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Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty

PURPOSE: To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. METHODS: DMEK transplants were prepared using two different methods: liquid bubble separation and...

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Autores principales: Bhogal, Maninder, Balda, Maria S, Matter, Karl, Allan, Bruce D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BMJ Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819631/
https://www.ncbi.nlm.nih.gov/pubmed/26740609
http://dx.doi.org/10.1136/bjophthalmol-2015-307534
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author Bhogal, Maninder
Balda, Maria S
Matter, Karl
Allan, Bruce D
author_facet Bhogal, Maninder
Balda, Maria S
Matter, Karl
Allan, Bruce D
author_sort Bhogal, Maninder
collection PubMed
description PURPOSE: To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. METHODS: DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface. RESULTS: Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04). CONCLUSIONS: Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group.
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spelling pubmed-48196312016-04-19 Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty Bhogal, Maninder Balda, Maria S Matter, Karl Allan, Bruce D Br J Ophthalmol Laboratory Science PURPOSE: To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. METHODS: DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface. RESULTS: Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04). CONCLUSIONS: Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group. BMJ Publishing Group 2016-04 2016-01-06 /pmc/articles/PMC4819631/ /pubmed/26740609 http://dx.doi.org/10.1136/bjophthalmol-2015-307534 Text en Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/ This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/
spellingShingle Laboratory Science
Bhogal, Maninder
Balda, Maria S
Matter, Karl
Allan, Bruce D
Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
title Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
title_full Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
title_fullStr Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
title_full_unstemmed Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
title_short Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
title_sort global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in descemet membrane endothelial keratoplasty
topic Laboratory Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819631/
https://www.ncbi.nlm.nih.gov/pubmed/26740609
http://dx.doi.org/10.1136/bjophthalmol-2015-307534
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