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. 2017 Nov;36(11):1295-1301.
doi: 10.1097/ICO.0000000000001301.

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

Meraf A Wolle  1 , David L DeMill, Lauren Johnson, Stephen I Lentz, Maria A Woodward, Shahzad I Mian
Affiliations

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

Meraf A Wolle et al. Cornea. 2017 Nov.

Abstract

Purpose: Availability of preloaded Descemet membrane endothelial keratoplasty (pDMEK) tissue may increase acceptance of DMEK in surgical management of endothelial disease. The goal of this study was to determine the safety of pDMEK grafts for 24 hours before surgery by analyzing endothelial cell loss (ECL) using 2 image analysis software programs.

Methods: A total of 18 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 9 corneas were left preloaded for 24 hours before 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 2 image analysis software programs.

Results: The control DMEK tissue resulted in 22.0% ± 4.0% ECL compared with pDMEK tissue, which resulted in 19.2% ± 7.2% ECL (P = 0.31). Interobserver 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. Intraobserver 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 method with increased efficiency and reproducibility compared with the previously validated Fiji.

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Conflict of interest statement

No financial disclosures of conflicts of interest.

Figures

Figure 1
Figure 1
Images of the catheter/injector set up used to load and store the DMEK grafts overnight (without Optisol and DMEK grafts).* * Pre-clinical experiment set up only.
Figure 2
Figure 2
A. 3 dimension projection image of calcein AM vital dye stained cornea. B. Color map of A representing the variations in depth of the tissue (blue represents signal closest to the glass bottom of the dish and red represents the deepest signals) C. Cross sections through the adjacent full cornea image showing the undulations of the tissue on the z axis. The numbered lines in A represent the XZ cross sections in C. Scale bars = 1 mm.
Figure 3
Figure 3
Final images after analysis showing ECL (hypofluorescent or black areas). A. MetaMorph (left) B. Fiji (right). Compared to C. Initial image obtained (bottom left).
Figure 4
Figure 4
The mean distribution of ECL amongst the 18 grafts analyzed using the two quantification methods discussed. In all grafts except for tissue #4, #8, and #9, MetaMorph ECL was slightly less than Fiji ECL. This difference was not statistically significant.
See this image and copyright information in PMC

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