Development of a selective, sensitive and robust oxyntomodulin dual monoclonal antibody immunoassay.

Background & Aim: Oxyntomodulin (Oxm) is a proglucagon-derived peptide agonist of both the GLP-1 and glucagon receptors and is a key regulator of gastric acid secretion and energy expenditure. Differential processing from proglucagon hinders assay immunoassay selectivity. Method & results: Antibody engineering was used to develop a sandwich immunoassay that selectively measures endogenous Oxm. The pre- and postprandial levels of Oxm from 19 healthy individuals over the course of 2 h were measured. Postprandial increases in Oxm occurred within minutes and levels significantly correlated with those obtained using previously published mass spectrometry assays. Conclusion: This sandwich immunoassay is appropriately sensitive and selective and is also amenable to high-throughput application for the reliable determination of endogenous levels of intact Oxm from human samples.

Evaluation of XSTAT® and QuickClot® Combat Gauze® in a Swine Model of Lethal Junctional Hemorrhage in Coagulopathic Swine.

BACKGROUND: Hemorrhage is associated with most potentially survivable deaths on the battlefield. Effective and field-tested products are lacking to treat junctional and noncompressible injuries. XSTAT® is a newly developed, U.S. Food and Drug Administration-approved product designed to treat junctional hemorrhage. The Committee on Tactical Combat Casualty Care has recently approved the product for use as part of its treatment guidelines, but data are lacking to assess its efficacy in different wounding patterns and physiologic states. METHODS: Dilutional coagulopathy was induced in 19 large (70-90kg), healthy, male swine by replacing 60% of each animal's estimated blood volume with room temperature Hextend ®. After dissection, isolation, and lidocaine incubation, uncontrolled hemorrhage was initiated by transection of both axillary artery and vein. Free bleeding was allowed to proceed for 30 seconds until intervention with either XSTAT or QuickClot® Combat Gauze® (CG) followed by standard backing. Primary outcomes were survival, hemostasis, and blood loss. RESULTS: XSTAT-treated animals achieved hemostasis in less time and remained hemostatic longer than those treated with CG. Less blood was lost during the first 10 minutes after injury in the XSTAT group than the CG group. However, no differences in survival were observed between XSTAT-treated and CG-treated groups. All animals died before the end of the observation period except one in the XSTAT-treated group. CONCLUSION: XSTAT performed better than CG in this model of junctional hemorrhage in coagulopathic animals. Continued testing and evaluation of XSTAT should be performed to optimize application and determine appropriate indications for use.

The Use of the Abdominal Aortic and Junctional Tourniquet During Cardiopulmonary Resuscitation Following Traumatic Cardiac Arrest in Swine.

BACKGROUND: Standard cardiopulmonary resuscitation (CPR) is ineffective in treating traumatic cardiac arrest (TCA) following hemorrhagic shock despite fluid resuscitation. CPR adjuncts, including abdominal compressions and external counter pressure, have shown some success in laboratory settings. The Abdominal Aortic and Junctional Tourniquet (AAJT) is a device that occludes both venous and arterial blood at the level of the aortic bifurcation and likely increases thoracic pressure when applied to the abdomen. We developed a swine model of controlled hemorrhage to induce a state of TCA to test the ability of the AAJT to improve the efficacy of CPR. METHODS: Twelve splenectomized, Yorkshire, male swine (70-90 kg) were randomized into two groups: presence or absence of AAJT placement. Controlled hemorrhage was performed at a rate of 2 mL/kg/min until systolic blood pressure reached below 10 mm Hg (defined as cardiac arrest). Following 3 minutes of arrest, the animals underwent CPR using a mechanical compression device along with either the presence or absence of the AAJT. Concurrently, 5 units of whole blood (2,500 mL) were delivered through the jugular vein at 500 mL/min. Efficacy of CPR was assessed by analyzing rates of return of spontaneous circulation (ROSC) and survival. Blood pressure, carotid blood flow, and other hemodynamic values were also compared. FINDINGS: No significant differences between groups were observed before treatments. The controlled hemorrhage resulted in an average loss of 2,654 ± 323 g of blood over 18.2 ± 3.9 minutes. All animals that had a ROSC survived to the end of the 1-hour observation period. Animals with AAJT survived 83% (5/6) compared to 17% (1/6) of animals without AAJT. Finally, blood pressure, carotid flow, mean pulmonary artery pressure, and end tidal carbon dioxide were all significantly different between groups at the end of the first 10-minute compression period. DISCUSSION/IMPACT/RECOMMENDATIONS: These results suggest that the AAJT could allow for increased CPR efficacy in cases of TCA when used in conjunction with rapid, massive blood transfusions.

