Preloaded Descemet Membrane Endothelial Keratoplasty Research Articles

Predicting Long-Term Endothelial Cell Loss after Preloaded Descemet Membrane Endothelial Keratoplasty in Fuchs’ Endothelial Corneal Dystrophy: A Mathematical Model

(1) Background: This study offers a biexponential model to estimate corneal endothelial cell decay (ECD) following preloaded “endothelium-in” Descemet membrane endothelial keratoplasty (DMEK) in Fuchs’ endothelial corneal dystrophy (FECD) patients; (2) Methods: A total of 65 eyes undergoing DMEK alone or combined with cataract surgery were evaluated. The follow-up period was divided into an early phase (first 6 months) and a late phase (up to 36 months). Endothelial cell count (ECC) and endothelial cell loss (ECL) were analyzed; (3) Results: The half time of the ECD was 3.03 months for the early phase and 131.50 months for the late phase. The predicted time-lapse interval to reach 500 cells/mm2 was 218 months (18.17 years), while the time-lapse interval to reach 250 cells/mm2 was 349 months (29.08 years). There was no statistically significant difference between the ECL in DMEK combined with cataract extraction and DMEK alone at 24 months (p ≥ 0.20). At the late phase, long-term ECL prediction revealed a lower ECC half time in patients undergoing DMEK combined with cataract surgery (98.05 months) than DMEK alone (250.32 months); (4) Conclusions: Based on the mathematical modeling, a predicted average half-life of a DMEK graft could reach 18 years in FECD. Moreover, combining cataract extraction with DMEK could result in excessive ECL in the long term.

Effect of Low-Temperature Preservation in Optisol-GS on Preloaded, Endothelium-Out DMEK Grafts.

Three groups of 15 DMEK grafts (five per group) were pre-loaded in the DMEK RAPID Mini and preserved in Optisol-GS for 72 h at different temperatures: group A at >8 °C, group B between 2-8 °C and group C at <2 °C. After stripping and preservation, the viability of the endothelium, cell loss and morphology were assessed through light microscopy following trypan blue and alizarin red staining. Overall mortality was 4.07%, 3.97% and 7.66%, in groups A, B and C, respectively, with percentages of uncovered areas of 0.31%, 1.36% and 0.20% (all p > 0.05). Endothelial cell density variation was 5.51%, 3.06% and 2.82% in groups A, B and C, respectively (p = 0.19). Total Endothelial Cell Loss (ECL) was 4.37%, 5.32% and 7.84% in groups A, B and C, respectively (p = 0.39). Endothelial cell morphology was comparable in all three groups. In the DMEK RAPID Mini, low temperatures (<2 °C) may affect the quality of pre-loaded grafts, inducing a higher ECL after 72 h of preservation, although no significant differences among groups could be proved. Our data would suggest maintaining grafts loaded in the DMEK RAPID Mini at temperatures between 2-8 °C for appropriate preservation.

Clinical Outcomes of Preloaded Descemet Membrane Endothelial Keratoplasty With Endothelium Inward: A 24-Month Comparative Analysis Between Fuchs Endothelial Corneal Dystrophy and Bullous Keratopathy.

The aim of this study was to compare long-term clinical outcomes of preloaded Descemet membrane endothelial keratoplasty (DMEK) between Fuchs endothelial corneal dystrophy (FECD) and bullous keratopathy (BK). In this single-center retrospective clinical case series, 71 eyes of 64 patients indicated with FECD (62%) or BK (38%) (with or without cataract) were treated with preloaded DMEK grafts between March 2018 and February 2020. Standard DMEK peeling, followed by manual folding of the tissue with endothelium-inward orientation and storing in a preloaded fashion inside a 2.2-mm intraocular lens cartridge. All tissues were delivered using a bimanual pull-through technique, followed by air tamponade. Graft unfolding time, endothelial cell loss, corrected distance visual acuity, central corneal thickness, rebubbling rate, and intraoperative and postoperative complications at 1, 3, 6, 12, and 24 months were recorded. The mean intraoperative graft unfolding time in FECD did not differ from the BK group ( P = 0.6061). Cystoid macular edema did not differ in either group ( P = 0.6866). The rebubbling rate was found to be significantly higher in FECD compared with the BK group ( P = 0.0423). Corrected distance visual acuity significantly improved at the first month after surgery ( P = 0.0012), with no differences between FECD and BK at 24 months ( P = 0.2578). Central corneal thickness was stable postoperatively and showed no differences between the groups ( P = 0.3693). Significantly higher endothelial cell counts were observed in the FECD group at 24 months ( P = 0.0002). Preloaded DMEK with "endothelium-in" offers acceptable intraoperative time, rebubbling rate, and clinical outcomes in both FECD and BK groups. Patients with FECD show better postoperative clinical outcomes even if the rebubbling rate is relatively high.

