Endothelial Cell Viability after DMEK Graft Preparation.

Aim: To evaluate the effect of graft preparation and organ-culture storage on endothelial cell density (ECD) and viability of Descemet membrane endothelial keratoplasty (DMEK) grafts.Materials and methods: DMEK grafts (n = 27) were prepared at Amnitrans EyeBank Rotterdam from 27 corneas (15 donors) that were eligible for transplantation but could not be allocated due to the Covid-19-related cancellation of elective surgeries. Cell viability (by Calcein-AM staining) and ECD of five grafts originally scheduled for transplantation were evaluated on the originally planned surgery day, whereas 22 grafts from paired donor corneas were evaluated either directly post-preparation or after 3-7 days of storage. ECD was analyzed by light microscopy (LM ECD) and Calcein-AM staining (Calcein-ECD).Results: Light microscopy (LM) evaluation of all grafts showed an unremarkable endothelial cell monolayer directly after preparation. However, median Calcein-ECD for the five grafts initially allocated for transplantation was 18% (range 92-73%) lower than median LM ECD. For the paired DMEK grafts, Calcein-ECD determined by Calcein-AM staining on the day of graft preparation and after 3-7 days of graft storage showed a median decrease of 1% and 2%, respectively. Median percentage of central graft area populated by viable cells after preparation and after 3-7 days of graft storage was 88% and 92%, respectively.Conclusion: Cell viability of most of the grafts will not be affected by preparation and storage. Endothelial cell damage may be observed for some grafts within hours after preparation, with insignificant additional ECD changes during 3-7 days of graft storage. Implementing an additional post-preparation step in the eye bank to evaluate cell density before graft release for transplantation may help to reduce postoperative DMEK complications.

Use of Corneas From Septic Donors for Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate the suitability of corneas from septic donors for transplantation by analyzing the discard rate in the eye bank and the clinical outcome of Descemet membrane endothelial keratoplasty (DMEK) using organ-cultured corneal grafts from septic versus nonseptic donors. METHODS: This retrospective study included 1554 corneas of which 456 corneas (29%) were from septic and 1072 corneas (69%) from nonseptic donors [for 26 corneas (2%) sepsis status was unknown]. The clinical outcome at 6 months after DMEK was evaluated for 82 grafts (26 from septic and 56 from nonseptic donors). Outcome measures were endothelial cell density, central corneal thickness, and postoperative complications. RESULTS: Primary discard rates were higher for corneas from septic than from nonseptic donors (32.9% vs. 24.5%, P = 0.001). The main discard reason was poor endothelial cell quality for both septic (13.8%) and nonseptic (11.8%) donor corneas. Eye bank contamination rates for septic and nonseptic donor corneas were 1.1% and 1.7%, respectively (P = 0.102). After DMEK, donor endothelial cell density at 6m postoperatively was comparable between grafts from septic and nonseptic donors (1410 ± 422 cells/mm vs. 1590 ± 519 cells/mm, P = 0.140). No differences in 6m central corneal thickness and in the rebubbling rate were observed between the 2 groups (P = 0.780 and P = 0.396, respectively). None of the cases had graft rejection nor endophthalmitis in both groups. CONCLUSIONS: Provided strict adherence to donor screening and evaluation protocols, the use of organ-cultured corneas from septic donors for DMEK does not seem to increase the risk for recipients and allows for expansion of the donor pool for corneal tissue.

