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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.
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Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Epiteliais/citologia , Células-Tronco/citologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Cocultura , Humanos , CamundongosRESUMO
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.
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Âmnio/transplante , Oftalmologia , Córnea/patologia , Feminino , Seguimentos , Humanos , Cuidados Pós-Operatórios , Gravidez , Resultado do Tratamento , Úlcera/patologia , Úlcera/terapiaRESUMO
PURPOSE: To describe a new proxy of the Eye Bank (EB) activity named "Eye Bank Efficiency Index" (EBEI), calculated as the ratio between the number of corneal tissues distributed by an EB within a certain time period, and the difference between the number of procured and discarded tissues. METHODS: To demonstrate the effectiveness of the new metric as compared to traditional statistics, an analysis was conducted using data from the largest Italian Eye Bank (Fondazione Banca degli Occhi del Veneto Venice, Italy). Collected data included: the number of corneas retrieved, the number of discarded grafts, and the number of distributed tissues. The analysis focused on three defined timeframes: January to December, March to May (the "Italian Lockdown period"), and June to December (the "Italian post-lockdown period"). RESULTS: In 2020, the annual variation of the EBEI showed a significant increase of up to 3.4% compared to the previous year (0.986 in 2019; 1.020 in 2020), but then gradually decreased to 0.993 in 2022. However, during the months of lockdown in 2020, there was a significant decline of -13.8% in the EBEI compared to the same period in the previous year. The variation in the EBEI during the post-lockdown months was minimal in 2020 and 2021, with the lowest EBEI value of 0.976 being reached in 2022 (-7.8% compared to 2019). CONCLUSION: The EBEI is a simple and reliable new measure of the EB activity. Its widespread adoption could ensure a more accurate and reliable analysis of EB data for academic, political, and economic purposes.
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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.
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COVID-19 , Obtenção de Tecidos e Órgãos , Humanos , COVID-19/epidemiologia , Pandemias/prevenção & controle , Estudos Retrospectivos , Controle de Doenças TransmissíveisRESUMO
OBJECTIVES: To assess to which extent the COVID-19 pandemic affected corneal transplantation by virtue of donor selection algorithms in different European countries. DESIGN: Survey. SETTING: 110 eye banks in 26 European countries. PARTICIPANTS: 64 eye banks covering 95% of European corneal transplantation activity. INTERVENTIONS: A questionnaire listing the number of corneas procured and distributed from February to May 2018-2020 was circulated to eye banks. MAIN OUTCOME MEASURES: The primary outcome was the number of corneal procurements. Additional outcomes were national algorithms for donor selection, classified according to their stringency (donors with COVID-19 history, suspected for COVID-19, asymptomatic, PCR testing) and the pandemic severity in each country. We calculated Spearman's correlation coefficient to determine, two by two, the relationship between the 3-month decline in eye banking activity (procurement), the stringency of donor selection algorithm and the grading of pandemic severity (cases and deaths). A partial correlation was run to determine the relationship between decline and stringency while controlling for pandemic severity. RESULTS: Procurements decreased by 38%, 68% and 41%, respectively, in March, April and May 2020 compared with the mean of the previous 2 years, while grafts decreased, respectively, by 28%, 68% and 56% corresponding to 3866 untreated patients in 3 months. Significant disparities between countries and the decrease in activity correlated with stringency in donor selection independent of pandemic severity. CONCLUSIONS: Our data demonstrate significant differences between countries regarding donor screening algorithms based on precautionary principles and, consequently, a decrease in the donor pool, already constrained by a long list of contraindications. Fundamental studies are needed to determine the risk of SARS-CoV-2 transmission by corneal transplantation and guide evidence-based recommendations for donor selection to justify their substantial medical and economic impact.
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COVID-19 , Córnea , Seleção do Doador , Doadores de Tecidos , COVID-19/epidemiologia , Transplante de Córnea , Europa (Continente)/epidemiologia , Bancos de Olhos , Humanos , Pandemias , Doadores de Tecidos/estatística & dados numéricosRESUMO
BACKGROUND: The European Directive on setting standards of quality and safety for human tissues and cells obliges tissue establishments to implement a quality management system (QMS), based on the principles of good practice, in order to assure a high level of protection with regard to the health of recipients of human tissues. METHODS: A systematic approach to quality management should be used and sustained. Although a number of quality systems can be employed, a recommended reference model is the ISO 9001:2000 standard which is particularly relevant to eye banking as it can be implemented to cover the entire donation-transplantation process to ensure that the tissues distributed, and services offered, by eye banks show uniform safety and quality. RESULTS: The adoption and correct management of a QMS is essential to maximise the benefits and minimise the risks for all those involved in the process. The performance and results of this system must be monitored and measured by appropriate parameters/indicators (positive and negative) which pertain to the structure (personnel, facilities, instruments), the process (the sequence of activities), the outcome (recipient health status or client satisfaction), the efficiency (the costs incurred to produce a certain outcome) and the effectiveness (frequency that the required results are attained). CONCLUSION: Careful attention must be paid to all aspects of the quality of donor tissues in order to maintain confidence in their safety and effectiveness. A well-managed QMS is a valuable and effective instrument to guarantee the required high standards for the donation, procurement, testing, processing, storage, distribution and traceability of ocular tissues as well as to facilitate the continuous improvement and the attainment of the objectives of an eye bank.
