Pre-dissected donor graft preparation for Pre-Descemet endothelial keratoplasty by the eye bank with 3D microscope system

Abstract

Purpose : To investigate the quality of pre-Descemet endothelial keratoplasty (PDEK) graft while viewing a three-dimensional image on a large display in a heads-up position.

Methods : Four donor corneas referred for disposal with endothelial cell density above 2000 cells/mm² were pre-dissected with Ngenuity 3D visualization system. The air injection was performed with a 30 gauges needle into the central cornea stroma and a type-1 big-bubble was successfully obtained. A small incision in bubble periphery was performed with a 15 degree knife and extended the cut with a scissor leaving a small hinge of one hour clock. S-stamp was performed in the anterior face of pre-Descemet layer and the graft was repositioned over the corneal stroma. It was fixed a weight in the scleral rim allowing the tissue to sink in the preservation media vial. After two days under refrigeration the tissue was completely dissected from the cornea and the features of PDEK graft roll in BSS solution were analyzed. For confocal fluorescence microscopy analysis the graft was flat mounting on a filter paper with the endothelial side up. Triple endothelial labeling was performed with Hoechst 33342, ethidium-homodimer (EthD-1) and calcein-AM and the analyzed variables were: density of nuclei stained in blue by Hoechst; density of nuclei stained in red by EthD-1; percentage of green-stained area by calcein-AM.

Results : The mean donor age was 63,3 years old. A no-touch preparing technique for pre-dissected PDEK graft was performed for the first time with 3D microscope system. Total time procedure was 14.7 minutes, the volume of 6.1 cm³ was injected and produced a 6.952 mm bubble diameter. The PDEK graft roll length was 7.532 mm with the central width of 2.257 mm. The density of endothelial cell nuclei stained in blue was 2407 cells/mm² while stained in red was 37 cells/mm². The percentage of viable area cells stained in green by calcein-AM was 86.7%, and the rest corresponded to denuded areas without fluorescence signal.

Conclusions : Pre-dissected PDEK graft processed by eye bank presented acceptable endothelial feasibility. The 3D microscope system enable the surgical team also have the same visualization of the surgeon.

This is a 2020 ARVO Annual Meeting abstract.

 

Upper left - needle into cornea stroma, upper right - cornea emphysema, bottom left - type-1 big-bubble, bottom right - ready graft.

 

Triple stained confocal fluorescence images.