Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK.

Maninder Bhogal, Chan N Lwin, Xin-Yi Seah, Elavazhagan Murugan, Khadijah Adnan, Shu-Jun Lin, Gary Peh, Jodhbir S Mehta

PloS one 2017

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To establish a method for assessing graft viability, in-vivo, following corneal transplantation. Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model. Global, macroscopic images of the entire graft and individual cell resolution could be attained by altering the magnification of a clinical confocal scanning laser microscope. Patterns of cell loss observed in situ were compared to those seen using standard ex-vivo techniques. Calcein AM showed a positive dose-fluorescence relationship. A dose of 2.67μmol was sufficient to allow clear discrimination between viable and non-viable areas (sensitivity of 96.6% with a specificity of 96.1%) and was not toxic to cultured endothelial cells or ex-vivo corneal tissue. Patterns of cell loss seen in-situ closely matched those seen on ex-vivo assessment with fluorescence viability imaging, trypan blue/alizarin red staining or scanning electron microscopy. Iatrogenic graft damage from preparation and insertion varied between 7-35% and incarceration of the graft tissue within surgical wounds was identified as a significant cause of endothelial damage. In-situ graft viability assessment using clinical imaging devices provides comparable information to ex-vivo methods. This method shows high sensitivity and specificity, is non-toxic and can be used to evaluate immediate cell viability in new grafting techniques in-vivo.

Citation

Maninder Bhogal, Chan N Lwin, Xin-Yi Seah, Elavazhagan Murugan, Khadijah Adnan, Shu-Jun Lin, Gary Peh, Jodhbir S Mehta. Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. PloS one. 2017;12(10):e0184824