Assessment of a closed wash system developed for processing living donor femoral heads.

NHS Blood and Transplant Tissue and Eye Services (TES) and Scottish National Blood Transfusion Services Tissues and Cells Directorate (TCD) currently bank whole, frozen femoral head bone from living donors who are undergoing primary hip replacement surgery. When required, the bone is issued to a surgeon still frozen on dry ice (- 79 °C). Consequently, the femoral head bone is not processed, is not sterilised and at the time of issue, it contains donor blood, bone marrow and associated cells. We have previously shown that, cut, shaped and washed bone from deceased donors can be processed to remove up to 99.9% of blood, bone marrow and associated cells (Eagle et al. 2015). However, cut and shaped bone is not suitable for some orthopaedic procedures and some orthopaedic surgeons do not wish to use irradiated bone; therefore in this report, a method has been developed in which whole femoral heads can be washed to remove donor blood and bone marrow components. Processing results in excess of 99% bone marrow component removal-soluble protein, haemoglobin and DNA; the procedure is performed inside a closed system, thereby eliminating the need for terminal sterilisation by irradiation. In addition, uniaxial testing demonstrated no difference in compressive strength between washed and unwashed bone. We suggest that this washed bone may be capable of improving incorporation after grafting without disturbing biomechanical properties of the graft.

Accurate and semi-automated analysis of bacterial association with mammalian cells.

To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria.

Disuse rescues the age-impaired adaptive response to external loading in mice.

UNLABELLED: We aimed to determine whether aged bone's diminished response to mechanical loading could be rescued by modulating habitual activity. By reducing background loading, aged bone's response to loading increased to a level no different to young mice. This suggests, given the right stimulus, that ageing bone can respond to mechanical loading. INTRODUCTION: Age-related decline in bone mass has been suggested to represent an impaired ability of bone to adapt to its mechanical environment. In young mice, the tibia's response to external mechanical loading has been shown to increase when habitual activity is reduced by sciatic neurectomy. Here we investigate if neurectomy can rescue bone's response to loading in old mice. METHODS: The effect of tibial disuse, induced by unilateral sciatic neurectomy (SN), on the adaptive response to a single peak magnitude of dynamic load-engendered mechanical strain was assessed in 19-month-old (aged) mice. In a second experiment, a range of peak loads was used to assess the load magnitude-related effects of loading on a background of disuse in young adult and aged mice. Bone architecture was analysed using micro-computed tomography (µCT) and dynamic histomorphometry. RESULTS: In the first experiment, SN in aged mice was associated with a significant periosteal osteogenic response to loading not observed in sham-operated mice (7.98 ± 1.7 vs 1.02 ± 2.2 % increase in periosteally enclosed area, p < 0.05). In the second experiment, SN abrogated the expected age-related difference in the bones' osteogenic response to peak strain magnitude (p > 0.05). CONCLUSIONS: These data suggest that bones' age-related decline in osteogenic responsiveness to loading does not originate in bone cells to either assess, or appropriately respond to strain, but rather is likely to be due to inhibitory "averaging" effects derived from the habitual strains to which the bone is already adapted. If such "strain averaging" is applicable to humans, it suggests that gentle exercise may degrade the beneficially osteogenic effects of short periods of more vigorous activity.

Collection, processing and testing of bone, corneas, umbilical cord blood and haematopoietic stem cells by European Blood Alliance members.

BACKGROUND AND OBJECTIVES: A questionnaire study was carried out in collaboration with the European Blood Alliance (EBA) Tissues and Cells (T&C) working group. The aim was to assess the level of involvement and commonality of processes on the procurement, testing and storage of bone, corneas, umbilical cord blood (UCB) and haematopoietic stem cells (HSC) in order to identify different practices and to explore whether recommendations can be made for harmonization. MATERIALS AND METHODS: An online questionnaire was used for data collection in 2011, and 43 replies were received covering 71 product answers from 13 countries. RESULTS AND CONCLUSIONS: Estimated percentages of tissue and cell banking covered by EBA member blood banks as a proportion of all collections of each individual country varied markedly. There were also major differences in the amounts of products collected and discarded and in proportions tissues provided for grafting. However, discarding of certain collections also reflects the practice of increasing the likelihood of the very best units being used for transplantation. Harmonization of possible practices should focus on matching supply with demand and on identifying the most efficient operators. This could allow for the development of practices for minimizing unnecessary collections.

