28†Validation of a closed system for dispensing serum eye drops.

INTRODUCTION: NHS Blood and Transplant Tissue and Eye Services (TES) offer a serum eyedrop (SE) service to patients suffering from severe ocular surface disease. SE are prepared from serum collected at blood donation sessions; the serum is diluted 1:1 with physiological saline. Formerly, 3ml aliquots of diluted serum were aliquoted into glass bottles in a Grade B clean room. Since this service was started, Meise Medizintechnik have developed an automatic closed filling system consisting of tubing-linked chains of squeezable vials. They can be heat-sealed closed, under sterile conditions, after the vials have been filled. MATERIALS AND METHODS: TES R&D were asked to validate the Meise system to increase the efficiency and speed of SE production. Validation of the closed system consisted of a process simulation assessment, using bovine serum and simulating each step of the filling process, freezing to -80oC, checking the integrity of each vial and packing the vials into storage containers. They were then put into transport containers and shipped on a round-trip journey to simulate delivery to patients. On return the vials were thawed and the integrity of each vial re-checked visually and by squeezing in a plasma expressor.Subsequently a shelf-life study was carried out on three batches of fully consented human allogeneic SE. The serum was dispensed into vials, frozen as above and stored for set time points 0, 1, 3, 6 and 12 months in a standard domestic freezer set at -15-20oC to mimic a patient's freezer. At each time point, 10 random samples of vials were removed, and the outer containers were tested for damage or deterioration, the vials for integrity and their contents for sterility and stability. Stability was assessed by measuring serum albumin concentrations and sterility by testing for microbial contamination. RESULTS: No structural damage or leakage was found in any of the vials, or the tubing evaluated, after thawing, at any time point. In addition, all samples tested negative for microbial contamination and serum albumin levels were always within the expected range (3 - 5 Dg/L) at each set time point. CONCLUSION: These results demonstrate that Meise closed system vials can successfully dispense SE drops and the vials can be stored frozen without affecting integrity, sterility or stability. These vials have been in use in TES for 3 years saving clean room space and greatly increasing the numbers of patients that can use the SE service.

3†Emergency salvage of time expired clinical corneas during the COVID-19 pandemic.

INTRODUCTION: Corneas for clinical use can be stored for a maximum of 28 days in organ culture medium after death. At the beginning of the COVID-19 pandemic in 2020 it became apparent that; a rare situation was arising in that clinical operations were being cancelled and that there would be a surplus of "clinical grade" corneas. Consequently, when the corneas reached the end of the storage period, if the tissue had appropriate consent, they were transferred to the Research Tissue Bank (RTB). However, University research had also stopped due to the pandemic and there was a situation where the RTB had good quality tissue without any users. Rather than discarding the tissue, a decision was made to store the tissue for future use by cryopreservation. MATERIALS AND METHODS: An established protocol for cryopreserving heart valves was adapted. Individual corneas were placed into wax histology cassettes then inside a Hemofreeze heart valve cryopreservation bag with 100 ml cryopreservation medium (10% Dimethyl sulphoxide)). They were frozen in a controlled rate freezer (Planer, UK) to below -150oC and stored in vapour phase over liquid nitrogen (VPLN) below -190oC. To assess morphology, six corneas were cut in half, one half was processed for histology whilst the other half was cryopreserved, stored for 1 week then thawed and processed for histology. The stains used were Haematoxylin and Eosin (H&E) and Miller's with Elastic Van Gieson (EVG). RESULTS: Comparative histological examination indicated that there were no visible, major, detrimental changes in morphology in the cryopreserved group as compared to the controls. Subsequently, a further, 144 corneas were cryopreserved. Samples were assessed for handling properties by eye bank technicians and ophthalmologists. The eye bank technicians felt that the corneas may be suitable for training purposes such a DSAEK or DMEK. The ophthalmologists said that they had no preference between the fresh or cryopreserved corneas, and both would be equally suitable for training purposes. CONCLUSION: Time expired, organ-cultured corneas, can be successfully cryopreserved using an established protocol by adapting the storage container and conditions. These corneas are suitable for training purposes and may prevent discard of corneas in future.

Development of a decellularised dermis.

The purpose of this investigation was to develop a decellularised human dermis suitable for allografting. Samples of human skin were obtained from deceased donors and taken through a series of steps to remove all cellular material. The steps were: chemical removal of the epidermis, disinfection, lysing of cells in hypotonic buffer, a detergent treatment and a nuclease buffer to remove residual nuclear material. Histological preparations of the decellularised dermis produced were then investigated. In addition residual DNA content, structural strength, collagen denaturation, cytotoxicity and in vivo tissue reactivity following implantation in a murine model were examined. For all donors tested there was no change in morphology as viewed by light microscopy. Mean DNA removal was evaluated at 92.1%. There were no significant changes in structural strength or evidence of collagen degradation. The tissue did not appear to be cytotoxic or elicit an immune response when implanted in the mouse model. A decellularised tissue has been developed that would appear to be suitable for a range of surgical procedures.

Morphometric effects of long-term exposure to latanoprost.

