Trypan-Assisted Automated Endothelial Cell Loss Measurements Compared With Specular Microscopy.

PURPOSE: 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 mm2) and automated (0.45 mm2) measurements of ACL after trypan staining underestimated mean ACL on specular imaging (0.54 mm2) (P < 0.01). However, on regression analysis, there was a good predictive correlation between automated trypan measurements and specular imaging (R2 = 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 mm2), 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.

Ex Vivo Safety and Efficacy of Paired Peripheral Incisions in Descemet's Membrane Endothelial Keratoplasty Grafts to Facilitate Unscrolling.

PURPOSE: To evaluate the ex vivo safety and efficacy of using paired peripheral incisions to achieve a triple scroll conformation that facilitates unscrolling in Descemet membrane endothelial keratoplasty (DMEK). METHODS: The safety of adding paired peripheral incisions to DMEK grafts was evaluated by assessing endothelial cell loss (ECL) and risk of tearing. ECL was measured using calcein-AM staining after incisions. The risk of tearing was evaluated by comparing incision lengths before and after simulated DMEK surgery using cadaveric eyes. Efficacy was evaluated by comparing the scrolling pattern and the width of grafts with different incision lengths (0.0 mm, 0.5 mm, and 1.0 mm). Surgical unscrolling times in simulated DMEK surgery by a novice DMEK surgeon were evaluated to determine whether incisions facilitate unscrolling in DMEK surgery. RESULTS: The mean ECL after adding incisions was 0.78% ± 0.23%. There was no significant change in incision length after simulated DMEK surgery (P = 0.6). In donor grafts aged less than or equal to 65 years, 60% (6/10) achieved a stable triple scroll with 0.5 mm incisions and 80% (8/10) achieved a stable triple scroll with 1.0 mm incisions. In donor grafts aged greater than 65 years, 0% (0/4) achieved a stable triple scroll. Mean graft width increased significantly after forming a triple scroll (5575 µm ± 1128 µm) compared with baseline (1563 µm ± 428 µm) (P < 0.001). In the hands of a novice DMEK surgeon, the mean unscrolling time was significantly shorter with incisions (2.61 min ± 1.41 min) versus without incisions (5.44 min ± 3.17 min) (P = 0.02). CONCLUSIONS: Paired peripheral incisions are safe and effective for inducing a triple scroll in DMEK grafts with donor age less than or equal to 65 years. Adding incisions may facilitate unscrolling for inexperienced DMEK surgeons.

Characterization of Endothelial Cell Loss in Pre-Descemet Endothelial Keratoplasty Graft Preparation.

PURPOSE: To characterize the pattern and factors affecting endothelial cell loss (ECL) in pre-Descemet Endothelial Keratoplasty (PDEK) graft preparation. METHODS: A prospective study was performed to characterize the pattern of ECL and the impact of inflation pressure in PDEK. Donor corneas were randomized to inflation with air versus Optisol GS storage media. PDEK preparation was performed under continuous pressure monitoring. Trypan blue was used to grade the tissue as acceptable (<25% ECL) or unacceptable (≥25% ECL). Rate of unacceptable ECL was correlated with injection media type and inflation pressure. A retrospective study was then performed of all attempted PDEK preparations at Lions Gift of Sight to evaluate impact of donor tissue factors on ECL. Donor age and tissue preservation time were evaluated and correlated with ECL with PDEK bubbling. RESULTS: Twenty-five corneas were tested prospectively. A reticular pattern of ECL that varied in severity occurred with bubbling. There was no difference in peak inflation pressure or mean expansion pressure between air (706.0, 510.7 mm Hg) and Optisol GS (852.9, 653.0 mm Hg). Increasing peak inflation pressure and mean expansion pressure were associated with an increased risk for unacceptable ECL. On retrospective evaluation of 131 attempted PDEKs, only 44.0% of cases with successful bubbles had acceptable endothelium after processing. Increasing donor age and decreasing preservation time were associated with increased rates of acceptable endothelium. CONCLUSIONS: PDEK processing can result in a reticular pattern of ECL. Higher inflation pressures are associated with greater ECL. Older donor tissues with shorter preservation times might be preferable for PDEK.

Peripheral Endothelial Cell Density in Descemet Membrane Endothelial Keratoplasty Grafts.

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/mm) (P = 0.03) for peeled corneas and 1.8% (51.5 cells/mm) (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.

Comparison of Donor Cornea Endothelial Cell Density Determined by Eye Banks and by a Central Reading Center in the Cornea Preservation Time Study.

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/mm greater by the EBs than by CIARC (N = 1286, P < 0.001) with 95% limits of agreement of (-644, 675 cells/mm). The limits of agreement in preoperative ECD were wider in the After-Lamellar-Dissection Group (-687, 683 cells/mm) than in the Before Shipping Group [(-505, 633 cells/mm); 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/mm vs. SD = 603 cells/mm; 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.

Technique for Preparing Ultrathin and Nanothin Descemet Stripping Automated Endothelial Keratoplasty Tissue.

PURPOSE: To describe and report outcomes of our single-pass microkeratome technique for preparation of ultrathin (UT, ≤100 µm) and nanothin (NT, ≤50 µm) Descemet stripping automated endothelial keratoplasty (DSAEK) grafts. METHODS: To prepare NT-DSAEK grafts, a pachymetry nomogram specific to each technician and individual microkeratome head was developed based on accumulated precut and postcut pachymetry data from previous DSAEK grafts. Mean graft thickness as well as precut and postcut endothelial cell counts (ECCs) of NT-DSAEK, UT-DSAEK, and Descemet membrane endothelial keratoplasty (DMEK) grafts between July 2015 and July 2017 were calculated and compared statistically. Endothelial cell loss was evaluated using calcein AM stains and ImageJ analysis. Postcut graft thickness and rates of perforation/tissue loss for NT-DSAEK grafts between May and July 2017 were calculated to determine overall graft preparation success rates. RESULTS: Mean postcut graft thickness for all grafts within the NT range was 41.0 ± 6.4 µm (range 26-50 µm). Mean ECC did not differ between NT-DSAEK, UT-DSAEK, and DMEK grafts (P = 0.759 and 0.633, respectively). The overall tissue loss rate from attempted NT-DSAEK was 4.8%. Excluding cases of perforation, the chance of achieving NT thickness was 60% and within the traditional UT range was 100%. CONCLUSIONS: 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.