The cyclooxygenase-2 selective inhibitor NS-398 does not influence trabecular or cortical bone gain resulting from repeated mechanical loading in female mice.

UNLABELLED: A single injection of the cyclooxygenase-2 (COX-2) selective inhibitor NS-398 reduces bone's osteogenic response to a single period of mechanical loading in female rats, while women taking COX-2 selective inhibitors do not have lower bone mass. We show that daily NS-398 injection does not influence bone gain from repeated loading in female mice. INTRODUCTION: Prostaglandins are mediators of bone cells' early response to mechanical stimulation. COX-2 expression is up-regulated by exposure of these cells to mechanical strain or fluid flow, and the osteogenic response to a single loading period is reduced by COX-2 inhibition. This study determined, in female mice in vivo, the effect of longer term COX-2 inhibition on adaptive (re)modelling of cortical and trabecular bone in response to repeated loading. METHODS: Nineteen-week-old female C57BL/6 mice were injected with vehicle or NS-398 (5 mg/kg/day) 5 days a week for 2 weeks. On three alternate days each week, the right tibiae/fibulae were axially loaded [40 cycles (7 min)/day] three hours after injection. Left limbs acted as internal controls. Changes in three-dimensional bone architecture were analysed by high-resolution micro-computed tomography. RESULTS: In control limbs NS-398 was associated with reduced trabecular number but had no influence on cortical bone. In loaded limbs trabecular thickness and cortical periosteally enclosed volume increased. NS-398 showed no effect on this response. CONCLUSION: Pharmacological inhibition of COX-2 by NS-398 does not affect trabecular or cortical bone's response to repeated mechanical loading in female mice and thus would not be expected to impair the functional adaptation of bone to physical activity in women.

The organization of tissue banking in Scotland.

Tissue banking in Scotland has developed significantly over the past 20 years or so. The range of issues procured has increased and so have their numbers. Initially, bone from live donors was the only tissue banked; later, tissues from multiorgan donors were procured; this was finally followed by the collection of tissues from donors following cardiac death. Bones, tendons, heart valves and skin are the main tissue types collected, stored and issued for clinical use. Much of our activity is based on identification of donors in two major accident and emergency departments followed by retrievals that take place in a dedicated mortuary by fully-trained staff. Tissues are released according to clinical need for Scottish patients and beyond. All of the tissue banking activity in Scotland takes place within the Scottish National Blood Transfusion Service, which is the preferred provider of tissues for Scottish patients. There is very close cooperation between our teams, the transplant teams and other clinical colleagues, including pathologists and anatomical technicians. The achievements in issue banking in Scotland are outlined along with the main clinical indications of the tissue procured. Diversification is now taking place into cellular therapy with the establishment of an islet processing programme and cell culturing techniques. The future is very exciting.

Estimating the potential of tissue donation in Scotland.

The Scottish National Blood Transfusion Service became the preferred provider of tissues in 2000. At that time, the only tissues procured were heart valves and some tendons from multiorgan donors. These sources and the range of tissues were not enough to provide for the clinical needs of Scottish patients. A systematic review was undertaken using the International Classification of Disease codes to assess what the potential pool of tissue donors would be by region, hospital and hospital department. Such data would enable us to focus our limited resources to maximize the yield of tissues to meet clinical demand. The data from this study were validated by a case-note audit in one hospital in Edinburgh. It was shown that the maximum tissue donor potential of approximately 48.5 per million population was in the central belt of Scotland in hospitals with large emergency medicine departments.

Prevention and control of childhood asthma and allergy in the EU from the public health point of view: Polish Presidency of the European Union.