Minimizing Graft Preparation Failure in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To report the failure rate of 2 graft preparation techniques for Descemet membrane endothelial keratoplasty (DMEK) and to evaluate how to minimize graft preparation failure. METHODS: Retrospective, nonrandomized study at an eye bank specialized in graft preparation for lamellar keratoplasty. For 1416 donor corneas, the DMEK graft preparation failure rate was evaluated for 2 different techniques, technique I: "Standardized traditional technique" (n = 341) and technique II: "Standardized no-touch technique" (n = 933), and for grafts that were converted from technique II to technique I during preparation (n = 142). RESULTS: The overall failure rate averaged 3.9% (55/1416): 7.0% (24/341) for technique I and 2.9% (31/1075) for technique II (P < 0.05). Tissue preparations which were converted from technique II to technique I failed in 13.4% (19/142), whereas for grafts that were entirely prepared by technique II, the failure rate was only 1.3% (12/933). The endothelial cell density decrease (before compared with after preparation) did not differ for both techniques (1.1% vs. 0.2%, P > 0.05). CONCLUSIONS: Various DMEK graft preparation techniques may provide failure rates of <4%. A "no-touch preparation" approach (technique II) may combine good graft quality (completely intact endothelial cell layer, ie, negligible preparation-induced endothelial cell density decrease) with low risk of dissection failure, leaving the possibility of conversion to "traditional preparation" (technique I) as a backup method.

Donor Tissue Preparation for Bowman Layer Transplantation.

PURPOSE: To describe and compare 2 preparation techniques for Bowman layer (BL) grafts for use in BL transplantation. METHODS: A retrospective evaluation of the 2 methods for preparing BL grafts was performed, that is, BL graft preparation from donor globes not eligible for penetrating keratoplasty or endothelial keratoplasty (technique I, n = 36) and BL graft preparation from previously excised corneoscleral buttons (technique II, n = 36) that could not be used for PK or had been denuded of Descemet membrane and endothelium for Descemet membrane endothelial keratoplasty graft preparation. BL graft preparation difficulties were recorded, and the preparation failure rate was examined and compared between the techniques. RESULTS: Overall, BL graft preparation was successful in 51 cases (51/72; 70.8%), of which 25 preparations were successful using technique I, and 26 using technique II, representing a success rate of 69.4% (25/36) and 72.2% (26/36) for techniques I and II, respectively. Reasons for discarding a BL graft were tearing of the BL tissue during the preparation (n = 19) and stroma attached to the graft (n = 2). CONCLUSIONS: Isolated BL grafts can be prepared from both whole donor globes and corneoscleral rims with equivalent success. Preparation from corneoscleral rims may offer the advantage that, from one donor cornea, the posterior layers can be used for Descemet membrane endothelial keratoplasty graft preparation and the anterior part for BL graft preparation.

Association Between Graft Storage Time and Donor Age With Endothelial Cell Density and Graft Adherence After Descemet Membrane Endothelial Keratoplasty.

IMPORTANCE: After retrospectively evaluating the clinical outcome of 500 consecutive cases after Descemet membrane endothelial keratoplasty (DMEK), we extended the analysis in this study by assessing the effect of donor-related parameters on endothelial cell density (ECD) decline and detachment rate in this group. OBSERVATIONS: This retrospective case series included 500 cases who had undergone DMEK from October 2007 to September 2012 at the Netherlands Institute for Innovative Ocular Surgery (NIIOS), Rotterdam, the Netherlands. Logistic regression analysis (n = 332 eyes) showed that donor age might be associated with a 3% increase in the risk for a detachment (odds ratio, 0.97; 95% CI, 0.94-1.00; P = .049) (ie, higher donor age seems to be associated with lower chances of a detachment). In addition, linear regression analysis indicated that graft storage time in medium was associated with ECD decrease (ie, the longer the storage time, the larger the decrease at 6 months after DMEK) (P = .01). CONCLUSIONS AND RELEVANCE: We showed an association between graft storage time and ECD decline after DMEK and possibly between donor age and graft detachment. Therefore, donor storage times should be kept as short as possible to improve short-term ECDs. More research is needed to draw definite conclusions on the possible effect of donor age on the chance of a detachment after DMEK.

Graft preparation for hemi-Descemet membrane endothelial keratoplasty (hemi-DMEK).