A comparison of Selective Aortic Arch Perfusion and Resuscitative Endovascular Balloon Occlusion of the Aorta for the management of hemorrhage-induced traumatic cardiac arrest: A translational model in large swine.

BACKGROUND: Survival rates remain low after hemorrhage-induced traumatic cardiac arrest (TCA). Noncompressible torso hemorrhage (NCTH) is a major cause of potentially survivable trauma death. Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) at the thoracic aorta (Zone 1) can limit subdiaphragmatic blood loss and allow for IV fluid resuscitation when intrinsic cardiac activity is still present. Selective Aortic Arch Perfusion (SAAP) combines thoracic aortic balloon hemorrhage control with intra-aortic oxygenated perfusion to achieve return of spontaneous circulation (ROSC) when cardiac arrest has occurred. METHODS AND FINDINGS: Male Yorkshire Landrace cross swine (80.0 ± 6.0 kg) underwent anesthesia, instrumentation for monitoring, and splenectomy. TCA was induced by laparoscopic liver lobe resection combined with arterial catheter blood withdrawal to achieve a sustained systolic blood pressure <10 mmHg, cardiac arrest. After 3 min of arrest, swine were allocated to one of three interventions: (1) REBOA plus 4 units of IV fresh whole blood (FWB), (2) SAAP with oxygenated lactated Ringer's (LR), 1,600 mL/2 min, or (3) SAAP with oxygenated FWB 1,600 mL/2 min. Primary endpoint was survival to the end of 60 min of resuscitation, a simulated prehospital phase. Thirty animals were allocated to 3 groups (10 per group)-5 protocol exclusions resulted in a total of 35 animals being used. Baseline measurements and time to cardiac arrest were not different amongst groups. ROSC was achieved in 0/10 (0%, 95% CI 0.00-30.9) REBOA, 6/10 (60%, 95% CI 26.2-87.8) SAAP-LR and 10/10 (100%, 95% CI 69.2-100.0) SAAP-FWB animals, p < 0.001. Survival to end of simulated 60-minute prehospital resuscitation was 0/10 (0%, 95% CI 0.00-30.9) for REBOA, 1/10 (10%, 95% CI 0.25-44.5) for SAAP-LR and 9/10 (90%, 95% CI 55.5-99.7) for SAAP-FWB, p < 0.001. Total FWB infusion volume was similar for REBOA (2,452 ± 0 mL) and SAAP-FWB (2,250 ± 594 mL). This study was undertaken in laboratory conditions, and as such may have practical limitations when applied clinically. Cardiac arrest in this study was defined by intra-aortic pressure monitoring that is not feasible in clinical practice, and as such limits the generalizability of findings. Clinical trials are needed to determine if the beneficial effects of SAAP-FWB observed in this laboratory study will translate into improved survival in clinical practice. CONCLUSIONS: SAAP conferred a superior short-term survival over REBOA in this large animal model of hemorrhage-induced traumatic cardiac arrest with NCTH. SAAP using an oxygen-carrying perfusate was more effective in this study than non-oxygen carrying solutions in TCA. SAAP can effect ROSC from hemorrhage-induced electrocardiographic asystole in large swine.

Hemodynamic effects of the Abdominal Aortic and Junctional Tourniquet in a hemorrhagic swine model.

BACKGROUND: Torso hemorrhage constitutes a leading cause of battlefield mortality. The Abdominal Aortic and Junctional Tourniquet (AAJT) uses a pneumatic bladder to compress the aorta reducing pelvic and lower extremity perfusion; however, concern exists over the risk of caval compression exacerbating hypotension after application. METHODS: Male swine (70-90 kg) were randomized into four groups of 10: presence or absence of hemorrhage and AAJT placement. After a 40% hemorrhage, a 15-min period of hypovolemia was observed before the AAJT application. All animals received two 500 mL boluses of Hextend separated by 30 min. Cardiovascular, pulmonary, and oxygenation values were compared among groups. RESULTS: The AAJT was effective in reducing blood flow to the femoral arteries in both hemorrhaged and nonhemorrhaged animals (P < 0.001 for both groups). Hemorrhage resulted in significant decrease in mean arterial pressure compared with sham controls (23.5 ± 2.4 versus 61.6 ± 7.8 mm Hg, respectively, P < 0.001). AAJT application, compared with untreated controls, resulted in a significant increase in mean arterial pressure and systemic vascular resistance but not in cardiac output, oxygenation, and central venous pressure. Furthermore, no indication of overresuscitation injury was present as evidenced by pulmonary artery pressure and pulmonary histology. CONCLUSIONS: AAJT application in an animal model of severe shock results in a favorable hemodynamic profile because of afterload support. The present study did not demonstrate any adverse consequences because of caval compression, bowel injury, or pulmonary dysfunction. In addition, there does not appear to be any particular intravenous fluid economy achieved by AAJT application.

