Optimizing Visualization of Descemet Membrane Endothelial Keratoplasty Tissue: Assessing the Impact of Trypan Blue Exposure on Stain Duration and Corneal Endothelial Cell Function

Abstract

Purpose: To assess how trypan blue staining affects Descemet membrane endothelial keratoplasty (DMEK) graft visibility and corneal endothelial cell (CEC) mitochondrial respiration. Methods: DMEK grafts (n = 20) were stained with trypan blue 0.06% for 1, 3, 5, or 10 minutes. Each graft was injected into an artificial anterior chamber. Surgery was simulated with tapping and sweeping motions on the corneal surface and injections of balanced salt solution (BSS). Graft visibility was assessed at 5, 10, 20, and 30 minutes. Effects of trypan blue on mitochondrial respiration were assessed using primary CECs cultured from donor corneas (n = 43). Treatment wells exposed to trypan blue 0.06% (1, 5, or 30 minutes) and donor-matched control wells to methylene blue 1% (1 minute) or BSS (1, 5, or 30 minutes) were assayed for key respiration parameters. Results: After 5 minutes of surgical manipulation, grafts stained for 5 minutes were significantly more visible than grafts stained for 1 or 3 minutes; there was no added benefit of staining for 10 minutes. After 10 minutes of surgical manipulation, grafts stained for 3 minutes were more visible than grafts stained for 1 minute, without additional benefits of staining ≥5 minutes. No visibility differences were observed after ≥20 minutes of surgical manipulation. CEC mitochondrial respiration did not change significantly following trypan blue exposure for all intervals tested compared to BSS. Conclusions: Staining DMEK grafts with trypan blue for 3 to 5 minutes optimizes visibility during surgical manipulation without mitochondrial impairment. Corneal surgeons learning DMEK will benefit from optimizing this critical step.

Corneal Endothelium Viability Assay Using Trypan Blue Dye After Preloaded DMEK Graft Preparation

“…Trypan blue is safe and widely used at eye banks during DMEK preparation. 10,11 We previously reported that trypan blue can become toxic to the corneal endothelium when applied for long durations especially at higher concentrations. 10,11 However, a standardized additional 30 seconds of application can likely be applied to most DMEK protocols without approaching the established limits.…”

“…Trypan blue dye at the concentration of 0.06% is safe for use on corneal endothelium and does not compromise corneal endothelial cell mitochondrial functioning. 10,11 Trypan blue staining pattern can provide a much more comprehensive view of endothelial damage that occurred during DMEK preparation when compared with specular microscopy. The purpose of this research was to validate an alternative method for endothelial assessment after DMEK graft preparation using 0.06% trypan blue dye and visual quantification of ECL by eye bank technicians.…”

Keratoplasty in the United States: Trends and Indications From 2015 to 2020

“…The use of prestripped and preloaded tissue grafts from eye banks has reduced operative time and tissue wastage. 14,15 Studies have demonstrated how optimal application of trypan blue dye can improve visibility and help surgeons with graft orientation, 16,17 and graft marking can prevent upside-down implantation and reduce primary graft failure. 18 Despite the rapid increase in DMEK prevalence in the United States, the technological and training requirements for DMEK procedures have led to wide variation in the uptake rate around the world.…”

The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues

“…Lutein/trypan blue staining persisted even if excited EDM tissues were extensively washed before DMEK grafting. This finding represents a great value as DMEK surgery involves the manipulation of EDM-stained grafts inside the anterior chamber for at least 7 min, the mean time required for the unrolling of the flap and its correct positioning in contact with the recipient’s cornea [ 17 , 18 ].…”