BACKGROUND/AIMS: There is a substantial shortage of donor tissue for endothelial keratoplasty worldwide. Using two endothelial grafts of one donor cornea may potentially increase the pool of suitable donor tissue for Descemet membrane endothelial keratoplasty (DMEK). This study evaluates the technical feasibility of obtaining two DMEK grafts with a different (semicircular) shape, but similar surface area as a 'conventional' 8-9 mm circular transplant from a single donor cornea and to evaluate the viability of these grafts stored in organ-culture medium up to 5 weeks after graft preparation. METHODS: Ten human corneas not suitable for transplantation were used for preparation of semicircular-shaped grafts (hemi-DMEK grafts). The viability of these hemi-DMEK grafts was measured by evaluating the endothelial cell density (ECD) with light microscopy before and immediately after preparation and during five additional weeks of organ culture. RESULTS: Hemi-DMEK preparation was successful in all cases. All grafts formed a roll. A small area denuded of cells was observed after preparation along the straight edge of the DM roll. The mean ECD was 2654 (±65) cells/mm(2) before and 2651 (±94) cells/mm(2) immediately after hemi-DMEK preparation and declined gradually from 2518 (±133) cells/mm(2) after 1 week to 1847 (±170) cells/mm(2) after 5 weeks of organ-culture storage. DISCUSSION: Preparation of two hemi-DMEK grafts from one single human donor cornea is technically feasible, and the grafts can be stored after preparation in organ-culture similar to standard circular DMEK grafts. Hemi-DMEK may have the potential to double the availability of donor endothelial tissue for DMEK.

Dehydration of corneal anterior donor tissue with polyethylene glycol (PEG)-enriched media.

Anterior donor grafts (including scleral rim, without Descemet membrane) increase in thickness and become hazy upon storage in organ culture (OC) medium. Transfer of these grafts to standard dehydration media just before transplantation does not reduce their thickness to normal. Therefore, we assessed the efficacy of different media enriched with polyethylene glycol (PEG) as dehydrating agents for organ-cultured anterior donor grafts. Grafts were harvested and stored in the commercial OC medium 'Max' (without dextran) for 1 week, and subsequently dehydrated in the standard commercial dehydration medium 'Jet' (with dextran) supplemented with 4-20% PEG3350, or 'Max' supplemented with 20% PEG6000 and PEG20.000, or 5-20% PEG35.000. Central corneal thickness (CCT), as assessed by anterior segment-optical coherence tomography, and transparency were evaluated before, and at 1, 4 and 7 days of dehydration. Transfer of grafts after 1 week of OC (average 1,200 µm) to 'Jet' supplemented with PEG3350 revealed a concentration-dependent effect of dehydration; CCT was restored to normal (500-600 µm) when 10% PEG3350 was added. However, transparency was only temporarily restored; after 1 day, the grafts turned hazy. In contrast, grafts transferred to 'Max' supplemented with 20% PEG35.000 were transparent throughout the evaluation period, but were dehydrated to beyond normal levels (average 300 µm). 'Max' supplemented with 5% PEG35.000 dehydrated grafts to normal values and restored transparency throughout. Thus, dehydration of anterior donor grafts prior to surgery in dextran-free OC medium supplemented with 5% PEG35.000 reduces graft thickness to normal and may facilitate anterior keratoplasty procedures.

Postmortem ultrastructural analysis of a cornea transplanted with Descemet membrane endothelial keratoplasty.

PURPOSE: The aim of this study was to describe the ultrastructure of the host-donor interface in the eye of a recently deceased patient, who had undergone Descemet membrane endothelial keratoplasty. METHODS: The eye was enucleated postmortem, and after standard decontamination, the corneoscleral button was excised, cut into 4 quadrants, and processed for light and transmission electron microscopy evaluation. RESULTS: Transmission electron microscopy revealed close attachment of the donor's Descemet membrane to the host's stroma and projection of stromal collagen fibers into the interfacial matrix, resembling a normal "virgin" corneal architecture. CONCLUSIONS: Ultrastructurally, an attached Descemet membrane endothelial keratoplasty graft closely resembles that of an unoperated, healthy eye with no appreciable adventitious or missing structures.

Standardized 'no-touch' donor tissue preparation for DALK and DMEK: harvesting undamaged anterior and posterior transplants from the same donor cornea.

