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Context.— Underuse of laboratory testing has been previously investigated in preselected populations, such as documented malpractice claims. However, these numbers might not reflect real-life situations. Objective.— To evaluate the underuse and misuse of laboratory follow-up testing in a real-life hospital patient population with microcytic anemia, using laboratory results ordered during routine patient care. Design.— From all patients in whom a microcytic anemia was detected during routine diagnostics in 2018, all available laboratory data were collected and screened for appropriateness of diagnostic workup of iron deficiency and thalassemia. Subgroup analysis was performed for patient groups with mean corpuscular volume values 75 to 79 μm3 (group 1), 65 to 74 μm3 (group 2), and Results.— A total of 2244 patients with microcytic anemia were identified. Follow-up testing for iron deficiency was not performed in 761 cases (34%). For inconclusive ferritin levels due to elevated C-reactive protein results (n = 336), reticulocyte hemoglobin content or soluble transferrin receptor levels were missing in 86 cases (26%). In patients with suspected thalassemia (n = 127), follow-up testing for hemoglobin variants was not performed in 70 cases (55%). Subgroup analysis showed that the frequency of underuse of iron status as well as thalassemia/hemoglobinopathy testing decreased from group 1 to group 3. When considering relevant preexisting anemia diagnoses, laboratory tests were underused in 904 cases (40.3%). Conclusions.— Because 40% (n = 904) of the patients with microcytic anemia were potentially not followed up correctly, laboratory specialists are advised to act by implementing demand management strategies in collaboration with clinicians to overcome underuse of laboratory tests and to improve patient safety.

Background Laboratory overutilization is associated with diagnostic error and potential patient risk. We applied a demand management strategy in collaboration with the local Department of Cardiology to reduce the cardiac markers high-sensitive troponin T (hsTropT) and N-terminal pro brain natriuretic peptide (NTproBNP) in laboratory ordering profiles (LOPs). The present study aimed to retrospectively evaluate the implemented strategies. Methods Strategies included educational measures and evidence-guided, active test de-selection from all cardiology ward LOPs, and/or permanent removal from LOPs. Tests remained available at all times. We evaluated overutilization by reductions in monthly orders, and assessed differences in 30-day all-cause readmission rate and length of patients’ hospital stay. Results Overall, we observed a mean reduction of 66.1% ± 7.6% (n = 277 ± 31) in hsTropT tests. Educational measures effectively reduced NTproBNP orders by 52.8% ± 17.7% (n = 60 ± 20). Permanent removal of tests from LOPs additionally decreased orders to a final extent of 75.8% ± 8.0% (n = 322 ± 31) in NTproBNP tests. The 30-day readmission rate and overall length of hospital stay did not increase. Conclusions Our results indicate that cardiac markers in routine care are subject to extensive overutilization when used within LOPs. Educational measures are an effective strategy to overcome the overutilization of cardiac markers but may be more effective when combined with the removal of cardiac markers from LOPs.

Background and Aims Anti‐mitochondrial antibodies (AMA) are closely linked to primary biliary cholangitis (PBC). The prevalence of AMA in the general population is low, and AMA positivity may precede PBC. We aimed to determine the natural history of subjects with positive AMA. Methods In total, 302 patients were tested AMA‐positive over a ten‐year period. Of these, immunoblotting confirmed specific AMA in 184 (29 male, 155 female, age 59.6 ± 14.1 years). These subjects were invited to our liver outpatient clinic for clinical and biochemical re‐evaluation. Detailed clinical history data were additionally collected from the hospital computer system and by telephone. The subsequent course with regard to mortality, liver‐related morbidity, extrahepatic co‐morbidities and effectiveness of PBC treatment was determined in 150 subjects (81.5%). Results After 5.8 ± 5.6 years of follow‐up (FU), of 184 AMA‐positive subjects, 28 subjects (15.2%; liver‐related mortality n = 5) were deceased, and 122 subjects (66.3%) completed FU while 34 subjects (18.5%) were not available for FU. The 122 patients who completed FU were 63 patients with established PBC, six de novo cases of PBC (10.2% of 59 initially at risk), 42 (34.4%) subjects were still AMA‐positive without PBC, and 11 (9.0%) subjects were AMA‐negative at FU. Conclusions Anti‐mitochondrial antibodies‐positive patients without PBC at baseline infrequently developed PBC over six years of FU. AMA positivity represented a transient serological autoimmune phenomenon in a significant proportion of subjects.

