Deep Anterior Lamellar Keratoplasty Using Dehydrated versus Standard Organ Culture-Stored Donor Corneas: Prospective Randomized Trial.

PURPOSE: To compare the outcomes of deep anterior lamellar keratoplasty (DALK) using dehydrated versus standard organ culture-stored donor corneas for eyes with keratoconus. DESIGN: Prospective, randomized, single-center trial conducted in Italy. PARTICIPANTS: Adult patients (age ≥ 18 years) with keratoconus scheduled for elective DALK. METHODS: Patients undergoing successful type 1 bubble pneumatic dissection using a standard DALK technique were randomized during surgery to receive either dehydrated (n = 30) or standard organ culture-stored (n = 30) donor corneas. MAIN OUTCOME MEASURES: The primary study outcome was best spectacle-corrected visual acuity (BSCVA) 12 months after surgery. Secondary outcomes were refractive astigmatism (RA), endothelial cell density (ECD), and complication rates. RESULTS: Postoperative BSCVA did not significantly differ between groups at both time points: mean difference at 6 months was 0.030 logarithm of the minimum angle of resolution (logMAR; 95% confidence interval [CI], -0.53 to 0.10 logMAR; P = 0.471) and at 12 months was -0.013 logMAR (95% CI, -0.10 to 0.08 logMAR; P = 0.764). No significant differences between groups were observed in terms of postoperative RA and ECD at all time points. In the first 3 days after DALK, an epithelial defect was present in 10 patients (33%) in the organ culture cornea group and in 29 patients (97%) in the dehydrated cornea group. Complete re-epithelialization was achieved by day 7 in all patients (100%) in both groups. CONCLUSIONS: The study provides evidence that the use of dehydrated corneas is noninferior to the use of standard organ culture donor corneas for DALK. Corneal tissue dehydration represents a viable solution that can allow long-term cornea preservation and avoid wastage of unused corneas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Comparison of rebubbling rate between preloaded endothelium-in and preloaded no-touch endothelium-out Descemet membrane endothelial keratoplasty transplantation.

BACKGROUND: To compare the difference in rebubbling rates between patients undergoing Descemet membrane endothelial keratoplasty (DMEK) with endothelium-in using a standard IOL cartridge and those with endothelium-out DMEK utilizing a no-touch technique with borosilicate glass cartridge transplantation. METHODS: This retrospective study included all eyes that underwent preloaded endothelium-in or endothelium-out DMEK transplantation from June 2019 to December 2023 at the Hanusch Hospital, Vienna, Austria. All DMEKs were harvested, prepared and preloaded at the European Eye Bank of Venice, Italy. DMEK surgeries were done by one experienced surgeon and the procedure was completed by air tamponade of the anterior chamber. RESULTS: Overall, 32 eyes each of 31 endothelium-out patients and of 29 endothelium-in patients were included. 32 preloaded endothelium-in procedures were followed by 32 preloaded endothelium-out procedures. Rebubbling rate for endothelium-in was 15/32 (47%) and for endothelium-out was 7/25 (28%) (p = 0.035, Pearson's chi-squared test). Donor age was the most important variable for rebubbling in a random forest algorithm model (ROC: 0.69). CONCLUSIONS: Rebubbling rate in endothelium-out DMEK was less than two-thirds compared to endothelium-in DMEK favoring no-touch endothelium-out DMEK as the preferred technique of DMEK transplantation.

Ultra-thin DSAEK using an innovative artificial anterior chamber pressuriser: a proof-of-concept study.

