) and foreign bodies.
2) Applanation tonometry: It has a major use in applanation (eg Goldmann and Perkins)
tonometry and is usually applied with a topical anaesthetic (e.g. proxymetacaine or
oxybuprocaine) or combined in a single preparation (e.g. with lidocaine).
3) Contact lens fitting: refer to your Anterior Eye Module. Although fluorescein is mainly
used as an aid in the fitting of rigid contact lenses it can also be used in the fitting of soft
 Ophthalmic Drugs Aston Lecture Course Notes Diagnostic Dyes 2

lenses but only in a high molecular weight special polymer form (i.e. Fluorexon) otherwise it
can enter the pore structure of the hydrogel material and remain within the lens for several
hours (this property however can be used to locate within conjunctival fornices a ‘lost’ or
‘split’ soft contact lens).
4) Tear BUT - as fluorescein makes the tear film more visible it can be used to assess the
dynamics of tear fragmentation over time - refer to your Anterior Eye Module.
5) Nasal duct patency – the rate at which fluorescein is dissipated from the conjunctival sac
can be used as an approximate measure of tear flow. It is occasionally used to demonstrate
the patency of the lacrimal drainage system; the appearance of fluorescein will be evident
via nasal discharge if the nasal duct passages are viable. For this application 1 or 2%
aqueous solutions of Minims fluorescein are generally used.

S4 Mode of Action:
The use of the term 'staining agent' for fluorescein is a misnomer as the consensus is that it
does not actually 'stain' tissues but is a pH dependent indicator dye by virtue of high visibility
in low concentrations. It should be noted however that the exact mechanism whereby
fluorescein actually 'stains' damaged cells is equivocal.

S5 Fluorescein paper strips are orange in colour but dilution by the precorneal tear film causes
the colour to change to yellow/green. The intact corneal epithelium (having a high lipid
content) resists penetration of the water soluble fluorescein and is not 'stained' by it as such.
Fluorescein has a high degree of ionization at physiological pH (i.e. is very water soluble).

S6 A corneal abrasion, however, causes a break in the epithelial barrier and permits access of
the fluorescein to areas around Bowman's membrane (not in the actual membrane which is
very tough but around the membrane) and one theory is that the greater concentration of
aqueous humour in these regions causes fluorescein to assume a bright green colour when
viewed under white light as the mix of tear fluid and aqueous humour is more alkaline than
the tear film (i.e. it has a pH of approximately 8 – which the optimum level for maximum
fluorescence of fluorescein).

The green colour is particularly enhanced when viewing through filters such as a colbolt blue
filter (which allows transmission of shorter wavelengths) – provided for example by a Burton
lamp - due to fluorescein’s property of fluorescence (although normal white light is also
effective).

Dilute concentrations of fluorescein have a peak absorption between 485 and 500nm. The
absorbed light energy excites the fluorescein molecules (known as fluorophores) which have
the property of re-emitting temporarily more light energy at a longer wavelength of between
 Ophthalmic Drugs Aston Lecture Course Notes Diagnostic Dyes 3

525-530nm (hence a bright green ‘fluorescence’ is observed). Generally the degree of

fluorescence will vary with the thickness, quality and quantity of the tear film.
When the epithelial surface regenerates the green colour disappears and the rate of change
of green appearance can be used to monitor recovery of the cornea.

S7 Irrigating the eye with normal saline solution to remove excess fluorescein can make the
damaged areas of the cornea more visible (as fluorescein has the property of high visibility
at low concentrations).
A short wait post-instillation (without irrigation) may also optimize the visibility of the ‘stained’
area as, over time and with blinking, excess fluorescein in the tears (which can mask
visibility of the area to be ‘stained’) is reduced; the volume of natural tears relative to
fluorescein thus increases i.e. the concentration of fluorescein decreases and hence its level
of dilution increases. Although now diluted, the residual amount of fluorescein present still
maintains its ability to produce fluorescence of the area of damaged tissue owing to its
property of high visibility under conditions of high dilution.

S8 A high proportion of the light transmitted through a colbalt-blue filter falls in the 485-500nm
range and will thus enhance the fluorescence effect.

S9 A yellow Kodax Wratten number 15 (or 12) barrier filter – interposed in the observation
system of the slit lamp - can also enhance visibility when used in conjunction with a colbalt
blue filter as, being yellow, it absorbs the blue light dispersed by the colbalt filter which can
reduce the visibility of the image.

S10 Adverse reactions:

Ocular adverse reactions to topical instillation of fluorescein are extremely rare. Systemic
adverse reactions are also very rare with one case of anaphylaxis* being reported in 2010 –
this was to a second instillation of 2% aqueous fluorescein being reported. Nevertheless, it
is always important to take a good medical and drug history for every patient. In particular, a
reported history of multiple drug and food allergies is a significant finding, especially if these
allergens have triggered anaphylaxis on previous occasions. The intravenous use of
fluorescein 10% in ophthalmology for retinal angiography can cause severe systemic
reactions including, on rare occasions, anaphylaxis.
*Anaphylaxis is a relatively rare but severe allergic reaction that can occur within minutes or
hours to a variety of agents (e.g. insect bites, foods, medications) and requires immediate
medical attention. It is a protective immune response (ana= ‘against’; phylaxis = ‘protection’)
that induces itchy skin rash, swelling of the throat and low blood pressure.
 Ophthalmic Drugs Aston Lecture Course Notes Diagnostic Dyes 4

S11 Fluorescein is often used when the eye is vulnerable and therefore paper strips are a
particularly effective way of using fluorescein in general optometric practice for the following
reasons:
1) fluorescein is particularly susceptible to the virulent pathogen pseudomonas aeruginosa
which is very resistant, if infection occurs, to a wide range of antibiotic treatments (although
all solutions are susceptible to pseudomonas aeruginosa to some degree). Aqueous-free
imprenated paper strips are therefore at less risk of contamination;
2) fluorescein is often used diagnostically when some degree of damage to the cornea has
occurred - hence the cornea is more susceptible to bacterial invasion and this is minimized
with paper strip administration;
3) Paper strips are a very economical way of instilling fluorescein;
4) Although fluorescein is invariably used in preservative-free preparations such as
fluorescein Minims it should be noted that the action of common preservatives in
ophthalmic solutions eg benzalkonium and chlorobutamol may be inhibited by fluorescein.

