Physical Examination of Potential Deceased Organ and Tissue Donors: An Overview of the European Landscape.

Physical examination (PE) of donors is essential to identify potential risks to the safety and efficacy of donated organs and tissues and is mandatory in the EU. However, no detailed guidance is available as to how PE should be performed. Health authorities (HA) and health professionals (HP) in member states of the European Committee on Organ Transplantation of the Council of Europe (CD-P-TO) and observer countries completed surveys relating to the regulatory requirements for PE and the professional practice of PE in their countries for organ and tissue donors. The HA survey addressed regulatory aspects, and the HP survey addressed professional practices, training, and respondents' opinions on the value of PE. These surveys revealed significant inter-country variation in the regulatory approach to PE and the performance of PE by professionals. Most respondents opined that PE was important and yielded valuable information in identifying contraindications to donation. There is no consensus at a regulatory or professional level as to how PE should be performed on organ and tissue donors. There is a requirement for agreed best practice guidelines in this area.

P22-A142†Validation of an improved closed system for dispensing serum eye drops.

PURPOSE: NHS Blood and Transplant Tissue and Eye Services provide a serum eye drop (SED) service to patients suffering from severe dry eye syndrome. Currently SED are dispensed using an automatic closed filling system (TF) manufactured by Meise Medizintechnik (Germany). An improved version (ATS) has recently been introduced by Meise, based on patient feedback on the TF system. ATS vials are easier to open, with a more secure, tamper evident closure and a better quality nozzle.To evaluate the suitability of ATS vials, a validation protocol, previously developed for TF vials, was repeated. It comprised assessment of their integrity following simulated storage and transport, and the stability and sterility of SED stored in them. METHOD: Firstly, a process simulation assessment was performed using bovine serum. Vials were filled, and frozen to -80oC. They were then removed from frozen storage and checked for damage, before being 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 checked visually and by application of a standard force.Subsequently a shelf-life study was carried out using three batches of human SED. The vials were initially frozen to -80oC, then stored for set time points of 1, 3, 6 and 12 months in a standard domestic freezer set at 20oC (to mimic a home freezer). At each time point, 10 vials were thawed and examined for integrity, and the sterility and stability of the contents. Stability was assessed by measuring serum albumin concentrations and sterility by testing for presence of microbial contamination, under aerobic and anaerobic conditions. RESULTS: No vial damage or leakage was found at any time point in the ATS vials. No microbial contamination was detected, and no change in albumin levels was detected in SED throughout the storage period. CONCLUSION: This study has demonstrated that the ATS vials are suitable for provision of SED for clinical use to patients. Feedback is now being gathered from a patient focus group relating to usability of the vials.

P21-A141†Quantification of bioactive factors in human serum eyedrops.

PURPOSE: NHS Blood and Transplant supply serum eye drops (SED) for the treatment of severe dry eye syndrome, however, understanding of what components of SED contribute to their activity is limited. SEDs are produced from a patient's own blood or from an allogeneic donor source. The serum component is separated from the whole blood which is then diluted 50/50 with sterile saline, and contains bioactive molecules that are believed to help heal and maintain the ocular surface. The objective of this study is to quantify the amount of bioactive molecules in donor serum, and to understand how processing variables effects these factors. METHODS: Samples of SEDs from 28 male allogenic donors were taken from ultra-low temperature storage and thawed. They were then centrifuged at 13,000 rpm at 4oC to remove potential contaminants such as residual red blood cells. Duplicate test samples were analysed for epidermal growth factor (EGF) and fibroblast growth factor (FGF) using ELISA kits. Analysis was carried out using Excel. RESULTS: The age range of the donors was 17 to 79 years (mean 47.9).Mean time from venepuncture to refrigerated storage was 6 hours 12 minutes with time ranging from 2 hours 40 minutes to 9 hours 35 minutes.The concentration of EGF found in the diluted serum ranged from 0.048 to 1.90 ng/ml (mean 0.87 ng/ml), and FGF concentration ranged from 4.88 to 39.50 pg/ml (mean 12.37 pg/ml).Analysis showed that there was no correlation between either age of the donor, or sample transfer time and growth factor concentration. CONCLUSION: Our study demonstrated that with both types of growth factors measured in the SED, a wide range of concentrations were found in the donor samples. Compared to published data EGF was at higher range while FGF was lower. Further analysis of other factors present in the donor serum is being undertaken to determine if any pattern can be found.

