BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

Optical magnetic imaging of living cells.

Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (for example, magnetic resonance imaging), or entail operating conditions that preclude application to living biological samples while providing submicrometre resolution (for example, scanning superconducting quantum interference device microscopy, electron holography and magnetic resonance force microscopy). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nanometres), using an optically detected magnetic field imaging array consisting of a nanometre-scale layer of nitrogen-vacancy colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the nitrogen-vacancy quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria. We also spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field microscopy allows parallel optical and magnetic imaging of multiple cells in a population with submicrometre resolution and a field of view in excess of 100 micrometres. Scanning electron microscope images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. Our results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks.

A feasability study of color flow doppler vectorization for automated blood flow monitoring.

An ongoing issue in vascular medicine is the measure of the blood flow. Catheterization remains the gold standard measurement method, although non-invasive techniques are an area of intense research. We hereby present a computational method for real-time measurement of the blood flow from color flow Doppler data, with a focus on simplicity and monitoring instead of diagnostics. We then analyze the performance of a proof-of-principle software implementation. We imagined a geometrical model geared towards blood flow computation from a color flow Doppler signal, and we developed a software implementation requiring only a standard diagnostic ultrasound device. Detection performance was evaluated by computing flow and its determinants (flow speed, vessel area, and ultrasound beam angle of incidence) on purposely designed synthetic and phantom-based arterial flow simulations. Flow was appropriately detected in all cases. Errors on synthetic images ranged from nonexistent to substantial depending on experimental conditions. Mean errors on measurements from our phantom flow simulation ranged from 1.2 to 40.2% for angle estimation, and from 3.2 to 25.3% for real-time flow estimation. This study is a proof of concept showing that accurate measurement can be done from automated color flow Doppler signal extraction, providing the industry the opportunity for further optimization using raw ultrasound data.

BSHI Guideline: HLA matching and donor selection for haematopoietic progenitor cell transplantation.

A review of the British Society for Histocompatibility and Immunogenetics (BSHI) "Guideline for selection and HLA matching of related, adult unrelated donors and umbilical cord units for haematopoietic progenitor cell transplantation" was undertaken by a BSHI appointed writing committee. Literature searches were performed, and the data extracted were presented as recommendations according to the GRADE nomenclature.

Autonomous and self-sustained circadian oscillators displayed in human islet cells.

AIMS/HYPOTHESIS: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. METHODS: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. RESULTS: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48 h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. CONCLUSIONS/INTERPRETATION: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell cultures.

Dressed-state resonant coupling between bright and dark spins in diamond.

Under ambient conditions, spin impurities in solid-state systems are found in thermally mixed states and are optically "dark"; i.e., the spin states cannot be optically controlled. Nitrogen-vacancy (NV) centers in diamond are an exception in that the electronic spin states are "bright"; i.e., they can be polarized by optical pumping, coherently manipulated with spin-resonance techniques, and read out optically, all at room temperature. Here we demonstrate a scheme to resonantly couple bright NV electronic spins to dark substitutional-nitrogen (P1) electronic spins by dressing their spin states with oscillating magnetic fields. This resonant coupling mechanism can be used to transfer spin polarization from NV spins to nearby dark spins and could be used to cool a mesoscopic bath of dark spins to near-zero temperature, thus providing a resource for quantum information and sensing, and aiding studies of quantum effects in many-body spin systems.

3-D PSF fitting for fluorescence microscopy: implementation and localization application.

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three-dimensional (3-D) point spread function models for the wide field microscope. We introduce a least-squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ.

Composition and function of the Galapagos penguin gut microbiome vary with age, location, and a putative bacterial pathogen.