PURPOSE: To describe a standardized 'no-touch' harvesting technique of anterior and Descemet membrane (DM) grafts for use in deep anterior lamellar keratoplasty (DALK) and Descemet membrane endothelial keratoplasty (DMEK), which provides undamaged anterior and posterior corneal grafts. METHODS: A retrospective evaluation was performed of our standard method for harvesting DM grafts and DALK grafts (Technique I; n = 31) versus a newly designed 'no-touch' technique (Technique II; n = 31), in which a peripheral ring of trabecular meshwork tissue is left in-situ, and the DM graft is trephined on an underlying soft contact lens. Endothelial cell density (ECD) before and immediately after DM stripping was used as the main outcome parameter. RESULTS: Endothelial cell density did not differ within Techniques I and II (before versus after DM stripping) (p = 0.75 and p = 0.54, respectively) or among Techniques I and II (p = 0.61). With the latter technique, anterior corneal grafts and posterior DM grafts could be harvested with negligible damage to the endothelial cell layer or the posterior stromal bed. All 93 grafts (62 DM grafts) were eligible for transplantation, and six months post-operatively all transplants used were functional. CONCLUSION: The new technique offers the following advantages: (i) production of 'undamaged' grafts for DALK and DMEK, (ii) better controlled tissue handling of the thin DM graft during DM stripping and (iii) an increase in availability of corneal grafts obtained from the same donor tissue pool.

Are polymegethism, pleomorphism, and "poor swelling" valid discard parameters in immediate postmortem evaluation of human donor corneal endothelium?

PURPOSE: To study the validity of endothelial polymegethism, pleomorphism, and "poor swelling" as tissue discard parameters in the immediate postmortem evaluation of human donor corneal endothelium. METHODS: We retrospectively evaluated the quality of the endothelium at first and second evaluations for all processed corneas exhibiting moderate polymegethism, pleomorphism, or "poor swelling" in our eye bank over a 5-year period. RESULTS: Out of 2008 eyes qualifying for our study, 422 corneas (21%) showed polymegethism, pleomorphism, or poor swelling at the first tissue evaluation immediately after excision of the corneoscleral button. In 363 (86%) of these corneas, a normal endothelial mosaic was observed at the second tissue evaluation after 7 to 21 days of organ culture, whereas only 59 (14%) still showed persistent polymegethism, pleomorphism, or "poor swelling" at that time point. CONCLUSIONS: A recovery of normal endothelial cell mosaic and "normal swelling" at the second evaluation suggests that cellular contour parameters do not relate to tissue viability, but rather to a cellular stress reaction. If so, the validity of endothelial cellular contour morphology as an early parameter in assessing the suitability of a donor cornea for transplantation may be reconsidered.

Combined chlorhexidine and PVP-I decontamination of human donor eyes prior to corneal preservation.

The aim of this study was to report the efficacy of adding chlorhexidine to the protocol for decontamination of human donor globes prior to excision of corneo-scleral rims for future keratoplasty procedures. In 2005, chlorhexidine was introduced by our eye bank as an additional step in the protocol for decontaminating human donor globes. After 5 years, we prospectively evaluated the number of contaminations. Out of 2,891 globes included in our study, 2,663 globes were processed, of which 36 (1.4%) were considered contaminated. Seventeen contaminations (0.6%) were detected by culturing limbal swabs, directly after decontamination, eight (0.3%) by visible discoloration of the culture medium carrying a corneo-scleral rim, and eleven (0.4%) after inoculation of the culture medium on blood agar plates. Importantly, after 4 weeks of incubation, none of the aerobic and anaerobic cultures taken from the secondary 'transport medium' (dextran containing medium used to transport corneal tissue to the transplantation centre) showed microbiological growth. In conclusion, the combined use of 0.02% chlorhexidine and 0.5% povidone-iodine may allow decontamination of donor globes to a level at which the risk of tissue contamination at the time of transplantation is minimized, while corneal viability is preserved.

Incidence of early allograft rejection after Descemet membrane endothelial keratoplasty.

