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Jeffrey D Holiman, Philip Dye, Chris Stoeger; A Technique for Quantification of Endothelial Cell Loss in Eye Bank Prepared DMEK Grafts Intended for Clinical Use. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1152.
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© ARVO (1962-2015); The Authors (2016-present)
Descemet membrane endothelial keratoplasty (DMEK) is a surgical technique to replace dysfunctional corneal endothelium. Damage to endothelial cells in the graft area can easily occur during the separation of Descemet membrane (DM) from stroma while preparing the graft. This damage may affect suitability for transplant by reducing the endothelial cell density (ECD). Trypan blue stain 0.06% (DORC, The Netherlands) is currently used in some preparation protocols to visualize the free edge of DM. This stain can be used to visualize cells with ruptured cell membranes and areas of denuded DM. The objective of this study is to determine if pan-endothelial cell loss (ECL) of the graft area can be determined conveniently and without compromising graft viability by using the trypan blue stain. Additionally, a more accurate measurement of ECD may be determined by combining percent ECL with known ECD as determined by specular microscopy.
Six human donor corneas unsuitable for transplant were prepared for DMEK in an eye bank setting under an operating microscope, age range 52-73 yrs, median age 54 yrs, with regular endothelium and avg ECD of 2457 cells/mm2 with range 2012-3040. Trypan blue staining for 90 seconds was used to visualize endothelium that lost viability using a comparison of pre-preparation image to post-preparation image. The central 8mm was digitally cropped and ECL was calculated using Image J with trainable segmentation plug-in to obtain a histogram with pixel counts of area of damage indicated by stain uptake divided by total area of endothelium in the central 8mm. Final ECD was calculated by subtracting the % ECL from the ECD.
All six DMEK grafts were able to be analyzed. The average total cell loss of graft area is 3.88% (range 2.0-5.9%) with the average net cell loss at 3.2% (range 1.6-5.0%). Final ECD average was 2365 (range 1906-2920).
In this small series the combined average ECL in 6 corneas prepared for DMEK in an eye bank setting was 3.2%. We have demonstrated that a more accurate final ECD may be determined than by specular microscopy alone which is the standard protocol in US eye banks. This measurement was obtained in a manner that does not compromise tissue quality.
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