Microbial colonization plays a direct role in host health. Understanding the ecology of the resident microbial community for a given host species is thus an important step for detecting population vulnerabilities like disease. However, the idea of integrating microbiome research into conservation is still relatively new, and wild birds have received less attention in this field than mammals or domesticated animals. Here we examine the composition and function of the gut microbiome of the endangered Galapagos penguin (Spheniscus mendiculus) with the goals of characterizing the normal microbial community and resistome, identifying likely pathogens, and testing hypotheses of structuring forces for this community based on demographics, location, and infection status. We collected fecal samples from wild penguins in 2018 and performed 16S rRNA gene sequencing and whole genome sequencing (WGS) on extracted DNA. 16S sequencing revealed that the bacterial phyla Fusobacteria, Epsilonbacteraeota, Firmicutes, and Proteobacteria dominate the community. Functional pathways were computed from WGS data, showing genetic functional potential primarily focused on metabolism-amino acid metabolism, carbohydrate metabolism, and energy metabolism are the most well-represented functional groups. WGS samples were each screened for antimicrobial resistance, characterizing a resistome made up of nine antibiotic resistance genes. Samples were screened for potential enteric pathogens using virulence factors as indicators; Clostridium perfringens was revealed as a likely pathogen. Overall, three factors appear to be shaping the alpha and beta diversity of the microbial community: penguin developmental stage, sampling location, and C. perfringens. We found that juvenile penguins have significantly lower alpha diversity than adults based on three metrics, as well as significantly different beta diversity. Location effects are minimal, but one site has significantly lower Shannon diversity than the other primary sites. Finally, when samples were grouped by C. perfringens virulence factors, we found dramatic changes in beta diversity based on operational taxonomic units, protein families, and functional pathways. This study provides a baseline microbiome for an endangered species, implicates both penguin age and the presence of a potential bacterial pathogen as primary factors associated with microbial community variance, and reveals widespread antibiotic resistance genes across the population.

Flow measurements in sewers based on image analysis: automatic flow velocity algorithm.

Discharges of combined sewer overflows (CSOs) and stormwater are recognized as an important source of environmental contamination. However, the harsh sewer environment and particular hydraulic conditions during rain events reduce the reliability of traditional flow measurement probes. An in situ system for sewer water flow monitoring based on video images was evaluated. Algorithms to determine water velocities were developed based on image-processing techniques. The image-based water velocity algorithm identifies surface features and measures their positions with respect to real world coordinates. A web-based user interface and a three-tier system architecture enable remote configuration of the cameras and the image-processing algorithms in order to calculate automatically flow velocity on-line. Results of investigations conducted in a CSO are presented. The system was found to measure reliably water velocities, thereby providing the means to understand particular hydraulic behaviors.

Vision-based system for the control and measurement of wastewater flow rate in sewer systems.

Combined sewer overflows and stormwater discharges represent an important source of contamination to the environment. However, the harsh environment inside sewers and particular hydraulic conditions during rain events reduce the reliability of traditional flow measurement probes. In the following, we present and evaluate an in situ system for the monitoring of water flow in sewers based on video images. This paper focuses on the measurement of the water level based on image-processing techniques. The developed image-based water level algorithms identify the wall/water interface from sewer images and measure its position with respect to real world coordinates. A web-based user interface and a 3-tier system architecture enable the remote configuration of the cameras and the image-processing algorithms. Images acquired and processed by our system were found to reliably measure water levels and thereby to provide crucial information leading to better understand particular hydraulic behaviors. In terms of robustness and accuracy, the water level algorithm provided equal or better results compared to traditional water level probes in three different in situ configurations.

Coupled tomography and distinct-element-method approach to exploring the granular media microstructure in a jamming hourglass.

We describe an approach for exploring microscopic properties of granular media that couples x-ray microtomography and distinct-element-method (DEM) simulations through image analysis. We illustrate it via the study of the intriguing phenomenon of instant arching in an hourglass (in our case a cylinder filled with a polydisperse mixture of glass beads that has a small circular shutter in the bottom). X-ray tomography provides three-dimensional snapshots of the microscopic conditions of the system both prior to opening the shutter, and thereafter, once jamming is completed. The process time in between is bridged using DEM simulation, which settles to positions in remarkably good agreement with the x-ray images. Specifically designed image analysis procedures accurately extract the geometrical information, i.e., the positions and sizes of the beads, from the raw x-ray tomographs, and compress the data representation from initially 5 gigabytes to a few tens of kilobytes per tomograph. The scope of the approach is explored through a sensitivity analysis to input data perturbations in both bead sizes and positions. We establish that accuracy of size--much more than position--estimates is critical, thus explaining the difficulty in considering a mixture of beads of different sizes. We further point to limits in the replication ability of granular flows away from equilibrium; i.e., the difficulty of numerically reproducing chaotic motion.

