What alternative dyes (e.g., methylene blue, indocyanine green, etc.) are there to trypan blue for use in ophthalmology cases that are safe and effective?

Comment by InpharmD Researcher

Though limited to indirect comparisons, NMA of various dyes (indocyanine green, trypan blue, brilliant blue G, triamcinolone acetonide) used in macular hole surgery with an adjuvant-assisted ILM peeling indicate that trypan blue, brilliant blue G, and triamcinolone acetonide outperformed in visual and functional outcomes according to ranking data. Direct comparative data suggest brilliant blue G exhibited equivalent staining efficiency to trypan blue without major perioperative complications.

Background

A 2021 systematic review and network meta-analysis (NMA) of 17 studies, including five randomized controlled trials and twelve retrospective trials, evaluated the efficacy of various dyes for assisting internal limiting membrane (ILM) peeling in patients with idiopathic macular hole (IMH). The analysis examined 1,492 participants undergoing ILM peeling assisted with chromovitrectomy dyes such as indocyanine green (ICG), trypan blue (TB), brilliant blue G (BBG), and triamcinolone acetonide (TA), comparing different concentrations of these dyes in terms of IMH closure rates and postoperative visual acuity (VA). The trials utilized a frequentist framework to calculate the mean difference and odds ratio for different dye concentrations, including 0.05% BBG, 0.15% TB, 0.5% ICG, 0.25% ICG, and 40 mg/ml TA, among others. The NMA aimed to establish the optimum dye concentration for improving anatomical outcomes such as IMH closure and functional recovery like VA. The analysis showed that the ILM peeling procedure assisted by 0.15% TB and 0.05% BBG demonstrated a significantly higher IMH closure rate compared to other concentrations, with SUCRA values indicating that these dyes ranked first and second in terms of efficacy. In contrast, 0.25% ICG showed inferior outcomes, ranking last in IMH closure probability. For postoperative visual acuity, peeling performed without any dye ranked as the most effective, suggesting better visual recovery without dye-related toxicity. Peeling with 0.5% ICG, while demonstrating modest anatomical success, resulted in poorer visual outcomes than TB, BBG, or no dye at all. The analysis acknowledges the potential retinal toxicity associated with chromovitrectomy dyes, and supports the use of lower-concentration dyes like 0.05% ICG or other alternatives like TB and BBG for safer outcomes. [1]

A 2020 systematic review and NMA (N= 27 studies incorporating 1,849 eyes) aimed to compare the outcomes of four adjuvant dyes— ICG, BBG, TA, and TB —used for ILM peeling in macular hole surgery. This analysis evaluated postoperative best-corrected visual acuity (BCVA) improvement and primary macular hole (MH) closure rates and used random-effects Bayesian models to allow for indirect comparisons across the four adjuvants. The results demonstrated that BBG and TA outperformed ICG, with BBG showing significantly greater BCVA improvement compared to ICG (weighted mean difference [WMD] 0.08; 95% credible interval [CrI] 0.01 to 0.16). TA was ranked highest in BCVA improvement, although this was not statistically significant when compared to ICG (WMD 0.09; 95%CrI -0.00 to 0.19). Regarding primary macular hole closure rates, BBG was found to be the most likely adjuvant to achieve favorable outcomes, with a higher probability of success than the other dyes. However, no significant differences were observed between any two groups in primary closure rates. These findings suggest that BBG and TA may provide superior visual outcomes, with BBG being particularly effective in promoting anatomical closure during ILM peeling for macular hole surgery. [2]

A 2014 poster abstract presented at the Association for Research in Vision and Ophthalmology (ARVO) annual conference compared the use of BBG and TB when performing lens capsule staining for cataract surgery. This pilot study randomized patients aged 40-80 years diagnosed with cataracts to undergo either TB 0.1%-assisted capsulorhexis or BBG 0.025%-assisted capsulorhexis. Results found BBG as effective as TB for staining the lens capsule and visualization during surgery. No significant differences were observed in VA, intraocular pressure, corneal edema, or inflammation of the anterior chamber. [3]