PURPOSE: To report the impact of establishing and maintaining a high intracameral pressure (ICP) of 200 mmHg on UT-DSAEK graft preparation using an artificial anterior chamber pressuriser (ACP) control unit (Moria SA, Antony, France). METHOD: Retrospective laboratory and clinical study. Four paired donor corneas were mounted on an artificial anterior chamber and subjected to 70 mmHg ("low") and 200 mmHg ("high") ICP using an ACP system. The central corneal thinning rate was measured after 5 min using AS-OCT and the endothelial cell viability was analysed using trypan blue and live/dead staining following 70 mmHg and 200 mmHg ICP. Visual outcomes and complications in a clinical case series of nine patients with bullous keratopathy who underwent UT-DSAEK using 200 mmHg ICP during graft preparation are reported. RESULTS: Laboratory outcomes showed 2 ± 1% and 2 ± 2% dead cells following 70 mmHg and 200 mmHg ICP respectively. Percentage viability in the 70 mmHg group (52.94 ± 5.88%) was not found to be significantly different (p = 0.7) compared to the 200 mmHg group (59.14 ± 10.43%). The mean corneal thinning rate after applying 200 mmHg ICP was 27 ± 13 µm/min centrally (7.2%/min). In the clinical case series, two cases were combined with cataract surgery. Re-bubbling rate was 11%. At the last follow-up (259 ± 109 days), graft thickness was 83 ± 22 µm centrally, endothelial cell density was 1175 ± 566 cell/mm2 and the BCVA of 0.08 ± 0.12 logMAR was recorded with no episodes of rejection. CONCLUSION: ACP control unit for UT-DSAEK graft preparation helps in consistently obtaining UT-DSAEK grafts without compromising endothelial cell viability.

Fate of endothelial cells after intrastromal implantation of Descemet's membrane-endothelial cell tissue.

In non-Descemet Stripping Automated Endothelial Keratoplasty (nDSAEK), the host DM and endothelium are not removed surgically before the introduction of the posterior lamellar graft; the result is that the patient has both the healthy donor endothelium and the diseased or residual host endothelium. Conversely, DSAEK tissues, that are inserted with inverted polarity (upside down), do not survive and the graft fails. While the mechanism of endothelial cell transplantation is clear, the fate of the endothelial cells retained between two stromal interfaces and their physiological role, if any, is not well understood. The aim of our study was therefore to evaluate the viability of a healthy endothelial-Descemet's membrane (EDM) graft after the insertion into a stromal pocket of a recipient donor cornea. Research corneas (n = 52) were divided into three groups: Group A, where an EDM (obtained from another cornea) with good endothelium was inserted in a stromal pocket endothelium side down; Group B, consisting of control corneas with a stromal pocket but without EDM insertion; and Group C, pre-stripped membranes resting on their stroma (not in a stromal pocket). The tissues were preserved in tissue culture medium for 21 days at 31 °C. Parameters including viability of endothelial cells, expression of tight junctions (ZO-1) and thickness were evaluated. After 21 days, all the membranes inserted within the stromal pocket of Group A survived, although an average endothelial cell loss of 30.1% (± 18.10) and a mortality of 10.2% (± 22.86) were recorded. Qualitative analysis using triple staining with Hoechst, ethidium homodimer and calcein AM confirmed the mortality. ZO-1 was expressed where the cells were present, showing good integrity of tight junctions. Group C showed an average endothelial cell loss of 1.9% (± 3.38), a mortality of 0.02% (± 0.07) and a higher expression of ZO-1. An EDM graft with endothelium facing downwards can survive in a stromal pocket for at least 3 weeks, with an overall cell mortality of 30%. Further studies are needed to evaluate the possible outcomes of the insertion of a healthy intrastromal EDMs with reverse polarity and in edematous corneas.

Banking of corneal stromal lenticules: a risk-analysis assessment with the EuroGTP II interactive tool.

