Patient-reported outcome measures for a large cohort of serum eye drops recipients in the UK.

INTRODUCTION: Serum eye drops (SED) are an important treatment for patients with chronic and severe ocular surface disease (OSD). Despite a long history of use, there is a paucity of information on patient-reported outcomes, particularly comparing autologous SED (Auto-SED) and allogeneic SED (Allo-SED). National Health Service Blood and Transplant is the national provider of SED service for patients in the UK. PURPOSE: To evaluate and compare patient-reported outcome measures (PROMs) in patients receiving Auto-SED and Allo-SED for severe OSD. MATERIALS AND METHODS: PROMs were retrospectively collected from all new patients commencing treatment with Auto-SED and Allo-SED between January 2017 and September 2018, using the Ocular Surface Disease Index (OSDI) 12-item questionnaire. A linear mixed model was used to evaluate the change in OSDI scores between baseline and follow-up. RESULTS: During the study period, 279 patients who received either Auto-SED (n = 71) or Allo-SED (n = 208) were included in the analysis. Baseline and follow-up OSDI scores were available for 161 of these (49 Auto-SED and 112 Allo-SED). There was a significant reduction in mean OSDI score for both Auto-SED (59.06-24.63, p < 0.001) and Allo-SED (64.21-34.37, p < 0.001). There was no significant difference between Auto-SED and Allo-SED patients in terms of the reduction in the OSDI score (p = 0.27). CONCLUSION: Both Auto-SED and Allo-SED were associated with improvements in the quality of life of patients with chronic and severe OSD. Auto-SED and Allo-SED were equally effective in relieving the symptoms of OSD.

Differential Modulation of Quorum Sensing Signaling through QslA in Pseudomonas aeruginosa Strains PAO1 and PA14.

Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite the isolates being closely related, PA14 exhibits increased virulence compared to that of PAO1 in various models. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected a transcriptomic approach through RNA sequencing. We found 2,029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by quorum sensing (QS), known to control most of the virulence factors in P. aeruginosa Among them, we chose to focus our study on QslA, an antiactivator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hypervirulence phenotype. After confirming that QslA protein was highly produced in PAO1 but not in the PA14 strain, we obtained evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics than PA14. Moreover, known virulence factors activated by QS, such as (i) pyocyanin production, (ii) H2-T6SS (type VI secretion system) gene expression, and (iii) Xcp-T2SS (type II secretion system) machinery production and secretion, were all lower in PAO1 than in PA14, due to higher qslA expression. However, biofilm formation and cytotoxicity toward macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Together, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginosa strains PAO1 and PA14.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen responsible for acute nosocomial infections and chronic pulmonary infections. P. aeruginosa strain PA14 is known to be hypervirulent in different hosts. Despite several studies in the field, the underlining molecular mechanisms sustaining this phenotype remain enigmatic. Here we provide evidence that the PA14 strain has faster quorum sensing (QS) kinetics than the PAO1 strain, due to the lack of QslA expression, an antiactivator of QS. QS is a major regulator of virulence factors in P. aeruginosa; therefore, we propose that the hypervirulent phenotype of the PA14 strain is, at least partially, due to the lack of QslA expression. This mechanism could be of great importance, as it could be conserved among other P. aeruginosa isolates.

Assessment of the permeability properties of cryopreservation outer bags used in NHSBT.

This study was carried out to investigate leakage/transport across the bag material of six outer cryopreservation bags in common use within NHS Blood and Transplant. In order to do this two different leak testing procedures; coloured dye and hydrogen tracer gas, were used. The data obtained show that a coloured dye cannot permeate through the materials both at room temperature and following storage at liquid nitrogen temperature (- 196 °C). In addition, when filled with the smallest elemental molecule, hydrogen, in the form of a tracer gas, all of the bags only allowed trace amounts of hydrogen to escape, either through the seal or the bag material. The data indicated that each of the bag materials tested would be capable of preventing bacterial or viral cross-contamination as long as the material remained intact.

Activity of four antimicrobial cocktails for tissue allograft decontamination against bacteria and Candida spp. of known susceptibility at different temperatures.