DMEK graft: One size does not fit all.

Descemet membrane endothelial keratoplasty (DMEK) is a popular procedure for the treatment of corneal endothelial diseases mainly targeting Fuchs endothelial corneal dystrophy (FECD) and pseudophakic bullous keratopathy (PBK). Although DMEK has multiple advantages, it is challenging in terms of graft preparation and delivery. One of the crucial factors of DMEK graft preparation is determining the size of the graft. Evaluating risks and benefits of transplanting larger or smaller grafts compared with the descemetorhexis performed following a standard DMEK procedure thus becomes important. Advanced techniques like pre-loaded DMEK requires pre-selection of graft diameter without physical examination of the eye making it more challenging. Therefore, recognizing the benefits of graft size and the number of transplanted endothelial cells becomes essential. Smaller DMEK grafts have been preferred and accepted for grafting. Larger diameter grafts have advantages but can be challenging due to higher detachment rates. We thus aim to review the challenges of preparing and delivering DMEK tissues with small or large diameter based on selected descemetorhexis area, discuss the outcomes based on different graft sizes, highlight related complications and suggest which cases may benefit from adopting smaller or larger graft size.

Factors Affecting the Success Rate of Preloaded Descemet Membrane Endothelial Keratoplasty With Endothelium-Inward Technique: A Multicenter Clinical Study

To evaluate factors affecting the outcomes of preloaded Descemet membrane endothelial keratoplasty (pl-DMEK) with endothelium-inward. Retrospective clinical case series and a comparative tissue preparation study. Participants: Fifty-five donor tissues for ex vivo study and 147 eyes of 147 patients indicated with Fuchs endothelial dystrophy or pseudophakic bullous keratopathy with or without cataract. Standardized DMEK peeling was performed with 9.5-mm-diameter trephination followed by second trephination for loading the graft (8.0-9.5 mm diameter). The tissues were manually preloaded with endothelium-inward and preserved for 4 days or shipped for transplantation. Live and dead assay and immunostaining was performed on ex vivo tissues. For the clinical study, the tissues were delivered using bimanual pull-through technique followed by air tamponade at all the centers. Tissue characteristics, donor and recipient factors, rebubbling rate, endothelial cell loss (ECL), and corrected distance visual acuity (CDVA) at 3, 6, and 12 months. At day 4, significant cell loss (P=.04) was observed in pl-DMEK with loss of biomarker expression seen in prestripped and pl-DMEK tissues. Rebubbling was observed in 40.24% cases. Average ECL at 3, 6, and 12 months was 45.87%, 40.98%, and 47.54%, respectively. CDVA improved significantly at 3 months postoperation (0.23 ± 0.37 logMAR) (P < .01) compared to the baseline (0.79 ± 0.61 logMAR). A significant association (P < .05) between graft diameter, preservation time, recipient gender, gender mismatch, and recipient age to rebubbling rate was observed. Graft loading to delivery time of pl-DMEK tissues in endothelium-inward fashion must be limited to 4 days after processing. Rebubbling rate and overall surgical outcomes following preloaded DMEK can be multifactorial and center-specific.