We evaluated the feasibility and performed a risk-benefit analysis of the storage and widespread distribution of stromal lenticules for clinical application using a new systematic tool (European Good Tissue and cells Practices II-EuroGTP II tool), specifically designed for assessing the risk, safety and efficacy of substances of human origin. Three types of potential tissue preparations for human stromal lenticules were evaluated: cryopreserved, dehydrated and decellularized. The tool helps to identify an overall risk score (0-2: negligible; 2-6: low; 6-22: moderate; > 22: high) and suggests risk reduction strategies. For all the three types of products, we found the level of risk to be as "moderate". A process validation, pre-clinical in vitro and in vivo evaluations and a clinical study limited to a restricted number of patients should therefore be performed in order to mitigate the risks. Our study allowed to establish critical points and steps necessary to implement a new process for safe stromal lenticule preparation by the eye banks to be used in additive keratoplasty. Moreover, it shows that the EuroGTP II tool is useful to assess and identify risk reduction strategies for introduction of new Tissue and Cellular Therapies and Products into the clinical practice.

Two-photon optical microscopy imaging of endothelial keratoplasty grafts.

PURPOSE: To investigate the microstructure of endothelial keratoplasty grafts using two-photon optical microscopy. METHODS: Six endothelial keratoplasty grafts obtained from human donor corneoscleral tissues and prepared by submerged hydrodissection technique were imaged by two-photon optical microscopy. In each graft, two liquid bubbles were created in order to investigate the presence of a conserved cleavage plane regardless of the volume of posterior stroma that remained attached to Descemet's membrane (DM); the first bubble (bubble A) was generated under DM and the second bubble (bubble B) injection was done in order to obtain a layer of deep stroma that kept the two bubbles separated. Six human donor corneoscleral tissues were used as controls. Second harmonic generation and two-photon emitted fluorescence signals were collected from each specimen. RESULTS: Dissection of stroma occurred along the posterior collagen lamellae at variable distance from DM, which ranged between 3 and 16 µm in bubble A and between 23 and 41 µm in bubble B. The residual stroma included, anteriorly, bands of collagen lamellae, and thin bundles of stromal collagen fibrils, posteriorly, which were tightly intertwining with the underlying DM. There was no anatomically distinct plane of separation between these pre-Descemetic stromal collagen bundles and the overlying collagen lamellae with this hydrodissection technique. CONCLUSIONS: Two-photon optical microscopy provided label-free high-resolution imaging of endothelial keratoplasty grafts, showing that the most posterior stroma changes organization at approximately 10 µm above the DM. The pre-Descemetic stromal collagen fibrils form an intertwined complex with DM, which cannot be separated using hydrodissection.

Polarization of human donor corneas.

To investigate the de-orientation effect of DSAEK grafts by observing the cross patterns and polarization power of human donor corneas using a polarizing device (Lumaxis(®)). Forty human donor corneas were placed in small petri-plates with epithelial side facing up. Polarizing power (arbitrary unit) and crosses were monitored and recorded by the software. The tissue was marked at 'Superior' position to ensure that the base and the polarizer are in alignment with each other after the cut. The anterior lamellar cut was performed using microkeratome. The lenticule was placed back in the same position as marked to mimic the alignment. The tissue was further rotated by 45° ensuring that the base of the cornea and the polarizer were in alignment. The polarization power and 'crosses' were identified at each step. The average of forty corneas from pre-cut to post-45° angular change showed statistically significant difference (p < 0.05) in terms of polarizing power. The cross-shaped pattern deformed and lost the sharpness towards 45° angle. However, multiple variances in terms of 'cross-patterns' were observed throughout the study. Lumaxis(®) was able to determine the worst quality tissue in terms of polarization (no black zone and crosses). Despite the quality of cross pattern which can be used as an additional objective parameter to evaluate the optical properties of the corneal tissue, this preliminary study needs to be further justified in terms of clinical relevance whether polarization changes with oriented or de-oriented grafts have any effects and consequences on the visual acuity.

A portable device for measuring donor corneal transparency in eye banks.