Characterization and quantification of oxyntomodulin in human and rat plasma using high-resolution accurate mass LC-MS.

BACKGROUND: A thorough understanding of the biological role of oxyntomodulin (OXM) has been limited by the availability of sensitive and specific analytical tools for reliable in vivo characterization. Here, we utilized immunoaffinity capture coupled with high-resolution accurate mass LC-MS detection to quantify OXM and its primary catabolites. RESULTS: Quantification of intact OXM 1-37 in human and rat plasma occurred in pre- and post-prandial samples. Profiles for the major catabolites were observed allowing kinetic differences to be assessed between species. CONCLUSION: A validated assay in human and rat plasma was obtained for OXM 1-37 and its catabolites, 3-37 and 4-37. The value of full scan high-resolution accurate mass detection without selected reaction monitoring for low-abundance peptide quantification was also demonstrated.

A validated LC-MS/MS method for neurotransmitter metabolite analysis in human cerebrospinal fluid using benzoyl chloride derivatization.

BACKGROUND: Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention. RESULTS: Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation. CONCLUSION: A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.

Comparison of novel hemostatic dressings with QuikClot combat gauze in a standardized swine model of uncontrolled hemorrhage.

BACKGROUND: Uncontrolled hemorrhage is the leading cause of preventable death on the battlefield. The development, testing, and application of novel hemostatic dressings may lead to a reduction of prehospital mortality through enhanced point-of-injury hemostatic control. This study aimed to determine the efficacy of currently available hemostatic dressings as compared with the current Committee for Tactical Combat Casualty Care Guidelines standard of treatment for hemorrhage control (QuikClot Combat Gauze [QCG]). METHODS: The femoral artery of anesthetized Yorkshire pigs was isolated and punctured. Free bleeding was allowed to proceed for 45 seconds before packing of QCG, QuikClot Combat Gauze XL (QCX), Celox Trauma Gauze (CTG), Celox Gauze (CEL), or HemCon ChitoGauze (HCG), into the wound. After 3 minutes of applied, direct pressure, fluid resuscitation was administered to elevate and maintain a mean arterial pressure of 60 mm Hg or greater during the 150-minute observation time. Animal survival, hemostasis, and blood loss were measured as primary end points. Hemodynamic and physiologic parameters, along with markers of coagulation, were recorded and analyzed. RESULTS: Sixty percent of QCG-treated animals (controls) survived through the 150-minute observation period. QCX, CEL, and HCG were observed to have higher rates of survival in comparison to QCG (70%, 90%, and 70% respectively), although these results were not found to be of statistical significance in pairwise comparison to QCG. Immediate hemostasis was achieved in 30% of QCG applications, 80% of QCX, 70% of CEL, 60% of HCG, and 30% of CTG-treated animals. Posttreatment blood loss varied from an average of 64 mL/kg with CTG to 29 mL/kg with CEL, but no significant difference among groups was observed. CONCLUSION: These results suggest that the novel hemostatic devices perform at least as well as the current Committee on Tactical Combat Casualty Care standard for point-of-injury hemorrhage control. Despite their different compositions and sizes, the lack of clear superiority of any agent suggests that contemporary hemostatic dressing technology has potentially reached a plateau for efficacy.

Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation.

There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.

Truncated careers in nuclear medicine technology: increased job control may improve retention.

OBJECTIVES: The aim of the study was to gain understanding of why nuclear medicine technologists (NMTs) leave and to compare workforce and service provision trends with diagnostic imaging professionals. DESIGN AND SETTING: A survey of all NMT professional body members in New South Wales, the Australian Capital Territory and Queensland was conducted. This paper reports on survey findings of those no longer working as a NMT. Analysis of 1996, 2001 and 2006 Australian Census data and Medicare statistics was made for NMTs, sonographers and radiographers. MAIN OUTCOME MEASURES: The five most influential reasons for leaving nuclear medicine were measured by survey. Census data measured workforce characteristics; size, sex, age. Medicare statistics measured national service provision. RESULTS: Primarily, limited career pathways and professional plateau influence retention of NMTs, with sonography a common career move. Nuclear medicine technologists are young (44.3% <30 years) compared with radiography (52.3% <40 years) or sonography (52.8% <40 years). From 2001 to 2006, service provision in nuclear medicine grew by 11.8% compared with 36% in ultrasound but the workforce size decreased by 4.9% whereas sonographers increased by 51.1%. CONCLUSIONS: Increasing the level of job control is the most likely factor in creating a positive change to the NMT job characteristics and improving retention.

