Shear Stress-Induced Total Blood Trauma in Multiple Species.

The common complications in heart failure patients with implanted ventricular assist devices (VADs) include hemolysis, thrombosis, and bleeding. These are linked to shear stress-induced trauma to erythrocytes, platelets, and von Willebrand factor (vWF). Novel device designs are being developed to reduce the blood trauma, which will need to undergo in vitro and in vivo preclinical testing in large animal models such as cattle, sheep, and pig. To fully understand the impact of device design and enable translation of preclinical results, it is important to identify any potential species-specific differences in the VAD-associated common complications. Therefore, the purpose of this study was to evaluate the effects of shear stress on cells and proteins in bovine, ovine, and porcine blood compared to human. Blood from different species was subjected to various shear rates (0-8000/s) using a rheometer. It was then analyzed for complete blood counts, hemolysis by the Harboe assay, platelet activation by flow cytometry, vWF structure by immunoblotting, and function by collagen binding activity ELISA (vWF : CBA). Overall, increasing shear rate caused increased total blood trauma in all tested species. This analysis revealed species-specific differences in shear-induced hemolysis, platelet activation, and vWF structure and function. Compared to human blood, porcine blood was the most resilient and showed less hemolysis, similar blood counts, but less platelet activation and less vWF damage in response to shear. Compared to human blood, sheared bovine blood showed less hemolysis, similar blood cell counts, greater platelet activation, and similar degradation of vWF structure, but less impact on its activity in response to shear. The shear-induced effect on ovine blood depended on whether the blood was collected via gravity at the abattoir or by venepuncture from live sheep. Overall, ovine abattoir blood was the least resilient in response to shear and bovine blood was the most similar to human blood. These results lay the foundations for developing blood trauma evaluation standards to enable the extrapolation of in vitro and in vivo animal data to predict safety and biocompatibility of blood-handling medical devices in humans. We advise using ovine venepuncture blood instead of ovine abattoir blood due to the greater overall damage in the latter. We propose using bovine blood for total blood damage in vitro device evaluation but multiple species could be used to create a full understanding of the complication risk profile of new devices. Further, this study highlights that choice of antibody clone for evaluating platelet activation in bovine blood can influence the interpretation of results from different studies.

Large Sequence Diversity within the Biosynthesis Locus and Common Biochemical Features of Campylobacter coli Lipooligosaccharides.

UNLABELLED: Despite the importance of lipooligosaccharides (LOSs) in the pathogenicity of campylobacteriosis, little is known about the genetic and phenotypic diversity of LOS in Campylobacter coli In this study, we investigated the distribution of LOS locus classes among a large collection of unrelated C. coli isolates sampled from several different host species. Furthermore, we paired C. coli genomic information and LOS chemical composition for the first time to investigate possible associations between LOS locus class sequence diversity and biochemical heterogeneity. After identifying three new LOS locus classes, only 85% of the 144 isolates tested were assigned to a class, suggesting higher genetic diversity than previously thought. This genetic diversity is at the basis of a completely unexplored LOS structural heterogeneity. Mass spectrometry analysis of the LOSs of nine isolates, representing four different LOS classes, identified two features distinguishing C. coli LOS from that of Campylobacter jejuni 2-Amino-2-deoxy-d-glucose (GlcN)-GlcN disaccharides were present in the lipid A backbone, in contrast to the ß-1'-6-linked 3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N)-GlcN backbone observed in C. jejuni Moreover, despite the fact that many of the genes putatively involved in 3-acylamino-3,6-dideoxy-d-glucose (Quip3NAcyl) were apparently absent from the genomes of various isolates, this rare sugar was found in the outer core of all C. coli isolates. Therefore, regardless of the high genetic diversity of the LOS biosynthesis locus in C. coli, we identified species-specific phenotypic features of C. coli LOS that might explain differences between C. jejuni and C. coli in terms of population dynamics and host adaptation. IMPORTANCE: Despite the importance of C. coli to human health and its controversial role as a causative agent of Guillain-Barré syndrome, little is known about the genetic and phenotypic diversity of C. coli LOSs. Therefore, we paired C. coli genomic information and LOS chemical composition for the first time to address this paucity of information. We identified two species-specific phenotypic features of C. coli LOS, which might contribute to elucidating the reasons behind the differences between C. jejuni and C. coli in terms of population dynamics and host adaptation.

Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry.

