Trusting Trypan Blue

The dye trypan blue (TB) has become an integral part of anterior segment surgery. It has been shown to help visualize cortical tissue attached to the capsular bag in cases of cortical cataract and for cortical clean-up, particularly when corneal haze is present. It helps to visualize the anterior capsule for capsulorhexis and reveal existing tears or other defects in eyes with traumatic cataracts. It also modifies the biomechanical properties of the human lens capsule, leading to a significant reduction in elasticity and increase in stiffness, and aids in visualizing and recovering dropped capsule tears in routine surgery. Other areas of application of TB are visualization of the donor tissue during corneal graft procedures, such as keratoplasty and Descemet’s membrane endothelial keratoplasty (DMEK) surgery and for visualization of the inflow/outflow of Schlemm’s canal during minimally invasive glaucoma surgery (MIGS).

TB is an anionic diazo dye first synthesized by Paul Ehrlich in 1904. The dye is excluded by living cells (that is, they stay colorless), while dead cells become dark blue on contact with it. The cell membrane of vital cells cannot be crossed by TB due to its high molecular weight. It selectively stains the basement membrane adjacent to the lens epithelial cell layer of the anterior lens capsule, but the lens cortex is not stained with TB. This enables surgeons to distinguish the lens capsule from the cortex and provides sufficient contrast for successful completion of continuous curvilinear capsulorhexis (CCC) during cataract surgery.

The first approved formulation of trypan blue, under brand name VisionBlue, was launched in Europe in 1999 and received US FDA approval in 2004. Since its launch, multiple competitive dyes have entered the European market, but Vision Blue is still the only FDA-approved anterior dye. To provide support for the safe use of these products, the characteristics of currently available CE-marked trypan blue ophthalmic solutions were compared in 33 samples from 20 suppliers (1). Significant differences were detected in the tested product purity profiles. The pH values ranged from 6.5 to 9.1, and the osmolality values ranged from 175 to 1043 mOsm/kg. The TB peak purity varied between 86 and 97 percent. There were significant differences between the TB concentration labeled and the TB concentration found (30–185 percent). The mono-azo41 dye content varied between 0.2 and 5.2 percent (see table here).

These tests show that there are clear differences between TB products on the market. In some cases, the quality of products from a specific manufacturer can vary from batch to batch. Ophthalmologists should be aware that any substance administered intraocularly can lead to complications, and they should know the source of all material used in surgery. There are reports of some ophthalmic TB solutions that include clumps, which can interfere with visualization of the tissue and increase risk of complication during capsulorhexis.

In addition to purity and safety considerations, it is important to stain the anterior capsule in a homogeneous manner, which cannot be achieved with all TB formulations. Permanent blue discoloration of hydrogel intraocular lenses by intraoperative TB are the only known adverse events when using high purity TB. It is concerning that, in some products, the measured values of parameters that have a decisive influence on biocompatibility can deviate significantly from physiological values. TB products with osmolality values of 175 mOsmol/kg or 1043 mOsmol/kg are far beyond isotonicity and can induce severe detrimental effects in the intraocular tissues. TB products with pH values of 6.5 or even 9.1 are far beyond the human physiological pH and can induce severe irritation in intraocular tissues.

Although there are ISO standards for ophthalmic viscosurgical devices (ISO 15798) and ocular endotamponades (ISO 16672), there is inconsistency in the international regulation and standards for intraocular dyes. In the USA, Canada, South Korea, and Japan, ocular dyes are regulated as pharmaceutical products with stricter standards, including reference-standard minimum purity, annual validation studies, and regular batch level purity testing. However, in other countries there is limited or no current regulation governing the purity of ocular dyes. As a result, there are multiple dyes available with no supporting information provided on their relative purity.

The take home message: clinicians should be aware of the varying concentrations and impurities in commercially available trypan blue. They should actively seek information from their suppliers on the purity of the dye they are using, work with their pharmacy teams to define minimum purity standards, and consider the relevance of using an FDA-approved dye for their surgery.