Preloaded descemet membrane endothelial keratoplasty grafts with endothelium outward: a validation study of the DMEK rapid device

AbstractPurposeTo validate a device (DMEK Rapid, Geuder AG, Germany) for preloading and transporting DMEK grafts with endothelium facing outwards.MethodsStripped DMEK tissues were divided into 2 groups: internal transportation simulations (study A) and cross‐country transport study (Study B). Study A was designed to investigate the influence of 2 types of media, tissue culture medium (TCM; n = 12) or transport medium (TM; n = 12), on DMEK tissues preloaded in the DMEK Rapid device. The tissues were placed on the laboratory rocker for 4 days at room temperature. For Study B, DMEK tissues (n = 9), preloaded with TM were shipped from Italy to the UK and analysed within 72 hr. For both studies, endothelial cell loss (ECL – trypan blue staining), morphology (alizarin red staining), viability (live/dead staining) and expression of biomarker (ZO‐1 immunostaining) were performed.ResultsPost‐stripping ECL was 7.5 ± 66.2% in TCM group, compared to 9.1 ± 64.9 from the TM group (p = 0.54). Post‐simulation ECL was 14.3 ± 63.8% from the TCM group and 11.5 ± 64.6% from the TM group (p = 0.08). Alizarin red (n = 4 in each group) showed the low polymorphism in both media groups. Weka segmentation of the live/dead analysis showed 78.83 ± 62.78% viability in TCM group and 80.83 ± 63.48% viable cells in TM group (p = 0.60). ZO‐1 immunostaining (n = 4 in each group) showed the presence of tight junctions in both the groups. In study B (n = 9), post‐stripping ECL was 10.1 ± 64.5% and 3.3 ± 66.6% after transporting the tissues from Italy to the UK (p = 0.05). Alizarin red (n = 3) showed proper tissue morphology and viability of 85.16 ± 65.33% was detected (n = 3). ZO‐1 (n = 3) was expressed in all tested grafts.ConclusionsPreloading DMEK tissue with endothelium outwards and transported within 72 hr using the DMEK Rapid device is safe and can be pursued for clinical application.

Viability of preloaded Descemet membrane endothelial keratoplasty grafts with 96-hour shipment

ObjectiveTo assess feasibility and compare the effects of 96-hour shipment of Descemet membrane endothelial keratoplasty (DMEK) grafts as a scroll or a tri-fold on cell viability.Methods and analysisDMEK grafts were...

Cost-effectiveness analysis of preloaded versus non-preloaded Descemet membrane endothelial keratoplasty for the treatment of Fuchs endothelial corneal dystrophy in an academic centre

AimsTo determine the cost-effectiveness of preloaded Descemet membrane endothelial keratoplasty (pDMEK) versus non-preloaded DMEK (n-pDMEK) for the treatment of Fuchs endothelial corneal dystrophy (FECD).MethodsFrom a societal and healthcare perspective, this...

Early Complications With Preloaded Descemet Membrane Endothelial Keratoplasty Are Not Dependent on Optisol-GS Washout or Trypan Blue Restaining.

To describe the intraoperative and early postoperative complications using preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts with intraocular injection of the graft in Optisol-GS and omission of trypan blue restaining. This is a retrospective case series of 132 consecutive eyes with Fuchs endothelial dystrophy or endothelial failure who underwent DMEK using preloaded donor tissue prepared as previously described. The graft was not restained with trypan blue by the surgeon, and Optisol-GS was injected with the graft into the eye instead of being rinsed from the injector. Early postoperative complications (0-8 wk) including intraoperative fibrin formation, intraocular inflammation, elevated intraocular pressure, partial graft detachment requiring rebubble, and early graft failure were recorded. No eyes developed intraoperative fibrin formation or postoperative inflammation (such as toxic anterior segment syndrome) or elevated intraocular pressure. For eyes with Fuchs corneal dystrophy, our rebubble rate was 21% (22/106 eyes). Early graft failure was noted in 2% (3/132 eyes), which is similar to previous reports. Our results suggest that injection of Optisol-GS into the anterior chamber during DMEK graft injection does not lead to increases in intraoperative or early postoperative complications. Trypan blue restaining is not necessary for intraoperative visualization. This simplification can reduce graft manipulation and save time and resources for this procedure.

Clinical outcomes of pre-loaded ultra-thin DSAEK and pre-loaded DMEK

ObjectiveTo compare clinical outcomes and complications between pre-loaded ultra-thin Descemet stripping automated endothelialkeratoplasty (pl-UT-DSAEK) and pre-loaded Descemet membrane endothelial keratoplasty (pl-DMEK).Methods and analysisComparative study in patients with endothelial dysfunction associated...