PURPOSE: To investigate the morphological and melanin granule changes in irides after variable-term exposure to latanoprost, where the latanoprost-induced iris darkening (LIID) side effect has been identified and photographically recorded. DESIGN: Experimental study. PARTICIPANTS: Fifteen LIID cases and 15 untreated controls. METHODS: Iridectomy specimens from LIID cases were collected from patients undergoing trabeculectomy, whereas before surgery they had been on topical latanoprost and there was clear evidence of iris color change from the treating ophthalmologist, which was recorded photographically. A control series of peripheral iridectomies were obtained from blue, heterogeneous, and brown irides. All the specimens were visualized by light and electron microscopy. Masked assessment was made of stromal cell number, cell atypia, anterior border thickness, presence of free stromal melanin, melanin proximity to blood vessels, and change in stromal melanocyte melanin granule numbers and size. For the melanin granule analysis, electron micrographs were subjected to detailed image analysis to quantify the number of melanin granules, size of the granules, and overall percentage filling of the iris stromal melanocytes with melanin. MAIN OUTCOME MEASURES: Iris morphology and melanin granule changes. RESULTS: There was no evident difference in stromal cellularity or anterior border thickness. Atypia, free stromal melanin, and melanin adjacent to blood vessel lumina were identified, but there was no difference between LIIDs and controls. Within stromal melanocytes, we found no change in the total number of melanin granules of the LIID cases, as compared with the brown and heterogeneously colored normals. However, melanin granules in the anterior border melanocytes of the LIID eyes were significantly larger than those in the controls. A trend towards bigger melanin granules was apparent in the deep stroma, but this difference did not reach significance. CONCLUSIONS: In the LIID cases that we examined, the darkening side effect does not seem to be associated with either proliferative or generative iris changes, nor with increases in number of granules. Instead, it appears to be due to small increases in the size of mature melanin granules, particularly in the anterior border region.

Melanin in the trabecular meshwork is associated with age, POAG but not Latanoprost treatment. A masked morphometric study.

We wished to conduct a light and electron microscopic investigation of pigmentation within the trabecular meshwork of normals and primary open angle glaucoma (POAG) patients. In particular we wished to get a precise determination of whether there was a relationship between pigmentation and age. In addition we wanted to know if there was a difference between normals and POAGs and whether trabecular meshwork hyperpigmentation was associated with topical latanoprost medication. A total of 25 sham trabeculectomies conducted on post mortem donor eyes provided the age-matched normals and there were 62 trabeculectomy specimens from POAG patients. These were masked and the meshwork subjected to qualitative and quantitative morphological investigation. Light and electron microscopy confirmed that most of the trabecular meshwork melanin was phagocytosed and within meshwork cells. The granules were measured and found to be of the large iris epithelial type. Light microscopic morphometric analysis showed that the number of meshwork cell profiles that contained melanin increased both in normals and POAGs with age. However there was nearly three times more pigmented meshwork cells in the POAGs than the normals. The POAGs were divided into three groups of (1) minimal or no medication prior to surgery, (2) maximal medical therapy and (3) maximum medical therapy including latanoprost (12 specimens). All groups were significantly greater that the normals but of the three it was the maximal medical therapy group (without latanoprost) that had the highest pigmentation. We concluded that pigmentation of the meshwork is age-related and it is elevated in POAG by mechanisms unknown. The melanin accumulation seems to be partly due to the disease process, partly as a consequence of chronic antiglaucoma medication but interestingly not due to latanoprost even in patients where there is iris darkening (four specimens).

Latanoprost-induced iris darkening: a morphometric study of human peripheral iridectomies.

PURPOSE: This microscopic study was undertaken to compare the melanocytes of peripheral iridectomy specimens from two eyes that had latanoprost-induced iris darkening (LIID) with iridectomies taken from the fellow untreated eyes. METHODS: The two patients in this study were the ones who underwent LIID in the latanoprost treated eye from a series of 17 patients requiring bilateral trabeculectomy. The first trabeculectomy procedure provided a control peripheral iridectomy for each patient, whereas the second eye was treated with once daily 50 microg ml(-1) latanoprost drops for 6 months. The four peripheral iridectomy specimens from the two LIID patients were subjected to quantitative morphometric analysis by light microscopy of iris cellularity, and electron microscopy of iris melanocyte immature melanosomes and mature melanin granules. RESULTS: There was no significant difference in stromal cellularity between the LIIDs and their respective controls nor were there significant differences in the numbers of immature melanosomes or melanin granules in the melanocytes. However, there was a significant increase in the diameter of melanin granules that was more pronounced in the anterior border layer than the deeper stroma. With the anterior border melanocytes, the increase in melanin granule size was associated with significant increases in granule area and the percentage of cell cytoplasm occupied by melanin (granularity). CONCLUSIONS: The only morphological change identified in two peripheral iridectomies that had LIID when compared to untreated fellow eye specimens was a modest increase in the size of stromal melanocyte melanin granules that was more pronounced in the cells of the anterior border region.

Direct comparison of the migration of three cell types involved in epiretinal membrane formation.

PURPOSE: The purpose of the present study was to develop an accurate and sensitive migration assay to compare the migratory capabilities of retinal pigment epithelial (RPE) cells, retinal glial (RG) cells, and fibroblasts (the cell types crucial in epiretinal membrane [ERM] formation) under identical microenvironmental conditions and thus to identify potential target areas in ERM management. METHODS: Cultured bovine RPE and RG cells and scleral fibroblasts (SFs) in both single and mixed cell type populations were induced to migrate in modified 48-well Boyden chambers. The labels used to distinguish between the cell types were latex microspheres and carmine particles. The chemoattractants used were fibronectin and PDGF, both of which are associated with epiretinal membrane development. RESULTS: When migrating independently, all three cell types showed a positive response to fibronectin at an optimal concentration of 10 microg/mL. The RG cells migrated in a significantly greater number than the RPE cells (P < 0.05), but the differences in number of migrating cells between RG cells and SFs and RPE cells and SFs were not significant. When the cells were labeled and migrating together, it became clear that the RG cells consistently migrated in a higher number than the SFs (P