PURPOSE: To report the incidence of early allograft rejection after Descemet membrane endothelial keratoplasty (DMEK), that is, transplantation of isolated Descemet membrane with its endothelium. METHODS: The first series of 120 eyes of 105 patients operated on for Fuchs endothelial dystrophy or pseudophakic bullous keratopathy, with an average 2 years of follow-up after 9.0- to 10.0-mm-diameter DMEK, enrolled in our study. RESULTS: During the entire study period, only 1 of the eyes showed any signs of a cellular immune response to the Descemet graft. A 76-year-old patient presented with discomfort, reduced visual acuity to counting fingers, corneal decompensation, and a Khodadoust line in the central cornea 4 months after (decentered) DMEK. Intensified topical corticoid therapy resulted in a complete visual recovery to 20/25 (0.8) within weeks. CONCLUSIONS: A "classic" allograft rejection (with an appearance similar to that after penetrating keratoplasty) can occur after DMEK. However, compared with the earlier (endothelial) keratoplasty procedures, DMEK may be associated with a lower rejection rate of ≤ 1%, despite transplant diameters of ± 9.5 mm. The apparent immune tolerance in DMEK may result from either less "upregulation" or more "downregulation" of the immune system.

Patterns of corneal endothelialization and corneal clearance after descemet membrane endothelial keratoplasty for fuchs endothelial dystrophy.

PURPOSE: To describe various endothelial migration healing patterns after Descemet membrane endothelial keratoplasty (DMEK), and to determine the contribution of the donor and host endothelium in the clearance of a transplanted cornea. DESIGN: Nonrandomized, prospective clinical study. METHODS: In a total of 150 consecutive eyes that underwent DMEK (ie, transplantation of an isolated Descemet graft, for Fuchs endothelial dystrophy), re-endothelialization patterns were studied. Of these eyes, 36 showed a "stromal gap" between the "descemetorhexis edge" and the graft, or (partial) graft detachment. Endothelialization patterns of the host posterior stroma were documented at 1, 3, 6, 9, 12, and 24 months after surgery with Pentacam imaging, specular microscopy, optical coherence tomography, confocal microscopy, and slit-lamp biomicroscopy. RESULTS: Complete corneal clearance was seen in 28 of 36 eyes (78%) with a stromal gap, or (partial) detachment, progressing from the periphery toward the center; and 27 of 34 eyes (79%) with normal visual potential reached a visual acuity of ≥20/40 (≥0.5) or better. In 3 eyes that had the Descemet graft implanted upside-down, a "reversed corneal clearance pattern" was observed (ie, persistent edema where the graft was attached), while the area overlying the detachment cleared. One case that had a "descemetorhexis" performed without endothelial graft implantation showed persistent stromal edema. CONCLUSION: The presence of donor endothelium in the recipient anterior chamber may be required for endothelial migration and/or recovery of corneal clarity. Re-endothelialization may be associated with massive endothelial migration and some form of cell signaling to draw donor endothelial cells toward the recipient posterior stroma ("homing").

Secondary DMEK for poor visual outcome after DSEK: donor posterior stroma may limit visual acuity in endothelial keratoplasty.

PURPOSE: To describe the clinical outcome of a secondary Descemet membrane endothelial keratoplasty (DMEK) to manage poor visual outcome after Descemet stripping endothelial keratoplasty (DSEK). METHODS: Three eyes of 3 patients that underwent DSEK for Fuchs endothelial dystrophy showed fluctuating and/or poor visual outcome ranging from 20/80 (0.25) to 20/40 (0.5). In a secondary procedure, 16-22 months after the initial DSEK, the DSEK graft was removed and replaced by a DMEK graft. The clinical outcome was evaluated by comparing the pre- to postoperative best-corrected visual acuity (BCVA), Pentacam imaging, and biomicroscopy. RESULTS: All secondary DMEK procedures were uneventful. Three months after secondary DMEK, all eyes had a BCVA of 20/25 (0.8) or better. Pentacam analysis showed a virtually stable anterior corneal curvature in all cases, but among cases, the transplant exchange induced variable refractive change at the posterior corneal surface. CONCLUSION: To manage DSEK cases with poor visual outcome, secondary DMEK may be a feasible procedure potentially resulting in full visual rehabilitation, as in primary DMEK. The presence of donor posterior stroma in DSEK, but not in DMEK grafts, may be a major factor in limiting the final BCVA in endothelial keratoplasty.

