174 Kerala Journal of Ophthalmology
O C U L A R
PHARMACOLOGY
Vital Dyes For Chromovitrectomy: Colours
for the Vitreoretinal Surgeon!!!
Dr. Meena Chakrabarti MS DO DNB, Dr. Valsa Stephen MS DO DNB, Dr. Sonia Rani John DNB,
Dr. Arup Chakrabarti MS DO
Chromovitrectomy 1 refers to the application of vital
dyes during retinal surgery to visualize preretinal tissues
and membranes. Chromovitrectomy arises from the
difficulty in visualizing and removing the several thin
and transparent tissues in the vitreoretinal interface
including the internal limiting membrane (ILM),
epiretinal membrane (ERM) and the vitreous. These
tissues are involved in the pathogenesis of several
macular disorders including macular holes and diabetic
macular oedema. Surgical manipulation of these poorly
visualized tissues have been shown to induce gliosis,
iatrogenic chorioretinopathy and phototoxicity. Staining
of these tissues with vital dyes may improve their visibility,
enhance the ability to peel them as well as ensure
complete removal of all tissues, which may lead to a better
visual result postoperatively with a lesser recurrence rate.
An ideal vital dye for chromovitrectomy should have
the “ability to selectively stain” the internal limiting
membrane and the epiretinal membrane, leaving the
retina unstained. It should provide adequate colour
difference between the stained ILM/ERM and the
normal retina. Other favorable characteristics are
1) rapid elimination from vitreous cavity 2)
photochemical stability 3) solubility in balanced salt
solution 4) absence of toxicity and 5) an adequate light
absorption profile.
The vitreoretinal interface staining agents have been
used since 2000. They can be classified into three
generations depending on the time of introduction.
Chakrabarti Eye Care Centre, Kochulloor, Trivandrum - 695 011
E-mail: tvm_meenarup@sancharnet.in
Vol. XX, No.
First Generation: (2000): Indocyanine Green2 (ICG)
Second Generation:(2003): Infracyanine Green 3
(IFCG), Trypan Blue 4 (TB) and Triamcinolone
accetonide 9 (TA)
Third Generation (2005): Brilliant Blue G5 (BBG),
Chicago Blue7, Bromophenol blue7, Patent blue8 (PB).
Table 1 gives the comparison of staining characteristics
and structures of various dyes used for chromo
vitrectomy.
Indocyanine Green (ICG)
ICG is a tricarbocyanine anionic dye with a molecular
formula of C43H47N2NaO6S2 and a molecular weight
of 775 Daltons. This green dye has amphiphilic
properties and hence interacts biochemically with
different human tissues. ICG demonstrates greatest
affinity to the extracellular matrix components10 of the
ILM, thereby exhibiting an ability to selectivity stain
the ILM.
Chromovitrectomy using ICG for dye assisted peeling
of ILM gained acceptance for the management of
macular holes11, 12, 13, 14. Its use was later extended to
improve visualisation of the glial ERM15, 16, proliferative
membranes of proliferative diabetic retinopathy (PDR)
and proliferative vitreoretinopathy (PVR)17.
Controversial reports on the toxic effects of ICG have
been published 18. These included Muller cell, RPE18
and ganglion cell damage; visual field defects and optic
atrophy 1, 2,17,18.
June 2008 M. Chakrabarti et al. - Vital Dyes
Histopathology of the peeled ILM after ICG assisted
ILM peel during chromovitrectomy revealed the
presence of cellular structures on and under the ILM19,20.
Detection of the presence of retinal elements (plasma
membrane of Mullers cells, myofibrocytes, astrocytes)
on the peeled ILM raised the issue of retinal damage
during peeling. Animal studies and in-vitro experiments
indicated a dose dependent ICG mediated toxicity to
retinal elements (Mullers cells, ganglion cells,
photoreceptors and RPE) 11, 21-24. The hypotonic ICG
solution has been shown to cause osmotic damage to
the retinal cells.
Exposure to ICG causes damage to the photoreceptors
and RPE cells leading on to apoptosis or necrosis due
to light induced damage 25-27. Subretinal injection of
ICG in rabbit models may result in RPE damage even
in concentration as low as 0.5 mg/ml.
There is paucity of published data comparing the effect
of ILM peeling with and without the use of ICG.
Majority of studies (Table 2) used ICG in higher
concentration and this factor could be responsible for
the toxicity. We suggest three safety measures when
using ICG for ILM peeling.