S12 Preparations available:

Fluorescein may be applied to the eye from Minims (B&L) 1 or 2% but much more
common is instillation via a sterile paper strip of fluorescein (1mg) – i.e. impregnated filter
paper (available as I-Dew ROSE (Mitron Medical, 1.0mg) or as Bio Glo U.S.P.) The strips
should be moistened with sterile saline solution (see earlier lecture on administration). High
molecular weight fluorexon (see above) is also available in sterile paper strip form as Soft
Glow Strips  U.S.P.(0.5mg fluorexon disodium) and, in the USA, as a single dose
preparation 0.35% aqueous preparation, Fluoresoft. Fluorescein (0.25%) is also available
in Minims (B&L) in a combined preparation with the topical anaesthetic lidocaine (4%).

S13 Lissamine Green

In recent years Lissamine Green has been introduced to UK general optometric practice
although its legal status for clinical use in the UK was previously (September 2016) unclear
but is now classed as a Medical Device (MD) in paper strip form.

S14 Uses:
Lissamine Green may be used in general optometric practice for dry eye assessment and
assessment of contact lens wearers (especially those with skin conditions associated with
dry eye such as eczema and rosacea). Its principal application is to demonstrate staining of
the conjunctiva in dry eye conditions rather than of the cornea – the dry eye condition has to
be relatively severe for Lissamine Green to stain the actual cornea. Lissamine Green is also
used to observe staining of the palpebral conjunctiva caused by excessive friction between
lid and conjunctival surface in ‘lid wiper epitheliopathy’.
 Ophthalmic Drugs Aston Lecture Course Notes Diagnostic Dyes 5

As Lissamine Green staining fades relatively quickly examination should be carried out
successively between eyes (i.e. one eye at a time). It is necessary to instil an adequate
volume (i.e. ~10-20ul, nb a standard eye drop is around 30ul) of Lissamine Green to permit
adequate staining to take place and, to avoid ‘bleaching’ of the stained area, not to view the
stained area with too bright a light source. At least a minute but no more than 4 minutes is
the period required for optimum staining to occur. The appearance of staining can be
enhanced by the use of a red Wratten 25 filter placed in the observation system.

S15 Mode of Action:

Lissamine Green is a synthetic dye that has similar staining characteristics to Rose Bengal
(see below) but causes significantly less stinging (and hence reflex tearing). It is more
specific to staining dead/devitalised cells than Rose Bengal. Lissamine Green will also stain
lipid-like structures such as mucous strands and can be utilized in filamentary keratitis.

Adverse reactions:
Lissamine Green is very well tolerated and when delivered via paper strips is of a
concentration much less likely to produce a toxic response than Rose Bengal.

Preparations available:
It is available in paper-impregnated sterile paper strips which are moistened with sterile
saline to provide an adequate volume.

S16 Rose Bengal

Rose Bengal is now rarely used in general optometric practice principally as it produces very
marked stinging and irritation, particularly in patients with dry eye (which is when it is most
likely to be used in specialist clinics). Rose Bengal is classed a Medical Device in paper strip
form but as a P medicine in aqueous form.

S17 Uses:
The main clinical use is to identify the conjunctival lesions of keratoconjunctivitis sicca (KCS)
which presents as an inter-palpebral limbus-based triangle of staining (with only minor
corneal involvements). As with other diagnostic dyes Rose Bengal is able to penetrate soft
contact lenses and therefore should not be used when soft contact lenses are worn.

Mode of Action:
Although Rose Bengal has a similar molecular structure to fluorescein in that it is also a
xanthene dye, it has significantly different staining properties. It is generally thought to
selectively 'stain' glycocalyx deficient and dead (i.e. devitalized) corneal cells, conjunctival
 Ophthalmic Drugs Aston Lecture Course Notes Diagnostic Dyes 6

epithelium cells and mucous strands a bright red colour. Hence in contrast to fluorescein the
cells and their nuclei are actually stained by Rose Bengal.

Adverse reactions:
Rose Bengal stings excessively and the resultant reflex lacrimation stains the dead
squamous epithelium present on the surface of a patient's cheek a persistent bright crimson
colour; hence it is advisable to use only the minimum amount necessary.

Preparations available:
Rose Bengal is no longer available in Minims form (1%) but is available in paper sterile
strip form as I-Dew ROSE (Mitron Medical, 1.5mg) and Rose Glow. The paper strips are
impregnated with 1.5mg of Rose Bengal and are moistened with sterile saline. As indicated
earlier Lissamine Green is generally preferred to Rose Bengal in general optometric
practice.

CLINICAL ARTICLE (access via Reading List on Blackboard):

Paramdeep Bilkhu, Shehzad Naroo and James Wolffsohn. Use of fluorescein in optometric
practice. in Optometry Today 25/04/2014.

S18 NEXT TIME

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