The leading priority for the Polish Presidency of the Council of the European Union was to reduce health inequalities across European societies, and, within its framework, prevention and control of respiratory diseases in children. This very important paper contain proposal of international cooperation on the prevention, early detection and monitoring of asthma and allergic diseases in childhood which will be undertaken by the EU member countries as a result of EU conclusion developed during the Polish Presidency of the Council of the European Union. This will result in collaboration in the field of chronic diseases, particularly respiratory diseases, together with the activity of the network of national institutions and NGOs in this area. Paper also contains extensive analysis of the socio-economic, political, epidemiological, technological and medical factors affecting the prevention and control of childhood asthma and allergy presented during Experts presidential conference organized in Warsaw-Ossa 21-22 September 2011.

Estrogen receptors α and ß have different gender-dependent effects on the adaptive responses to load bearing in cancellous and cortical bone.

To determine the effect of estrogen receptors (ER) α and ß on bones' adaptive response to loading, we subjected the right tibiae of mice lacking ERα or ERß activity to either axial loading or to disuse. Adaptive changes in architecture were assessed by comparing differences between the right (treated) and left (control) tibiae in these genotypes as assessed by microcomputed tomography. In female ERα(-/-) mice, the net-osteogenic response to loading was lower in cortical bone compared with their wild-type littermates (11.2 vs. 20.9% in ERα(+/+)), but it was higher in both cortical and cancellous bone of male ERα(-/-) mice (cortical 20.0 vs. 4.6% in ERα(+/+); cancellous 30.0 vs. 5.3% in ERα(+/+), P < 0.05). In ERß(-/-) male and female mice, the net-osteogenic response to loading was higher in cortical bone (males 10.9 vs. 3.9% in ERß(+/+); females 18.5 vs. 15.8% in ERß(+/+), P < 0.05) but no different from controls in cancellous bone. The bone loss in response to disuse was less in cancellous bone of ERα(-/-) mice than in controls (-15.9 vs. -21.3%, respectively, P < 0.05) but no different at any other site or between any other groups. Our conclusion is that functional ERα enhances the net-osteogenic response to loading in cortical but not cancellous bone in female mice but reduces it in males. ERß decreases the response to loading in cortical bone of males and females but has no effect in cancellous bone. Bone loss due to disuse in cortical bone is unaffected by ER status, but in cancellous bone, functional ERα contributes to greater disuse-related bone loss.

Intra-operative washing of morcellised bone allograft with pulse lavage: how effective is it in reducing blood and marrow content?

The use of unprocessed bone carries a risk of transmission of blood borne diseases. Although models of infectivity are unproven, a theoretical risk of transmission of variant Creutzfeld-Jakob Disease, a human prion disease, exists as probable blood borne transmission has been reported in three cases. The aim of our study was to determine the effectiveness of standard operating theatre pulse lavage in removing protein, fat and double stranded Deoxyribonucleic acid (dsDNA) from morcellised bone allograft. Twelve donated femoral heads were divided into halves and milled into bone chips. One half of the bone chips were washed with pulse lavage, whereas, the other half acted as control. In order to determine the amount of protein, fat and dsDNA present in the washed and unwashed samples, a validated multistep washing protocol was used. Using the validated technique, simple intra-operative washing of morcellised unprocessed bone allograft removed a significant amount of the protein (70.5%, range: 39.5-85%), fat (95.2%, range: 87.8-98.8%) and DNA (68.4%, range: 31.4-93.1%) content. Intra-operative washing of morcellised bone allograft with pulse lavage may thereby reduce the theoretical risk of prion and other blood borne disease transmission. Combined with the known improved mechanical characteristics of washed allograft, we would recommend pulse lavage as a routine part of bone allograft preparation.

Hygiene behaviour of adolescents in the Pacific: associations with socio-demographic, health behaviour and school environment.

This study presents findings on health-related hygiene behaviours of 9,013 adolescents from Vanuatu, Tonga and Pohnpei in the Federal States of Micronesia as part of the Health Behaviour of Pacific Youth Life surveys. We examined the prevalence of and relationship between tooth brushing, hand washing before eating, hand washing after toileting and a range of psychosocial factors such as sociodemographic charac-teristics, health behaviour and school affiliation. The results showed that patterns of prevalence and relationships were consistent for all countries, with weaker associations observed in Pohnpei. In general, adolescents reported moderate levels of optimal hygiene practice. Girls reported significantly more frequent optimal hygiene practice than boys. Logistic regression analysis confirmed that frequent hygiene behaviours were significantly associated with gender, parental occupations and high levels of school affiliation. These findings suggest the need to consider psycho-social and economic factors when examining potential influences on hygiene behaviour to ensure effective interventions.

