Case Report:Current Dyes in Chromovitrectomy: Pros and Cons

Current Dyes in Chromovitrectomy: Pros and Cons

The application of dyes to stain preretinal tissues during chromovitrectomy has become a widespread technique among vitreoretinal surgeons.1 The introduction of the first vital dye in chromovitrectomy, indocyanine green (ICG), facilitated the visualization of the fine and transparent internal limiting membrane (ILM).2 Subsequently, trypan blue (TB) was proposed as a helpful tool in identifying several epiretinal membranes (ERM),3 and the intravitreal steroid triamcinolone acetonide (TA) was found to stain the vitreous.4 Recently, other dyes, including infracyanine green (IfCG), patent blue (PB), and sodium fluorescein (SF), were proposed for use as alternative dyes during chromovitrectomy.

The intraoperative utilization of vital dyes for staining of human tissues has been established for more than 3 decades.5 The use of vital dyes in ophthalmology to stain retinal tissues dates back to the last century. Beginning with Sorsby in 19396 and later Gifford in 1940,7 ophthalmologists began injecting Kiton-fast-green V, Xylene-Fast-green B, and fluorescein intravenously. In 1969, Kutschera began injecting PB intravitreally to stain retinal tissue and determine retinal breaks in retinal detachments.8

Chromovitrectomy was motivated by the difficulty in visualizing the several thin and transparent tissues in the vitreoretinal interface. The ILM, ERM, and the vitreous are involved in the pathogenesis of macular diseases, including idiopathic macular hole and diabetic macular edema. Excessive surgical vitreoretinal manipulation during peeling of the preretinal membranes has been shown to induce gliosis formation, iatrogenic chorioretinopathy, and light toxicity.9 Staining of the ILM, ERM, and the vitreous with a dye may assist in the visualization of those fine transparent tissues, thereby leading to improvement in the postoperative visual acuity (VA).

This review presents the current knowledge involving the use of dyes in chromovitrectomy, the advantages and disadvantages of each dye for chromovitrectomy, the surgical recommendations to minimize the risk of toxic effects, and the introduction of a new instrument for a selective application of dyes in chromovitrectomy.

Indocyanine Green

Indocyanine green is a sterile tricarbocyanine with the chemical formula C43H47N2NaO6S2 and molecular weight 774.96 Daltons. The dye has amphiphilic properties, which interact with different molecular species and manifest different light emission spectra. Indocyanine green is applied in ophthalmologic angiography for imaging retinal and choroidal tissues.

Indocyanine green was first reported to stain the anterior capsule of the lens for the capsulorrhexis. Experiments in cadaveric eyes have demonstrated ICG to promote visualization of the ILM,2 and since then many articles were published on the use of ICG for chromovitrectomy.10-12 Indocyanine green-assisted macular hole surgery demonstrated variable closure rates (76.4%­100%) and VA improvements (18%­94.1%). Studies regarding the use of ICG in the treatment of idiopathic epiretinal membranes showed conflicting visual outcomes varying from 54.5%­93%.13

For intraocular application, the ICG powder must be dissolved in a glucose/water solution prior to injection, as dilution in sodium chloride induces dye precipitation. The usual dilution of ICG for intravitreal injections is 0.5%, which is achieved by dissolving it in 5 mL water, then 1 mL of this concentration is diluted in a 4 mL balanced salt solution. Viscoelastic material may also be used instead of fluid as an ICG-carrying medium to be prepared with a final concentration of 0.175%.

The target tissues of ICG in chromovitrectomy are well established. Most clinical and histologic evidence support that ICG stains the ILM. In contrast, the cellular environment of ERM should not allow ICG to bind to its surface, and the highly cellular ERM stains negatively or irregularly by the hydrophilic ICG. If the ERM is thick, a green color may come from an extra-cellular accumulation of ICG on the ERM. The advantage of ICG staining for surgical treatment of ERM is the staining and peeling of adjacent ILM around the ERM, as well as the ability to completely remove the entire ILM-ERM-complex.14