Culturing Discarded Peripheral Human Corneal Endothelial Cells From the Tissues Deemed for Preloaded DMEK Transplants.

To investigate if the peripheral corneal endothelium that is discarded after the preparation of preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts for transplantation could be successfully used for corneal endothelial cell culture. Complete Descemet membrane-endothelial complex (11.00 mm) was peeled from research-grade tissues (n = 15). The periphery (2.75 mm) of clinical-grade tissues (n = 15) deemed for preloaded DMEK transplants was gently peeled and preserved for 48 hours in tissue culture media, followed by centrifugation at 1000 rpm for 5 minutes. After enzymatic digestion, the cells from each group were plated in 2 different wells of an 8-well chamber slide. Media were refreshed and the confluence rate was monitored every alternate day. Live/dead staining and the expression of ZO-1, Tag1A3, Tag2A12, and Ki-67 markers were used to assess the viability, morphology, tight-junctions, cell area, and number of proliferative cells. The Wilcoxon and Student's t test were applied, where P < 0.05 was deemed statistically significant. Average endothelial cell density at confluence was 2,352 cells/mm2 from complete endothelium and 2,510 cells/mm from peripheral endothelium (P = 0.0351). The confluence rate (%), hexagonality (%), polymorphism (%), cell area (μm), and Ki-67 positivity (%) did not differ between both groups (P > 0.05). All the antibodies were expressed in both groups at confluence. The discarded peripheral endothelial cells obtained after preparing a preloaded DMEK graft for clinical application has a huge reservoir of healthy endothelial cells having proliferative potential. Using these discarded tissue pieces from donor tissues will significantly increase the primary source of healthy donor endothelial cells for regenerative treatments, which are otherwise difficult to obtain.

Safety of Long-Term Storage and Shipping of Prestripped, Prestained, and Preloaded Descemet Membrane Endothelial Keratoplasty Tissue.

The purpose of this study was to determine the safety of long-term storage and shipping of prestripped, prestained, and preloaded Descemet membrane endothelial keratoplasty (pDMEK) grafts. A total of 33 cadaveric corneas were prestripped, prestained, and preloaded using modified Jones tube injectors as pDMEK. The corneas were masked to groups that were prepared <9 hours (control), 48 hours, and 72 hours before unloading and analysis. The 48- and 72-hour tissues were shipped by airfreight on each day before arrival to simulate domestic and international shipping. The corneas were then stained using Calcein AM vital dye (Molecular Probes, Eugene, OR) and imaged using an inverted confocal microscope. Primary outcome measures were endothelial cell loss (ECL, %) and sustainability of staining. MetaMorph software (Molecular Devices, Downingtown, PA) was used to quantify ECL, and staining was evaluated subjectively using all-or-none rating. There was no difference in the mean ECL for the control, 48-hour, and 72-hour groups, which were 25.1% ± 8.8%, 26.4% ± 17.5%, and 19.2% ± 11.5%, respectively (P = 0.45; Kruskal-Wallis test). In all tissues of each group, no loss of staining was identified at each time point of analysis. ECL in pDMEK tissue prepared 48 and 72 hours in advance and shipped using standard methods is similar to that in pDMEK tissue prepared on the same day. These findings support the safety of domestic and international shipping of pDMEK grafts.

Comparison of Tri-folded and Scroll-based Graft Viability in Preloaded Descemet Membrane Endothelial Keratoplasty.