Ovine and bovine blood is used heavily within the development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs), for which blood cell damage needs to be monitored during in vitro testing. Hematology analyzers provide cell counts but no information about cell viability. The anthraquinone DNA dyes CyTRAK Orange™ and DRAQ7™ have practical and spectral properties rendering them suitable for multicolor assays. Compared to other DNA dyes such as Vybrant Dyecycle, CyTRAK Orange enables a faster staining protocol and does not require incubation at +37°C. Compared to traditional viability dyes such as propidium iodide and 7AAD, DRAQ7's unique spectral profile of excitation in both blue and red lasers and far-red emission enables identification of dual positive dead cell events and frees up detectors for use with other reagents. CyTRAK Orange and DRAQ7 could be used in combination with absolute counting bead standards to provide cell counts and viability but the combination of these dyes has previously only been used for microscopy on rodent cells. The purpose of this study was to evaluate the use of these dyes in combination in large animal blood samples for flow cytometry. A viability and cell counting protocol for bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was prepared. Four different counting bead standards were evaluated using the Navios and FACSAria cytometers and compared to counts obtained from hematology analyzers. CyTRAK Orange successfully detected CD45(+) leukocytes in all species. The DRAQ7 single-stained dead cell counts correlated well with the CyTRAK Orange/DRAQ7 double-stained dead cell counts in human and bovine blood, but not in ovine blood, which could be related to the blood source. In conclusion, for human and bovine blood, this method works well for viability counts using different flow cytometers and bead standards. © 2016 International Society for Advancement of Cytometry.

Evaluation of Four Veterinary Hematology Analyzers for Bovine and Ovine Blood Counts for In Vitro Testing of Medical Devices.

Small affordable automated hematology analyzers that produce rapid and accurate complete blood cell counts are a valuable tool to researchers developing blood-handling medical devices, such as ventricular assist devices, for in vitro safety assessments. In such studies, it is common to use the blood of large animals such as cattle and sheep. However, the commercially available instruments have not been evaluated for their ability to measure the blood counts of these animals. In this study, we compare, for the first time, four veterinary analyzers for blood counts on bovine and ovine blood samples. We look at ease of use, repeatability and agreement with a view to inform researchers of the benefits of these instruments in routine measurement of ovine and bovine bloods during in vitro testing. Complete blood cell counts and a three-part differential (granulocytes, monocytes, and lymphocytes) were measured by each of the instruments, and the results compared to those obtained from two additional analyzers used in a reference laboratory. Repeatability and agreement were evaluated using the Bland-Altman method; bias and 95% limits of agreement between the instruments, and between the instruments and two reference instruments, were used to evaluate instrument performance. In summary, there are advantages and disadvantages with all instruments. Of the four instruments tested, the repeatability and agreement was fairly similar for all instruments except one instrument which cannot be recommended for bovine or ovine blood counts.

The CentriMag centrifugal blood pump as a benchmark for in vitro testing of hemocompatibility in implantable ventricular assist devices.

Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge-to-transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10(-5) ), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10(-5) ) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.

The effect of shear stress on the size, structure, and function of human von Willebrand factor.

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.

Widening Excess Mortality During the COVID-19 Pandemic in Individuals Who Self-Harmed.

Background: Studies on COVID-19 pandemic-associated changes in mortality following self-harm remain scarce and inconclusive. Aims: To compare mortality risks in individuals who had self-harmed to those for individuals who had not, before and during the COVID-19 pandemic (Waves 1 and 2) in Wales, the United Kingdom, using population-based routinely collected data. Method: We linked whole population health data to all-cause mortality following an episode of self-harm between April 2016 and March 2021. Propensity score matching, Cox regression, and difference-in-differences were applied to compute changes in excess mortality (as ratios of hazard ratios, RHRs) before and during the pandemic for individuals who self-harmed. Results: The difference in mortality for individuals who self-harmed compared to those who did not widened during Wave 1 (RHR = 2.03, 95% CI: 1.04-4.03) and Wave 2 (RHR = 2.19, 95% CI: 1.12-4.29) from before the pandemic. Stratification by sex and age group produced no significant subgroup differences although risk for younger than 65 years group were higher. Limitations: Limitations include small sample size and incomplete data on cause-specific deaths during the pandemic. Conclusion: Our results underscore continuous monitoring of mortality of individuals who self-harm and effective interventions to address any increases in mortality.

Association of school absence and exclusion with recorded neurodevelopmental disorders, mental disorders, or self-harm: a nationwide, retrospective, electronic cohort study of children and young people in Wales, UK.