Soon after the introduction of ICG for the treatment of IMH, criticism and doubts replaced enthusiasm, as Gandorfer and colleagues postulated in 2001 that intravitreous ICG-assisted ILM-peeling may damage the retinal tissue.15 Another concern related to ICG use refers to a possible higher incidence of retinal pigment epithelium (RPE) defects and postoperative persistent intraocular ICG staining after surgery.16 Currently, several studies exist that compare the functional and anatomical outcomes in macular hole surgery with vs. without ICG application.13,17 The histopathologic analysis of ILM tissue harvested during ICG-assisted vitreomacular surgery revealed contradictory findings regarding the persistence of cellular structures on the ILM.18,19

The current reports on ocular ICG toxicity remain controversial assuming the following different mechanisms for possible toxicity or damage:

Direct toxicity by ICG itself. ICG dye or its metabolites is proposed to be toxic either to the outer retinal layers/ RPE-cells, or to the inner retinal layers/ganglion cells.20

pH or hypo-osmolarity. Intravitreal ICG injections may change the osmolarity in the vitreous cavity, thus damaging either the neurosensory retina or the RPE-cells directly.21

Latrogenic surgical trauma. Several clinical reports have raised the hypothesis that ICG-assisted ILM peeling may induce an iatrogenic trauma to the inner retinal tissue, as a high incidence of vitreoretinal hemorrhage and visual field defects have been observed.18

Light toxicity potentiated by ICG dye. ICG exhibits characteristics of a photosensitizer because it absorbs light in the near infrared spectrum.13

Recent reports have shown a dose-dependent toxic effect of ICG to the retina.11,12,22 Various concentrations (between 0.025% and 0.5%) and volumes (from 3 drops to 2 mL) of ICG solution have been advocated for chromovitrectomy.10-18 The ICG concentration prepared for injection is not the same as the concentration in the entire vitreous cavity. As the dye mixes with the fluid content of the vitreous cavity, the result is the so-called "intravitreous-ICG concentration." We found a strong predictive value of the "intravitreous-ICG concentration" with surgical outcomes and complications.13 Lochhead and colleagues reported good visual improvements after using low "intravitreous-ICG concentration," no higher than 0.0125%.17

Based on recent ICG-toxicity information, emphasis should be made on:

  • reducing the concentration of the dye in contact to the retina
  • avoidance of repeated ICG injections onto bare retina (ie, areas where ILM was already removed)
  • injection of the dye far from the macular hole to prevent dye contact with the RPE
  • quick aspiration of ICG from the vitreous cavity to diminish the concentration in contact with retinal tissue (Figure 1)12
  • concentrations as low as 0.05% to distinguish the light greenish ILM from the underlying reddish retina
  • a distance from the light pipe to the retina far enough to minimize ICG-related photosensitizer effects, as postulated by several authors.23

Figure 1. Injection of ICG in low concentration and directed to the macular prevents staining of the retina elsewhere, and minimizes the amount of dye left intravitreous.

In summary, ICG is the first pioneer dye of chromovitrectomy. The dye facilitated the previously difficult task of peeling the ILM. The main technical advantages of chromovitrectomy are related to its high biochemical affinity to ILM, as the surgeon may assure total removal of the ILM plus its overlying epiretinal tissue. Better ILM visualization should reduce the incidence of intraoperative traumatic damage caused by the surgical instruments or endoillumination. Disadvantages of ICG are its poor affinity to the ERM and vitreous, and the controversial findings regarding its safety.

Infracyanine Green

Infracyanine green is the modified iodine-free version of ICG. The latter contains up to 5% sodium iodide, a substance used to prepare the lyophilized form necessary to achieve solubility. Recent evidence suggests dilution of IfCG in glucose 5% generates an iso-osmotic solution.24 Based on the theory that retinal toxicity may be a consequence of the hypotonicity generated by the dilution of ICG in saline solution, IfCG dye may decrease the chance of dye-induced toxicity. Similar to ICG, IfCG has demonstrated to stain avidly to the ILM.25

