Color Me Green, White, and Blue: Chromovitrectomy in Surgery

An overview of vital dyes for vitreoretinal surgery


Color Me Green, White, and Blue: Chromovitrectomy in Surgery

An overview of vital dyes for vitreoretinal surgery

Ryan N. Vogel, MD • Judy E. Kim, MD

Chromovitrectomy is a term used to describe pars plana vitrectomy with the use of vital dyes to help identify ocular tissues that are otherwise difficult to visualize due to transparency.1 These transparent tissues include vitreous, epiretinal membrane, and the internal limiting membrane. In cases of proliferative vitreoretinopathy, removal of fibrous tissues may also be aided by staining.

While it is possible to remove all of these tissues without staining, the process is significantly easier and more complete after staining. This is especially true in certain cases, such as ILM peeling in highly myopic eyes or in cases of severe diabetic macular edema. Thus, chromovitrectomy may allow for more effective and potentially less traumatic handling of these intraocular tissues.

Many dyes have been used for chromovitrectomy, most commonly indocyanine green (ICG),2 trypan blue (TB),3 brilliant blue G (BBG),4 and triamcinolone acetonide.5 More recently, lutein-based dyes have been described to improve vitreous visualization, often used in conjunction with BBG.6

Ideal vital dyes or coloring agents should selectively stain intraocular tissues of interest, be safe for intraocular use without visual or anatomic toxicity, and be rapidly eliminated from the eye after surgery.

Numerous studies have been performed to evaluate the efficacy and safety of various dyes and to determine their role in vitreoretinal surgery. We will discuss some of the salient points regarding each of these vital dyes.

Ryan N. Vogel, MD, and Judy E. Kim, MD, serve on the faculty of the Department of Ophthalmology and Visual Sciences at the Medical College of Wisconsin in Milwaukee. Neither author reports any financial interests in products mentioned in this article. Dr. Kim can be reached via e-mail at


Indocyanine green is a hydrophilic cyanine dye that binds to both cellular and acellular elements of tissue. Due to its fluorescent properties, it is used as a contrast agent to visualize the retinal and choroidal vasculature during angiography.

Figure 1. Intraoperative ICG stains the ILM a noticeable green color that helps differentiate it from the underlying retina.

Membrane Peeling

Since 2000, ICG has also been used intraoperatively to stain the ILM. Currently it is one of the most widely used vital dyes in chromovitrectomy, especially for macular hole (MH) repair and ERM removal, although it remains off label for intraoperative use.

Surgical management of MH and ERM involves PPV, removal of the posterior hyaloid, peeling of ERM and often peeling of the ILM, especially in the cases of MH. Although controversy exists over the necessity of ILM peeling, it is generally thought to reduce the amount of retinal striae and to decrease recurrence of ERM by ensuring more complete removal of the membrane. It is also thought to improve the anatomical closure rate in MH surgery.7

The ILM is a transparent basement membrane measuring 2.5 µm in thickness; therefore, it can be difficult to visualize and to remove completely. Vital dyes, such as ICG, can facilitate visualization of the ILM, resulting in more efficient and effective removal.

Burk et al2 demonstrated on cadaveric eyes that ICG stained the ILM without staining the underlying retina. There have been multiple reports of the off-label use of ICG-assisted ILM peeling for management of MH,9 DME,10 and ERM.11 The Preferences and Trends (PAT) surveys from the American Society of Retina Specialists indicated that ICG is the most commonly used vital dye for ILM staining in the United States.

Figure 2. White triamcinolone crystals deposited onto the posterior hyaloid membrane improves visualization of the nearly transparent structure and facilitates its removal.

Toxicity Concerns

However, multiple studies have raised concerns about the cellular toxicity of ICG.12 In vitro studies of human retinal pigment epithelium cells have shown that brief exposure to ICG induced dose-dependent signs of cellular toxicity,13,14 which was also exacerbated by light exposure.15-17 This may be due to ICG being able to uptake light approximately 800 nm in wavelength.

In vivo studies using rat or rabbit models have shown that subretinal injections of ICG induced RPE and photoreceptor damage,18 which may be worse with hypo-osmolar solutions.19 Enaida et al20 showed that prolonged exposure to very low concentrations of ICG (0.025 mg/mL) in rats resulted in decreased amplitudes of a- and b-waves on electroretinography.

In vivo studies on humans have also demonstrated potentially concerning findings. Observational studies in patients undergoing ICG-assisted ILM peeling have shown atrophic RPE changes,21 visual field defects,11,22 and optic nerve atrophy.22

However, in many instances, it is not clear whether these complications were due to use of ICG or other factors related to PPV. In a large retrospective study comparing the ILM peeling with and without the use of ICG, best-corrected visual acuity was found to be lower in the ICG group, suggesting possible subtle toxicity of the dye.23 However, the hole closure rate was higher in the ICG group.