Several antimicrobial cocktail solutions of differing composition and concentrations are widely used to decontaminate viable banked tissue allografts at different temperatures and times of exposure. We compared the efficiency of four cocktails comprising nine antimicrobials to kill suspensions of a panel of 27 strains of 13 bacterial species, and 3 Candida spp. at 4, 22 and 37 °C for 24 h. All but one bacterial strains were susceptible to one or more of the agents tested individually at concentrations at least fourfold below the recommended susceptibility breakpoint minimum inhibitory concentrations for drug/species combinations. Candida lusitaniae was resistant to nystatin and amphotericin. The concentrations of several of the cocktail constituents were often greatly in excess (50-1,000-fold) of that required to inhibit the growth of susceptible strains. All cocktails were ineffective against a pan-resistant strain of Enterococcus faecium and one of the four cocktails failed to kill two strains of methicillin resistant Staphylococcus aureus. Each cocktail was most efficient at 37 °C, less so at 22 °C, and poorly active at 4 °C. We conclude that the practice of decontamination of tissues with antimicrobials at low temperatures is not supported by in vitro susceptibility tests.

A standardised protocol for the validation of banking methodologies for arterial allografts.

The objective of this study was to design and test a protocol for the validation of banking methodologies for arterial allografts. A series of in vitro biomechanical and biological assessments were derived, and applied to paired fresh and banked femoral arteries. The ultimate tensile stress and strain, suture pullout stress and strain, expansion/rupture under hydrostatic pressure, histological structure and biocompatibility properties of disinfected and cryopreserved femoral arteries were compared to those of fresh controls. No significant differences were detected in any of the test criteria. This validation protocol provides an effective means of testing and validating banking protocols for arterial allografts.

The implementation of nucleic acid amplification technology testing for living tissue donors.

There is a significant requirement within the United Kingdom for tissue grafts from living donors. To ensure safety, blood samples from these donors are tested for pathogens at donation, and at 180 days post donation. Nucleic acid amplification technology (NAT) permits more sensitive detection of pathogens in blood samples than serum antigen testing. NAT testing can be applied to samples from living tissue donors to eliminate the need to re-test these donors 180 days post-donation before grafts can be implanted. This has major financial and operational advantages for a tissue bank, and this manuscript describes how NAT testing was assessed and implemented by NHSBT Tissue Services. When compared to traditional serum antigen testing, NAT testing was more cost effective, more convenient for donors and resulted in a greater proportion of donated grafts being made available for transplant.

Sterilisation of skin allograft with gamma irradiation.

The primary surgical requirement of skin allografts within the UK is for cryopreserved viable allografts as these engraft to the wound bed and gain a vascular supply, thus providing true wound closure and a superior clinical performance. Consequently the only disinfection treatment the skin receives is exposure to an antibiotic cocktail. However, antibiotic treatment does not reliably decontaminate skin allografts and 22% of cryopreserved skin fails microbial acceptance criteria and cannot be used clinically. We describe here a study which was carried out to determine a means of saving and using the microbiologically failed skin. Four different treatment regimens were investigated; treatment with 20%, 50% and 85% glycerol followed by 25 kGy irradiation at -80 degrees C, and treatment with 85% glycerol at ambient (30-40 degrees C) temperature and irradiation. Following treatment, the grafts were evaluated for their histological structure, in vitro cytotoxicity and handling properties. The radioprotective effects of the different glycerol concentrations and temperatures on microorganisms were also determined. The data indicate that 25 kGy irradiation of deep-frozen skin in 20% glycerol sterilised the tissue without any histological, cytotoxicological or physical alterations compared to normal cryopreserved skin. In contrast, irradiation of all other glycerol concentrations elicited some cytotoxicity and/or histological effect. These non-viable grafts can be made available for surgical use when cryopreserved viable grafts are not available or required.

Validation of radiation dose received by frozen unprocessed and processed bone during terminal sterilisation.

Fresh frozen femoral heads (FH) and frozen processed bone (FP) are widely used as a source of allograft bone. The FP bone and some of the FH are terminally sterilised by the National Blood Service Tissue Services (NSBTS), via application of a minimum 25 kGy gamma radiation dose. To comply with the Guidelines for the Blood Transfusion Services in the United Kingdom (2002), frozen musculoskeletal tissue must be maintained below -40 degrees C during storage and transit. In practice, NBSTS stores bone long-term in -80 degrees C freezers. During transport for irradiation, a temperature of circa -79 degrees C is maintained by packing the bone in dry ice. An evaluation of the radiation dose received by bone has previously been made via dosimeters located within the tissue and dry ice, however, some evidence suggests that low temperature can influence the accuracy of the dosimeter readings. The aim of this study was to determine the actual radiation dose received by FH and FP bone during the irradiation process. This was accomplished by comparing radiation dose readings from dosimeters placed in dry ice with dosimeters placed in a dry ice substitute of similar dimensions and density i.e., polytetrafluoroethylene (PTFE) at ambient temperature. New packing formats were developed for both FH and FP bone such that 15 FH or 3 kg of FP bone could be irradiated in one transport box at any given time in a standardised fashion. The data show that low temperature consistently increased dosimeter readings 10--27%, and that radiation dose always fell within the range of 25--40 kGy (FH=25.1--35.7 kGy; FP bone=25.2--32.4 kGy).