To compare corneal endothelial damage associated with 2 techniques for preloaded Descemet membrane endothelial keratoplasty (DMEK): a tri-folded graft stored in a plastic cartridge designed for DMEK and a scrolled graft stored in a modified Jones Tube, at the time of preparation and after shipping. DMEK grafts were prepared at the Rocky Mountain Lions Eye Bank. The grafts were either tri-folded and loaded in a plastic cartridge or scrolled and loaded into a modified Jones Tube. In each group, the grafts were then either immediately removed from the cartridges or shipped for 48 hours. The grafts were then stained with Calcein AM and imaged using a fluorescent microscope. Endothelial cell loss (ECL) was determined using trainable segmentation in Fiji by 2 graders. At each time point, rates of ECL loss were compared across the 2 groups. To explore the role of donor characteristics, a multivariable regression model was produced to account for method (tri-folding vs. scroll), donor age, donor gender, death-to-preservation time, death-to-preparation time, and shipping. A total of 40 grafts were prepared, processed, imaged, and analyzed. No significant difference in cell loss was seen between groups at either time point alone. In the multivariate model, no significant increase in cell loss was associated with either tri-folding (3.7% less ECL; P = 0.051) or shipping (4.3% less ECL; P = 0.049). All techniques used resulted in clinically acceptable levels of ECL. Tri-folded tissue in a plastic cartridge did not result in ECL inferior to a scroll when prepared either immediately or preloaded for 48 hours.

Minimizing Endothelial Cell Loss Caused by Orientation Stamps on Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

To quantify endothelial cell loss (ECL) caused by orientation stamps on prestripped and preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts, and to examine a method for reducing ECL using a smaller stamp. Ten prestripped and 10 preloaded DMEK grafts were prepared with S-stamps. Ten additional preloaded DMEK grafts were prepared with both an S-stamp and a smaller F-stamp in different paracentral areas of the graft. The footprint of each stamp was measured using ink on cardstock. DMEK grafts were stored in viewing chambers filled with 20 mL of Optisol-GS for 3 days at 4°C. ECL was quantified using Calcein-AM staining and FIJI Weka Segmentation. S-stamps on prestripped DMEK grafts contributed an average ECL of 1.1% ± 0.5% (range: 0.6%-2.2%) toward total graft damage, whereas S-stamps on preloaded DMEK grafts contributed approximately twice that amount (average ECL: 2.0% ± 0.7%, range: 1.3%-3.1%, P = 0.004). Overall ECL for prestripped grafts (average: 7.1% ± 3.3%, range: 3.3%-13.7%) and preloaded grafts (average: 11.3% ± 4.2%, range: 6.9%-19.4%) was similar to previous reports. The footprint of the S-stamp was approximately 45% larger than that of the F-stamp. In 10 preloaded grafts marked with both stamps, the S-stamp caused an average ECL of 1.9% ± 0.6% (range: 1.2%-3.2%), whereas the smaller F-stamp caused an average ECL of 1.0% ± 0.2% (range: 0.8%-1.4%, P = 0.0002). Loss of endothelial cells associated with graft-stamping was greater in preloaded tissue than in prestripped tissue and was less with a smaller F-stamp than with a larger S-stamp. Using a smaller stamp could help minimize ECL in prestripped and preloaded DMEK grafts.

Clinical Outcomes of Preloaded Descemet Membrane Endothelial Keratoplasty Grafts With Endothelium Tri-Folded Inwards

To evaluate the initial outcomes and complications of Descemet membrane endothelial keratoplasty (DMEK) using donor tissues tri-folded with the endothelium inwards, preloaded at the Eye Bank, and delivered with bimanual pull-through technique. Prospective, noncomparative, interventional case series. Setting: Eye bank and tertiary care eye department. Forty-six consecutive eyes of 41 patients with Fuchs endothelial dystrophy with or without cataract operated between November 2016 and March 2017. DMEK tissues prepared with SCUBA technique and punched to a diameter of 8.25mm were preloaded with the endothelium tri-folded inwards in an intraocular lens (IOL) cartridge with a 2.2-mm opening filled with the same tissue culture medium contained in the vial used for shipment to the surgeon. Standardized DMEK was performed as a single procedure (n= 15) or in combination with phacoemulsification and IOL implantation (n= 31) within 48 hours from preparation using a bimanual pull-through technique. Preparation and surgical times, intraoperative and postoperative complications, best spectacle-corrected visual acuity (BSCVA), endothelial cell density (ECD), and graft detachment rate. Preparation time averaged 26.2 ± 4.1minutes (range 17-36minutes), while the surgical time from opening of the stoppers to air fill of the anterior chamber never exceeded 9minutes (range 3-9minutes). Surgery was uneventful in all cases. Postoperative complications included graft detachment in 9 of 46 cases (19.6%), successfully managed in all cases by single rebubbling within 6days from surgery, and glaucoma irresponsive to conservative treatment in 1 of 46 cases (2.1%). In all eyes without comorbidities (35 of 40 eyes) BSCVA was 20/25 (0.097 logMAR) or better as early as 3months after surgery. Six months postoperatively, ECD was available in 24 of 25 eyes with an endothelial cell loss calculated as a percentage of the preoperative value determined at the eye bank (ranging from 2500 to 2800 cells/mm2) of 29.5% ± 14.8% (range 8.3%-52.1%). Delivering a preloaded DMEK tissue, tri-folded with the endothelium inwards, minimizes surgical time and costs without negatively affecting the outcomes ofthe procedure.