Dementia with Lewy bodies (DLB) represents a huge medical need as it accounts for up to 30% of all dementia cases, and there is no cure available. The underyling spectrum of pathology is complex and creates a challenge for targeted molecular therapies. We here tested the hypothesis that leukotrienes are involved in the pathology of DLB and that blocking leukotrienes through Montelukast, a leukotriene receptor antagonist and approved anti-asthmatic drug, might alleviate pathology and restore cognitive functions. Expression of 5-lipoxygenase, the rate-limiting enzyme for leukotriene production, was indeed elevated in brains with DLB. Treatment of cognitively deficient human alpha-synuclein overexpressing transgenic mice with Montelukast restored memory. Montelukast treatment resulted in modulation of beclin-1 expression, a marker for autophagy, and in a reduction in the human alpha-synulcein load in the transgenic mice. Reducing the protein aggregation load in neurodegenerative diseases might be a novel model of action of Montelukast. Moreover, this work presents leukotriene signaling as a potential drug target for DLB and shows that Montelukast might be a promising drug candidate for future DLB therapy development. Electronic supplementary material The online version of this article (10.1007/s13311-020-00836-3) contains supplementary material, which is available to authorized users.

Prognostication after cardiopulmonary resuscitation (CPR) is complex. Novel biomarkers like soluble suppression of tumorigenicity 2 (sST2) may provide an objective approach. A total of 106 post-CPR patients were included in this single-center observational prospective study. Serum sST2 levels were obtained 24 h after admission. Individuals were assigned to two groups: patients below and above the overall cohort’s median sST2 concentration. Primary outcome was a combined endpoint at 6 months (death or Cerebral Performance Category > 2); secondary endpoint 30-day mortality. A uni- and multivariate logistic regression analysis were conducted. Elevated sST2-levels were associated with an increased risk for the primary outcome (OR 1.011, 95% CI 1.004–1.019, p = 0.004), yet no patients with poor neurological outcome were observed at 6 months. The optimal empirical cut-off for sST2 was 46.15 ng/ml (sensitivity 81%, specificity 53%, AUC 0.69). Levels above the median (> 53.42 ng/ml) were associated with higher odds for both endpoints (death or CPC > 2 after 6 months: 21% vs. 49%, OR 3.59, 95% CI 1.53–8.45, p = 0.003; death after 30 days: 17% vs. 43.3%, OR 3.75, 95% CI 1.52–9.21, p = 0.003). A positive correlation of serum sST2 after CPR with mortality at 30 days and 6 months after cardiac arrest could be demonstrated.

Introduction Intoxications are common in intensive care units (ICUs). The number of causative substances is large, mortality usually low. This retrospective cohort study aims to characterize differences of intoxicated compared to general ICU patients, point out variations according to causative agents, as well as to highlight differences between survivors and non-survivors among intoxicated individuals in a large-scale multi-center analysis. Methods A total of 105,998 general ICU patients and 4,267 individuals with the admission diagnoses “overdose” and “drug toxicity” from the years 2014 and 2015 where included from the eICU Collaborative Research Database. In addition to comparing these groups with respect to baseline characteristics, intensive care measures and outcome parameters, differences between survivors and non-survivors from the intoxication group, as well as the individual groups of causative substances were investigated. Results Intoxicated patients were younger (median 41 vs. 66 years; pppppppppp=0.26). In sensitivity analysis (interactions for age, sex, ethnicity, hospital category, maximum initial lactate, mechanical ventilation, and vasopressor use), a trend towards lower mortality in intoxicated patients persisted in all subgroups. Conclusion This large-scale retrospective analysis indicates a significantly lower mortality of intoxicated individuals compared to general ICU patients.

