Endothelial Keratoplasty Tissue Research Articles

Novel Trephine for Descemet Membrane Endothelial Keratoplasty Surgery Improves Surgical Preparation Time and Graft Quality: Pilot Data From the "Number 7" Endothelial Keratoplasty Trephine.

To report the safety and efficacy of a novel asymmetric marking trephine for Descemet membrane endothelial keratoplasty (DMEK) tissue preparation. The trephine design incorporates optimized curves to distribute mechanical forces and minimize the risk of tears without loss of total endothelial cell volume transplanted compared with a standard 8.00 mm circular transplant. A retrospective case series of 40 consecutive DMEK operations comparing 20 cases using the "Number 7" trephine against 20 using a modified shark fin technique as standard. Patient and donor characteristics, tissue preparation time and complications, total surgical time, intraoperative and postoperative complications, and endothelial cell counts at 3 months postsurgery are reported. DMEK tissue preparation was significantly faster using the "Number 7" trephine (5.4 vs. 7.6 minutes, P = 0.008). Further, fewer complications arose during tissue preparation (0 vs. 2 complications) despite the faster preparation time. Total surgical time was similar between techniques; however, time spent manipulating the graft was reduced (5.1 vs. 6.8 minutes, P = 0.007) primarily because of the lower tissue preparation complications. Fewer postoperative complications were observed (1 vs. 3, of which 1 in the standard technique group requiring a redo-DMEK), again due to the reduced tissue preparation complications. The 3-month endothelial cell count was comparable between techniques (P > 0.05). Use of the "Number 7" trephine led to faster and safer DMEK tissue preparation, which in turn reduced graft manipulation times and postoperative complications without compromising total endothelial cell volume transplanted. This novel device is ideal for use by surgeons who prepare their own DMEK tissue and within tissue banks who provide prestripped and preloaded DMEK tissue.

Microkeratome versus manually dissected donor tissue for Descemet stripping endothelial keratoplasty: A randomized prospective study.

To compare the visual outcomes, postoperative complications, and graft asymmetry between precut and manually dissected donor tissues for Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet stripping endothelial keratoplasty (DSEK) procedures, respectively. Seventy eyes of 70 patients undergoing DSEK/DSAEK at a tertiary eyecare center in eastern India were included in this prospective randomized control study. The 70 eyes were divided equally into two groups. Group A (n = 35) and Group B (n = 35) underwent DSEK and DSAEK procedures, respectively. the operating surgeons prepared the manually dissected corneal grafts using lamellar dissectors. the eye bank technicians prepared the precut corneal grafts using a microkeratome. The patients were investigated postoperatively after 1 day, 1 week, 1 month, 3 months, and 6 months. The data were compared for visual outcomes [uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), spherical and cylindrical refraction, and spherical equivalent], incidence of postoperative complications, and pachymetry. The mean age was 56.5 (±17.8) years, and the male-to-female ratio was 1.3:1. The most common indications were pseudophakic bullous keratopathy (45.71%, 32/70), failed graft (32.86%, 23/70), and Fuchs' endothelial corneal dystrophy (10%, 7/70). The indications for surgery (P = 0.732) and donor (P = 0.258) and host pachymetry (P = 0.986) were comparable between both groups. There was no significant difference in change of UCVA, BCVA, and postoperative refraction (mean spherical refraction, P = 0.849; mean cylindrical refraction, P = 0.516; spherical equivalent, P = 0.518) between the two groups. Postoperative complications like graft detachment (P = 0.179), graft failure (P = 0.513), graft infiltrate (P = 0.428), and endophthalmitis (P = 0.493) were higher in the DSAEK group; however, the difference between the groups was insignificant. The graft asymmetry index was higher in DSAEK than in DSEK; the difference was significant till the 1-month follow-up (P = 0.005). The outcome was similar in both groups. Therefore, manual donor dissection can be an alternative for microkeratome-assisted dissection in low-resource centers and countries to achieve good results.

