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.

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

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

5†The impact of the pandemic on ocular tissue procurement : the experience of fondazione banca degli Occhi del veneto.

INTRODUCTION: Since 21 February 2020, the day that the first Italian COVID-19 case was identified, the organizational and regulatory conditions for ocular tissue donation have undergone numerous changes in order to guarantee safety and quality. Herewith we report the key responses of the procurement programme to these challenges. MATERIALS AND METHODS: A retrospective analysis of the ocular tissue procured between 1 January 2020 and 30 September 2021 is reported. RESULTS: 9224 ocular tissues were procured during the study period (weekly average: 100 ± 21 tissues, mean ± SD; down to 97 ± 24, if only 2020 is considered). During the first wave, the weekly average reached 80 ± 24 tissues, a significant reduction if compared to the first 8 weeks of the year (124 ± 22 tissues/week, p<0.001), falling to 67 ± 15 tissues/week during the lock-down period. Considering the ocular tissues collected in the Veneto Region alone, the weekly mean was 68 ± 20, a reduction when compared to the first 8 weeks of the year (102 ± 23, p<0.001), arriving at 58 ± 15 tissues/week during the lock-down period. The percentage of healthcare professionals who tested positive during the first wave was on average 12% of the positive cases in the whole country, and equal to 18% in the Veneto Region alone. During the second wave, the mean weekly recovery of ocular tissue was 91 ± 15 and 77 ± 15 in the Veneto Region, compared to positive cases of healthcare professionals of 4% across Italy and in the Veneto Region. During the third wave, the overall weekly mean recovery rate was 107 ± 14, and 87 ± 13 in the Veneto Region, with only 1% of positive cases among healthcare professionals in Italy and in the Veneto Region. CONCLUSIONS: The most dramatic decrease of ocular tissue recovery occurred during the first wave of COVID-19, notwithstanding the lower number of infected people. This phenomenon can be attributed to different factors: a high percentage of positive cases and/or contacts among potential donors; the number of infections among healthcare professionals, favoured by the lack of personal protection equipement and the still partial knowledge of the disease; the exclusion of donors with bilateral pneumonia. Subsequently, the system was better organized with the assimilation of new knowledge about the virus, overcoming the initial fears about transmission and thus guaranteeing the resumption and maintenance of donations.

Descemet Membrane Endothelial Keratoplasty Learning Curve for Graft Preparation in an Eye Bank Using 645 Donor Corneas.

PURPOSE: To investigate the learning curve of Descemet membrane endothelial keratoplasty (DMEK) graft preparation in an eye bank. METHODS: Four operators prepared 645 DMEK grafts using the stripping technique between 2014 and 2017 at the Veneto Eye Bank Foundation, Italy. Endothelial cell loss (ECL) and tissue wastage were recorded retrospectively after DMEK preparation and correlated with the number of tissues prepared each year by each operator. On average, our operators performed 1 donor preparation a week over the course of this study. Only donors older than 60 years were used in this study, and approximately 10% of donors had diabetes. The Wilcoxon test for paired data and 1-way ANOVA were used for checking statistical significance with the Tukey test as post hoc analysis. P < 0.05 was considered statistically significant. RESULTS: ECL did not change significantly over time from operator 1. Significant ECL drop was noted from operator 2 between years 2014-2016 (P = 0.0049) and 2017 (P = 0.0094); from operator 3 between years 2015-2016 (P = 0.0288) and 2017 (P = 0.0097); and from operator 4 between 2015-2016 (P = 0.0469) and 2017 (P = 0.0331). Operators 1 and 3 did not show a significant difference, considering every 50 grafts prepared by each operator. Operator 2 showed significant ECL drop between 1 to 50 and 51 to 100 (P = 0.0002) and 1 to 50 and 101 to 150 (P = 0.0001) grafts. Operator 4 showed significant ECL drop between 1 to 50 and 101 to 150 (P = 0.002) and 51 to 100 and 101 to 141 (P = 0.0207) grafts. No intraoperator difference was observed per 50 grafts (P > 0.05). CONCLUSIONS: There is a learning curve for DMEK graft preparation. ECL and tissue wastage can be reduced with practice and skills. However, each operator may be limited to his or her own learning capability.

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction.

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum "XerumFree™ XF205" (XF); (3) CnT-20 medium supplemented with "XerumFree™ XF205" (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ∆Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ∆Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ∆Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.

Amniotic membranes in ophthalmology: long term data on transplantation outcomes.

The use of amniotic membrane (AM) is a widespread clinical practice for eye surgeries and the treatment of an increasing number of ocular surface pathologies. Here we describe the AM collection methods and donor selection criteria adopted by our tissue bank to distribute 5349 amniotic membrane patches over the last 12 years for the treatment of several ocular pathologies. Specific quality control measures are described and the long term results attained using the reported procedure are presented. A case of AM utilized to treat severe ocular ulceration is also described as an example of AM transplantation. Collective data for the total amniotic membrane patches deployed to treat various ocular diseases are discussed and success rates for AM transplantations are reported. An extensive follow-up is illustrated. The results suggest that the procedures and protocols used by the Treviso Tissue Bank Foundation and Veneto Eye Bank Foundation for collection, preservation, distribution and follow-up are of an optimal standard. Accordingly, the authors conclude that the safety and efficiency of the proposed procedure for the therapeutic use of AM to treat various ocular pathologies are reproducible, with additional evidence favoring the use of AM as an alternative to conventional medical treatment for certain ocular conditions.

Effect of postmortem interval on the graft endothelium during preservation and after transplantation for keratoconus.

PURPOSE: To investigate the effect of postmortem intervals and prognostic factors on endothelial cell density (ECD) of human donor corneas during preservation and at 1 and 3 years after transplantation in patients transplanted for keratoconus. METHODS: Two different studies were performed: (1) with 733 donor corneas selected for the preservation study and (2) 64 patients with keratoconus selected retrospectively from 2 hospital clinics. The corneas were evaluated on the basis of the ECD during preservation, study A, and at 1 and 3 years after transplantation, study B. The effect of ≥ 10 hours of postmortem interval on the percentage of corneal endothelial cell loss (ECL) was determined. RESULTS: The multiple regression showed no statistical significance (P = 0.827) of postmortem interval on ECL during preservation. However, for patients with keratoconus, the postmortem interval was statistically significant at both 1 year (P < 0.0001) and 3 years after transplantation (P < 0.0001). CONCLUSIONS: The postmortem interval has no influence on the ECD during preservation. However, it has a statistically significant effect on the ECL after transplantation for patients transplanted for keratoconus, and therefore, it becomes eligible to be one of the potential factors affecting the ECD apart from surgical trauma.