Most studies demonstrated a safe profile of IfCG for chromovitrectomy.24 In vitro studies with IfCG disclosed no disruption of cellular elements of the neural retina.25 Moreover, ILM specimens excised with IfCG staining did not induce neural experiments or significant damage by IfCG to glial cells.27

Trypan Blue

Trypan blue is an azo dye, and has a large hydrophilic tetrasulfonated anionic molecule with the chemical formula C34H24N6Na4O14S4. Trypan blue may stain a tissue either by binding to degenerated cell elements or being taken in by phagocytosis.28 Trypan blue is utilized as a fluorescent tracer of cell populations, and it was introduced in 1998 to stain the anterior capsule to facilitate capsulorrhexis for cataract surgery.29

Trypan blue arouses subsequently to ICG as the second generation of vital dyes for chromovitrectomy.30 The vital stain is injected intravitreally in concentrations ranging from 0.06%­0.20%, although its application in concentrations of 0.06% produced only a faint ERM staining. The introduction of TB not only changed the color, but also added the ERM as a novel target tissue. The better visualization of the ERM with TB could minimize the mechanical trauma to the retina when the ERM is peeled, and staining ERM with TB usually reveals the extent of ERM to be larger than suspected clinically.31 While it has been suggested that TB may stain both ILM and ERM,32 the dye most likely stains primarily the ERM, possibly due to a strong binding affinity to glial-cell elements.33

Most clinical studies suggest a safe profile of TB for chromovitrectomy. Commercially available TB 0.2% was shown to cause no toxic effects to the retina in a few case series. Teba and colleagues found TB to be a useful tool to better visualize the size and extent of ERMs in macular pucker surgery in 50 patients (macular pucker, macular holes, diabetic maculopathy, and proliferative vitreoretinopathy), and VA improvement was achieved in most patients.34

In vitro studies have shown conflicting results on the safety of intravitreal TB injections. Trypan blue demonstrated little or no toxic effects on cultured RPE and glial cells.27 Further in vivo investigations in cats and rabbits determined that TB 0.2% caused partial damage to the lower retina, while in concentrations of 0.06% a safe profile was achieved in animal studies.7 However, Narayanan and colleagues recently published data on significant toxic effects of TB to cultured glial cells.35

In summary, TB has been established as the current most appropriate stain for ERM during chromovitrectomy, while the ILM may only be faintly stained with TB. The poor visualization of ILM with TB may induce longer operation time, trauma to the superficial retina at the time of peeling, and risk of light toxicity. In contrast, ICG in low concentrations allows a quick identification and peeling of the fine ILM. Stalmans and colleagues described a double staining of retinal structures with both TB (for ERM) and IfCG (for ILM) in the treatment of macular pucker.26 Therefore, current evidence suggests ICG or IfCG as appropriate stains for the ILM, whereas TB may be indicated for ERM identification.

Sodium Fluorescein

Sodium fluorescein is anionic hydrophilic xanthene presenting with the chemical formula C20H10Na2O5. The dye has a moderately sized, nonplanar aromatic system and is applied in medicine as a marker for the study of motility of bile canaliculi. The dye was found to be highly safe for fundus angiography in a concentration of 5%­25% solution, even when leakage through the retina occurred. The transparent vitreous should play a role in the pathogenesis of several macular diseases, and its complete removal during chromovitrectomy is frequently warranted. Because of its hydrophilic properties, SF is highly absorbed by the vitreous gel. Das and Vedantham showed that intravitreal SF 0.6% out of 20% injectable dye improved the visualization of clear vitreous fibers through a green coloring during chromovitrectomy, and no complications were noticed in their clinical series.36 To date, the main indication of SF in chromovitrectomy remains in the vitreous, while future clinical investigations should determine its role in ILM staining.

Patent Blue

Patent blue is an anionic triarylmethane dye with the chemical formula of C27H31N2NaO6S2. The dye is orange in acid conditions and blue in alkali. Patent blue has a hydrophilic anion with an aromatic system, and has long used as a marker for aiding accurate excision of lymph nodes and as a stain to recognize fungi.37 The dye has been recently certified for capsule staining during cataract surgery at the concentration of 2.4 mg/mL. The carcinogenic and mutagenic effects described with systemic use of TB have not been seen for PB.