Chamber shallowing technique for challenging DMEK cases: Tucking cellulose spears under the speculum to augment posterior pressure.

Some anterior chambers do not readily shallow because of insufficient posterior pressure and/or very deep anterior chamber anatomy, which can make unscrolling descemet membrane endothelial keratoplasty (DMEK) tissue more challenging with an unmodified tap technique. We present a hands-free method for augmenting posterior pressure by temporarily tucking cellulose sponges under the blades of the eyelid speculum. The sponges transfer some of the eyelid speculum's weight onto the bulbar surface posterior to the iris, thereby indenting the sclera and causing the iris diaphragm to bulge further forward. This hands-free technique can transform a potentially challenging DMEK case into a more straightforward one by facilitating both a shallow anterior chamber and a bimanual unscrolling technique. However, it only works in bicameral eyes with a vitreous body (e.g., an eye with penetrating keratoplasty, vitreous syneresis, and axial myopia) and will not work in unicameral eyes after vitrectomy (e.g., an eye with an Anterior Chamber Intraocular Lens (ACIOL)).

Evaluating the forces involved in bubble management in DMEK surgery: mathematical and computational model with clinical implications.

To model postoperative forces involved in Descemet membrane endothelial keratoplasty (DMEK) tissue adherence and bubble management, including the impact of surface tension on graft support, with a view towards clinical applications. Tennent Institute of Ophthalmology, Glasgow, and James Watt School of Engineering, University of Glasgow, Glasgow, United Kingdom. Mathematical modelling and computer simulation. Theoretical modelling of biphasic flow and interaction of gas, liquid and tissue within the anterior chamber for static horizontal scenario A (adherent DMEK with mobile bubble) and dynamic vertical scenario B (release of bubble due to pupil block following DMEK). The model assumed incompressibility for both fluids within realistically achievable pressure ranges. Cahn-Hilliard Navier-Stokes equations were discretised through the application of the Finite Element Method. Mathematical modelling and computer simulation showed bubble size, corneal curvature and force intensity influences surface tension support for DMEK tissue in scenario A. Scenario B demonstrated complex, uneven distribution of surface pressure on the DMEK graft during uncontrolled bubble release. Uneven pressure concentration can cause local tissue warping, with air/fluid displacement via capillary waves generated on the fluid-air interface adversely impacting DMEK support. We have quantitatively and qualitatively modelled the forces involved in DMEK adherence in normal circumstances. We have shown releasing air/gas can abruptly reduce DMEK tissue support via generation of large pressure gradients at the liquid/bubble/graft interfaces, creating negative local forces. Surgeons should consider these principles to reduce DMEK graft dislocation rates via optimised bubble size to graft size, longer acting bubble support and avoiding rapid decompression where possible.

Using a Cone-Shaped Glass Funnel Adapter Reduces Endothelial Cell Loss Caused by Preloading Descemet Membrane Endothelial Keratoplasty Tissue.

The aims of this study were (1) to compare "front" and "rear" methods for loading Descemet membrane endothelial keratoplasty (DMEK) tissue into both micro-Jones and standard-Jones tubes and (2) to evaluate the efficacy of a cone-shaped glass funnel adapter designed to make loading DMEK tissue safer for corneal endothelial cells. The corneal endothelium was stained with 0.06% trypan blue to confirm equivalence between mate corneas. The tissues were then processed using the Iowa Lions Eye Bank standard DMEK protocol. In comparison 1, one mate was loaded into the rear of a micro-Jones or standard-Jones tube and the other was loaded into the front of the same tube. In comparison 2, one mate was loaded into the front of the micro-Jones tube and the other was loaded through the cone-shaped funnel adapter into the rear. All tissues were ejected through the front of the modified Jones tubes and assessed for endothelial cell loss (ECL) with calcein AM staining, FIJI, and Trainable Weka Segmentation; scroll widths were measured digitally. There were no statistically significant differences in ECL between front and rear loading [micro (N = 6 pairs): front 15.74% vs. rear 17.95%; standard (N = 6 pairs): front 19.58% vs. rear 19.17%; all P > 0.05]. DMEK scrolls loaded with the funnel adapter exhibited lower ECL compared with scrolls loaded through the front [micro (N = 8 pairs): front 13.53% vs. loading funnel 2.40%; P < 0.001]. Loading with the adapter was not faster (front 6.66 seconds vs. loading funnel 5.52 seconds; P = 0.24). Using a cone-shaped DMEK loading funnel may reduce ECL sustained during preloading.