Na(v)1.7 and Na(v)1.3 are the only tetrodotoxin-sensitive sodium channels expressed by the adult guinea pig enteric nervous system.

The types of sodium channels that are expressed by neurons shape the rising phase of action potentials and influence patterns of action potential discharge. With regard to the enteric nervous system (ENS), there is uncertainty about which channels are expressed, and in particular it is unknown whether Na(v)1.7 is present. We designed specific probes for the guinea pig Na(v)1.7 alpha subunit as well as for the other tetrodotoxin (TTX)-sensitive alpha subunits (Na(v)1.1, Na(v)1.2, Na(v)1.3, and Na(v)1.6) in order to perform in situ hybridization (ISH) histochemistry on guinea pig myenteric ganglia. We established that only Na(v)1.7 mRNA and Na(v)1.3 mRNA are expressed in these ganglia. The ISH signal for Na(v)1.7 transcripts was found in seemingly all the myenteric neurons. The expression of the Na(v)1.3 alpha subunit was confirmed by immunohistochemistry in a large proportion (62%) of the myenteric neuron population. This population included enteric sensory neurons. Na(v)1.6 immunoreactivity, absent from myenteric neurons, was detected in glial cells only when a high anti-Na(v)1.6 antibody concentration was used. This suggests that the Na(v)1.6 alpha subunit and mRNA are present only at low levels, which is consistent with the fact that no Na(v)1.6 mRNA could be detected in the ENS by ISH. The fact that adult myenteric neurons are endowed with only two TTX-sensitive alpha subunits, namely, Na(v)1.3 and Na(v)1.7, emphasizes the singularity of the ENS. Both these subunits, known to have slow-inactivation kinetics, are well adapted for generating action potentials from slow excitatory postsynaptic potentials, a mode of synaptic transmission that applies to all ENS neuron types.

Evaluating team-based inter-professional advanced life support training in intensive care-a prospective observational study.

Recent focus on national standards within Australian hospitals has prompted a focus on the training of our staff in advanced life support (ALS). Research in critical care nursing has questioned the traditional annual certification of ALS competence as the best method of delivering this training. Simulation and team-based training may provide better ALS education to intensive care unit (ICU) staff. Our new inter-professional team-based advanced life support program involved ICU staff in a large private metropolitan ICU. A prospective observational study using three standardised questionnaires and two multiple choice questionnaire assessments was conducted. Ninety-nine staff demonstrated a 17.8% (95% confidence interval 4.2-31, P=0.01) increase in overall ICU nursing attendance at training sessions. Questionnaire response rates were 93 (94%), 99 (100%) and 60 (61%) respectively; 51 (52%) staff returned all three. Criteria were assessed by scores from 0 to 10. Nurses reported improved satisfaction with the education program (9.4 to 7.1, P <0.001), as well as improvement in role understanding (8.7 and 9.1 versus 7.9 and 8.2, P <0.001) and confidence (8.4 and 8.8 versus 7.4 and 7.8, P <0.001) during ALS provision (outside ICU and inside ICU) following the course when compared to before the program. Doctors' only statistically significant improvement was in their confidence in ALS provision outside ICU (8.7 versus 8.1, P=0.04). The new program cost approximately an extra $16,500 in nursing salaries. We concluded that team-based, inter-professional ALS training produced statistically significant improvements in nursing attendance, satisfaction with ALS education, confidence and role understanding compared to traditional ALS training.

Marrow transplants from matched unrelated donors for aplastic anaemia using alemtuzumab, fludarabine and cyclophosphamide based conditioning.

