Purpose: With new compounding pharmacy laws, the only economically feasible approach to using lissamine is through dye-impregnated strips. This research aims to determine the concentration of lissamine that can be obtained using a single commercially available lissamine strip. With the optimal vital staining requiring 1% concentration of lissamine, we sought to obtain this concentration using supplies in an ordinary ophthalmology clinic.
Methods: A standard curve was generated using compounded lissamine green 1% solution. Serial dilutions were made with 3 different diluents and measured using a spectrophotometer at a wavelength of 633 μm. Combinations of the number of strips, amount of solvent, and absorption time were performed to obtain a 1% solution. Cost analyses were performed to select the most economical method.
Results: Single lissamine strips wetted with any of the diluents produced 0.17% 0.05% (95% confidence interval) lissamine solution, a 5-fold weaker concentration than the optimal for vital staining. Combinations of 4 strips in 200 μL (4 drops) for 1 minute and 2 strips in 200 μL for 5 minutes were found to reach concentrations of 1%. Cost analysis showed that the 2 strip/4 drops/5 minutes method costs $0.67 and the 4 strips/4 drops/1 minute method $1.27.
Conclusions: Use of a single lissamine strip leads to suboptimal concentrations for vital staining. With only the addition of disposable microcentrifuge tubes to the clinical setting, ophthalmologists can make 1% solutions of lissamine. This solution is both more economical and in compliance with both state and national compounding laws.
Purpose: To investigate the performance of lissamine green strips from different manufacturers. Additionally, the repeatability, need for sequential dye instillation and impact of repeated lid evertion on lid wiper staining were assessed.
Methods: Study 1 was a prospective, randomised cross-over study where controlled volumes of lissamine green solution prepared from strips (Biotech, Lissaver, GreenGlo, OPGreen) were instilled (right eye: single; left eye: double instillation) on five different days, with OPGreen being tested twice. Lids were everted and digital photographs taken, which were later assessed by a masked observer. Study 2 was an investigator-masked, randomised, controlled study testing the impact of single versus repeated lid evertion. Lid wiper staining was graded (0 to 3 in 0.5 steps).
Results: Lid wiper staining differed significantly between lissamine green solutions, with GreenGlo showing the highest amount of staining, and Lissaver the least (all p>0.009). There were no differences in lid wiper staining over two days, using the OPGreen solution (all p>0.05). The number of drops instilled (single versus double) did not significantly affect lid wiper staining (all p>0.05). Repeated lid evertion increased lid wiper staining (p=0.007 when combined with double drop instillation). Light absorbance patterns and measured concentrations aligned with clinical findings.
Conclusion: There were significant differences in performance between lissamine green solutions. Lid wiper staining was impacted by repeated lid evertion but sequential instillation and use of the Korb grading scale provided little advantage over simpler methods Clinicians must consider this when investigating lid wipers, especially when interpreting a negative finding.
Dyes are used in ophthalmology for a variety of diagnostic purposes, in the clinic on an out-patient basis; and as an integral part of ocular procedures and surgeries. In clinical practice, the use of individually wrapped, sterile, dye-impregnated paper strips is the preferred staining technique for the ocular surface. Dyes used for posterior segment surgeries are used to stain tissues that are difficult to see such as the internal limiting membrane or anterior capsule. Here, we discuss the various dyes being used in ophthalmology.
Double vital staining : Use of 1% flourescein + 1% lissamine green together in diagnosis of ocular surface disorders. This is done for combined assessment of corneal and conjunctival surface evaluation. Both the strips are wet and applied at the same time, but as more lissamine green is required, usually two strips of lissamine green are used with one strip of flourescein. 
The use of diagnostic dyes represents one of the most efficient, objective, non-invasive, and directly visible means we have of identifying and tracking ocular-surface changes at the cellular level. Though they're particularly useful in dry-eye diagnosis and clinical trials, the utility of these dyes also extends far beyond dry eye to numerous other ocular surface conditions that affect corneal and conjunctival cells.The three dyes with the highest visibility in the eye-care practitioner's office today are fluorescein, rose bengal and lissamine green. In this month's column, we'll explore each of these three dyes, looking at their merits and drawbacks, with a particular focus on lissamine green, an underappreciated tool.