Isolated Bowman layer transplantation to manage persistent subepithelial haze after excimer laser surface ablation.

In a 21-year-old patient with persistent dense subepithelial haze after photorefractive keratectomy, unresponsive to retreatment, a stromal flap, 9.0 mm in diameter and approximately 60 microm in thickness, was excised and an unsutured 9.0 mm donor Bowman layer was transplanted onto the stromal bed. The corrected distance visual acuity improved from 20/40 (0.5) before surgery to 20/18 (1.2) with a scleral-supported contact lens 2 months after transplantation. With optical coherence tomography, the transplanted Bowman layer was seen as a fine white line bordering the anterior host stroma. No recurrence of stromal haze was seen throughout the 6-month follow-up. Isolated Bowman layer transplantation may be a new technique for the management of anterior stromal opacities or complicated epithelial wound healing such as persistent corneal haze after excimer laser surface ablation.

Back-up procedure for graft failure in Descemet membrane endothelial keratoplasty (DMEK).

AIM: To evaluate the efficacy of a secondary Descemet stripping endothelial keratoplasty (DSEK) as a back-up procedure for managing graft failure after primary Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Non-randomised prospective clinical study. METHODS: A first group of 50 cases with Fuchs endothelial dystrophy underwent DMEK. Two to five weeks after the DMEK, 10 cases showed no corneal clearance, so a secondary DSEK was performed. To evaluate the eyes of these 10 cases, best corrected visual acuity (BCVA) and endothelial cell density at 6 and 12 months were used as outcome parameters. RESULTS: At 6 months after secondary DSEK, 87% of the cases had a BCVA of > or = 20/40 (> or = 0.5) and one eye reached 20/25 (> or = 0.8). Donor DSEK grafts endothelial cell densities averaged 2617+/-152 cells/mm2 before surgery, 1510+/-799 cells/mm2 at 6 months and 1602+/-892 cells/mm2 at 12 months after surgery. CONCLUSION: In the event of a DMEK graft failure, a secondary DSEK may be an effective back-up procedure, as it may give a clinical outcome similar to that after a primary DSEK. Particularly during the surgeon's learning curve, patient information may be provided not only on visual outcomes after DMEK, but also after DSEK. TRIAL REGISTRATION NUMBER: NCT00521898.

Endothelial cell density after descemet membrane endothelial keratoplasty: 1- to 2-year follow-up.

PURPOSE: To evaluate donor endothelial cell density (ECD) after Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Nonrandomized, prospective clinical study. METHODS: From a larger group of patients who underwent DMEK for Fuchs endothelial dystrophy or pseudophakic bullous keratopathy, complete ECD measurements were available of 26 patients with 6 and 12 months of follow-up, of whom 7 also had 24 months of follow-up. RESULTS: For the group with 24 months of follow-up, ECD averaged 2700 (+/- 260) cells/mm(2) before surgery, 2200 (+/- 460) cells/mm(2) at 6 months after surgery, 2050 (+/- 330) cells/mm(2) at 12 months after surgery, and 1780 (+/- 390) cells/mm(2) at 24 months after surgery. For the group with 12 months of follow-up, ECD averaged 2620 (+/- 210) cells/mm(2) before surgery, 1850 (+/- 540) cells/mm(2) at 6 months after surgery, and 1680 (+/- 550) cells/mm(2) at 12 months after surgery. In both groups, the ECD decreased significantly between the preoperative and 6-month measurement (P < .05). CONCLUSIONS: Similar to earlier endothelial keratoplasty techniques, DMEK may be associated with a decrease in donor ECD of approximately 25% in the early postoperative phase.