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Commissioned Article
Use of dyes in ophthalmology
Atul Kumar, M B Thirumalesh
Dyes are used in ophthalmology, both as diagnostic and therapeutic aid. The use of diagnostic dyes represents one
of the most efficient, objective, non-invasive, and directly visible means we have of identifying and tracking ocular
structures at the cellular level. They particularly are useful as both diagnostic modalities and as therapeutic adjutants
in both anterior and posterior segment disorders.
Key words: Brilliant blue G, flourescein, triamcinolone, trypan blue
Dyes are used in ophthalmology, both as diagnostic and
therapeutic aid. The use of diagnostic dyes represents one of
the most efficient, objective, non-invasive, and directly visible
means we have of identifying and tracking ocular structures at
the cellular level. They particularly are useful as both diagnostic
modalities and as therapeutic adjutants in both anterior and
posterior segment disorders. Since the advent of flourescein,
the diagnosis and management of retinal vascular disorders has
been revolutionized; however, it has been used in various other
disorders in anterior segment also. In this review article, we would
like to give a brief note on the uses of dyes in ophthalmology.
In dry-eye diagnosis and clinical trials, the utility of these
dyes also extends far beyond dry eye to numerous other ocular
surface conditions that affect corneal and conjunctival cells. The
three dyes used most commonly in the eye-care practitioner’s
office today are fluorescein, rose bengal, and lissamine green.[1]
Fluorescein: It is particularly valuable as an assessment tool
in clinical studies of dry eye. The water-soluble dye molecules
diffuse into the intercellular spaces between living cells. The
intensity of the stain is increased in areas of cellular degeneration
or death, where the damage to cells, cell membranes, and cell-
to-cell junctions allow for the intracellular spaces to be more
highly penetrated by the dye. Standardized grading of corneal
and conjunctival fluorescein staining as well as measurement
of tear-film breakup times, which have been made more clearly
visible using fluorescein, have given this dye broad applicability
as a dry-eye diagnostic test.
Fluorescein is also used in identifying and monitoring corneal
epithelial defects, corneal ulcers.
It is also used in applanation tonometry while using either
Department of Ophthalology, AIIMS Institution, New Delhi, India
Address for correspondence: Dr. Atul Kumar, Room # 494, 4th Floor,
Dr. R. P. Centre AIIMS, Ansari Nager, New Delhi, India. E-mail:
atul56kumar@yahoo.com
Manuscript received: 19.10.2012; Revision accepted: 07.11.2012
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Goldmann tonometer/Perkins hand-held tonometer.
Seidel’s test: Concentrated fluorescein dye (from a moistened
fluorescein strip) is applied directly over the potential site of
perforation/bleb in cases of operated trabaculectomy while observing
the site with the slit lamp. If a perforation and leak exist, the
fluorescein dye will be diluted by the aqueous and will appear as a
green (dilute) stream within the dark orange (concentrated) dye. The
stream of aqueous is best seen with the blue light of the slit lamp.
Fluorescein dye can be used in Jones dye disappearance test
for assessment of lacrimal passage functional potency.
This dye can be injected in the lacrimal apparatus using a
syringe for identification of canlicular ends in traumatic laceration
of the lid margins and for repair of the canaliculus.
Rose Bengal: Rose bengal is actually a derivative of fluorescein.
It’s been used for the evaluation of numerous other ocular
pathologies including herpetic corneal epithelial dendrites,
superficial punctate keratitis, meibomian gland dysfunction,
and dysplastic or squamous metaplastic cells of conjunctival
squamous neoplasms. Research on rose bengal has revealed that
it’s blocked from staining the ocular surface where molecules
such as mucins, albumin, or even an artificial tear compound
such as carboxymethylcellulose are present. Rose bengal has also
been discovered to have intrinsic cellular toxicity. Studies have
shown that rose bengal has a dose-dependent, toxic effect on
human corneal epithelial cells in vitro that is further enhanced by
light exposure. Lastly, it’s widely known that patient discomfort,
particularly stinging upon instillation, which can become severe,
is often a deterrent from using rose Bengal.[2]
Lissamine green: It preferentially stains membrane-damaged
or devitalized cells, and, like rose bengal, localization of the dye to
the cell nucleus has been noted. However, lissamine is unique in
this group of three in that it has not been shown to stain healthy
ocular surface cells. Evaluation of lissamine green staining in both
rabbit and human corneal epithelial cells in vitro revealed that it
doesn’t stain healthy, proliferating cells and has a minimal effect
on cell viability.[2] There is no stinging or discomfort such as that
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Kumar and Thirumalesh: Use of dyes in ophthalmology
associated with rose bengal.