Advances in eye banking and corneal tissue processing.

Eye bank processing of donor corneal tissue has helped to revolutionize and popularize newer corneal transplantation surgeries. In particular, Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) have benefited from eye banks preparing donor corneal tissue in advance of the surgery. As a result of these eye banking advances, surgeons have been able to rapidly adopt these new techniques. This article reviews the techniques that are now being utilized to prepare donor tissue for endothelial keratoplasty (EK) with a focus on Ultrathin-DSAEK, prestamped, prestained, preloaded DMEK tissue, and advancements to improve the safety of donor corneal tissue. Collaborative efforts between surgeons and eye banks have been at the core of advances that have been made in EK over the past decade. Corneal surgery starts in the eye bank, and it is important for corneal surgeons to understand the process and appreciate the efforts that have been made to provide them with suitable and safe donor corneal tissue.

Glasses-Assisted 3D Display System-Guided Descemet Membrane Endothelial Keratoplasty Tissue Preparation.

The aim of this study was to evaluate the feasibility of Descemet membrane endothelial keratoplasty (DMEK) tissue preparation using a glasses-assisted 3-dimensional (3D) display system and to compare it with a conventional surgical microscope. Healthy pairs of human corneas suitable for penetrating keratoplasty surgery were selected for this study. The tissues were randomly divided into 2 groups. Each pair of corneas had 1 cornea (group 1) prepared with NGENUITY (Alcon) with a 5-second staining time with vision blue, and the fellow cornea (group 2) was prepared using a OPMI Lumera 700 surgical microscope (Carl Zeiss Meditec, Jena, Germany) with a 30-second staining time. DMEK graft preparation time, speed of stripping, graft width, and endothelial cell loss were evaluated. Twenty-eight pairs of corneas were included in this study. The graft preparation time was significantly higher in the 3D group than in the conventional group (498 ± 147 vs. 418 ± 85 seconds, P value = 0.031). The mean speed of stripping was 0.59 ± 0.081 mm/s in group 1 and 0.089 ± 0.005 mm/s in group 2 ( P value = 0.024). The mean endothelial cell density in group 1 and group 2 before tissue preparation was 2162 ± 115.21 and 2153 ± 122.45, respectively ( P value > 0.1). After tissue preparation, the endothelial cell density reduced to 1911 ± 150.72 in group 1 and 1998 ± 90.72 in group 2 ( P value = P value > 0.05). The graft width was 5.05 ± 0.71 mm in group 1 and 4.92 ± 0.23 mm in group 2 ( P value > 0.05). DMEK tissue preparation with 3D display system NGENUITY is feasible with a slightly increased preparation time. The improved visualization allows a reduced staining time that could be beneficial for eye banks because it may reduce the toxic effect of staining colorants.

Eye bank versus surgeon prepared Descemet stripping automated endothelial keratoplasty tissues: Influence on adhesion force in a pilot study.