Originally Lipid droplets (LDs) were considered as being droplets for lipid storage only. Increasing evidence, however, demonstrates that LDs fulfill a pleiotropy of additional functions. Among them is the modulation of protein as well as lipid homeostasis. Under unfavorable pro-oxidative conditions, proteins can form aggregates which may exceed the overall proteolytic capacity of the proteasome. After stress termination LDs can adjust and support the removal of these aggregates. Additionally, LDs interact with mitochondria, specifically take over certain proteins and thus prevent apoptosis. LDs, which are loaded with these harmful proteins, are subsequently eliminated via lipophagy. Recently it was demonstrated that this autophagic process is a modulator of longevity. LDs do not only eliminate potentially dangerous proteins, but they are also able to prevent lipotoxicity by storing specific lipids. In the present study we used the model organism Saccharomyces cerevisiae to compare the proteome as well as lipidome of mitochondria and LDs under different conditions: replicative aging, stress and apoptosis. In this context we found an accumulation of proteins at LDs, supporting the role of LDs in proteostasis. Additionally, the composition of main lipid classes such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylglycerols, triacylglycerols, ceramides, phosphatidic acids and ergosterol of LDs and mitochondria changed during stress conditions and aging. Electronic supplementary material The online version of this article (10.1007/s11357-019-00103-0) contains supplementary material, which is available to authorized users.

Background Published evidence on the risk of additive carryover during phlebotomy remains elusive. We aimed to assess potential carryover of citrated and heparinized blood and the relative volume needed to bias clinical chemistry and coagulation tests. Methods We simulated standardized phlebotomies to quantify the risk of carryover of citrate and heparin additives in distilled water, using sodium and lithium as surrogates. We also investigated the effects of contamination of heparinized blood samples with increasing volumes of citrated blood and pure citrate on measurements of sodium, potassium, chloride, magnesium, total and ionized calcium and phosphate. Likewise, we studied the effects of contamination of citrated blood samples with increasing volumes of heparinized blood on heparin (anti-Xa) activity, lithium, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). We interpreted these results based on measurement deviations beyond analytical, biological and clinical significance. Results Standardized phlebotomy simulations revealed no significant differences in concentration of surrogate markers. Clinically significant alterations were observed after contamination of heparinized blood samples with volumes of citrated blood beyond 5–50 μL for ionized calcium and beyond 100–1000 μL for sodium, chloride and total calcium. Investigations of pure citrate carryover revealed similar results at somewhat lower volumes. Heparinized blood carryover showed clinically significant interference of coagulation testing at volumes beyond 5–100 μL. Conclusions Our results suggest that during a standardized phlebotomy, heparin or citrate contamination is highly unlikely. However, smaller volumes are sufficient to severely alter test results when deviating from phlebotomy guidelines.