Graft failure, regimen-related toxicity and graft-versus-host disease (GVHD) are the critical barriers to unrelated donor transplants for aplastic anaemia (AA). We investigated the use of a novel conditioning regimen consisting of alemtuzumab (humanized CD52 antibody), fludarabine and cyclophosphamide in seven patients with AA, who underwent bone marrow transplant procedure using matched unrelated donors. The aetiology of AA was acquired (n=3), Fanconi's (n=3) and congenital (n=1). Median age was 13 years (range 8-35). All the donors were fully matched for HLA class I and II antigens using high-resolution typing. All the patients engrafted at a median of 18 days (range 13-35). Two patients died of transplant-related complications: one of adenovirus disease and the other developed extensive chronic GVHD of skin followed by cytomegalovirus (CMV) disease. Three patients developed Grade II acute GVHD disease (GVHD); none had Grade III-IV acute GVHD. Of the six evaluable patients, only one developed chronic GVHD. We conclude that this conditioning regimen for unrelated donor transplants for AA is sufficiently immunosuppressive to allow stable engraftment and appears to have a favourable impact on the incidence and severity of GVHD, warranting further investigation.

Covalent linking of haptens, proteins and nucleic acids to a modified polystyrene support.

Biological molecules of various molecular weights were successfully linked to polystyrene microtitration plates bearing carbonyl and hydroxyl reactive groups. Cross-linking of proteins, nucleic acids and haptens was carried out with water soluble carbodiimide. The covalent nature of the binding reaction was demonstrated by the immunodetection of a hapten linked to the surface through a disulphide spacer arm and its subsequent release by cleavage of the bridge. The amount of protein fixed per surface unit could be correlated to molecular weight. Nanograms of biotinylated nucleic acids and synthetic polynucleotides could also be retained on the solid support.

UHG crossmatching. A comparison with PCR-SSO typing in the selection of HLA-DPB1-compatible bone marrow donors.

The technique of universal heteroduplex generator (UHG) crossmatching has been developed to permit comparison of HLA-DPB1 alleles between two or more individuals. It offers a rapid and simple method of screening prospective bone marrow donors for HLA-DPB1 compatibility with the recipient. We present the nucleotide sequence and describe the method of construction of the DPB1 UHG. To test its effectiveness, 56 patient-bone marrow donor pairs previously HLA-DPB1-typed by PCR-SSO probing, were tested by UHG crossmatching. In 52/56 (93%) pairs there was concordance between PCR-SSO typing and UHG crossmatching. All 32 pairs that were defined as mismatched by PCR-SSO typing were also mismatched by UHG crossmatching. We conclude that UHG crossmatching is a simple, sensitive, and cost effective method of HLA-DPB1 matching for the rapid selection of compatible bone marrow donors.

Reduction of postoperative mortality and morbidity with epidural or spinal anaesthesia: results from overview of randomised trials.

OBJECTIVES: To obtain reliable estimates of the effects of neuraxial blockade with epidural or spinal anaesthesia on postoperative morbidity and mortality. DESIGN: Systematic review of all trials with randomisation to intraoperative neuraxial blockade or not. STUDIES: 141 trials including 9559 patients for which data were available before 1 January 1997. Trials were eligible irrespective of their primary aims, concomitant use of general anaesthesia, publication status, or language. Trials were identified by extensive search methods, and substantial amounts of data were obtained or confirmed by correspondence with trialists. MAIN OUTCOME MEASURES: All cause mortality, deep vein thrombosis, pulmonary embolism, myocardial infarction, transfusion requirements, pneumonia, other infections, respiratory depression, and renal failure. RESULTS: Overall mortality was reduced by about a third in patients allocated to neuraxial blockade (103 deaths/4871 patients versus 144/4688 patients, odds ratio=0.70, 95% confidence interval 0.54 to 0.90, P=0. 006). Neuraxial blockade reduced the odds of deep vein thrombosis by 44%, pulmonary embolism by 55%, transfusion requirements by 50%, pneumonia by 39%, and respiratory depression by 59% (all P<0.001). There were also reductions in myocardial infarction and renal failure. Although there was limited power to assess subgroup effects, the proportional reductions in mortality did not clearly differ by surgical group, type of blockade (epidural or spinal), or in those trials in which neuraxial blockade was combined with general anaesthesia compared with trials in which neuraxial blockade was used alone. CONCLUSIONS: Neuraxial blockade reduces postoperative mortality and other serious complications. The size of some of these benefits remains uncertain, and further research is required to determine whether these effects are due solely to benefits of neuraxial blockade or partly to avoidance of general anaesthesia. Nevertheless, these findings support more widespread use of neuraxial blockade.