BACKGROUND: Poor attendance at school, whether due to absenteeism or exclusion, leads to multiple social, educational, and lifelong socioeconomic disadvantages. We aimed to measure the association between a broad range of diagnosed neurodevelopmental and mental disorders and recorded self-harm by the age of 24 years and school attendance and exclusion. METHODS: In this nationwide, retrospective, electronic cohort study, we drew a cohort from the Welsh Demographic Service Dataset, which included individuals aged 7-16 years (16 years being the school leaving age in the UK) enrolled in state-funded schools in Wales in the academic years 2012/13-2015/16 (between Sept 1, 2012, and Aug 31, 2016). Using the Adolescent Mental Health Data Platform, we linked attendance and exclusion data to national demographic and primary and secondary health-care datasets. We identified all pupils with a recorded diagnosis of neurodevelopmental disorders (ADHD and autism spectrum disorder [ASD]), learning difficulties, conduct disorder, depression, anxiety, eating disorders, alcohol or drugs misuse, bipolar disorder, schizophrenia, other psychotic disorders, or recorded self-harm (our explanatory variables) before the age of 24 years. Outcomes were school absence and exclusion. Generalised estimating equations with exchangeable correlation structures using binomial distribution with the logit link function were used to calculate odds ratios (OR) for absenteeism and exclusion, adjusting for sex, age, and deprivation. FINDINGS: School attendance, school exclusion, and health-care data were available for 414 637 pupils (201 789 [48·7%] girls and 212 848 [51·3%] boys; mean age 10·5 years [SD 3·8] on Sept 1, 2012; ethnicity data were not available). Individuals with a record of a neurodevelopmental disorder, mental disorder, or self-harm were more likely to be absent or excluded in any school year than were those without a record. Unadjusted ORs for absences ranged from 2·1 (95% CI 2·0-2·2) for those with neurodevelopmental disorders to 6·6 (4·9-8·3) for those with bipolar disorder. Adjusted ORs (aORs) for absences ranged from 2·0 (1·9-2·1) for those with neurodevelopmental disorders to 5·5 (4·2-7·2) for those with bipolar disorder. Unadjusted ORs for exclusion ranged from 1·7 (1·3-2·2) for those with eating disorders to 22·7 (20·8-24·7) for those with a record of drugs misuse. aORs for exclusion ranged from 1·8 (1·5-2·0) for those with learning difficulties to 11·0 (10·0-12·1) for those with a record of drugs misuse. INTERPRETATION: Children and young people up to the age of 24 years with a record of a neurodevelopmental or mental disorder or self-harm before the age of 24 years were more likely to miss school than those without a record. Exclusion or persistent absence are potential indicators of current or future poor mental health that are routinely collected and could be used to target assessment and early intervention. Integrated school-based and health-care strategies to support young peoples' engagement with school life are required. FUNDING: The Medical Research Council, MQ Mental Health Research, and the Economic and Social Research Council. TRANSLATION: For the Welsh translation of the abstract see Supplementary Materials section.

A Retrospective Review of the Clinical Characteristics and Blood Glucose Data from Cellnovo System Users using Data Collected from the Cellnovo Online Platform.

Technological advances have led to innovative insulin delivery systems for patients with type 1 diabetes mellitus. In particular, the combination of miniature engineering and software algorithms contained in continuous subcutaneous insulin infusion (CSII) system pumps provide the user and the healthcare practitioner with an opportunity to review and adjust blood glucose (BG) levels according to system feedback, and to modify or programme their regimen according to their needs. While CSII pumps record a number of data parameters such as BG level, carbohydrate intake, activity and insulin delivered, these data are generally 'locked in' and can only be accessed by uploading to a cloud-based system, thus information is not contemporaneous. The Cellnovo Diabetes Management System (Cellnovo, Bridgend, UK) allows data to be transmitted securely and wirelessly in real time to a secure server, which is then retrieved by an online platform, the Cellnovo Online platform, enabling continuous access by the user and by clinicians. In this article, the authors describe a retrospective review of the patient data automatically uploaded to the Cellnovo Online platform. Baseline clinical and demographic characteristics collected at the start of pump therapy are shown for all patients, and BG data from a sub-cohort of patients who have been using the system for at least 6 months and who take and record an average of three BG level tests per day are presented to demonstrate glycaemic data over time.

Combat Trousers as Effective Improvised Pelvic Binders A Comparative Cadaveric Study.