Purpose:To evaluate and compare the biomechanical properties of the eye bank-prepared and surgeon prepared Descemet stripping automated endothelial keratoplasty (DSAEK) tissues.Methods:In this laboratory study, corneal tissues for research were randomly allocated in the following groups: a) surgeon-cut DSAEK and b) eye bank-prepared (pre-cut and pre-loaded) DSAEK. Endothelial cell loss (ECL), immunostaining for tight junction protein ZO-1, elastic modulus, and adhesion force were investigated.Results:ECL was not found to be significantly different between surgeon-cut DSAEK (7.8% ±6.5%), pre-cut DSAEK (8.6% ±2.3%), and pre-loaded DSAEK (11.1% ±4.8%) (P = 0.5910). ZO-1 was expressed equally across all groups. Surgeon-cut DSAEK grafts showed a significantly higher elastic modulus compared to pre-cut and pre-loaded DSAEK groups (P = 0.0047 and P < 0.0001, respectively). Adhesion force was significantly greater in the surgeon-cut DSAEK compared to pre-cut (P < 0.0001) or pre-loaded DSAEK groups (P = 0.0101).Conclusion:The laboratory data on the biomechanics of DSAEK grafts suggests that surgeon-cut DSAEK grafts present higher elastic modulus and adhesion force compared to eye bank-prepared DSAEK grafts.

Descemet's membrane endothelial keratoplasty tissue type and endothelial cell loss over time

To investigate endothelial cell loss after Descemet's membrane endothelial keratoplasty (DMEK) when surgery is performed with surgeon-loaded tissue versus preloaded tissue. The endothelial cell count over time of patients who underwent the last DMEK surgery by a single surgeon with surgeon-loaded tissue from 2014-2017 was compared to that of patients who underwent the first DMEK surgery by different single surgeon with preloaded tissue from 2017-2020. Baseline characteristics, including age, sex, and baseline endothelial cell count of DMEK grafts, were not statistically different between the groups. Endothelial cell loss both immediately after surgery and continuing over a 4-year period after surgery was greater in the surgeon-loaded DMEK group compared to the preloaded DMEK group based on log-regression curve analysis. Endothelial cell loss was statistically significantly greater at 1 year, 2 years, 3 years, and 4 years after DMEK surgery with surgeon-loaded tissue compared to preloaded tissue on analysis of variance testing. Preloaded DMEK tissue has significantly less endothelial cell loss both initially after surgery and over time compared to surgeon-loaded DMEK tissue, providing support for use of preloaded DMEK tissue for surgery.

Trends in Early Graft Failure Leading to Regrafting After Endothelial Keratoplasty in the United States.

The purpose of this study was to report trends in the prevalence of early graft failure after endothelial keratoplasty in the United States. Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) graft volumes were collected from records maintained by 6 major eye banks in the United States from January 1, 2013, to December 31, 2018. The prevalence and presumed cause of early graft failures (defined as a graft with persistent edema or regrafted within 8 weeks after keratoplasty) each year were sourced from surgeon-reported adverse events. Failed graft cases from the 3 eye banks were compared with nonfailures at the donor and recipient levels to perform subset analysis of factors associated with early graft failure. A total of 51,887 endothelial keratoplasty tissues were distributed during the study period; 72% were DSAEK grafts. The total number of early graft failures reported was 168 of 14,284 (1.18%) for DMEK and 322 of 37,603 (0.86%) for DSAEK. Early DMEK failures decreased from 2013 (7.69%) to 2018 (0.68%). In generalized linear mixed model analyses adjusting for donor tissue characteristics, recipient age, and diagnosis, an association of borderline significance was found between higher donor age and early failure [odds ratio (95% confidence interval): 1.03 (1.00-1.05); unit change of 1 yr] and DSAEK [odds ratio 1.02 (1.00-1.04); unit of change 1 yr] cases. The proportion of early graft failures in DMEK decreased over time and was comparable with failure rates in DSAEK at the end of the study period. The surgical learning curve might have played a role.

Cleavage plane after liquid-bubble preparation of Descemet's membrane.