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.

2†Impact of COVID-19 on a national serum eyedrops programme in the UK.

NHSBT run a Serum Eyedrops programme for the UK, providing Autologous (AutoSE) and Allogenic (AlloSE) eyedrops for patients affected by severe dry eyes. The service is based within the Eye & Tissue Bank in Liverpool.In February 2020 (pre-pandemic within the UK) there were 1052 patients on the programme. 34% received AutoSE and 66% AlloSE. Due to a recent change in central funding, referrals for AlloSE had increased, creating a waiting list; in March 2020 the list had 72 patients.In March 2020 government guidelines were introduced to reduce the spread of COVID-19. These measures presented a number of challenges for NHSBT and our ability to maintain the supply of Serum Eyedrops: i) Many AutoSE patients could not attend donation appointments, as they were clinically vulnerable and needed to shield. This issue was addressed by temporarily providing them with AlloSE. This was done with agreement between patients and consultants. As a result, the proportion of patients receiving AlloSE increased to 82%.ii) There was a reduced supply of AlloSE donations due to a general reduced attendance at blood donation centres. To deal with this, additional donor centres were recruited to collect AlloSE. In addition, the postponing of much elective surgery during the pandemic meant the demand for blood for transfusion reduced, enabling us to build up stock in anticipation of blood stocks reducing as the pandemic developed.iii) Our service was also impacted by reduced staffing levels, due to staff needing to shield or self-isolate, and the need to implement workplace safety measures. To address these problems, a new laboratory was created, allowing staff to dispense eyedrops and adhere to social distancing. It was also possible to re-allocate staff from other areas within the Eye Bank due to a reduction in demand for other grafts during the pandemic.iv) There were initial concerns over the safety of blood and blood products as to whether the transmission of COVID-19 was possible through blood. Following a stringent risk assessment by NHSBT clinicians, and implementation of additional safeguards around blood donation, it was agreed that provision of AlloSE was safe and could continue.Despite all the challenges created by the pandemic, the measures we implemented enabled us to maintain our SE service for existing patients, provide treatment for new referrals and accommodate a significant increase (25% in the 12 months following the beginning of the pandemic) in the number of patients requiring treatment.

Study protocol for a multicentre, randomised controlled trial to compare the use of the decellularised dermis allograft in addition to standard care versus standard care alone for the treatment of venous leg ulceration: DAVE trial.

INTRODUCTION: Venous leg ulceration (VLU), the most common type of chronic ulcer, can be difficult to heal and is a major cause of morbidity and reduced quality of life. Although compression bandaging is the principal treatment, it is time-consuming and bandage application requires specific training. There is evidence that intervention on superficial venous incompetence can help ulcer healing and recurrence, but this is not accessible to all patients. Hence, new treatments are required to address these chronic wounds. One possible adjuvant treatment for VLU is human decellularised dermis (DCD), a type of skin graft derived from skin from deceased tissue donors. Although DCD has the potential to promote ulcer healing, there is a paucity of data for its use in patients with VLU. METHODS AND ANALYSIS: This is a multicentre, parallel group, pragmatic randomised controlled trial. One hundred and ninety-six patients with VLU will be randomly assigned to receive either the DCD allograft in addition to standard care or standard care alone. The primary outcome is the proportion of participants with a healed index ulcer at 12 weeks post-randomisation in each treatment arm. Secondary outcomes include the time to index ulcer healing and the proportion of participants with a healed index ulcer at 12 months. Changes in quality of life scores and cost-effectiveness will also be assessed. All analyses will be carried out on an intention-to-treat (ITT) basis. A mixed-effects, logistic regression on the outcome of the proportion of those with the index ulcer healed at 12 weeks will be performed. Secondary outcomes will be assessed using various statistical models appropriate to the distribution and nature of these outcomes. ETHICS AND DISSEMINATION: Ethical approval was granted by the Bloomsbury Research Ethics Committee (19/LO/1271). Findings will be published in a peer-reviewed journal and presented at national and international conferences. TRIAL REGISTRATION NUMBER: ISRCTN21541209.

Multiple Interventions for Diabetic Foot Ulcer Treatment Trial (MIDFUT): study protocol for a randomised controlled trial.