The leukotriene receptor antagonist Montelukast (MTK) is an approved medication for the treatment of asthma and allergic rhinitis. The existing marketed tablet forms of MTK exhibit inconsistent uptake and bioavailability, which partially explains the presence of a significant proportion of MTK low- and non-responders in the population. Besides that, tablets are suboptimal formulations for patients suffering from dysphagia, for example, seen in patients with neurodegenerative diseases such as Alzheimer&rsquo, s disease, a disease with increasing interest in repurposing of MTK. This, and the need for an improved bioavailability, triggered us to reformulate MTK. Our aim was to develop a mucoadhesive MTK film with good safety and improved pharmacological features, i.e., an improved bioavailability profile in humans as well as in a mouse model of Alzheimer&rsquo, s disease. We tested dissolution of the MTK mucoadhesive film and assessed pharmacoexposure and kinetics after acute and chronic oral application in mice. Furthermore, we performed a Phase I analysis in humans, which included a comparison with the marketed tablet form as well as a quantitative analysis of the MTK levels in the cerebrospinal fluid. The novel MTK film demonstrated significantly improved bioavailability compared to the marketed tablet in the clinical Phase 1a study. Furthermore, there were measurable amounts of MTK present in the cerebrospinal fluid (CSF). In mice, MTK was detected in serum and CSF after acute and chronic exposure in a dose-dependent manner. The mucoadhesive film of MTK represents a promising alternative for the tablet delivery. The oral film might lower the non-responder rate in patients with asthma and might be an interesting product for repurposing of MTK in other diseases. As we demonstrate Blood-Brain-Barrier (BBB) penetrance in a preclinical model, as well as in a clinical study, the oral film of MTK might find its use as a therapeutic for acute and chronic neurodegenerative diseases such as dementias and stroke.

Serum levels of FGF23 have been associated with adverse outcomes in cardiovascular diseases in patients with and without impaired renal function. Hence, this study aimed to explore the prognostic relevance of intact FGF23 (iFGF23) and its derivate C-terminal FGF23 (cFGF23) in patients undergoing transcatheter aortic valve replacement (TAVR) with regard to renal function.A total of 274 patients undergoing transfemoral TAVR were enrolled in this study. Blood samples were obtained preinterventionally and analyzed for iFGF23 and cFGF23 by means of enzyme linked immunosorbent assay (ELISA). Follow-up was obtained for 12 months.Serum levels of cFGF23 and iFGF23 both correlated positively with serum creatinine and inversely with estimated glomerular filtration rate (eGFR). Cox regression analysis revealed a significant association of cFGF23 with 1-year-mortality in patients with eGFR ≥45ml/min/1.73m², but not in patients with an eGFR45ml/min/1.73m². A cut-off was calculated for cFGF23 (6.82 pmol/l) and patients with eGFR ≥45ml/min/1.73m² were retrospectively divided into two groups (above/below cut-off). Patients above the cut-off had a significantly worse 1-year-mortality than patients below the cut-off (33.3% vs. 19.6%; OR 2.05 (95%CI 1.03-4.07), p= 0.038). The association of cFGF23 with 1-year-mortality in patients with eGFR ≥45ml/min/1.73m² remained statistically significant even after correction for possible confounders in a multivariate Cox regression analysis.cFGF23 could be an individual risk factor for mortality in patients undergoing TAVR with an eGFR ≥45ml/min/1.73m².

Introducti on The appropriate use of laboratory diagnostics is increasingly at stake. The aim of this study was to depict some paradigmatic examples of under- and overutilization, as well as possible solutions across Europe.METHODS: We collected six examples from five European countries where a rise or decline of orders for specific laboratory parameters was observed after organizational changes but without evidence of changes in patient collective characteristics as source of this variation.RESULTS: The collected examples were the following: 1-Germany) Switch from a Brain-Natriuretic-Peptide assay to NT-pro Brain-Natriuretic-Peptide assay, resulting in a 374% increase in these analytics; 2-Spain) Implementation of a gatekeeping strategy in tumor marker diagnostics, resulting in a 15-61% reduction of these diagnostics; 3-Croatia) Stepwise elimination of creatine-kinase-MB assay from the laboratory portfolio; 4-UK) Removal of γ-glutamyl transferase from a "liver function" profile, resulting in 82% reduction of orders; 5-Austria) Implementation of a new device for rapid Influenza-RNA detection, resulting in a 450% increase of Influenza testing; 6-Spain) Insourcing of 1,25-(OH)2-Vitamin D measurements, leading to a 378% increase of these analyses.CONCLUSION: The six paradigmatic examples described in this manuscript show that availability of laboratory resources may considerably catalyze the demand, thus underscoring that inappropriate use of laboratory resources may be commonplace in routine laboratories all across Europe and most probably beyond. They also demonstrate that the application of simple strategies may assist in overcoming this issue. We believe that laboratory specialists need to refocus on the extra-analytical parts of the testing process and engage more in interdisciplinary patient-care.