Dyes used for Posterior Segment Diagnosis
and Evaluation
Fluorescein is also used extensively in photographic retinal
vasculature imaging during fundus flourescein angiography.
The dye sodium fluorescein can be used either in concentration
of 10% or 20% in the form of intravenous bolus injection.It is
used to image retinal, choroidal, optic disc, or iris vasculature,
or a combination of these. It is used diagnostically as well as in
planning for many retinal laser procedures. It has a very important
role in management of diabetic retinopathy, vein occlusion, and
age-related macular degeneration. It is also used extensively in
diagnosis of macular ischemia.
Indocyanine green angiography (ICG: tricaboxycyanine
dye) isphotographic method of ocular angiography similar to
fluorescein angiography; however, the contrast agent is ICG
rather than fluorescein. It is used for imaging choroidal and
retinal bloodvessels. Compared with fluorescein angiography,
ICG provides better resolution of choroidal vasculature, but lesser
resolution of retinal blood vessels. ICG is used in evaluating
suspected occult choroidalneovascular membranes (CNVMs), and
can also be used to identify recurrence of CNVM after treatment
and choroidal polyps (PCV: polypoidalchoroidalvasculopathy).
Dosages up to 40 mg IC-GREENÔ (tricarbocyanine) dye in 2 mL
of aqueous solvent have been found to give optimal angiograms,
depending on the imaging equipment and technique used. The
antecubital vein injected IC-GREENÔ (tricarbocyanine) dye bolus
should immediately be followed by a 5 mL bolus of normal saline.
Dyes used in Ophthalmic Surgery
Two of the basic tenets of ophthalmic surgery are exposure
and visualization. In ophthalmic surgery, we often take these
two principles for granted because the eye is readily accessible
and we can usually see the pathology directly. This is especially
true when we are performing corneal, cataract, and retinal
procedures. However, any clouding of the ocular media can
interfere with the quality of our view during intraocular surgical
manipulations. Ophthalmic surgical dyes have become valuable
tools and are now widely used for both anterior and posterior
segment indications.
Dyes may be designated vital when they are used to stain living
tissues or cells. In ophthalmology, vital dyes have become effective
and useful surgical tools for identifying ocular tissues.
Cataract Surgery
The continuous curvilinear capsulorrhexis is the most difficult
as well as the most critical step in modern cataract surgery. A
properly constructed capsulorrhexis maintains the IOL implant
in the proper position and resists radial tears of the capsular bag.
An adequate red reflex is necessary for the surgeon to visualize
the leading edge of the curvilinear capsulorrhexiswith retro-
illumination as the capsulorhexis is created. The capsulorrhexis
edge may be difficult or unable to be seen if the red reflex is
poor or absent. In the case of a mature cataract, not only is there
no red reflex but also milky liquid cortex can escape into the
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anterior chamber, further clouding an already poor view of the
anterior lens capsule. Therefore, prior to capsular dyes, we would
struggle to complete a capsulorrhexis in such instances and would
sometimes have to convert to a can-opener style capsulotomy.
Capsular dyes[3] have dramatically improved our ability to
perform the capsulorrhexis in these circumstances. In fact,
they have eliminated the problem of visualizing the capsule.
Indocyanine green (ICG) has been used but requires preparation
(reconstitution and dilution); it also should be filtered to prevent
undissolved particles from entering the eye. Trypan blue 0.06%
ophthalmic solution is FDA-approved for intraocular surgery
and is available in ready-to-use preloaded syringes for cataract
procedures. The dye is injected onto and painted over the anterior
lens capsule under an air bubble. This produces a blue-stained
capsule that is clearly identifiable throughout surgery. In addition
to cases of a poor red reflex, capsular dyes are extremely helpful
in cases of weak zonules. The use of dye reduces the risk of
capsule-related complications because any radial tear or shift
of the capsular bag is readily apparent from the clearly outlined
capsulorrhexis.
Triamcinolone can be used if a posterior capsular rent occurs
during cataract surgery to know if any vitreous strands are left
in AC after anterior vitrectomy.
Corneal Surgery
Trypan blue 0.06% ophthalmic solution is also used to stain
Descemet’s membrane during DSAEK (Descemet’s stripping
endothelial keratoplasty). It is also used in staining and stripping
the endothelium from the donor lenticule in DALK (deep anterior
lamellar keratoplasty).