BACKGROUND: Improvised explosive devices and landmines can cause pelvic fractures, which, in turn, can produce catastrophic hemorrhage. This cadaveric study compared the intrapelvic pressure changes that occurred with the application of an improvised pelvic binder adapted from the combat trousers worn by British military personnel with the commercially available trauma pelvic orthotic device (TPOD). METHODS: Six unembalmed cadavers (three male, three female) were used to simulate an unstable pelvic fracture with complete disruption of the posterior arch (AO/OTA 61-C1) by dividing the pelvic ring anteriorly and posteriorly. A 3-4cm manometric balloon filled with water was placed in the retropubic space and connected to a 50mL syringe and water manometer via a three-way tap. A baseline pressure of 8cm H2O (average central venous pressure) was set. The combat trouser binder (CTB) and TPOD were applied to each cadaver in a random sequence and the steady intrapelvic pressure changes were recorded. Statistical analysis was performed using the Wilcoxon rank-sum test and a paired t test depending on the normality of the data to determine impact on the intrapelvic pressure of each intervention compared with baseline. RESULTS: The median steady intrapelvic pressure achieved after application of the CTB was 16cm H2O and after application of the TPOD binder was 18cm H2O, both of which were significantly greater than the baseline pressure (ρ < .01 and .036, respectively) but not significantly different from each other (ρ > .05). CONCLUSION: Pelvic injuries are increasingly common in modern theaters of war. The CTB is a novel, rapidly deployable, yet effective, method of pelvic binding adapted from the clothes the casualty is already wearing. This technique may be used in austere environments to tamponade and control intrapelvic hemorrhage.

Intra-pelvic pressure changes after pelvic fracture: A cadaveric study quantifying the effect of a pelvic binder and limb bandaging over a bolster.

INTRODUCTION: Unstable pelvic fractures can be life-threatening due to catastrophic haemorrhage. Non-invasive methods of reducing and stabilising these injuries include pelvic binder application and also lower limb bandaging over a knee-flexion bolster. Both of these methods help close the pelvic ring and should tamponade bleeding. This study aimed to quantify the intra-pelvic pressure changes that occurred with 3 different manoeuvres: lower limb bandaging over a bolster; a Trauma Pelvic Orthotic Device (T-POD) pelvic binder, and a combination of both. METHODS: Following a pilot study with 2 soft embalmed cadavers, a formal study with 6 unembalmed cadavers was performed. For each specimen an unstable pelvic injury was created (OA/OTA 61-C1) by dividing the pelvic ring anteriorly and posteriorly. A 3-4cm manometric water-filled balloon was placed in the retropubic space and connected to a 50ml syringe and water manometer via a 3-way tap. A baseline pressure of 8cmH2O (equating to the average central venous pressure) was used for each cadaver. Steady intra-pelvic pressures (more reliably reflecting the pressures achieved following an intervention) were used in the subsequent statistical analysis, using R statistical language and Rstudio. Paired t-test or Wilcoxon's rank sum test were used (depending on the normality of the dataset) to determine the impact of each intervention on the intra-pelvic pressure. RESULTS: The mean steady intra-pelvic pressures were significantly greater than the baseline pressure for each intervention. The binder and limb bandaging over a bolster alone increased the mean steady pelvic pressures significantly to 24 (SE=5) (p<0.036) and 15.5 (SE=2) (p<0.02)cmH2O respectively. Combining these interventions further increased the mean steady pressure to 31 (SE=7)cmH2O. However, this was not significantly greater than pressures for each of the individual interventions. DISCUSSION: Both lower limb bandaging over a bolster and pelvic binder application significantly increased intra-pelvic pressure above the baseline pressure. This was further increased through combining these interventions, which could be useful clinically to augment haemorrhage control in these fractures. CONCLUSION: Lower-limb bandaging over a bolster, and pelvic binder application, both significantly increased intra-pelvic pressures, and were greatest in combination. These findings support the use of these techniques to facilitate non-surgical haemorrhage control.

Recombination-Mediated Host Adaptation by Avian Staphylococcus aureus.

Staphylococcus aureus are globally disseminated among farmed chickens causing skeletal muscle infections, dermatitis, and septicaemia. The emergence of poultry-associated lineages has involved zoonotic transmission from humans to chickens but questions remain about the specific adaptations that promote proliferation of chicken pathogens. We characterized genetic variation in a population of genome-sequenced S. aureus isolates of poultry and human origin. Genealogical analysis identified a dominant poultry-associated sequence cluster within the CC5 clonal complex. Poultry and human CC5 isolates were significantly distinct from each other and more recombination events were detected in the poultry isolates. We identified 44 recombination events in 33 genes along the branch extending to the poultry-specific CC5 cluster, and 47 genes were found more often in CC5 poultry isolates compared with those from humans. Many of these gene sequences were common in chicken isolates from other clonal complexes suggesting horizontal gene transfer among poultry associated lineages. Consistent with functional predictions for putative poultry-associated genes, poultry isolates showed enhanced growth at 42 °C and greater erythrocyte lysis on chicken blood agar in comparison with human isolates. By combining phenotype information with evolutionary analyses of staphylococcal genomes, we provide evidence of adaptation, following a human-to-poultry host transition. This has important implications for the emergence and dissemination of new pathogenic clones associated with modern agriculture.