A comparison of the impact of hospital reform on medical subcultures in some Australian and New Zealand hospitals.

This article examines similarities and differences in the way that hospital staff in Australia and New Zealand are evaluating efforts to improve quality, clinical effectiveness and service integration, and to strengthen clinical accountability. We draw on data from a cross-national study of hospital staff in Australia and New Zealand. The results highlight the way in which respondents' views about reform are influenced by the interplay of two factors: the impact of respondents' occupational backgrounds (our findings point to differences in the profession-based subcultures of medicine, nursing and general management and the way that these are reflected in respondents' assessments of particular aspects of reform); and the way that the impact of professional subcultures may be mitigated by differences between the systems in which respondents were located, including differences between the programs of reform that have been pursued in each country. The implications of these findings are discussed.

HLA antibody-incompatible kidney transplantation between jehovah's witnesses--a case report.

Desensitization before HLA antibody-incompatible (HLAi) transplantation involves nonspecific apheresis of HLA antibodies. Clotting factors and albumin are also removed and have to be replaced. This makes transplantation difficult because it increases the risk of bleeding. Such risk is further compounded when certain blood products are refused on religious grounds. We present a case of successful HLAi transplantation in a Jehovah's Witness across a positive-flow cytometric HLA crossmatch from a live donor who was also a Jehovah's Witness. This was achieved by giving rituximab 1 month before transplantation and starting prednisolone, tacrolimus, and mycophenolate mofetil 10 days before surgery. In preparation, the patient also underwent 4 sessions of double-filtration plasma exchange each followed by low-dose intravenous immunoglobulin. The night before transplantation, the fibrinogen was low, requiring 2 pools of cryoprecipitate. The organ was retrieved through laparoscopic hand-assisted retroperitoneoscopic nephrectomy and transplanted into the recipient with no complications. In addition, the patient received basiliximab during surgery. Sixteen months after transplantation the serum creatinine was 70 µmol/L (0.79 mg/dL) and there were no rejection episodes. To our knowledge this is the world's first live-related kidney transplant across the HLAi barrier between 2 Jehovah's Witnesses. This case may allow further HLAi transplants to be carried out in Jehovah's Witnesses in the future around the world.

Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy.

Mechanical properties of the adventitia are largely determined by the organization of collagen fibers. Measurements on the waviness and orientation of collagen, particularly at the zero-stress state, are necessary to relate the structural organization of collagen to the mechanical response of the adventitia. Using the fluorescence collagen marker CNA38-OG488 and confocal laser scanning microscopy, we imaged collagen fibers in the adventitia of rabbit common carotid arteries ex vivo. The arteries were cut open along their longitudinal axes to get the zero-stress state. We used semi-manual and automatic techniques to measure parameters related to the waviness and orientation of fibers. Our results showed that the straightness parameter (defined as the ratio between the distances of endpoints of a fiber to its length) was distributed with a beta distribution (mean value 0.72, variance 0.028) and did not depend on the mean angle orientation of fibers. Local angular density distributions revealed four axially symmetric families of fibers with mean directions of 0°, 90°, 43° and -43°, with respect to the axial direction of the artery, and corresponding circular standard deviations of 40°, 47°, 37° and 37°. The distribution of local orientations was shifted to the circumferential direction when measured in arteries at the zero-load state (intact), as compared to arteries at the zero-stress state (cut-open). Information on collagen fiber waviness and orientation, such as obtained in this study, could be used to develop structural models of the adventitia, providing better means for analyzing and understanding the mechanical properties of vascular wall.