To develop a portable device for measuring the donor corneal transparency and validate its efficacy for corneal evaluation in the eye-banks and for research. The transparency device (TD) has a light source, a detachable system for corneal insertion and a base for light transmission. The probe detects the transmitted light which is measured by a lux-meter. A contact lens was set as 'control' to reduce the light scattering concern, an empty petri-plate as 'blank' and the cornea as 'sample'. Two experts and non-experts (masked) observed the corneas for subjective analysis which was then compared using the TD. The parameters observed were scars, foreign-body, stromal-deformities, folds, thickness and opacity which were then converted to a relative overall percentage by the observer. Twenty corneas were evaluated for correlation, five tissues to obtain standard-deviation and twenty-four pairs for a comparative study. Experts mimicked the eye-banks with long-term experience while non-experts mimicked the emerging eye-banks. Subjective values by the experts closely resembled the measurements by TD. The average correlation between the experts and the non-experts to TD was 0.985 and 0.960 respectively. TD showed higher reproducibility than experts followed by the non-experts. The comparative study showed that increase in thickness reduces the transparency. TD is portable, easy, efficient, maintains sterility and less expensive hence the emerging eye-banks and researchers can use to raise their standards and evaluate the transparency for in vitro tests and comparative studies. The suitable transparency for the cornea deemed for clinical applications was found to be >75 %.

Preloaded DMEK with endo-in technique: Standardizing and minimizing the learning curve over 5 years using 599 corneal tissues.

PURPOSE: To report the outcomes of standardizing pre-loaded DMEK with endothelium-inwards and its associated learning curve. METHODS: Between 2017 and 2021, a total of 599 tissues were stripped using 'trephine and strip' method and loaded by folding the tissue as a taco-fold with endothelium-inwards. The folded tissues were pulled inside the funnel of a 2.2 mm IOL cartridge and stored for the desired number of days in organ culture media supplemented with dextran. Donor characteristics, endothelial cell loss (ECL) and mortality assessed by trypan blue positivity before and after stripping, and eventful cases during stripping/loading were recorded. RESULTS: The tissues found unsuitable for transplant after stripping (6.7%) were significantly higher compared with loading (0.67%). Central or peripheral tears, fragility of the tissues, and insufficient endothelial cell density mainly attributed towards the discard rate. Mean ECL from pre-stripping to post-stripping was 0.27% with endothelial cell mortality of 0.64% at the end of stripping. Cumulative endothelial mortality fold change (pre-strip to post-strip) was high in the first two years of operation (18.9%), which reduced to 5.1% in the following three years with significant difference (p = 0.0352). Average tissue wastage (3 operators) from first 1-150 tissues was 3%, which significantly reduced to 0.9% after achieving the learning curve (151-250) (p = 0.0492). CONCLUSION: DMEK graft preparation requires a learning curve. However, an operator with DMEK stripping skills can easily adapt to pre-loading a DMEK graft in endothelium-inwards fashion with minimal learning curve.

Performance outcomes from a DMEK peeling and preparation wet lab.

OBJECTIVE: To evaluate the Descemet membrane endothelial keratoplasty (DMEK) preparation performance of trainee surgeons in an ex vivo human donor cornea DMEK wet lab simulation setting. METHODS: Human donor corneoscleral rims unsuitable for transplantation were obtained from Moorfields Lions Eye Bank. At the wet lab, graft stripping was performed by scoring the peripheral endothelium. The trypan blue positive cells (TBPC) and cell density (cells/mm2-reticule count) were counted manually before and after stripping. The procedural time, peripheral and central tears and complete peel-off were also recorded and analysed. RESULTS: Eight trainee surgeons attended the wet lab each attempting three DMEKs. Between the first and last attempts a significant decrease was seen in the procedural time (17.6 min vs 10.6 min (p<0.05)) and the TBPC % (12.9% vs 3.8% (p<0.05)). The percentage of tears peripherally and centrally also reduced between the first and the last trials (50% vs 13% (p=0.2226) and 38% vs 0% (p=0.1327)). A significant correlation was found between longer peeling times and higher TBPC % (p<0.001) with a 0.7% endothelial mortality increase for each additional minute required to complete the peel. CONCLUSIONS: DMEK wet labs provide a controlled risk-free learning opportunity for trainee surgeons to improve confidence and competence. Wet labs improve the success rate of DMEK graft preparation as well as flatten the learning curve. This emphasises the importance of continued support for the expansion of this valuable learning resource, promoting wider uptake of DMEK surgery.