To investigate the ultrastructure of the cleavage plane of human cornea after liquid-bubble-prepared tissue for Descemet's membrane endothelial keratoplasty (DMEK). Experimental study with scanning electron microscopy (SEM) and block-face SEM of 18 corneal specimens. Fresh human research donor corneoscleral discs (n=12) were prepared with liquid-bubble technique or examined as untreated controls (n=3). In addition, Descemet's membrane samples, n=3, were obtained in DMEK surgery. The cleavage plane after liquid-bubble Descemet's membrane (DM) preparation was consistently located between interfacial matrix and posterior stromal collagen lamellae, providing a largely smooth surface exposing the amorphous interfacial zone without any significant amounts of adherent stromal remnants. No demarcation of a distinct pre-DM layer could be detected. The DMEK graft preparation performed by liquid-bubble technique showed a smooth cleavage plane and could not reveal any demarcation of a distinct pre-DM layer.

Optimizing Visualization of Descemet Membrane Endothelial Keratoplasty Tissue: Assessing the Impact of Trypan Blue Exposure on Stain Duration and Corneal Endothelial Cell Function.

To assess how trypan blue staining affects Descemet membrane endothelial keratoplasty (DMEK) graft visibility and corneal endothelial cell (CEC) mitochondrial respiration. DMEK grafts (n = 20) were stained with trypan blue 0.06% for 1, 3, 5, or 10 minutes. Each graft was injected into an artificial anterior chamber. Surgery was simulated with tapping and sweeping motions on the corneal surface and injections of balanced salt solution (BSS). Graft visibility was assessed at 5, 10, 20, and 30 minutes. Effects of trypan blue on mitochondrial respiration were assessed using primary CECs cultured from donor corneas (n = 43). Treatment wells exposed to trypan blue 0.06% (1, 5, or 30 minutes) and donor-matched control wells to methylene blue 1% (1 minute) or BSS (1, 5, or 30 minutes) were assayed for key respiration parameters. After 5 minutes of surgical manipulation, grafts stained for 5 minutes were significantly more visible than grafts stained for 1 or 3 minutes; there was no added benefit of staining for 10 minutes. After 10 minutes of surgical manipulation, grafts stained for 3 minutes were more visible than grafts stained for 1 minute, without additional benefits of staining ≥5 minutes. No visibility differences were observed after ≥20 minutes of surgical manipulation. CEC mitochondrial respiration did not change significantly following trypan blue exposure for all intervals tested compared to BSS. Staining DMEK grafts with trypan blue for 3 to 5 minutes optimizes visibility during surgical manipulation without mitochondrial impairment. Corneal surgeons learning DMEK will benefit from optimizing this critical step.

Transport and Preservation Comparison of Preloaded and Prestripped-Only DMEK Grafts

This study compares the effect of the transport of conventionally prestripped Descemet membrane endothelial keratoplasty (DMEK) tissue with the DMEK revolutionary advanced Preloadable Injection Device (RAPID) preloaded transport system from Geuder AG (Heidelberg, Germany). Endothelial cell loss, tissue integrity, endothelial cell phenotype, and viability were assessed and compared. Twelve DMEK grafts were prestripped by the cornea bank and transported using the following 2 conditions: conventional flask (n = 6) or a preloaded transport cartridge (DMEK RAPID, n = 6). After transport, tissues were analyzed for cell density; denuded areas; immunolocalization of corneal endothelial markers, such as ZO-1, CD166, and Na/K ATPase; histology analysis; and cell viability staining with Hoechst, calcein AM, and ethidium homodimer. Endothelial cell loss (10.35% vs. 9.15%) did not differ between transport conditions. Histological analysis confirmed the integrity of the Descemet membrane and endothelial cell layer with both transport conditions. Similarly, the corneal endothelial cell mosaic was conserved in both conditions. The ZO-1 tight junctions confirmed the integrity of the confluent corneal endothelial cell monolayer. CD166 and Na/K ATPase detection with immunofluorescence was also comparable. A similar percentage of dead cells was reported in both conditions (18.1% vs. 16.73%). Moreover, the surface covered with calcein-positive cells (59.02% vs. 61.95%) did not differ between transport conditions. Our results suggest that DMEK grafts can be prestripped or preloaded into a novel transport cartridge and shipped to the clinic with comparable endothelial cell loss, phenotypical marker expression, and viability to the conventional prestripped donor tissue.