Background: Heart failure (HF) remains one of the leading causes of death to date despite extensive research funding. Various studies are conducted every year in an attempt to improve diagnostic accuracy and therapy monitoring. The small cytoplasmic heart-type fatty acid-binding protein (H-FABP) has been studied in a variety of disease entities. Here, we provide a review of the available literature on H-FABP and its possible applications in HF. Methods: Literature research using PubMed Central was conducted. To select possible studies for inclusion, the authors screened all available studies by title and, if suitable, by abstract. Relevant manuscripts were read in full text. Results: In total, 23 studies regarding H-FABP in HF were included in this review. Conclusion: While, algorithms already exist in the area of risk stratification for acute pulmonary embolism, there is still no consensus for the routine use of H-FABP in daily clinical practice in HF. At present, the strongest evidence exists for risk evaluation of adverse cardiac events. Other future applications of H-FABP may include early detection of ischemia, worsening of renal failure, and long-term treatment planning.

Laboratory analyses are crucial for diagnosis, follow-up and treatment decisions. Since mistakes in every step of the total testing process may potentially affect patient safety, a broad knowledge and systematic assessment of laboratory errors is essential for future improvement. In this review, we aim to discuss the types and frequencies of potential errors in the total testing process, quality management options, as well as tentative solutions for improvement. Unlike most currently available reviews on this topic, we also include errors in test-selection, reporting and interpretation/action of test results. We believe that laboratory specialists will need to refocus on many process steps belonging to the extra-analytical phases, intensifying collaborations with clinicians and supporting test selection and interpretation. This would hopefully lead to substantial improvements in these activities, but may also bring more value to the role of laboratory specialists within the health care setting.

Introduction Too frequent HbA1c measurements may lead to unnecessary treatment modifications of diabetic patients. The aim of this study was to estimate the percentage of falsely elevated HbA1c results in two hospitals, Landeskrankenhaus/Uniklinikum Salzburg (LKH) and Landesklinik St. Veit (STV), as well as to retrospectively investigate the effect of an automated and an educative 60-day re-testing interval (RTI). Methods The amount of estimated falsely elevated results (eFER), based on odds calculated using the baseline and the follow-up values and the time between these measurements, the number of HbA1c re-testings within 60 days as well as the overall number of ordered and performed HbA1c analyses were calculated. In LKH, an automated algorithm cancelling inappropriate HbA1c testing was applied, and in STV, educational actions were taken. Results Before RTI- implementation, eFER were 0.9% and 2.1% and within-60-days-re-testing were 15.0% and 7.4% of cases in LKH and STV, respectively. After RTI- implementation, these numbers decreased to 0.2% (p < .001) and 1.8% (p = .869) and within- 60-days- re-testing decreased to 1.1% (p < .001) and 3.6% (p = .003) in LKH and STV, respectively. Median monthly HbA1c measurements decreased by 15.8% (p < .001) and 21.1% (p = .002) in LKH and STV, respectively. Conclusion Both the educational and the automated 60-day-RTI were proven to be efficient in reducing overall HbA1c measurements, re-testing within 60 days and eFER.