INTRODUCTION: Diabetes affects more than 425 million people worldwide with a lifetime risk of diabetic foot ulcer (DFU) of up to 25%. Management includes wound debridement, wound dressings, offloading, treatment of infection and ischaemia, optimising glycaemic control; use of advanced adjuvant therapies is limited by high cost and lack of robust evidence. METHODS AND ANALYSIS: A multicentre, seamless phase II/III, open, parallel group, multi-arm multi-stage randomised controlled trial in patients with a hard-to-heal DFU, with blinded outcome assessment. A maximum of 447 participants will be randomised (245 participants in phase II and 202 participants in phase III). The phase II primary objective will determine the efficacy of treatment strategies including hydrosurgical debridement ± decellularised dermal allograft, or the combination with negative pressure wound therapy, as an adjunct to treatment as usual (TAU), compared with TAU alone, with patients randomised in a 1:1:1:2 allocation. The outcome is achieving at least 50% reduction in index ulcer area at 4 weeks post randomisation.The phase III primary objective will determine whether one treatment strategy, continued from phase II, reduces time to healing of the index ulcer compared with TAU alone, with participants randomised in a 1:1 allocation. Secondary objectives will compare healing status of the index ulcer, infection rate, reulceration, quality of life, cost-effectiveness and incidence of adverse events over 52 weeks post randomisation. Phase II and phase III primary endpoint analysis will be conducted using a mixed-effects logistic regression model and Cox proportional hazards regression, respectively. A within-trial economic evaluation will be undertaken; the primary economic analysis will be a cost-utility analysis presenting ICERs for each treatment strategy in rank order of effectiveness, with effects expressed as quality-adjusted life years.The trial has predefined progression criteria for the selection of one treatment strategy into phase III based on efficacy, safety and costs at 4 weeks. ETHICS AND DISSEMINATION: Ethics approval has been granted by the National Research Ethics Service (NRES) Committee Yorkshire and The Humber - Bradford Leeds Research Ethics Committee; approved 26 April 2017; (REC reference: 17/YH/0055). There is planned publication of a monograph in National Institute for Health Research journals and main trial results and associated papers in high-impact peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN64926597; registered on 6 June 2017.

EuroGTP II: a tool to assess risk, safety and efficacy of substances of human origin.

A systematic methodology, able to assess risk and predict clinical safety and efficacy of Substances of Human Origin' (SoHO) has been developed. The model consists of a risk based approach taking into account factors such as novelty of the product, preparation process, clinical indication, and its technical complexity.

Bone allograft in the U.K.: perceptions and realities.

Bone allografts are widely used in the U.K. in joint revision surgery. Despite this widespread usage, there remain concerns among the surgical community regarding the safety of allografts, in terms of the risk of transmission of infection, together with a persistent misconception that allografts are in limited availability. In this paper we discuss the precautions taken to ensure that allografts are safe, and review the residual risks. We also demonstrate that the availability of allograft in the U.K., both actual and potential, greatly exceeds the current clinical demand.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts.

Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage varphix174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated > 6.1 log(10) of the model virus, and gamma irradiation (at 25 -40 kGy and applied to frozen allografts) inactivated 3.6 - 4.0 log(10) of the model virus and > 4 log(10) of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.

Radiofrequency-generated glow discharge treatment: potential benefits for polyester ligaments.

This multicenter study has revealed that treating a woven polyethylene terephthalate (polyester) ligament with a radiofrequency (RF)-generated glow discharge (RFGD) produces marked benefits in terms of increased cell attachment and proliferation on the implant surface. In vitro tests of the same material revealed that the number of synovial fibroblasts attached to the treated samples after 14 days was four times that of the untreated material. Many of the cells were spread over the surface of a single filament, and some formed bridges between one filament and the next. The incorporation of [(3)H]-thymidine by synovial stromal cells (a measure of the amount of cell division) growing on the treated material was five times that on the untreated samples. The amount of DNA present on the treated material was also found to be almost an order of magnitude greater than that on untreated samples. This increase in cell attachment and proliferation is almost certainly related to a notable increase in wettability of the polyester surface induced by treatment. Mechanical tests revealed that, for ligaments with a nominal ultimate tensile strength of 2100 N, RF-generated glow treatment reduced the ligament's strength by 12% but increased its stiffness by 15%. After a medium-term fatigue test (10.8 million cycles), however, there appeared to be recovery of the mechanical properties, with the strength and stiffness of untreated and treated samples being essentially the same. After exhaustive fatigue tests (more than 62 million cycles) the residual strength of the treated ligaments was only 9% lower than that of the unfatigued and untreated ligaments.