Comparison of prevalence rates of microbiological markers between bone/tissue donations and new blood donors in Scotland.

BACKGROUND AND OBJECTIVES: Blood-borne virus prevalence rates of samples accompanying tissue donors are not widely available. This article compares the rates in Scottish bone/tissue donors with those of new blood donors for the 7-year period, 1998-2004. MATERIALS AND METHODS: Data were collated from existing internal reports. Age distributions of the donor populations were obtained by extracting information from existing computer databases. RESULTS: Scottish bone/tissue donors were found to have a fourfold higher prevalence for hepatitis B virus (HBV), a 1.6-fold higher prevalence for hepatitis C virus (HCV), an 11-fold higher prevalence for human T-cell lymphotropic virus (HTLV) and a 34-fold higher prevalence for syphilis compared with new blood donors. Excluding confirmed positives, the repeat-reactive rates for bone/tissue donors were similar to those of new blood donors. CONCLUSIONS: The data demonstrated that the prevalence of blood-borne viruses in Scottish bone/tissue donors is higher than in new blood donors. We believe that the different age profiles of the two donor populations plays a significant role.

Processing of whole femoral head allografts: validation methodology for the reliable removal of nucleated cells, lipid and soluble proteins using a multi-step washing procedure.

Following the major outbreak of BSE in the United Kingdom in the mid 1980's and the first reported cases of the human disease equivalent, vCJD, in 1996 a number of measures were taken to reduce the risk of secondary transmission of vCJD by blood or surgical instruments. Two cases of probable transmission of vCJD by blood components have now been reported. The Department of Health has recently undertaken a vCJD risk analysis for bone. Although significant uncertainties still exist in the assumptions on which the assessment is based, a few important recommendations were made. These include a recommendation that bone should be processed to remove blood and marrow and that pooling of donations is never advantageous. This study aimed to establish a method of processing whole femoral heads using disposable equipment, with the purpose of removing as much of the marrow components as possible. We are able to remove 98.2% (range 94.5-99.9%) of the nucleated cells and 98.7% (range 97.8-99.4%) of the soluble protein content from within an intact femoral head. This work confirms that the majority of blood and marrow components can be removed without the need for morselising the bone prior to washing.

Clinical effectiveness of processed and unprocessed bone.

Bone allografts have been used clinically for a number of years. Understanding the biology of bone healing and the impact that bone banking has on this helps to improve the methodologies used in increasing the quality and safety of banked bone. Banked bone in its various forms has been used in a variety of surgical procedures, and although there is no doubt that it is clinically effective, most of the studies have been retrospective and non-randomized. The review attempts to summarize some of the data in this area and highlights some of the difficulties encountered in such work. Although there is no doubt that bone banking is nowadays better controlled, there are ever-increasing pressures to produce bone that is as safe as possible with the least impact on its effectiveness. This can only be achieved if the requirements of the providers and users of bone are better understood.

Global priority setting for Cochrane systematic reviews of health promotion and public health research.

BACKGROUND: Systematic reviews of health promotion and public health interventions are increasingly being conducted to assist public policy decision making. Many intra-country initiatives have been established to conduct systematic reviews in their relevant public health areas. The Cochrane Collaboration, an international organisation established to conduct and publish systematic reviews of healthcare interventions, is committed to high quality reviews that are regularly updated, published electronically, and meeting the needs of the consumers. AIMS: To identify global priorities for Cochrane systematic reviews of public health topics. METHODS: Systematic reviews of public health interventions were identified and mapped against global health risks. Global health organisations were engaged and nominated policy-urgent titles, evidence based selection criteria were applied to set priorities. RESULTS: 26 priority systematic review titles were identified, addressing interventions such as community building activities, pre-natal and early infancy psychosocial outcomes, and improving the nutrition status of refugee and displaced populations. DISCUSSION: The 26 priority titles provide an opportunity for potential reviewers and indeed, the Cochrane Collaboration as a whole, to address the previously unmet needs of global health policy and research agencies.