Retinal Surgery
Trypan blue is also a helpful aid for posterior segment surgeons
and is utilized for retinal procedures. Visualization of membranes
overlying the retina can be difficult [Figure 1], so trypan blue
0.15% ophthalmic solution (RetiBlue, Auro Labs, Madurai,
India) is useful for identifying and delineating them to allow
complete removal. The dye stains the posterior hyaloid, internal
limiting membrane, and epiretinal membranes blue, making
these structures highly visible against the unstained retina. This
facilitates macular hole and macular pucker surgery, and makes
these procedures safer. The dye stains the pre-retinal membranes
and ILM under air (the dye is injected after fluid air exchange) as
the dilution of the dye is prevented.
Trypan blue now has a well-established role in ocular surgery.
It is readily available, simple to use, and extremely effective. There
is no reason to struggle with poor visualization any more.
Vital Dyes
Triamcinolone acetonide. The state-of-the-art staining agent
for identifying the vitreous is the white steroid triamcinolone
acetonide.[4] Its crystals bind avidly to the vitreous gel, enabling
visualization of a clear contrast between empty portions of
the vitreous cavity and areas, in which vitreous fibers are still
present.
Triamcinolone acetonide is injected into the vitreous cavity
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Kumar and Thirumalesh: Use of dyes in ophthalmology
toward the area to be visualized (0.1 to 0.3 mL, 40 mg/mL [4%]
concentration). Injecting this steroid during vitrectomy for the
management of retinal detachment may prevent fibrin reaction
and proliferative vitreoretinopathy postoperatively. The steroid
improves identification of tissue through the deposition of
crystals, which helps the surgeon achieve complete detachment
and removal of the posterior hyaloid [Figure 2] and improves
the results of primary vitrectomy for management of retinal
detachment[5] and diabetic retinopathy in young patients.
Indocyanine green. Indocyanine green[6] (ICG) and infracyanine
green may be considered the gold standard dyes for staining and
visualizing the ILM in surgical therapy for macular hole and diabetic
macular edema. These dyes possess a great affinity for the matrix
components of the ILM, such as collagen type 4 and laminin.[4]
ICG-guided chromovitrectomy [4] first gained worldwide
popularity, and a number of studies showed ICG-guided peeling
to be easier and less traumatic than surgery without ICG,
demonstrating good clinical results in macular hole surgery.
However, subsequent studies have revealed that ICG may be
toxic to the retina. Clinical data showed that ICG can remain
Figure 1: Non-dye stained ERM peel
Figure 3: BBG stained ILM prior to peeling
Journal of Clinical Ophthalmology and Research - Jan-Apr 2013 - Volume 1 - Issue 1
intravitreally or deposit persistently on the optic disc after surgery
for macular hole. Studies also suggest that ICG can diffuse into
the sub-retinal space through a macular hole, causing damage to
the retinal pigment epithelium.
It has been postulated that the use of ICG at low concentrations
in ILM peeling could be a safer alternative because lower rates of
RPE abnormalities have been observed with ICG at a concentration
of 0.5 mg/mL (0.05%) or less, and an osmolarity of approximately
290 mOsm.
There are many hypotheses about why and how ICG may
induce retinal damage. Intravitreal ICG injections may change
the osmolarity in the vitreous cavity, thereby damaging either
the neurosensory retina or the RPE cells directly. Investigations
in various animal models have shown that ICG may be hazardous
to the RPE or neuroretinal cells. Moderate to high doses (2.5
[0.25%] to 25 mg/mL [2.5%]) of intravitreal ICG were toxic to
retinochoroidal cells, and impairment of retinal function was
described even at low doses of ICG (0.025 mg/mL [0.0025%].
An ICG molecule has approximately 5% iodine in its final
Figure 2: Induction ofposterior hyaloid detachment using Triamcinolone
crystals (preservative –free)
Figure 4: Injection of BBG dye into fluid –filled vitreous for helping
stain the Macula
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Kumar and Thirumalesh: Use of dyes in ophthalmology
solution and no sodium or calcium. Nevertheless, it is has been
suggested that removing sodium from the saline solution used
for diluting the dye may decrease the risk of RPE damage. It has
been speculated that ICG injection into the vitreous cavity may
absorb light; this interaction may lead to a photodynamic effect
that induces retinal damage. It was demonstrated that sub-retinal
ICG injection plus light exposure in rabbits can result in functional
retinal damage and RPE changes.
