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

Abstract

Purpose: The purpose of this study was to establish a validated method, consistent with Eye Bank Association of America medical standards, for evaluating endothelial cell loss (ECL) from an entire Descemet membrane endothelial keratoplasty (DMEK) graft using trypan blue dye as an alternative to specular microscopy. Method: Twenty-nine corneas were prepared for preloaded DMEK by a single technician, and the endothelium was stained with trypan blue dye for 30 seconds. The technician estimated total cell loss as a percentage of the graft and captured an image. Images were evaluated by a blinded technician using ImageJ software to determine ECL and compared with endothelial cell density from specular microscopy. Tissue processing intervals were analyzed for 4 months before and after implementation of this method. Results: For the 29 grafts, there was no statistically significant difference (t test, P = 0.285) between ECL estimated by a processor (mean = 5.8%) and ECL calculated using an ImageJ software (mean = 5.1%). The processor tended to estimate greater ECL than the actual ECL determined by ImageJ (paired t test, P = 0.022). Comparatively, postprocessing endothelial cell density measured by specular microscopy were higher compared with the preprocessing endothelial cell density (mean = 4.5% P = 0.0006). After implementation of this evaluation method, DMEK graft processing time intervals were reduced by 47.9% compared with specular microscopy evaluation (P < 0.001). Conclusions: Our results show that visual ECL estimation using trypan blue staining by a DMEK graft processor is a reliable and efficient method for endothelial assessment. Unlike specular microscopy, this method achieves comprehensive visualization of the entire endothelium, reduces total time out of cold storage, and decreases total time required to prepare and evaluate DMEK grafts.