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Transplante de Córnea , Bancos de Olhos/normas , Gestão da Qualidade Total/normas , Europa (Continente) , Humanos , Controle de Qualidade , Doadores de Tecidos , Coleta de Tecidos e Órgãos/normas , Obtenção de Tecidos e Órgãos/normasRESUMO
BACKGROUND: Inter-laboratory proficiency schemes are widely used to control the performance of clinical and forensic toxicology laboratories. In 2016 the Laboratory of Environmental Hygiene and Forensic Toxicology - Venice (Italy) initiated an inter-laboratory proficiency test of blood-alcohol analysis. The number of participating laboratories gradually increased from 26 to 36. Furthermore, a few clinical laboratories were included if gas chromatographic (GC) methods were used for blood alcohol analysis. PROCEDURE: Whole blood was obtained from the Blood Transfusion Centre of the Venice Hospital and a mixture of sodium fluoride and potassium oxalate was added as a preservative and anticoagulant, respectively. Aliquots of the blood were spiked with certified pure ethanol to obtain target blood-alcohol concentrations (BACs) ranging from 0 to 5.0g/L. Two blood samples (4mL each) were included in each shipment to the participating laboratories. The laboratories were asked to provide information about number of replicate BAC determinations they made, the types of ethanol reference standards used, and inherent measurement uncertainty. The aim of the testing was to obtain a mean consensus value for the target BAC and to assess inter-laboratory imprecision. All procedures for registration and submission of results were done on-line. A confidential report and statistical evaluations were returned to the participants one week later. ANALYTICAL METHODS: All participants used head-space GC (HS-GC) for the analysis of ethanol in blood. More than 85% of participants used HS-GC with flame-ionization detection, whereas the others used mass spectrometry (MS) as a detector. More than 40% of the participating laboratories kept the blood samples frozen (-20°C) prior to analysis, whereas the others used refrigeration (+4°C). The preliminary validation tests showed that there were no statistically significant differences between BAC in frozen or refrigerated samples for a period of 20 days. RESULTS AND CONCLUSION: The statistical evaluation of results was done using an iterative procedure known as Algorithm A (ISO 13528:2015, C.3.1). This provides robust estimates for mean and standard deviation between laboratories and these were used as consensus values. More than 85% of participants provided satisfactory results (z-score <1) and 94% of laboratories were within z-score <2, based on five control samples. When a blood sample without any alcohol (blank) was sent for analysis, laboratories reported this as zero, 0.00g/L, below limit of detection (LOD) or not detected. Some type of consensus should be reached for reporting blank samples.
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Concentração Alcoólica no Sangue , Laboratórios/normas , Detecção do Abuso de Substâncias/normas , Cromatografia Gasosa/métodos , Cromatografia Gasosa/estatística & dados numéricos , Temperatura Baixa , Toxicologia Forense/normas , Toxicologia Forense/estatística & dados numéricos , Humanos , Itália , Laboratórios/estatística & dados numéricos , Espectrometria de Massas/estatística & dados numéricos , Manejo de Espécimes/métodos , Manejo de Espécimes/estatística & dados numéricosAssuntos
COVID-19 , Transplante de Córnea , Obtenção de Tecidos e Órgãos , Córnea , Humanos , SARS-CoV-2 , Doadores de TecidosRESUMO
Retinal degenerative diseases are one of the main clinical causes of incurable and severe visional impairment. Thus, extensive research effort is put into the development of new causal therapeutic options. Promisingly, a number of studies showed regenerative capacity in specific retinal regions (the ciliary epithelium, retinal pigmented epithelium, iris, and Müller glia cells). However, most recent research studies are based on animal models or in vitro cultured cells, probably because of the limited availability of human posterior eye tissues (vitreous, retina, and choroid). To address this, we showed in our previous reports that eye banks with large numbers of globes collected yearly could set up biorepositories/biobanks where these precious tissues are isolated, quality controlled, and finally stored for scientists and clinicians wanting to access human tissues and test their own hypotheses. These precious human posterior eye tissues could be used for further research purposes, epidemiological studies, and target validation of newly developed drugs. In addition, this could be a promising and challenging option to retrieve potential retinal stem and progenitor cells from different parts of the retina and could be a breakthrough in the future delivery of ex vivo prepared customized (histocompatible) retinal tissue on scaffolds for transplantation purposes. In this Perspective, we will consider how the biorepositories could influence the future strategies for retinal stem cell therapies.
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Bancos de Olhos , Retina/transplante , Degeneração Retiniana/terapia , Transplante de Células-Tronco , Animais , Corpo Ciliar/patologia , Corpo Ciliar/transplante , Humanos , Neuroglia/transplante , Retina/patologia , Degeneração Retiniana/patologia , Neurônios Retinianos/patologia , Neurônios Retinianos/transplante , Células-TroncoRESUMO
Descemet membrane endothelial keratoplasty (DMEK) is a corneal surgical technique which selectively replaces the damaged posterior part of the cornea with a healthy donor graft retaining the rest of the tissue intact. There is a need to validate and standardize the donor tissue before grafting due to certain issues that can lead to consequences such as graft failure due to poor endothelial cell count, higher mortality, detachment of the graft, or increased surgical expenses, time, and effort. Thus, prospective potential surgeons and eye banks should now aim at developing new improved surgical techniques in order to prepare the best suited, validated, precut, preloaded, and easy to transplant tissue to reduce pre- and postsurgical complications. This could be achieved by defining parameters like graft thickness, accepted mortality threshold of the endothelial cells, and behavior of grafts during preservation and transportation along with using more sophisticated instruments like microkeratome and femtosecond lasers for graft preparation. Thus, a rapport between the eye banks and the surgeons along with the advanced instruments can overcome this challenge to find the best possible solution for endothelial keratoplasty (EK).
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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.