We propose the term "NT-DSAEK" for grafts ≤50 μm. The described nomogram allows for standardized creation of NT grafts with a low tissue loss rate. This technique is safe and does not result in significant ECC loss compared with UT-DSAEK and DMEK grafts. Further studies are necessary to corroborate the postsurgical results of NT grafts.

Purpose: To evaluate agreement between eye banks (EBs) and a reading center on endothelial cell density (ECD) determinations in the Cornea Preservation Time Study. Methods: The Cornea Image Analysis Reading Center (CIARC) performed variable frame image analysis on EB-obtained–preoperative central endothelial images (after lamellar dissection for Descemet stripping automated endothelial keratoplasty by the EBs or before shipping, if surgeon prepared) to determine ECD. The EBs performed their usual method of ECD determination. The CIARC and EBs also provided ECD determinations from screening central endothelial images taken by the EBs during donor evaluation. Two independent masked CIARC readers determined ECD with measurements averaged. Results: The mean preoperative ECD was 15 cells/mm2 greater by the EBs than by CIARC (N = 1286, P < 0.001) with 95% limits of agreement of (−644, 675 cells/mm2). The limits of agreement in preoperative ECD were wider in the After-Lamellar-Dissection Group (−687, 683 cells/mm2) than in the Before Shipping Group [(−505, 633 cells/mm2); P = 0.03]. The EBs-determined preoperative ECD was within 10% of the CIARC-determined ECD for 886 (69%) image sets, with 236 (18%) higher by >10% and 164 (13%) lower by >10%. Excellent agreement appeared between the EBs and CIARC when 100–300 cells could be analyzed in contrast to <100 cells (SD = 308 cells/mm2 vs. SD = 603 cells/mm2; P < 0.001). Conclusions: The mean ECD by the EBs and CIARC were similar, but there was considerable variability between determinations for individual corneas. Agreement improved between the 2 measurements when more than 100 cells were able to be analyzed.

The aims of this study were 1) to compare area of cell loss (ACL) on trypan staining with ACL on specular imaging and 2) to evaluate the use of automated software for measuring ACL on trypan staining.Methods: Donor corneas with transplant-grade endothelium were mechanically injured with an 18-gauge cannula and a Fogla deep anterior lamellar keratoplasty dissector tip to create an easily identifiable "bullseye" pattern of cell death. Each cornea was then stained with trypan blue 0.06% for 90 seconds and imaged at 2• magnification. ACL on staining was measured using manual (ImageJ, National Institute of Health, Bethesda, MD) versus automated software tools (custom-built Aphelion macro, ADCIS, S.A., Saint-Contest, France). The bullseye was then imaged using specular microscopy, and ACL was measured by tracing the dead cell borders. ACL was then compared between both modalities.Results: Eleven donor corneas were evaluated. Both manual (0.42 mm 2 ) and automated (0.45 mm 2 ) measurements of ACL after trypan staining underestimated mean ACL on specular imaging (0.54 mm 2 ) (P , 0.01). However, on regression analysis, there was a good predictive correlation between automated trypan measurements and specular imaging (R 2 = 0.99, residual SE = 0.0044, P , 0.01). When ACL on specular imaging was measured by tracing cell nuclei along the margin of injury (rather than cell borders) (0.45 mm 2 ), there was no statistically significant difference between specular and automated trypan measurements (P = 0.95).Conclusions: Trypan-assisted automated measurements of ACL correlated well with ACL on specular imaging, suggesting that automated software may be a useful tool for evaluating endothelium in donor corneas.

Purpose: To compare the variation in corneal endothelial cell density (ECD) from the center to the periphery in unpeeled and peeled donor corneas and to determine the impact of eccentric trephining on total endothelial cells in Descemet membrane endothelial keratoplasty (DMEK) grafts. Methods: Mated donor cornea pairs were obtained. One cornea from each pair was peeled for DMEK, whereas the other was left unpeeled. Alizarin Red was used to stain the endothelial cells. High-resolution images at fixed magnification were obtained for the center, midperiphery (2.5 mm from the center), and the periphery (5 mm from the center). The cells were then counted, and ECD was calculated by a masked evaluator using ImageJ software. Regression analysis was then performed to evaluate the change in ECD as a function of radius (distance from the corneal center). The impact of eccentric trephining on total endothelial cells in a given DMEK graft was then calculated using numerical integration. Results: Ten pairs of corneas were evaluated. ECD increased by 1.4% (40.0 cells/mm2) (P = 0.03) for peeled corneas and 1.8% (51.5 cells/mm2) (P < 0.01) for unpeeled corneas for each millimeter from the center. There was no difference between peeled and unpeeled corneas in the mean central (P = 0.98) or peripheral (P = 0.35) ECD. Based on the increase in ECD as a function of radius, eccentric trephining of a 7.5-mm DMEK graft by 2.25 mm yields 0.95% more total endothelial cells per graft. Conclusions: Corneal ECD increases from the center to the periphery in both peeled and unpeeled corneas. Eccentric trephining increases the number of transplanted endothelial cells per graft.