The Influence of Speed During Stripping in Descemet Membrane Endothelial Keratoplasty Tissue Preparation.

To evaluate whether the speed of stripping a Descemet membrane endothelial keratoplasty graft influences the graft scroll width. Human corneas suitable for research were selected for the study. Pairs of corneas were randomly divided into 2 groups: 1 cornea was stripped with a slow speed (group 1) and the contralateral with a fast speed (group 2). Slow speed was defined as the total time greater than 150 seconds or speed <0.057 mm/s. Fast peeling was defined as less than 75 seconds or speed >0.11 mm/s. The grafts acquired were evaluated by microscopy for the graft scroll width and endothelial cell density change pre- and post-preparation. Twenty corneas of 10 donors were included in the analysis. The mean donor age was 68.6 ± 7.58 years. The mean total time of the tissue preparation in group 1 was 282.7 ± 28 seconds and in group 2 was 126 ± 50 seconds (P-value = 0.00000047). The mean speed of stripping in group 1 was 0.045 ± 0.006 mm/s and in group 2 was 0.266 ± 0.093 mm/s (P-value = 0.000027). The graft width in group 1 was 6.4 ± 0.92 mm and in group 2 was 2.87 ± 0.32 mm (P-value = 0.00000014). The mean endothelial cell loss in group 1 was 389 ± 149 cells/mm and in group 2 was 186 ± 63.44 cells/mm (P-value = 0.00134). We found a correlation between the speed of stripping, scroll width, and endothelial cell loss. Slow-peeled Descemet membrane endothelial keratoplasty grafts result in a wider scroll width but were associated with a greater reduction in endothelial cell density.

Proposed risk factors for failure of transplanted eye bank-prepared descemet stripping automated endothelial keratoplasty tissues.

To investigate the possible risk factors for failure of transplanted eye bank-prepared Descemet stripping automated endothelial keratoplasty (DSAEK) tissues. In a retrospective study between March 2011 and March 2019, all the failed DSAEK cases (131 cases) reported to the Central Eye Bank of Iran were compared with a surgeon-matched successful DSAEK group (control, 126 cases) in terms of the donor, DSAEK tissue, and recipient characteristics. Univariate analysis indicated that the DSAEK tissue preparation from excised corneoscleral tissues (OR 2.17; P = 0.026) and donor conjunctival hyperemia (OR 1.62; P = 0.042) were more common in the re-DSAEK group than in the controls. Other donor and recipient characteristics as well as other DSAEK tissue criteria were not significantly different between the re-DSAEK and control groups. DSAEK lenticules in both re-DSAEK and control groups demonstrated symmetric contours. Subgroup analyses in the re-DSAEK group revealed a higher rate of surgeons' low to moderate experience in comparison to those of high experience (P < 0.001). Additionally, failed DSAEK was observed more commonly in donors aged over 50years than among those under 30years (P < 0.001). Our study showed that DSAEK tissues prepared from excised corneoscleral tissues and from donors with conjunctival hyperemia were associated with higher rates of re-DSAEK due to failed DSAEK. An increasing trend of re-DSAEK was observed with shorter surgeons' experience and more advanced donor's age.

Increasing Povidone-Iodine Exposure in Endothelial Keratoplasty Tissue Processing and Fungal Infection Impact.