Figure 2. Initial experience with PB for macular surgery demonstrated staining of the fine ERM associated with advanced macular hole. 

We performed preclinical studies in enucleated pig eyes to determine the binding properties of PB to the ILM, ERM, and vitreous tissue. The results have shown a moderate affinity of PB to ERM and vitreous, but poor staining to the ILM,38 which was confirmed by histologic examination. Based on these preliminary data, we evaluated the feasibility and the efficacy of chromovitrectomy with PB (unpublished data). Patent blue staining was evaluated in 5 patients with idiopathic epiretinal membrane (ERM) (n=2), proliferative vitreoretinopathy (n=2), and macular hole (n=1) (Figure 2). Patent blue provided better visualization of the ERM in comparison to the ILM, and no visual field defects or visible RPE-changes were observed postoperatively.

In conclusion, PB may play a role in the future as an alternative dye in chromovitrectomy. Preliminary data have shown PB to stain the vitreous and ERM thereby facilitating tissue visualization and delamination without any clinical signs of toxicity. Future studies with PB-dye application should clarify advantages and disadvantages of this novel vital dye in chromovitrectomy.

Triamcinolone Acetonide

Triamcinolone acetonide is a corticosteroid with the chemical formula C24H31FO6 and molecular weight of 434.5 Daltons. Triamcinolone acetonide is a relatively insoluble steroid that is used for local treatment of several ocular diseases, such as macular edema and age-related macular degeneration.39

Triamcinolone acetonide was added as an alternative stain for chromovitrectomy based on experience from other ophthalmic applications.40 Triamcinolone acetonide injections for chromovitrectomy have been performed with 0.5­1 mL of the 40 mg/mL commercial product. The white steroid is used to visualize the vitreous gel and posterior vitreous cortex. The high affinity of TA to the vitreous particles and the fine ILM was postulated as the result of the steroid precipitation. Besides aiding in the visualization of preretinal tissues, TA application in chromovitrectomy may improve surgical outcomes by decreasing the break down of blood-ocular barrier and reducing the chance of preretinal fibrosis.41

Complications associated with TA injections include endophthalmitis and glaucoma.42 During chromovitrectomy, TA was found to deposit in the macular or submacular space for several days after its intravitreal application, although no clinical signs of retinal damage were observed. Researchers have proposed that the toxic substance in TA is the vehicle, rather than the steroid itself. In order to minimize the risks, TA should be filtered to generate a vehicle-poor suspension. Hernaez-Ortega and Soto-Pedre applied density gradient centrifugation as a fast and simple technique to remove the vehicle from the TA suspension.43

While TA was proposed for visualization of the ILM and vitreous, further investigation on its exact target tissue during chromovitrectomy is required. Intraoperative identification of the preretinal membranes may be hindered by the deposition of TA particles. This steroid has shown no toxic effects in chromovitrectomy. This lack of toxicity remains the most remarkable advantage of staining preretinal membranes with TA.

Figure 3. A new "painting" instrument developed for selective application of dyes (ICG in this case) during chromovitrectomy. The flexible tube was gently exposed to the retinal surface in a circular movement, applying the dye to a minimal area of essential interest. The circular movement created a small greenish rim, strictly preventing any dye at the center of the macula and in the periphery of the fundus. The painted area was limited to the planned edge of the ILM-rhexis.


Several different surgical approaches have been used to inject vital dyes in the vitreous cavity during chromovitrectomy. One technique has been named the "dry method" or "air-filled technique." By either name, this technique consists of removing the fluid in the vitreous cavity by a fluid-gas exchange before dye injection.44 While the technique has the advantage of concentrating the dye in the posterior pole and avoiding contact at the posterior capsule of the lens,13 it may expose the retinal surface to a higher concentration of dye to the vitreoretinal interface.