Preloaded Descemet Membrane Endothelial Keratoplasty Donor Tissue: Surgical Technique and Early Clinical Results.

To describe the technique, advantages, and early complication rates of using Descemet membrane endothelial keratoplasty (DMEK) donor tissue that is prestained and preloaded into an injector at the eye bank and delivered in a storage medium to the surgeon for transplantation 1 to 2 days later. A total of 111 eyes with endothelial failure underwent DMEK using donors that were prestripped, prestained, S-stamped, and preloaded into a Straiko modified Jones tube and delivered in an Optisol-filled viewing chamber 1 to 2 days later. Scroll tightness, time to unscroll and center the tissue, postoperative rebubble rate, and graft failure rate were recorded. Endothelial cell density was measured at 3 and 6 months. All tissues remained well stained with easy visualization at the time of surgery (n = 111). The mean scroll tightness was 2.2 (range: 1-4). The mean time to center and unscroll the tissue was 3.5 minutes (range: 0.5-11.25 min). There was no primary graft failure. There were 16 cases with the placement of another bubble postoperatively (with a 14.4% rebubble rate). Of those 16 cases, 2 required a second rebubble. Endothelial cell loss at 3 and 6 months postoperatively was 26.7% (n = 63 eyes) and 30.9% (n = 67 eyes), respectively. This is the first report of the clinical use of prestained, preloaded tissue for DMEK. The characteristics and handling of the tissue were not different from those of surgeon-loaded tissue. Because punching, staining, and loading the graft intraoperatively is not necessary, the surgery time and risk of damaging donor tissue are reduced when using preloaded tissue.

Comparison of preservation and transportation protocols for preloaded Descemet membrane endothelial keratoplasty

Background/aimsDescemet membrane endothelial keratoplasty (DMEK) preparation is technically demanding and is a limiting factor for uptake of this kind of surgery. Supply methods that simplify the procedure for surgeons are...

Prestained and Preloaded DMEK Grafts: An Evaluation of Tissue Quality and Stain Retention.

To examine endothelial cell damage and stain retention of prestained preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts. DMEK grafts were stained with trypan blue or left unstained before loading into a Straiko modified Jones tube. A protocol to stain preloaded grafts inside the modified Jones tube and resulting cell loss was also examined. Endothelial cell loss was determined by vital dye staining after 3 days of cold storage and compared between the 3 groups. Eight additional grafts were stained, loaded, and shipped from Oregon to New York to assess stain stability. Stain retention and the ability to successfully inject and open these "prestained preloaded" grafts in cadaveric donor eyes were also evaluated. Unstained preloaded DMEK grafts showed an overall cell loss of 15% ± 3% [95% confidence interval (CI), 11%-18%]. Prestained grafts had a cell loss of 16% ± 4% (95% CI, 10%-22%) and were not significantly different from unstained grafts (P = 1). Grafts stained while inside the modified Jones tubes had an average cell loss of 18% ± 2% (95% CI, 16%-20%, Pprestained = 0.15, Punstained = 0.09). All grafts shipped across the country and examined 3 days after preparation showed a decrease in stain intensity but were still deemed suitable for transplantation by an experienced DMEK surgeon. It is possible to prestain and preload DMEK grafts without inducing additional endothelial cell loss. Consideration should be given to the interval between staining and surgery because stain can fade.

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

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. 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. 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. 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.