Table 1. Properties of dyes used for chromo vitrectomy
175
1. Perform a fast surgical procedure in order to minimize
the duration of contact with RPE cells and to minimize
the ICG exposure to light from endo illuminator.
2. Use ICG concentrations lower than 0.5 mg/ml to
minimize the risk of RPE damage and possible
retinal toxicity.
3. Avoid ICG injection direct through the macular hole
by any method to control ILM staining (slow
injection, use of the 20 gauge, prototype painting
brush called vitreo retinal internal limiting
membrane color enhancer (VINCE) 31, or by use of
perfluorocarbons over the macular hole etc).
Infracyanine Green (IfCG)
IfCG possesses two well recognized pharmacological
differences from ICG, which vouches for its safe profile
in chromovitrectomy 32-35.
1. IfCG is produced in a synthesis mode without sodium
iodine. High dose of topical or intraocular iodine can
induce severe corneal and retinal damage.
2. the presence of sodium iodine in the ICG solution
requires dilution in water resulting in a hypotonic
176 Kerala Journal of Ophthalmology Vol. XX, No. 2

Table 2. Meta analysis of Articles on ICG use.

Sl.No Author Year Procedure Effect Complication
1 Sheidow et al 10
2003 MHS Worse visual acuity
when ICG used as adjuvant
2 Gass et al13 2003 MHS Worse visual acuity when
ICG used as adjuvant
3 Ando et al14 2004 MHS Worse visual acuity when
ICG used as adjuvant Optic Atrophy
4 HillenKamp J et al16 2006 ERM No difference with and
without ICG Optic Atrophy
5 Lochead et al28 2004 MHS No adverse effects
6 Slaughter and Lec29 2004 MHS No adverse effects
7 Rodrigues and Meyer30. 2007 MHS No difference in anatomic
outcome. Worse functional outcome

solution of 248-275 m mol/kg. The iodine free IfCG is
dissolved in 5 % glucose solvent generating an iso
osmotic solution of 294 – 314 m mol /kg 36,37.
IfCG in concentrations of 0.5 mg/ml selectively stains
the ILM just like ICG and has a much safer profile for
intravitreal use.
Trypan Blue
The anionic hydrophilic azo dye trypan blue has a
chemical formula C34 H24 N6 O14 S4 and a molecular
weight of 960 Daltons.
This vital stain traverses the cell membrane in dead
cells thereby staining the dead tissues blue. In the 1990s
Trypan blue was first used intraocularly to stain the
anterior lens capsule to facilitate capsulorrhexis for
cataract surgery 38 . The use of trypan blue in
chromovirectomy is limited to the staining of ERM. It
stains the ILM minimally sometimes necessitating
repeated application to facilitate visualization. Trypan
blue staining of ERM helps mark out its entire extent
thereby ensuring complete removal.
Clinical studies 39,40 have clearly demonstrated that
Trypan blue exerted little or no toxic effects on the
retina. Experimental data, however disclosed evidence
of retinal toxicity following trypan blue staining. Luke
et al 41 reported irreversible damage to the retina after
exposure to trypan blue in a bovine model. In contrast
to this report Jin et al42, and Narayanan et al 43, showed
that damage to the rodent neurosensory cells was dose
dependent and the toxicity could be eliminated at lower
doses. A new indication for the use of trypan blue was
to stain the edges of an open retinal tear on subretinal
administration 44 facilitating its identificaion during
vitrectomy.
Triamcinolone Acetonide (TA)
Triamcinolone Acetonide (TA) is a synthetic insoluble
corticosteroid, with a chemical formula C24H31 FO6 and
a molecular weight of 434 daltons. The white steroid
suspension has been used for chromovitrectomy since
2003 to visualize the transparent vitreous gel45 and the
posterior vitreous cortex. Guo 46 et al compared the
effectiveness of four biostains (triamcinolone acetonide,
indocyanine green, trypan blue and sodium fluoroscein)
in delineating the vitreous and reported the best
visibility and contrast following use of triamcinolone
acetonide. In addition triamcinolone crystals get
deposited on the ERM and ILM making their
identification and peeling easier. The safety of
triamcinolone accetonide 47-50 to the retina has been
demonstrated by several invivo and in- vitro studies.
Marison VL 46 et al demonstrated that the vehicle
(benzyl alcohol) can induce toxicity to the retina in
rabbit models and hence the use of preservative free
triamcinolone acetonoide is recommended.