A 2011 experimental study evaluated the capsule-staining properties and biocompatibility of the triarylmethane dyes methyl blue and aniline blue compared to the established dyes patent blue and TB, utilizing cultured human corneal endothelial cells. Corneal endothelial cell cultures were derived from human donor cells and exposed to varying concentrations of the four dyes (0.025 to 5.0 mg/mL). Cytotoxicity was assessed using an MTT assay after 24 hours of incubation, with calcein live cell staining conducted concurrently. Additionally, lens capsule staining was performed in vitro using pig lenses soaked in varying concentrations of the dyes (0.5, 1.5, and 2.5 mg/mL), and staining properties were evaluated using a semiquantitative scoring system. The results revealed that methyl blue and patent blue showed no significant cytotoxicity at any tested concentration, whereas aniline blue exerted significant cytotoxic effects at concentrations of 1.5 mg/mL or higher and trypan blue at concentrations of 2.5 mg/mL or higher. In terms of capsule-staining properties, the triarylmethane dyes yielded sufficient staining intensity for capsulorhexis creation, although at higher concentrations compared to TB. Notably, TB provided superior staining intensity at lower concentrations, while methyl blue demonstrated quicker fading after washing than other dyes. These findings suggest that methyl blue and patent blue offer a more favorable safety profile regarding corneal endothelial cell viability, positioning them as viable alternatives for cataract surgery. [4]

References:

[1] Li SS, Li M, You R, et al. Efficacy of different doses of dye-assisted internal limiting membrane peeling in idiopathic macular hole: a systematic review and network meta-analysis. Int Ophthalmol. 2021;41(3):1129-1140. doi:10.1007/s10792-020-01656-2
[2] Wang XW, Long Y, Gu YS, Guo DY. Outcomes of 4 surgical adjuvants used for internal limiting membrane peeling in macular hole surgery: a systematic review and network Meta-analysis. Int J Ophthalmol. 2020;13(3):481-487. Published 2020 Mar 18. doi:10.18240/ijo.2020.03.17
[3] Magana-García D, Ramos-Espinoza K, Bonilla-Barraza C, Romo-Garcia E. Brilliant Blue G as an alternative to Trypan Blue for conducting continuous curvilinear capsulorhexis in cataract surgery. ARVO Annual Meeting Abstract. 2014;55(13):2537.
[4] Thaler S, Hofmann J, Bartz-Schmidt KU, Schuettauf F, Haritoglou C, Yoeruek E. Methyl blue and aniline blue versus patent blue and trypan blue as vital dyes in cataract surgery: capsule staining properties and cytotoxicity to human cultured corneal endothelial cells. J Cataract Refract Surg. 2011;37(6):1147-1153. doi:10.1016/j.jcrs.2010.12.051

Literature Review

A search of the published medical literature revealed 2 studies investigating the researchable question:

What alternative dyes (e.g., methylene blue, indocyanine green, etc.) are there to trypan blue for use in ophthalmology cases that are safe and effective?

Level of evidence

A - Multiple high-quality studies with consistent results  Read more→



Please see Tables 1-2 for your response.


A COMPARISON OF BRILLIANT BLUE G, TRYPAN BLUE, AND INDOCYANINE GREEN DYES TO ASSIST INTERNAL LIMITING MEMBRANE PEELING DURING MACULAR HOLE SURGERY
Design

Comparative, interventional case series;

N= 50 eyes of 50 patients

Objective To compare surgical outcomes with three dyes, brilliant blue G (BBG), trypan blue (TB), and indocyanine green (ICG), used to facilitate internal limiting membrane peeling during macular hole surgery
Study Groups

BBG (n= 15)

TB (n= 20)

ICG (n= 15) 

Inclusion Criteria Senile idiopathic macular holes, visual symptoms solely attributed to macular hole, willingness to follow-up for at least 6 months, and OCT documentation of a full-thickness macular hole
Exclusion Criteria History or evidence of ocular trauma, inflammatory or degenerative disease, any concomitant disease likely to compromise outcomes, lack of OCT documentation, inability to maintain prone position for 1 week, and any previous intraocular surgery within 6 months of enrollment
Methods Vitrectomy with internal limiting membrane peeling using BBG (0.05%), TB (0.15%), or ICG (5 mg/mL). Dyes were used as supplied by the manufacturer. internal limiting membrane (ILM) was stained, peeled, followed by fluid-air exchange and gas injection. Patients maintained a facedown position for 1 week post-surgery.
Duration Follow-up: 6 months 
Outcome Measures

Primary: Macular hole closure

Secondary: Visual improvement

Baseline Characteristics   Mean Age, years Preoperative BCVA
BBG (n= 15) 59.47 ± 7.27 0.20 ± 0.13
TB (n= 20) 58.8 ± 7.65 0.19 ± 0.09
ICG (n= 15)  58.73 ± 7.91 0.18 ± 0.08
Results Final Outcome (BCVA/MH Closure) BBG (%) TB (%) ICG (%) p-value
Improved 80 85 33.3 0.010
20/40 or better 33 30 6.67 0.197
Worsened 6.67 5 40 0.049
MH closure 100 95 86 0.480
BCVA, best-corrected visual acuity; MH, macular hole
Adverse Events 8 patients lost ≥1 Snellen lines of BCVA, predominantly in the ICG group. No retinal detachments, reopening of the MH, or macular pucker were reported.
Study Author Conclusions Brilliant blue G was comparable with TB in optimizing visual and functional outcomes, while it was similar to ICG in ease of internal limiting membrane peeling. BBG appears to combine ease of use intraoperatively and good surgical outcomes postoperatively.
Critique The study provides a valuable comparison of three dyes used in macular hole surgery, highlighting BBG's advantages. However, it is limited by its partly retrospective design and small sample size. The lack of evaluation for visual field defects is another limitation.
References:

Shukla D, Kalliath J, Neelakantan N, Naresh KB, Ramasamy K. A comparison of brilliant blue G, trypan blue, and indocyanine green dyes to assist internal limiting membrane peeling during macular hole surgery. Retina. 2011;31(10):2021-2025. doi:10.1097/IAE.0b013e318213618c

Comparison of Trypan Blue and Brilliant Blue G for Staining of the Anterior Lens Capsule During Cataract Surgery: Short-term Results

Design

Single-center, prospective, randomized study

N= 150 eyes (117 patients)

Objective To evaluate the potential corneal endothelial cell toxicity of trypan blue (TB) and Brilliant Blue G (BBG) during cataract surgery
Study Groups

Control (n= 35 eyes)

TB group (n= 36 eyes)

BBG group (n= 40 eyes)

Inclusion Criteria Patients undergoing cataract surgery at Yokohama Minami Kyosai Hospital in Japan between April and September 2015
Exclusion Criteria History of internal eye surgery, corneal diseases, corneal endothelial count less than 2000 cells/mm², mature cataracts, extracapsular or intracapsular cataract extraction, discontinuation during 6-month follow-up    
Methods TB and BBG 1.0 mL of 0.1% were injected directly in the anterior chamber in both intervention groups while 1.0 mL of balanced salt solution was injected in the control group. Preoperative and postoperative evaluations of CDVA, corneal endothelial cell counts, and central corneal thickness were evaluated.     
Duration 6 months follow-up with evaluations at 1, 3, and 6 months post-surgery
Outcome Measures

Primary: Corneal endothelial cell count

Secondary: Corrected distance visual acuity (CDVA), central corneal thickness (CCT)

Baseline Characteristics  

Control

(n= 35)

TB

(n= 36)

BBG

(n= 40)

p-value
Age, years 75.9 75.1 74.5 0.68
Male/female 13/22 16/20 16/24 0.82
Eye (R/L) 19/16 18/18 19/21 0.84
CDVA (logMAR) -0.16 0.20 0.15 0.43
ECD 2810 2809 2784 0.84
CCT 530 533 529 0.88
Nuclear grade 2.3 2.4 2.5 0.21
Results  

Control

(n= 35)

TB

(n= 36)

BBG

(n= 40)

p-value

CDVA (logMAR)

Pre-op

Post-op 1M

Post-op 3M

Post-op 6M

 

0.156 ± 0.154

0.001 ± 0.095

- 0.026 ± 0.061

- 0.020 ± 0.070

 

0.203 ± 0.207 

0.023 ± 0.145

0.029 ± 0.145

0.017 ± 0.142

 

0.152 ± 0.197

0.019 ± 0.154 

- 0.007 ± 0.169

- 0.002 ± 0.196

 

0.430

0.749

0.217

0.565

Corneal endothelial cell loss, %

Post-op 1M

Post-op 3M

Post-op 6M

 

- 4.092 ± 7.256

- 6.023 ± 17.47

- 3.172 ± 5.860

 

- 3.774 ± 6.416

- 1.713 ± 6.659

- 2.195 ± 6.523

 

- 5.992 ± 14.50

- 5.057 ± 12.01

- 3.547 ± 9.792

 

0.594

0.326

0.737

The corneal endothelial cell counts were 2,711 ± 225, 2,748 ± 251, and 2,680 ± 284 cells/mm2, for control, TB, and BBG, respectively, and the central corneal thicknesses were 524.3 ± 35.5, 532.2 ± 36.1, and 531.4 ± 33.0 μm, respectively, 6 months after surgery.

Adverse Events: No major perioperative complications were observed during surgery.

Study Author Conclusions: Neither TB nor BBG was associated with detectable toxicity to corneal endothelial cells during cataract surgery. BBG exhibited equivalent staining efficiency to TB.

Critique: Despite the randomized design and direct comparison of TB and BBG, potential limitations include small sample size, lack of immediate postoperative ECD and CCT measurements, exclusion of patients with corneal endothelium vulnerability.

References:

Nagashima T, Yuda K, Hayashi T. Comparison of trypan blue and Brilliant Blue G for staining of the anterior lens capsule during cataract surgery: short-term results. Int Ophthalmol. 2019;39(1):33-39. doi:10.1007/s10792-017-0779-9