Environmental influences on helminthiasis and nutritional status among Pacific schoolchildren.

This paper describes a study undertaken to: (1) determine the prevalence of Ascaris lumbricoides, Trichuris trichiura and hookworm infections and nutritional status among Pacific Island school children; (2) identify factors influencing helminthiasis; (3) identify interventions to improve school health. A total of 3,683 children aged 5-12 years attending 27 primary schools in 13 Pacific Island countries were surveyed along with school environmental data. Stool samples were collected from 1996 children (54.2%) and analysed for ova and helminths. Total prevalence of helminthiasis was 32.8%. Anaemia prevalence was 12.4%. Children with helminthiasis and anaemia were found to be 8.7 times more likely to be stunted and 4.3 times more likely to be underweight than non-anaemic and non-infected children. Four significant environmental influences on helminthiasis were identified: (1) an inadequate water supply; (2); availability of a school canteen; (3) regular water/sanitation maintenance regimes; and (4) overcrowded classrooms. Helminthiasis was found to be strongly associated with anaemia, stunting and underweight and environmental influences identified. Although mass anti-helminthic drug administrations (MDA) have been taking place, reinfection is common as drug therapy alone is not enough. Programme effectiveness depends upon upgrading school environments to include an adequate water supply, controlled food preparation/provision, well-maintained water/sanitation facilities and class sizes of 30 students or less.

Hepatitis B vaccinees may be mistaken for confirmed hepatitis B surface antigen-positive blood donors.

BACKGROUND AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) test sensitivities have gradually increased, and neutralizable weak HBsAg-positive donations, with no other hepatitis B virus (HBV) markers, have occasionally been found in our donor population. On investigation, these donors have admitted to receiving hepatitis B vaccine up to 5 days previously. A study was therefore initiated to monitor HBsAg reactivity amongst volunteers after receiving their first dose of hepatitis B vaccine. MATERIALS AND METHODS: Eight volunteers were tested using three HBsAg assays (Abbott Auszyme, Ortho HBsAg-3 and Abbott/Murex GE34/36) on days 0, 3, 5, 7 and 10 after administration of hepatitis B vaccine. RESULTS: Two HBsAg tests (Abbott Auszyme and Ortho HBsAg-3) did not detect HBsAg reactivity amongst the volunteers, although the Abbott Auszyme test results reached 70-80% of the manufacturer's cut-off at day 3 in two volunteers. The most recently launched assay (Abbott/Murex GE 34/36) detected seven (87%) of the eight volunteers as HBsAg reactive on day 3, and two (25%) volunteers were still reactive on day 5. CONCLUSION: The Abbott/Murex GE 34/36 assay demonstrated HBsAg reactivity in most volunteers on day 3 and in some on day 5 after vaccination. It is therefore recommended that individuals who have recently been vaccinated with hepatitis B be deferred from blood donation for at least 7 days.

Utilisation of bone allograft by orthopaedic surgeons in Scotland.

The use of bone allograft in orthopaedic surgery has been predicted to increase, particularly in joint revision surgery. This has led to a potential problem with supply. Questionnaires were distributed to all 146 Consultant Orthopaedic surgeons working in Scotland in 2000. They were asked to indicate their current usage of bone and tissue allograft, any problems encountered with supply and if alternatives to allograft, such as processed bone, might be used. The questions asked were very similar to those asked in a previous study in 1995 to enable comparisons to be made. Replies were received from 125 Consultants (87%) of whom 93 reported using bone allograft. Forty-one consultants (46%) predicted an increase in their requirement for bone allograft, and 23 (26%) felt they could currently use more bone if this was available. Sixty percent of surgeons would consider using processed bone as an alternative. In comparison with figures from 1995, an increasing number of surgeons are prepared to use processed bone as an alternative to fresh frozen allograft.