Development and characterization of a rat model of nonpenetrating liver trauma.

The aim of this study was to develop and characterize a rodent model of liver trauma suitable for preclinical evaluation of new treatments and diagnostic technologies. Liver trauma was created by dropping a steel cylinder through a plastic tube onto the abdomen of supine, anesthetized rats. Internal hemorrhage in the absence of liver trauma was simulated by instilling fresh blood into the peritoneum. Platelet counts were elevated significantly after liver trauma but not simulated hemorrhage. Liver trauma and simulated internal hemorrhage both increased blood levels of the factor growth-regulated oncogene-Kupffer cell. Transcription of plasminogen activator inhibitor 1, heat shock protein 70, and suppressor of cytokine syntheses 3 was increased 77-, 22-, and 27-fold, respectively, 2 h after liver trauma but was unaltered by simulated internal hemorrhage. Levels returned to pretrauma levels by 24 h after trauma. Transcript levels for hypoxia-inducible transcription factor 1alpha were increased 2.8-fold at 24 h but not 2 h after trauma and were not affected by simulated hemorrhage. Production of heat shock protein 70 and inducible nitric oxide synthase in liver was limited to a penumbra surrounding areas of necrosis associated with trauma. The rat model described produces lesions similar to those that occur in humans after blunt trauma.

The work environment and its effect on engagement and retention of nuclear medicine technologists: differences between public and private sector workers.

OBJECTIVE: The retention of Australian nuclear medicine technologists (NMTs) is poor with the future workforce size in question. As a consequence, the primary aim of this study was to determine Australian NMTs' level of work engagement and the factors influencing this to identify the issues surrounding retention. METHODS: The job demands resource model assumes that each job has its own demands and resources and the balance between these can influence the level of work engagement. Lower levels of work engagement are predictive of an intention to leave. Work engagement levels can be measured using the Utrecht work engagement scale. This study used the Utrecht Work Engagement Scale in a self-report questionnaire with additional open-ended and closed-ended items designed to evaluate satisfying job characteristics. Members of the professional body in specific geographical locations of Australia were invited to participate. RESULTS: A 49.6% response rate was achieved (n=201); of these, 164 were practicing NMTs. Public sector workers had significantly lower total mean scores (P=0.05) on the Utrecht Work Engagement Scale and the subscale of dedication (P=0.005) compared with private NMTs. Seven of the 14 job satisfaction closed-ended items were statistically significantly lower for public NMTs: the level of decision making; feelings of importance with the tasks performed; feedback on tasks and roles; and relationships with physicians, staff and the organization. CONCLUSION: To improve the retention of NMTs, changes in the job resources and demands are needed. Advanced practice roles may improve retention by enhancing the job resources.

A profile of Australian nuclear medicine technologist practice.

BACKGROUND: Nuclear medicine in Australia has encountered significant change over the past 30 years, with a move to privately owned practices, technological advances and the transfer of education of the nuclear medicine technologist (NMT) from technical college apprenticeships to university degrees. Currently, shortages of nuclear medicine technologists are reported in some states of Australia. It is not known whether changes in NMT practice or the type of centre in which an NMT works have an influence on retention of staff. AIM: The primary objective of this survey was to establish a profile of NMT practice in Australia, with the aim of producing baseline data that could be used in further research to establish levels of retention and job satisfaction. METHODS: Chief technologists in three states of Australia were invited to respond to a written questionnaire. The questionnaire included data about staffing levels, imaging modalities, procedures performed, and movement of staff. Findings presented will relate to the profile of practice data only. RESULTS: Forty-eight (54%) chief technologists responded to the questionnaire with 73% working in privately owned practices. The majority of centres employ up to two full-time equivalent nuclear medicine technologists and have two gamma cameras and one full-time equivalent nuclear medicine physician. Most centres perform a limited range of studies with bone scans predominating. More than half the centres make some use of a centralized radiopharmacy service. CONCLUSION: Further research is required to determine how these changes may impact on workplace satisfaction and in turn, on retention.