The second proposed technique to inject dyes is called the "wet method" or "fluid-filled technique." In this approach, the intravitreal fluid (usually balanced salt solution) is left inside the vitreous cavity, while the surgeon injects the dye. The amount of dye in contact with the retinal surface becomes much lower because it is immediately washed out by the fluid in the vitreous cavity.45 The disadvantage to this technique is the possible dispersion of the dye leading to unwanted staining of the retina elsewhere. Czajka and colleagues compared the 2 methods in a porcine model and concluded that the air-filled technique induces a higher incidence of RPE atrophy and outer retinal degeneration than the fluid-filled technique.44

One alternative technique consists of using viscoelastic to control where the dye settles on the retinal surface to avoid staining outside the macular region. A further concern after dye administration may be incubation time of the dye on the retinal surface. Since early dye washout may minimize the dye exposure on retinal tissue, there is a recent trend for washing out the dye no later than a few seconds after its injection.12


To avoid an unnecessary and nonselective staining of the entire retina, we developed a new applicator for chromovitrectomy. A brush-like prototype was constructed on a metal cannula encoded by an adjustable silicone tube giving the instrument an outer diameter of 20 g. Its proximal end contained multiple long silk filaments. This prototype created a stream limited to the planned edge of the ILM rhexis in animal experiments (Figure 3). Subsequently, a commercially available applicator instrument called VINCE (Vitreoretinal INternal limiting membrane Color Enhancer, Dutch Ophthalmic) was produced. It consists of a modified backflush needle, containing an adjustable silicone tube, which is surrounded by metal cannula. The tube is connected to a reservoir in the handpiece filled with vital dye. The customized cartridge loading system contains the readily prepared and diluted vital dye. The advantage of the VINCE is the cartridge loading system, which avoids a time consuming preparation of the vital dye during surgery. In our preliminary experiments, we obtained favorable results by selectively painting the retinal surface. This new device may provide a better visualization of fine, delicate, semitransparent preretinal tissues, avoiding the uncontrolled staining of the RPE in the macular hole and peripheral retina.46

Figure 4. Preclinical studies with several biological stains were performed. Eosin dye provided an orange-red coloring of enucleated fresh vitreous material of pigs.


Chromovitrectomy has become a popular approach to visualize preretinal structures. While there is a consensus that chromovitrectomy facilitates macular surgery, some issues still remain unclear. Regarding ICG, the dye may be toxic in high concentrations and under certain techniques, but is otherwise safe. Nevertheless, it remains unknown whether the benefits of tissue visualization after ICG staining outweigh the recently described adverse effects in chromovitrectomy. The same concerns of ICG-related toxicity may be extrapolated for the other dyes available for chromovitrectomy.

Future perspectives in chromovitrectomy include new instruments and alternative dyes. Alternative vital dyes (eg, congo red or eosin) may possess an advanced affinity to selected vitreoretinal structures with no side effects on the retina (Figure 4). Intravitreous dye injection will be improved by further innovations in instrumentation to improve the selective placement of the dye on the retina, as well as better visualization of fine delicate semitransparent tissues on the retinal surface. Chromovitrectomy is an approach in vitreoretinal surgery that promises exciting frontiers ahead.


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41. Enaida H, Hata Y, Ueno A, et al. Possible benefits of triamcinolone-assisted pars plana vitrectomy for retinal diseases. Retina. 2003;23:764-770.

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44. Czajka MP, McCuen BW, Cummings TJ, et al. Effects of indocyanine green on the retina and retinal pigment epithelium in a porcine model of retinal hole. Retina. 2004;24:275-282.

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46. Meyer CH, Rodrigues EB. A novel applicator for the selective painting of pre-retinal structures during vitreoretinal surgery. Graefes Arch Clin Exp Ophthalmol. In press.

Eduardo Rodrigues, MD and Dr. Meyer are from the Department of Ophthalmology, Philipps-University, Marburg, Germany. Dr. Rodrigues is also from the Retina Department, Ophthalmology Service, Hospital Regional Sao Jose, Instituto de Olhos Florianopolis, Florianopolis, Brazil. The dye applicator VINCE is disclosed to Dutch Ophthalmics, Netherlands. Drs. Meyer and Rodrigues have a patent pending on the VINCE in conjunction with Dutch Ophthalmics, Netherlands.