Comparison of Wash-out Properties after Use of the Vital Dye Trypan Blue in the Form of an Ophthalmic Dye and Bound in a Sodium Hyaluronate by Raman Spectroscopy

Abstract

In cataract surgery, the creation of the anterior capsulorhexis as one of the critical steps is of most importance for the surgical success. In challenging initial situations (e.g. in eyes with pseudoexfoliation syndrome, mature, brunescent cataract, juvenile cataract, corneal opacities, or in post-traumatic and postuveitic cases), vital dyes are used as a routine to increase the visibility and plasticity of the ocular structures in the anterior chamber. The range of applications of the vital dye trypan blue (TB) has expanded considerably in recent years due to its excellent staining properties.1 However, its use in ophthalmology as an effective and useful tool requires that the dye has no adverse effects on the cell structures of the eye. As two laboratory studies on cell cultures showed, the time of exposure to TB plays an important role in addition to concentration.2,3 The in vitro studies by Chang et al. with rabbit corneal endothelial cell cultures, and van Dooren et al.3 demonstrated no toxicity of TB with a maximum concentration of 0.4% after 1 minute in cell cultures of human corneal fibroplasts. A significant toxicity of a TB concentration of 0.01% or higher after exposure was observed. At 24-hour exposure, a TB concentration of 0.005% was found to be the threshold for a significant cytotoxicity index. In principle, it is important to note that trypan blue can be cytotoxic at a certain concentration. Monoazo, the most toxic of known impurities found in trypan blue dyes can be carcinogenic. However, the TB concentrations used in eye surgery do not have undesirable effects on the cell structures of the eye and are therefore generally considered safe.3,4 However, a case report showed a transient retinal toxic reaction in the form of transient visual field defect following the entry of TB into the vitreous body space.5 In modern intraocular procedures viscoelastic substances (OVDs, ophthalmic viscoelastic devices) are widely used. Since their introduction in the 1970s, they have been routinely used in cataract surgery and serve to protect sensitive eye structures from mechanical injuries or to create and maintain anatomical spaces such as the anterior chamber or the capsular bag. They increase safety during the procedure and can also shorten overall surgery time by improving visibility and simplifying some surgical steps for the surgeon.6 A shorter surgery time is associated with a lower degree of trauma and a lower risk of complications, and may ultimately be associated with faster recovery and a better final outcome and satisfaction for the patient. In addition, from an economic point of view, the time saving factor is particularly important for high-volume facilities. After the introduction of Healon® in 1979, sodium hyaluronate became the most widely used biopolymer for OVDs in intraocular surgery. Since then, the pharmacological, physiological, and clinical aspects of sodium hyaluronate for ophthalmic applications have been assessed in a large number of studies.7,8 Recently, a combination of a viscoelastic with the vital dye TB has been introduced (Pe-Ha-Blue®PLUS, Albomed, Schwarzenbruck, Germany) and has already been clinically investigated in a prospective case series of 52 eyes with pseudoexfoliation syndrome.6 In addition to a significantly shorter surgery time (due to fewer individual surgical steps) with cost- and safety-relevant advantages of Pe-Ha-Blue®PLUS compared to separate administration of OVD (POLY-HYL® 1. 6%; Polytech Domilens GmbH) and TB (Vision Blue®; DORC, Holland/Blue Color Caps®), the surgeon gains better control over whether the OVD is removed completely at the end of surgery by using the blue OVD. This should also reduce postoperative complications such as hypertension due to OVD residues remaining in the eye. The aim of the present in vitro study was to determine by Raman spectroscopy the amount of residue of the TB dye that remains on a slide during the routine application of two commercial products (TB dye Vision Blue® and Pe-Ha-Blue®PLUS). In Raman spectroscopy, the interaction of light and matter is used to investigate, for example, the properties of a material or to enable the microscopic examination of materials. Excited by monochromatic light, the sample emits scattered light with a specific frequency shift. The frequency shift (the so-called Raman shift) contain information about the vibrational states of the molecules and thus about the chemical composition and structure of the sample. This phenomenon was discovered by Sir C. V. Raman in the early 20th century. Since the beginning of the 20th century, the method, today mostly stimulated by a laser light source, is widely used in fields such as industry, chemistry, archaeology or for the qualitative and quantitative analysis of products in the pharmaceutical industry