Effects of a peracetic acid disinfection protocol on the biocompatibility and biomechanical properties of human patellar tendon allografts.

Patellar tendon allografts, retrieved from cadaveric human donors, are widely used for replacement of damaged cruciate ligaments. In common with other tissue allografts originating from cadaveric donors, there are concerns regarding the potential for disease transmission from the donor to the recipient. Additionally, retrieval and subsequent processing protocols expose the graft to the risk of environmental contamination. For these reasons, disinfection or sterilisation protocols are necessary for these grafts before they are used clinically. A high-level disinfection protocol, utilising peracetic acid (PAA), has been developed and investigated for its effects on the biocompatibility and biomechanics of the patellar tendon allografts. PAA disinfection did not render the grafts either cytotoxic or liable to provoke an inflammatory response as assessed in vitro . However, the protocol was shown to increase the size of gaps between the tendon fibres in the matrix and render the grafts more susceptible to digestion with collagenase. Biomechanical studies of the tendons showed that PAA treatment had no effect on the ultimate tensile stress or Young's modulus of the tendons, and that ultimate strain was significantly higher in PAA treated tendons.

Application of a high-level peracetic acid disinfection protocol to re-process antibiotic disinfected skin allografts.

Skin allografts, derived from cadaveric donors, are widely used for the treatment of burns and ulcers. Prior to use in clinical situations, these allografts are disinfected using a cocktail of antibiotics and then cryopreserved. Unfortunately, this antibiotic disinfection procedure fails to decontaminate a significant proportion and these contaminated grafts can not be used clinically. We have investigated whether it is possible to apply a second, more potent disinfection procedure to these contaminated grafts and effectively to re-process them for clinical use. Cadaveric skin grafts, treated with antibiotics and cryopreserved, were thawed and a peracetic acid (PAA) disinfection protocol applied. The grafts were then preserved in a high concentration of glycerol or propylene glycol, and properties thought to be essential for successful clinical performance assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. PAA disinfection, in conjunction with either glycerol or propylene glycol preservation, did not render the grafts cytotoxic, pro-inflammatory, or increase their susceptibility to collagenase digestion. The rates of penetration of glycerol and propylene glycol into the re-processed skin were comparable to those of fresh skin. This study has demonstrated that PAA disinfection combined with immersion in high concentrations of either glycerol or propylene glycol was an effective method for re-processing contaminated skin allografts, and may justify their clinical use.

Assessment of the biological properties of human split skin allografts disinfected with peracetic acid and preserved in glycerol.

Skin allografts derived from cadaveric human donors are widely used in the treatment of serious burn injuries and other conditions, such as ulcers. In order to render these allografts safe for clinical use, and to enable them to be preserved and banked for long periods, effective methods of decontamination and preservation are required. These methods must not adversely affect graft properties essential for clinical performance. We have investigated the application of a peracetic acid (PAA) disinfection protocol, coupled with preservation in either glycerol or propylene glycol to achieve these goals. An effective decontamination procedure, comprising of a 3h exposure to 0.1% (v/v) PAA in phosphate buffered saline (PBS) at pH 7.0, was developed and had no significant detrimental effects on the structure of skin. Cadaveric skin allografts were then treated with this disinfection protocol and subsequently preserved in either 85% (v/v) glycerol or propylene glycol in PBS, and the biological properties of the allografts thought to be essential to successful clinical performance were assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. Neither the disinfection protocol nor either of the preservation techniques rendered the grafts cytotoxic or pro-inflammatory. The PAA disinfection and glycerol preservation protocol had no effects on collagenase susceptibility, whereas the disinfection protocol in combination with propylene glycol rendered some of the test samples significantly more susceptible to collagenase digestion. Therefore, this study has demonstrated that PAA disinfection combined with glycerol preservation is suitable for skin allografts. The use of propylene glycol as a preservation agent for skin requires further development.

An evaluation of the capacity of differently prepared demineralised bone matrices (DBM) and toxic residuals of ethylene oxide (EtOx) to provoke an inflammatory response in vitro.