To evaluate the effect on donor rim cultures and postoperative infections of doubling the povidone-iodine exposure time during corneal tissue recovery before its use in keratoplasty. Consecutive donor cornea recoveries were evaluated for positive donor corneal rim cultures and postoperative infections before and after a protocol change of doubling the exposure time of povidone-iodine during donor preparation. In 631 consecutive cornea donor recoveries, 18 (2.9%) had positive fungal rim cultures and 41 (6.5%) had positive bacterial rim cultures. Three (0.48%) developed postoperative fungal infections, and no bacterial infections occurred. After doubling the povidone-iodine exposure time during the recovery process, 725 consecutive corneas were reviewed. Four (0.6%) had positive fungal rim cultures, and 29 (4.0%) had positive bacterial rim cultures. No postoperative fungal or bacterial infections occurred. No noticeable increase in epithelial toxicity developed between the 2 groups. Increasing the povidone-iodine exposure time during the donor cornea recovery process decreased the rate of positive donor corneal rim fungal cultures (P = 0.001), positive donor corneal rim bacterial cultures (P = 0.04), and postoperative fungal infections (P = 0.06).

Trephine- and dye-free technique for eye bank preparation of pre-stripped Descemet membrane endothelial keratoplasty tissue.

We describe a rapid and easy technique for the preparation of pre-stripped Descemet membrane (DM) endothelial keratoplasty (DMEK) tissue in the eye bank, without the use of dye or trephining by the eye bank technician. After drawing 2 radial marks 90° apart on the scleral rim of the cornea, a circumferential incision was made internal to the trabecular meshwork and the edge of the DM opposite to the marked area was scored and gently peeled towards the marked area. Finally, the peeled DM was gently unrolled and attached to the posterior stromal bed via absorbing excess liquid from the periphery. The prepared DMEK tissue while reattached on the posterior aspect of the cornea was subjected to slit-lamp biomicroscopic and specular microscopic examinations. In case of proper preparation of DMEK tissue and suitability of endothelial cells for DMEK surgery, the tissue was distributed for transplantation. At the time of surgery, trypan blue staining was performed by the cornea surgeon before DM trephination. Duration of DMEK tissue preparation, uneventful preparation, post-preparation endothelial cell loss, surgeon's satisfaction, and intra- and post-operative attachment of the tissue were investigated variables in this study. Over a 2-year period, 28 DMEK tissues were successfully prepared from 30 corneas using our new technique. The median duration of DMEK preparation was 100s. The area of endothelial cell loss was 1.8% ± 1.2% centrally and 6.2% ± 1.8% in areas close to the forceps contacts. Seven out of the 28 (25%) DMEK tissues were ruined by surgeons during surgery and the overall rate of re-bubbling in 19 transplanted DMEK tissues was 16%. The presented method, which uses no trephining or vital dye, has simplified and expedited the preparation process of pre-stripped DMEK tissue in the eye banks.

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.

Elastin Content and Distribution in Endothelial Keratoplasty Tissue Determines Direction of Scrolling

Descemet membrane endothelial keratoplasty (DMEK) and pre-Descemet endothelial keratoplasty (PDEK) tissues always scroll with the endothelial cells (EC) outside. We designed a study to understand the reason for this behavior. Experimental study. Elastin content in Descemet membrane (DM), pre-Descemet layer (PDL), central and peripheral stroma, sclera, and trabecular meshwork were measured by the Fastin elastin assay kit. Distribution of elastin in DM, PDL, and anterior lens capsule (ALC) were examined by immunohistology. The effect of recombinant elastase enzyme and the effect of complete removal of EC and epithelial cells on the scrolling of DM and ALC, respectively, were studied. PDL showed the highest elastin content among the different tissues studied. Elastin localized as a distinct anterior band in the DM and was uniformly distributed in the PDL demarcating the latter from corneal stroma. Enzymatic treatment of DM with elastase reversed scrolling and corresponded with degradation or disappearance of elastin. Removal of EC did not affect the direction of scrolling. ALC behaved in the same manner with regard to distribution of elastin, scrolling, and removal of epithelial cells. This pattern of elastin distribution in DM explains why DMEK and PDEK tissues always scroll with the EC outside. This behavior is not influenced by the EC. High elastin content and uniform distribution in the PDL suggest a structural difference from the posterior stroma.

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

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. 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. 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%. 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.