FluoresceinThis yellow-colored dye was first used on the eye in 1882 when researchers discovered it could reveal corneal epithelial defects.1 Fluorescein is now used in various areas of ophthalmology, especially in retinal vasculature imaging and as a systemic marker in numerous pharmacokinetic studies. From the ocular-surface perspective, the water-soluble dye molecules diffuse into the intercellular spaces between living cells. The intensity of the stain is increased in areas of cellular degeneration or death, where the damage to cells, cell membranes and cell-to-cell junctions allow for the intracellular spaces to be more highly penetrated by the dye. This property makes the dye most useful for observing permeability in corneal epithelial and endothelial cells. However, in conjunctival staining it may become more difficult to make precise observations because of the dye's presence in both cells and intercellular spaces.2Localization of the dye in this manner allows the practitioner to identify areas of desiccated or injured cells and areas where ocular-surface damage has occurred, such as epithelial defects and corneal abrasions.Standardized grading of corneal and conjunctival fluorescein staining as well as measurement of tear-film breakup times, which have been made more clearly visible using fluorescein, have given this dye broad applicability as a dry-eye diagnostic test. It is particularly valuable as an assessment tool in clinical studies of dry eye. Research has indicated that fluorescein stains some types of healthy cells in vitro, though this typically is not visible except by using fluorescence microscopy.3One challenge with the use of fluorescein staining is that the presence of the dye in the tear film may obscure readings of ocular-surface staining.4 A method developed at Ophthalmic Research Associates to circumvent this difficulty is to first assess tear-film related measurements (e.g., TFBUT) immediately upon instillation of micro-quantities of the dye while a portion of the dye is concentrated in the tear film. Once the dye diffuses from the tear film to the ocular surface, ocular-surface related measurements (e.g., corneal and conjunctival staining) can be evaluated. The portion of fluorescein that has penetrated to the ocular surface and localized in areas of cell damage will then become visible. Using controlled micro-quantities of fluorescein has been shown to greatly contribute to the repeatability and precision of such measures.5 The level of standardization achieved using this method has allowed TFBUT, fluorescein staining and, in some cases, fluorophotometry to become standards of evaluation for testing the efficacy of novel dry-eye therapies.Rose bengal, though useful in some situations, may have too many drawbacks to be the dye of choice in dry-eye diagnosis.Fluorescein is still an effective diagnostic dye for dry-eye management, but could yield to lissamine green in some situations. It may be time to give lissamine green a go, given its excellent safety profile and sensitivity in detecting the signs of dry eye.The use of a Wratten #12 yellow filter also enhances the visibility of fluorescein and is recommended when observing TFBUT and staining. In fluorophotometry, the instillation of a calculated amount of fluorescein onto the ocular surface allows measurement of tear volume immediately and over time in order to determine the tear turnover rate, a useful way to diagnose dry eye.
Rose BengalThe second of these three dyes, rose bengal, is actually a derivative of fluorescein. Both dyes are hydroxyxanthines, though they differ structurally: Eight more halogens, four chlorines and four iodines are present on the rose-bengal molecule. Its instillation in the eye was first documented in 1914 and later popularized by Swedish physician Henrik Sjögren in the 1930s for diagnosing keratoconjunctivitis sicca; he noted a distinctive staining pattern following rose bengal instillation in the eyes of patients having the disorder.6 Since that time it's been used for the evaluation of numerous other ocular pathologies including herpetic corneal epithelial dendrites, superficial punctate keratitis, meibomian gland dysfunction and dysplastic or squamous metaplastic cells of conjunctival squamous neoplasms.3 Researchers have hypothesized that the differences in molecular structure between these two dyes confer the functional differences observed between the two molecules.3 However, our understanding of the functionality of rose bengal has shifted in recent years. Unlike fluorescein and lissamine green, it cannot be termed a \"vital\" dye. Rose bengal stains not only dead or dying cells as previously thought, but actually stains normal, healthy, living cells.3 How then do we explain its selective staining patterns in dry eye or Sjögren's syndromeResearch on rose bengal has revealed that it's blocked from staining the ocular su