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Year : 2013  |  Volume : 1  |  Issue : 1  |  Page : 55-58

Use of dyes in ophthalmology

Department of Ophthalology, AIIMS Institution, New Delhi, India

Date of Submission19-Oct-2012
Date of Acceptance07-Nov-2012
Date of Web Publication22-Jan-2013

Correspondence Address:
Atul Kumar
Room # 494, 4th Floor, Dr. R. P. Centre AIIMS, Ansari Nager, New Delhi
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Source of Support: None, Conflict of Interest: None

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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.

Keywords: Brilliant blue G, flourescein, triamcinolone, trypan blue

How to cite this article:
Kumar A, Thirumalesh M B. Use of dyes in ophthalmology. J Clin Ophthalmol Res 2013;1:55-8

How to cite this URL:
Kumar A, Thirumalesh M B. Use of dyes in ophthalmology. J Clin Ophthalmol Res [serial online] 2013 [cited 2022 Jul 4];1:55-8. Available from: https://www.jcor.in/text.asp?2013/1/1/55/106288

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 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 More Details 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 associated with rose bengal.

  Dyes used for Posterior Segment Diagnosis and Evaluation Top

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 Top

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 Top

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 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 Top

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 Top

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.
Figure 1: Non-dye stained ERM peel

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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 Top

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 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.
Figure 2: Induction ofposterior hyaloid detachment using Triamcinolone crystals (preservative – free)

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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 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 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].
Figure 3: BBG stained ILM prior to peeling

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Figure 4: Injection of BBG dye into fluid – filled vitreous for helping stain the Macula

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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 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) Top

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.

  References Top

1.Kim J. The use of vital dyes in corneal disease. Curr Opin Ophthalmol 2000;11:241-7.  Back to cited text no. 1
2.Abelson MB, Ousler GW, Nally LA, Emory TB. Dry eye syndromes: Diagnosis, clinical trials, and pharmaceutical treatment' improving clinical trials.' Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3. In: Sullivan D, et al., editors. Kluwer Academic/Plenum Publishers; 2002. p. 1079-86.  Back to cited text no. 2
3.Jacobs DS, Cox TA, Wagoner MD, Ariyasu RG, Karp CL. Capsule staining as an adjunct to cataract surgery: A report from the American Academy of Ophthalmology. Ophthalmology 2006;113:707-13.  Back to cited text no. 3
4.Rodrigues EB, Maia M, Meyer CH, Penha FM, Dib E, Farah ME. Vital dyes for chromovitrectomy. Curr Opin Ophthalmol 2007;18:179-87.  Back to cited text no. 4
5.Peyman GA, Cheema R, Conway MD, Fang T. Triamcinolone acetonide as an aid to visualization of the vitreous and the posterior hyaloid during pars planavitrectomy. Retina 2000;20:554-5.  Back to cited text no. 5
6.Rodrigues EB, Meyer CH, Farah ME, Kroll P. Intravitreal staining of the internal limiting membrane using indocyanine green in the treatment of macular holes. Ophthalmologica 2005;219:251-62.  Back to cited text no. 6
7.Kumar A, Gogia V, Shah VM, Nag TC. Comparative evaluation of anatomical and functional outcomes using brilliant blue G versus triamcinlone assisted ILM peeling in macular hole surgery in Indian population. Graefes Arch Clin Exp Ophthalmol 2011;249:987-95.  Back to cited text no. 7
8.Feron EJ, Veckeneer M, Parys-Van Ginderdeuren R, Van Lommel A, Melles GR, Stalmans P. Trypan blue staining of epiretinal membranes in proliferative vitreoretinopathy. Arch Ophthalmol 2002;120:141-4.  Back to cited text no. 8


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]


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