Quality assurance in corneal transplants: Donor cornea assessment and oversight.

The cornea is the most frequently transplanted human tissue, and corneal transplantation represents the most successful allogeneic transplant worldwide. In order to obtain good surgical outcome and visual rehabilitation and to ensure the safety of the recipient, accurate screening of donors and donor tissues is necessary throughout the process. This mitigates the risks of transmission to the recipient, including infectious diseases and environmental contaminants, and ensures high optical and functional quality of the tissues. The process can be divided into 3 stages: (1) donor evaluation and selection before tissue harvest performed by the retrieval team, (2) tissue analysis during the storage phase conducted by the eye bank technicians after the retrieval, and, (3) tissue quality checks undertaken by the surgeons in the operating room before transplantation. Although process improvements over the years have greatly enhanced safety, quality, and outcome of the corneal transplants, a lack of standardization between centers during certain phases of the process still remains, and may impact on the quality and number of transplanted corneas. Here we detail the donor screening process for the retrieval teams, eye bank operators. and ophthalmic surgeons and examine the limitations associated with each of these stages.

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

The aim of the study was to assess different temperature ranges for the preservation of pre-loaded Descemet Membrane Endothelial Keratoplasty (DMEK) grafts in the DMEK RAPID Mini device. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

DMEK surgical training: An instructional guide on various wet-lab methods.

Descemet membrane endothelial keratoplasty (DMEK) is a partial-thickness corneal transplantation procedure that involves selective transplantation of the Descemet membrane and endothelium. DMEK offers significant advantages over other keratoplasty techniques, such as faster visual rehabilitation, better final visual acuity due to minimal optical interface effects, lower risk of allograft rejection, and less long-term dependence on topical steroids. Despite all its advantages, DMEK has been found to be more challenging than other corneal transplantation techniques, and its steep learning curve appears to be an obstacle to its widespread use and adoption by corneal surgeons worldwide. DMEK surgical training laboratories (wet labs) provide a window of opportunity for surgeons to learn, prepare, manipulate, and deliver these grafts in a risk-free environment. Wet labs are a significant learning tool, especially for those institutions that have limited tissue availability in their local centers. We provide a step-by-step guide for preparing DMEK grafts using different techniques on human and nonhuman models with instructional videos. This article should eventually help the trainees and the educators understand the requirements for performing DMEK and conducting a DMEK wet lab and develop their skills and interests from a wide variety of available techniques.

Preoperative surgeon evaluation of corneal endothelial status: the Viability Control of Human Endothelial Cells before Keratoplasty (V-CHECK) study protocol.

INTRODUCTION: The success of keratoplasty strongly depends on the health status of the transplanted endothelial cells. Donor corneal tissues are routinely screened for endothelial damage before shipment; however, surgical teams have currently no means of assessing the overall viability of corneal endothelium immediately prior to transplantation. The aim of this study is to validate a preoperative method of evaluating the endothelial health of donor corneal tissues, to assess the proportion of tissues deemed suitable for transplantation by the surgeons and to prospectively record the clinical outcomes of a cohort of patients undergoing keratoplasty in relation to preoperatively defined endothelial viability. METHODS AND ANALYSIS: In this multicentre cohort study, consecutive patients undergoing keratoplasty (perforating keratoplasty, Descemet stripping automated endothelial keratoplasty (DSAEK), ultra-thin DSAEK (UT-DSAEK) or Descemet membrane endothelial keratoplasty) will be enrolled and followed-up for 1 year. Before transplantation, the endothelial viability of the donor corneal tissue will be evaluated preoperatively through trypan blue staining and custom image analysis to estimate the overall percentage of trypan blue-positive areas (TBPAs), a proxy of endothelial damage. Functional and structural outcomes at the end of the follow-up will be correlated with preoperatively assessed TBPA values. ETHICS AND DISSEMINATION: The protocol will be reviewed by the ethical committees of participating centres, with the sponsor centre issuing the final definitive approval. The results will be disseminated on ClinicalTrials.gov, at national and international conferences, by partner patient groups and in open access, peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05847387.