Once diluted in any solvent and exposed to light, ICG may
undergo various chemical reactions by self-sensitized oxidation
because it is chemically unstable; this phenomenon is called
decomposition. It was demonstrated that, independent of light
exposure, singlet oxygen (photodynamic type 2 reaction) is
generated by ICG, leading to dioxetanes by cycloaddition of singlet
oxygen. Furthermore, dioxetanes thermally decompose into
several carbonyl compounds. Decomposition of ICG was blocked
by sodium azide, a quencher of singlet oxygen. This supports
the rationale for future use of quenchers in chromovitrectomy.
Infracyanine green. Iodine and its derivates may be toxic to
the RPE. Therefore, infracyaninegreen (IFCG), a dye free of iodine
in its formulation either as free ion or as part of the dye moiety,
is believed to have less potential for RPE toxicity than ICG. With
this presumably safer profile, IFCG may represent an alternative
to ICG during ILM peeling in chromovitrectomy due to the lack
of sodium iodine in its formulation and physiologic osmolarity.
Brilliant blue G (BBG), also known as Coomassie or acid blue,
has recently reported to be a safe tool for chromovitrectomy. It
has good ILM staining property and is a non-fluorescent dye. In
humans, brilliant blue causes adequate ILM staining [Figure 3] in an
isosmolar solution of 0.25 mg/mL (0.025%) to 0.50 mg/mL (0.05%)
with good clinical results and no signs of toxicity on multifocal
electroretinogram. This stain has become a good alternative to
ICG and IFCG in chromovitrectomy because of its remarkable
affinity for the ILM. Toxicity data regarding its application are
limited, so further investigations to confirm these observations
are warranted. However, we consider this dye the best one for
ILM peeling in macular hole surgery.[7] It is also used worldwide
despite the fact that there are no clinical trials to support its use
(unpublished data). This dye is injected into post-vitrectomy fluid
filled eyes directly [Figure 4].
Trypan blue. This dye may not enable ILM visualization as well
as ICG, but this blue dye remains an alternative. In order to enhance
the staining properties of trypan blue, the dye may be injected
into the posterior pole after fluid air exchange, or it may be mixed
with glucose 5% to 10% to create a “heavy” trypan blue, which
is denser than balanced salt solution. However, higher glucose
concentrations should be avoided because glucose 50% has a highly
toxic osmolarity of 2020 mOsm/L. It is recommended that trypan
blue be used mainly for ERM staining. Trypan blue has an affinity
for epiretinal glial tissues such as the ERM.[8] Therefore, we consider
trypan blue the best dye for staining the ERM. It is suggested to
mix 0.3 mL of trypan blue with 0.1 mL of glucose 10%, resulting
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in a 1 mg/mL (0.1%) solution with an osmolarity of 300 mOsm.
MembraneBlue-Dual is a dye to stain both the ILM as well as
the ERM and PVR membrane, without compromising the staining
effect within one injection. MembraneBlue-Dual is a 100% stable
mix MEMBRANEBLUE-DUAL: Dye for staining ILM, ERM,and PVR
membranes consists of: Combination of TrypanBlue 0,15%+
BBG 0,025% + 4% PEG. It stains the ILM at the same level as
ILM-BlueÒ and stains the ERM and PVR at a higher level than
the classic MembraneBlue. Due to a new integrated carrier 4%
PEG solution,MembraneBlue-DualÒ can be injected in a BSS-filled
eyeand sinks immediately as a cohesive ball without diffusion
throughout the whole globe.
Double Staining Technique (in macular
ERM eyes)
The double-staining technique is an elegant procedure that may
facilitate negative stain” of the overlying ERM, which does not
stain, but is visible against a surrounding blue-stained ILM. The
non-stained ERM is first peeled off, and then BBG dye is injected a
second time to stain the unstained ILM, which lay directly under
the now peeled ERM, and is now peeled off.
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3. Jacobs DS, Cox TA, Wagoner MD, Ariyasu RG, Karp CL.
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5. Peyman GA, Cheema R, Conway MD, Fang T. Triamcinolone
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6. Rodrigues EB, Meyer CH, Farah ME, Kroll P. Intravitreal staining
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Cite this article as: Citation will be included before issue gets online***
Source of Support: Nil. Conflict of Interest: No.
Journal of Clinical Ophthalmology and Research - Jan-Apr 2013 - Volume 1 - Issue 1