Staining of the ILM by ICG is dose dependent. While higher ICG concentrations may stain the ILM more readily, it is important to use the lowest concentration that allows for visualization of ILM to improve safety. This is especially important in the cases of MH repair, in which bare RPE is exposed at the fovea.

ICG concentrations as low as 0.05% can effectively stain the ILM, and concentrations this low have been shown to have minimal or no signs of RPE toxicity.8,24 Murata et al25 showed increased Bcl-2 expression (a marker of apoptosis) at ICG concentrations of 0.5 mg/mL but not at 0.05 mg/mL.

Tips for Safer Use

Based on these studies, multiple methods to reduce ICG toxicity have been proposed, although few have been studied in depth in vivo. These methods include using the lowest volume of dye needed to stain, quickly aspirating ICG from the eye, avoiding dye injection aimed at the fovea, avoiding use after fluid-air exchange, which may concentrate the dye at the retinal surface, diluting ICG with dextrose to reduce the hypo-osmolarity, minimizing foveal light exposure, and using a light source <620 nm, which is further from the peak of the ICG absorption spectrum at 800 nm.23

Covering bare RPE in the fovea during MH surgery with blood, viscoelastic, or perfluorocarbon liquid has also been suggested to mitigate direct toxicity from the dye.26 In addition, one should consider not injecting ICG into the eye in the cases of retinal tears near the macula to avoid subretinal migration of ICG.

The current consensus among retina specialists is that ICG is an effective vital dye for ILM staining, but it must be used with caution given its dose-dependent toxicity to the RPE. By taking precautions that include those listed above, ICG-related toxicity can be minimized during vitreoretinal surgery.

ICG also has been used to stain the ILM during ERM surgery. While ICG stains the ILM, it does not stain ERM. In effect, the use of ICG results in “negative staining” of ERM, outlining the extent of ERM by visualizing the bare ILM that lies beyond the ERM. Unlike MH surgery, ERM cases generally have no exposed RPE, which is the main target of potential toxicity with ICG. Therefore, slightly higher concentration of ICG may be used compared, to cases of MH with central bare RPE.

Nevertheless, even in ERM cases, one should avoid prolonged peeling times, excessive light pipe exposure on the macula, or the use of ICG after any iatrogenic posterior retinal tear to minimize potential toxic effects.


BBG was introduced in 2006 as a dye used for the intraoperative staining of the ILM.4 Currently, BBG is not approved for human use in the US by the FDA, but it may be available through a few compounding pharmacies. However, it is used widely in Europe for chromovitrectomy.

BBG is a triphenylmethane dye that binds nonspecifically to multiple types of proteins. Similar to ICG, it has a high affinity for the ILM but does not stain ERMs as well. As a result, it can be particularly effective at staining and removing ILM remnants during ERM peeling.27,28

The ILM staining chromaticity of BBG is less intense compared to ICG, although it is still sufficient to permit complete removal of the ILM in the vast majority of cases.29,30 Recently, intraoperative filters have been developed to improve the visibility of the dye.31

The primary advantage of BBG over ICG is that it is considered less toxic to the neurosensory retina and RPE.32,33 A retrospective study comparing ICG and BBG in patients undergoing MH repair showed that the BBG group had better postoperative VA and better retinal sensitivity in the central 2°, as measured by microperimetry. The group treated with BBG also demonstrated faster restoration of the ellipsoid zone seen on optical coherence tomography and had thicker central foveal thickness.34

Additionally, BBG is readily removed from the eye following irrigation. The vitreous concentration measured postoperatively has been found to be very low (34.5 ng/mL), and the dye was not detected in the plasma several hours or days following surgery.35


TB is an azo dye that crosses the cell membranes of dead tissue or cells, staining them blue. It has been widely used as a vital dye since the late 1990s to stain the anterior capsule during cataract surgery. More recently, TB has been used during chromovitrectomy because it is ideal for staining ERM by binding to degenerated cell elements within the membranes.36,37

Several studies in animal eyes have demonstrated signs of retinal toxicity with TB, although not at low concentrations.38-40 Compared to ICG, TB has shown less retinal toxicity following subretinal injections in rabbits.19

Studies in humans comparing ILM peeling with and without TB use showed no adverse effects or visual field defects in eyes treated with TB,36,37 although disorganized inner retinal architecture was observed at undiluted concentrations in human cadaveric eyes.41

At the concentration being used for posterior-segment surgery, TB stains ERM more effectively than the ILM. Therefore, some surgeons have used TB after fluid-air exchange to increase TB concentrations at the macula and to improve the staining of the ILM.