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

PURPOSE: To evaluate factors affecting the outcomes of preloaded Descemet membrane endothelial keratoplasty (pl-DMEK) with endothelium-inward. DESIGN: Retrospective clinical case series and a comparative tissue preparation study. METHODS: 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. INTERVENTION: 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. MAIN OUTCOME MEASURES: Tissue characteristics, donor and recipient factors, rebubbling rate, endothelial cell loss (ECL), and corrected distance visual acuity (CDVA) at 3, 6, and 12 months. RESULTS: 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. CONCLUSION: 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.

Synthetic media for preservation of corneal tissues deemed for endothelial keratoplasty and endothelial cell culture.

PURPOSE: To compare the difference between various endothelial graft preparation methods and endothelial cell culture from tissues that are preserved in serum-based and synthetic medium. METHODS: In a randomized masked study, the tissues (n = 64) were preserved in Cornea Max (serum-based) and Cornea Syn (synthetic) series for 36 days at their respective preservation conditions. Following organ culture, corneal tissues (n = 48) were used to prepareDescemet stripping automated endothelial keratoplasty (DSAEK), preloaded ultra-thin (UT) -DSAEK, prestripped Descemet membrane endothelial keratoplasty (DMEK), free-floating DMEK, and preloaded DMEK with endothelium inward and outward grafts. These tissues were preserved for another 4days at room temperature in dextran supplemented media following which they were subjected to trypan blue, alizarin red, live/dead and Zonula Occludens-1 (ZO-1) staining. A separate set of tissues (n = 16) from both the series was used for human corneal endothelial cell (HCEnC) culture. At confluence, the proliferation and cell doubling rate was calculated and the cultured cells were subjected to live/dead, ZO-1, 2A12 and Ki-67 staining. Mann-Whitney test was performed with p < 0.05 deemed statistically significant. RESULTS: After preparation and preservation of the tissues for endothelial keratoplasty, alizarin red showed standard endothelial morphology from both the groups. Endothelial cell loss, hexagonality and uncovered areas did not show statistically significant differences (p > 0.05) between both groups. For HCEnC, cell doubling rate was 4.7 days (p > 0.05). All the antibodies were expressed in both the groups. Hexagonality, polymorphism, cell area, viable/dead cells and Ki-67 positivity were not statistically significant (p > 0.05). CONCLUSIONS: Complete synthetic organ culture series is safe and advantageous for carrying out advanced endothelial keratoplasty graft preparation procedures and for HCEnC culture as it is free from animal or animal-derived products.

Preloaded Descemet Membrane Endothelial Keratoplasty Grafts With Endothelium Outward: A Cross-Country Validation Study of the DMEK Rapid Device.