Demineralised bone matrix (DBM) is a form of allogeneic tissue graft widely used in oral and maxillofacial procedures. There is a long history of controversy relating to the suitability of ethylene oxide gas (EtOx) as a terminal sterilisation agent for this graft, relating to its effects on the clinical performance of the grafts. Furthermore, the generation of a toxic residual chemical (ethylene chlorohydrin, ECl) during the ethylene oxide sterilisation of patellar tendon allografts has been implicated in the failure of these grafts owing to the induction of a localised inflammatory response. In this study we have investigated the capacity of a range of different DBM preparations, and ECl dilutions, to induce the production of three pro-inflammatory cytokines, interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumour necrosis factor alpha (TNF-alpha) from human peripheral blood mononuclear cells (PBMNCs). The levels of EtOx and ECl in EtOx terminally sterilised DBM and mineralised bone grafts were measured by gas chromatography. It was found that the only factor capable of rendering DBM pro-inflammatory was the presence of small (<20 micrometre diameter) DBM particles. No other processing or sterilisation technique resulted in the DBM becoming pro-inflammatory. Although it was also found that DBM, when EtOx-sterilised, retained more ECI than mineralised bone grafts following a standard EtOx sterilisation protocol, ECl did not provoke an inflammatory response in vitro at levels up to and including those which are cytotoxic to PBMNCs.

Processing of whole femoral head allografts: a method for improving clinical efficacy and safety.

Femoral heads removed during primary hip replacement surgery are widely utilised as a source of allograft bone. Despite evidence that processing these grafts to remove blood and marrow elements improves both the clinical performance and safety of these allografts, many are transplanted without any processing being applied at all. The goal of this study was to investigate the efficiency of an allograft processing protocol which incorporates pasteurisation, (3 h, 56-60 degrees C) centrifugation, (1850g, 2 x 15 min, 40 degrees C) sonication, and repeated washing in warm (56-60 degrees C, 19 h) distilled, sterile water to remove blood and marrow elements from the graft. The protocol also involves applying heat treatment to the grafts which has been demonstrated to inactivate many pathogenic viruses. Following the processing procedure, the grafts are lyophilised and sterilised with ethylene oxide gas. The amount and rate of removal of 4 different components of blood and marrow from 6 whole femoral head allografts were measured. These were lipid, soluble protein, elastase and chloride ions. Lipid removal was assessed gravimetrically by solvent extraction of dried samples, soluble protein by the Bradford assay, elastase by radioimmunoassay and choride ion content by a modified commercially available colorimetric assay. Removing lipid from grafts has been shown to increase the rate of incorporation when the graft is used clinically. Elastase was studied as a marker of leukocyte removal, as evidence suggests the majority of potentially infective transmissible spongiform encephalopathy (TSE) activity resides in a sub-population of leukocytes. Soluble protein was studied as a marker of plasma removal, as a smaller amount of TSE infectivity resides here. Chloride removal was measured as this is a necessary pre-requisite to terminal sterilisation with ethylene oxide. The results showed that the protocol removed 74.5% (range: 68.0-90.8) of the lipid content, 96.4% (range: 94.8-98.4) of the soluble protein content, 97.7% (range: 97.1-100) of the elastase content and 98.8% (range: 98.0-99.2) of the chloride ion content. We have shown that processing designed to improve the clinical efficiency and safety of bone allografts can be accomplished without compromising the structural and biological properties of the graft.

Assessment of functional ability in patients with scleroderma: a proposed new disability assessment instrument.

OBJECTIVE: To develop a simple assessment of functional ability in patients with scleroderma and to examine its reliability both as a patient self-administered instrument and when administered by a trained observer. METHODS: An 11 item, 4 grade, functional assessment questionnaire was developed after extensive consultation with patients, physiotherapists (PT), and occupational therapists (OT) with the aim of including all functional areas of relevance. The instrument was self-administered by patients after an interval of 7 days. In the interval, the patients were assessed using the same instrument by direct observation from both a PT and an OT. Forty-seven patients with scleroderma, of varying severity, were recruited from 2 centers. Results were similar for both centers and data were pooled for analysis. RESULTS: Agreement between the patients' first and 2nd assessment was good for all questions (estimated kappas 0.69 to 0.94) with no evidence of an order effect. Agreement was also good between therapists (estimated kappas 0.47 to 0.81). There was poor agreement between patients and therapists, with patients rating their disability substantially higher compared to the standardized therapist assessment. CONCLUSION: This assessment schedule has high face and content validity and has excellent reliability both between trained therapists and within patients over a short time period. Its administration either as a self-report or by a therapist depends, in part, on the type of investigation undertaken.