Triamcinolone is a synthetic corticosteroid composed of insoluble white crystals that can be deposited in the transparent vitreous gel to improve visualization. The use of triamcinolone in vitrectomy was described as early as 2000,5 and it remains the most widely used vital dye to stain the vitreous and posterior hyaloid.

In a comparative study of four vital dyes (fluorescein, ICG, triamcinolone, and TB), triamcinolone was found to be best for visualizing the posterior hyaloid.42 By improving visualization of the vitreous, triamcinolone may facilitate a complete posterior vitreous detachment and decrease the risk of surgical complications, such as intraoperative or postoperative retinal detachment.43

While triamcinolone can cause a transient increase in intraocular pressure,44 the anti-inflammatory effects may reduce postoperative blood-retinal barrier breakdown and preretinal fibrosis.45

Preservative-free formulations of triamcinolone are readily available and have been shown to produce less inflammatory reactions compared with formulations containing preservatives.46 However, due to the short period of time that triamcinolone is used intraoperatively and the cost of preservative-free formulations, triamcinolone acetonide injectable suspension with benzyl alcohol preservative (Kenalog, Bristol-Myers Squibb, New York, NY) is most commonly used.

We have found the 10 mg/mL concentration to be more useful than the 40 mg/mL concentration due to less need for dilution, less time needed to aspirate the drug from the eye, and better spread on the retina, with less clumping of the triamcinolone crystals.

Triamcinolone has also been used in MH surgery to visualize indirectly and to assist in the peeling of the ILM or ERMs.44,47 The white crystals blanket the membrane so that areas that have not been peeled remain covered in white specks.

In a retrospective comparison of eyes undergoing MH repair with triamcinolone-assisted or ICG-assisted ILM peeling, the triamcinolone group was found to have significantly better VA one year after surgery.48 However, the sample sizes in these studies were small, and larger studies are needed. While the use of triamcinolone in the eye is deemed safe, many find that it is difficult to visualize the ILM or ERM because the crystals cover both membranes on the retina.


Less commonly used vital dyes in chromovitrectomy include patent blue49 and bromphenol blue,50 which have been used to stain ERM effectively. Infracyanine green is a cyanine dye, similar to ICG but without iodine. It precipitates in water and therefore is typically dissolved in a 5% glucose solution.51

Penha et al52 found less retinal damage with infracyanine green compared to ICG, even at 10-fold higher concentrations, although this outcome may be related to the osmolarity differences between the two solutions used in the study.

Fluorescein can be administered orally in the preoperative period to give the vitreous a uniform green appearance. This method improves visualization of the vitreous and is well tolerated by patients.53

While all of these less commonly used vital dyes appear to be effective and well tolerated for use in chromovitrectomy, they are not as well studied as the previously mentioned dyes, do not appear to offer significant advantages, and are not readily available.


Recently, lutein-based dyes have been developed for chromovitrectomy. These dyes contain golden, micronized lutein/zeaxanthin crystals, which act similarly to triamcinolone crystals. They become deposited within the vitreous gel to improve visualization, especially at the posterior hyaloid and vitreous base. The dye can be combined with BBG, resulting in simultaneous double staining of the vitreous and ILM.6,54,55 The advantages and role of this type of vital dye are not yet well established.

Several other vital dyes have been investigated recently as alternatives for chromovitrectomy. Acid violet is an arylmethane dye that has recently been tested in a rabbit model. It was shown to stain the ILM adequately, and it did not induce significant anatomical or functional toxicity.56 Other new vital dyes that have been tested include Trisodium, Orangell, and Methyl violet, which showed no signs of toxicity in preliminary animal studies.57


In summary, multiple vital dyes have been studied for use in vitreoretinal surgery, which has been termed chromovitrectomy. Most of these dyes are used off-label, but they significantly improve visualization and facilitate the removal of various transparent tissues in the eye. They may also improve safety by allowing for more efficient and less traumatic peeling of the posterior hyaloid, ILM, and ERM.

ICG and BBG are commonly used to stain the ILM, although toxicity concerns have been raised with ICG. TB has also been frequently used in chromovitrectomy, primarily to stain ERMs. Triamcinolone is the most commonly used dye to improve visualization of the vitreous, and it is also used to aid in the removal of the ILM and ERM.

While all of these dyes are effective at visualizing various retinal structures, and they appear to be reasonably safe, they have not been extensively tested in controlled clinical trials. Multiple factors, such as concentration, osmolarity, and the duration of exposure, may play roles in safe use of these agents in the eye. Different tissues may also be more susceptible to damage, such as the exposed RPE in MH surgery. Furthermore, the duration, intensity, and wavelength of light used during surgery may play roles in cellular damage.

We should continue to be vigilant when using these vital dyes and should study specific factors that can be optimized to reduce toxicity and ultimately improve surgical and visual outcomes. RP


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