Patent Blue
Patent Blue is a hydrophilic anionic triylmethane dye
with a chemical formula of C27 H31N2NaO6S2 and a
molecular weight of 582 Daltons. This dye has been
used to stain the anterior capsule during cataract
surgery 17 in a concentration of 0.24 % . Patent blue
exhibit minimal systemic toxicity, carcinogenicity and
June 2008 M. Chakrabarti et al. - Vital Dyes
Table 3. The techniques used for staining the ILM.
Sl.No Technique Procedure
1 Air- filled Technique FAE to remove fluid from
vitreous cavity before dye injection.
2 Fluid- filled technique Inj of dye intravitreously into the
BSS/ RL filled eye.
3 VINCE (Vitreoretinal Painting brush constructed of
Internal limiting a silicone tube connected to a
membrane color 20 G metal cannula, Diluted
enhancer.) dye in silicone cartridge.
mutagenicity 51. Preliminary clinical data demonstrates
a moderate affinity of patent blue to ERM and vitreous
and a poor affinity to the ILM 52. Toxicity studies
revealed conflicting reports on retinal toxicity of patent
blue. Luke et al 53 demostrated that patent blue
exhibited mild and reversible retinal toxicity, whereas
Westermeier et al 54 showed that RPE cells exposed in
vitro to patent blue showed no toxicity. Analysis of
available data indicate a safer profile for patent blue in
comparison to trypan blue, however the exact safe
dosage of patent blue for intravitreal injection remains
unclear.
Brilliant Blue G
Briliant blue G, also known as Coomassie or acid blue,
is a blue biostain with a chemical formula
C47H48N3S2O7Na and a molecular weight of 854 Daltons.
Human and animal studies on the use of BBG for
chromovitrectomy and anterior lens capsular staining
55,56
were published in 2006. These study results indicate
a safe clinical profile for both capsular staining and
chromovitrectomy. Absence of corneal endothelial cell
damage, no significant retinal pathological changes on
light and electron microscopy, no reduction in ERG
waves, and no clinical evidence of long term toxicity,
were the hallmark results of these studies 55,56,57.
The remarkable affinity to the ILM and absence of
toxicity makes it a first real alternative to ICG and IfCG.
Bromophenol Blue (BrB)
(Tetrabromophenosulfonaphthalein) has a molecular
weight of 670 Daltons and a chemical formula
C19H10Br4O5S.
In cataract surgery BrB represents an appropriate
177
Advantages Disadvantages
Concentrates dye at Higher retinal toxicity.
posterior pole.
Immediate dye washout Less staining as dye is
lesser retina toxicity. washed out rapidly.
Selective staining Not patented.
biostain at a concentration of 1.2 % to stain the anterior
lens capsule intensively facilitating easy removal.
Preclinical experiments on six novel vital dyes for
chromovitrectomy (Light green yellowish, E68, BrB,
Chicagoblue, Rhodamine, Rhodulin blau- basic)
showed that BrB stained the ERM and ILM better, and
was free of toxicity at concentration of 1.2 % and
0.02 % 58. Similar reports have been obtained from
histopathological studies 59,60. At a higher concentration
of 1 % and 2 % enhanced ILM colouring was possible.
Further human clinical data on its safety profile and
defining its dosage and indications in chromovitrectomy
is awaited.
Sodium Fluorescein
Sodium Fluorescein is a hydrophilic xanthene dye with
a chemical formula C20 H 10 Na 2O5 and a molecular
weight of 376 Daltons. Abrams And coworkers 61 in
1978 demonstrated the efficacy of intravitreally injected
sodium fluroscein in staining the vitreous thereby aiding
its complete removal. The toxicity of sodium fluroscein
to the retina has not been reported.
Fluoromethalone Acetate (FMA)
Fluoromethalone Acetate (FMA) is a synthetic
fluorinated glucocorticosteriod with an empirical
formula C24 H31 FO5 and a molecular weight of 418
Daltons. Hata et al 62 performed pre-clinical
investigations of flurometholone acetate as a potential
new adjuvant during vitreous surgery. They found
neither reduction in ERG or histological changes
following the use of Fluromethalone acetate and
concluded that FMA could be used as an alternative to
TA during chromovitrectomy.
178 Kerala Journal of Ophthalmology
Key issues
1. Chromovitrectomy improved the visualisation of
preretinal structures in vitreoretinal surgery.
2. Intravitreal injection of dyes appear to be the most
feasible approach to stain the vitreous and preretinal
tissues.
3. Lower dye concentration and shorter exposure time
can limit side effects.
4. Various technique are available to stain ILM (Table 3)
Newer vital dyes exhibiting selective staining of
preretinal tissue, and no retinal staining are less toxic
to the retina.
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