PURPOSE: To validate the "Descemet membrane endothelial keratoplasty (DMEK) Rapid" device for the cross-country transportation of preloaded DMEK grafts preserved with endothelium outward. METHODS: DMEK grafts were stripped and loaded in the DMEK Rapid device with tissue culture medium (TCM) or transport medium (TM) with endothelium outward. The device was mounted in a 40-mL flask and preserved for 4 days on a rocker to simulate transportation (study A, n = 24) or shipped in the TM from Italy to the United Kingdom (study B, n = 9) and evaluated within 72 hours. All the tissues were stained with Alizarin red. Viability of the cells was checked postsimulations and posttransportation and was confirmed using live/dead staining. Expression of tight junction proteins was evaluated. RESULTS: In study A, the endothelial cell loss observed from the TCM group was 20.8% (±5.2) compared with 19.5% (±6.7) from the TM group (P = 0.41) after transport simulation. Alizarin red showed minimal uncovered areas in both groups. There were no statistical differences in viability between the TM (80.83%) and TCM groups (78.83%). In study B, 12.9% (±7.8) endothelial cell loss was observed after transporting the tissues from Italy to the United Kingdom with no significant difference between prestrip and posttransportation (P = 0.05). Alizarin red staining did not show any uncovered area. Live/dead analysis showed 85.16% cell viability after transportation. zonula occludens-1 (ZO-1) was expressed in all tissues. CONCLUSIONS: The DMEK Rapid device is safe for preloading and shipping DMEK grafts internationally with endothelium outward within 72 hours when preserved in the transport media.

Eye Bank Management of Irregular Descemet Stripping Automated Endothelial Keratoplasty Lenticules.

PURPOSE: To report the management of precut Descemet stripping automated endothelial keratoplasty (DSAEK) lenticules unsuitable for transplantation because of irregular anterior profile after microkeratome cutting. METHODS: After preparation for DSAEK, 20 tissues were considered unsuitable for transplantation because of nonhomogeneous posterior stromal thickness. To convert them into suitable tissues for surgery, manual stromal delamination was performed by removing the excess stromal layers after the indications obtained through optical coherence tomography. These tissues were further transplanted as ultrathin DSAEK. RESULTS: Nineteen tissues were delaminated successfully. The average reduction in thickness in the center (63 ± 69 µm; P = 0.0101) and periphery (129 ± 39 µm; P < 0.0001) before and after delamination was significantly different. One tissue showed signs of perforation during manual dissection and therefore considered unsuitable for transplantation. Primary graft failure was reported in one case, but it was not correlated with the tissue preparation. No other clinical complications were observed after surgery. CONCLUSIONS: Manual delamination of the stroma because of irregular microkeratome cutting is a viable option to obtain a uniform graft thickness required for DSAEK surgeries. This technique can further reduce tissue wastage that is observed after microkeratome cutting errors.

Rebubbling rate in preloaded versus surgeon prepared DSAEK.

PURPOSE: To compare the clinical outcomes of eye bank preloaded Descemet stripping automated endothelial keratoplasty (DSAEK) grafts and surgeon prepared. METHODS: In this retrospective study, the data were obtained from two groups (a) surgeon cut DSAEK where tissue was prepared by the surgeon immediately before surgery, and (b) preloaded DSAEK tissue shipped to the surgeon after preparation by the eye bank. Standard DSAEK preparations using Moria microkeratome with single pass method were performed. For the tissues prepared by the eye banks, they were preloaded in an iGlide device and shipped in transport media. Standard DSAEK surgery using bimanual pull-through technique was performed for all the grafts. Air was used as a tamponade. Main outcome measures included best corrected visual acuity (BCVA) and rebubbling rate. RESULT: Out of 107 eyes of 101 patients that underwent DSAEK surgery, 33 tissues were prepared by the surgeon (sc-DSAEK), while 74 were prepared by the eye bank (pl-DSAEK). sc-DSAEK showed a rebubbling rate of 9.1%, compared to the 16.2% for the preloaded DSAEK (p = 0.11). There was no statistical difference in postoperative BCVA between the two groups. Logistic regression analysis showed no association between detachment rate and cataract surgery, graft preparation method, graft diameter and reason for graft. CONCLUSION: Preloaded grafts have similar rebubbling rate and visual acuity achieved compared with surgeon prepared grafts.

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

PURPOSE: To evaluate whether the speed of stripping a Descemet membrane endothelial keratoplasty graft influences the graft scroll width. METHODS: 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. RESULTS: 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). CONCLUSION: 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.