Quantitative Analysis of Endothelial Cell Loss in Preloaded Descemet Membrane Endothelial Keratoplasty Grafts

Abstract

Purpose Availability of preloaded DMEK (pDMEK) tissue may increase acceptance of DMEK in surgical management of endothelial disease. The goal of this study is to determine the safety of pDMEK grafts for 24 hours prior to surgery by analyzing endothelial cell loss (ECL) using two image analysis software programs. Methods Eighteen cadaveric corneas were prepared for DMEK using a standardized technique and loaded in a modified jones tube injector. Nine of the corneas were injected into Calcein AM vital dye after 1 minute (controls) and the remaining nine corneas were left preloaded for 24 hours prior to injection into vital dye for staining. The stained corneas were imaged using an inverted confocal microscope. ECL was then analyzed and quantified by 2 different graders using two image analysis software programs. Results The control DMEK tissue resulted in 22.0 ± 4.0 % ECL compared to pDMEK tissue which resulted in 19.2 ± 7.2% ECL (p=0.31). Inter-observer agreement was 0.93 for MetaMorph and 0.92 for Fiji. The average time required to process images with MetaMorph was 2±1 minutes, and with Fiji was 20±10 minutes. Intra-observer agreement was 0.97 for MetaMorph and 0.93 for Fiji. Conclusions Preloading DMEK tissue is safe and may provide an alternative technique for tissue distribution and surgery for DMEK. The use of Metamorph software for quantifying ECL is a novel and accurate imaging software with increased efficiency and reproducibility compared to the previously validated Fiji.

Quantitative analysis of cornea endothelial cell damage from enucleation, corneal buttoning, and storage in donor corneas using trypan blue dye staining

“…The mean corneal endothelial cell damage rate was 16.8 ± 5.9% in the donor corneas previously loaded into a Jones tube and 9.3 ± 5.9% in non-preloaded corneas. Wholle et al [10] evaluated the endothelial cell damage of preloaded DMEK grafts to determine the safety of preloaded DMEK grafts for 24 hours before surgery by analyzing endothelial cell loss. They loaded 18 corneas into a modified Jones’ tube that had been filled with Optisol.…”

“…Since their report, many researchers have reported the degree of corneal endothelial cell damage from experimental donor punches, donor preparations, and lamellar keratoplasty procedures using various corneal staining solutions for donor corneas not suitable for keratoplasty in patients. [5–10]…”

<p>Perceived difficulties and barriers to uptake of Descemet’s membrane endothelial keratoplasty among surgeons</p>

“…In their study involving 31 DMEK grafts, Tran et al reported that tissue preloading incurred more endothelial cell loss than simply prestripping tissue 13. Studies have also found greater cell loss in the S-stamp area in preloaded DMEK grafts, often with complete loss of all cells in the stamped area 14. DMEK presents a steep learning curve and surgical skills that are entirely different from DSEK 8.…”

Viability of Descemet Membrane Endothelial Keratoplasty Grafts Folded in the Eye Bank

“…The rate of cell loss in the samples peeled and removed as a scroll are consistent with the 22.0% and 19.2% rates of cell loss in groups measured by Wolle and colleagues using a comparable methodology of assessment. 4 It is notable that tri-folding resulted in significantly less cell loss in this study, which we believe may be due to the protection of an endothelium-in morphology during manipulation and staining. The data suggest that at the minimum, the additional manipulation of the periphery of the graft required for tri-folding does not appear to contribute to increased cell loss in the tri-folding group.…”