Colorectal cancer (CRC) is a major public health burden and one of the leading causes of cancer-related deaths worldwide. Screening programs facilitate early diagnosis and can help to reduce poor outcomes. Serum metabolomics can extract vital molecular information that may increase the sensitivity and specificity of colonoscopy in combination with histopathological examination. The present study identifies serum metabolite patterns of treatment-naïve patients, diagnosed with either advanced adenoma (AA) or CRC in colonoscopy screenings, in the framework of the SAKKOPI (Salzburg Colon Cancer Prevention Initiative) program. We used a targeted flow injection analysis and liquid chromatography-tandem mass spectrometry metabolomics approach (FIA- and LC-MS/MS) to characterise the serum metabolomes of an initial screening cohort and two validation cohorts (in total 66 CRC, 76 AA and 93 controls). The lipidome was significantly perturbed, with a proportion of lipid species being downregulated in CRC patients, as compared to AA and controls. The predominant alterations observed were in the levels of lyso-lipids, glycerophosphocholines and acylcarnitines, but additionally, variations in the quantity of hydroxylated sphingolipids could be detected. Changed amino acid metabolism was restricted mainly to metabolites of the arginine/dimethylarginine/NO synthase pathway. The identified metabolic divergences observed in CRC set the foundation for mechanistic studies to characterise biochemical pathways that become deregulated during progression through the adenoma to carcinoma sequence and highlight the key importance of lipid metabolites. Biomarkers related to these pathways could improve the sensitivity and specificity of diagnosis, as well as the monitoring of therapies.

Inappropriate laboratory test selection in the form of overutilization as well as underutilization frequently occurs despite available guidelines. There is broad approval among laboratory specialists as well as clinicians that demand management strategies are useful tools to avoid this issue. Most of these tools are based on automated algorithms or other types of machine learning. This review summarizes the available demand management strategies that may be adopted to local settings. We believe that artificial intelligence may help to further improve these available tools.

Objectives Regular aerobic exercise provides beneficial effects on human health and reduces all-cause mortality. Aerobic exercise has profound metabolic effects, and specific metabolites may reflect physiological changes. We aimed to identify endogenous metabolites that distinguish the trained from the untrained state to increase the spectrum of analytes amenable for hypothesis testing and to expand understanding of putative beneficial pathways. Design Cross sectional laboratory repeated measures study. Methods Exercise testing was performed in 37 healthy male participants and serum samples were obtained before and after completion of a ten-week standardized exercise program. Samples were analyzed for routine clinical parameters and for 188 endogenous metabolites by LC–MS/MS. Results Indicating the effectiveness of the intervention program, parameters of sport physiology were different after training. After correcting for multiple testing, serum concentrations of several metabolites differed between the trained and untrained state. Serine and glutamate decreased in response to exercise, whereas sarcosine and kynurenine increased. Phosphatidylcholines showed a mixed response in that four species increased and three decreased. However, all seven lysophosphatidylcholines and all four plasmalogens that differed between the trained and untrained state, increased. One short-chain acylcarnitine also decreased. In receiver operator characteristics analyses, sarcosine displayed the highest AUC value (0.839; 95% CI: 0.734–0.926) in discriminating the pre- from the post-trained state. Conclusions Our study detected metabolites that clearly differentiate the trained from the untrained state. These metabolites may be targeted in mechanistic studies to understand underlying biochemical pathways and could serve to improve the design, monitoring and individualization of training programs.

// Silvia Vidali 1 , Sepideh Aminzadeh-Gohari 1 , Rene Gunther Feichtinger 1 , Renaud Vatrinet 2 , Andreas Koller 1 , Felix Locker 1 , Tricia Rutherford 3 , Maura O’Donnell 3 , Andrea Stoger-Kleiber 3 , Bridget Lambert 3 , Thomas Klaus Felder 4 , Wolfgang Sperl 5 and Barbara Kofler 1 1 Laura Bassi Centre of Expertise-THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria 2 Department of Pharmacy and Biotechnology (FABIT), University of Bologna, Bologna, Italy 3 Clinical Nutrition, Vitaflo International Ltd, Liverpool, UK 4 Department of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria 5 Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria Correspondence to: Barbara Kofler, email: b.kofler@salk.at Keywords: renal cell carcinoma, mitochondria, ketogenic diet, metabolism, Warburg effect Received: March 10, 2017 Accepted: June 27, 2017 Published: July 17, 2017 ABSTRACT The ketogenic diet (KD), a high-fat low-carbohydrate diet, has shown some efficacy in the treatment of certain types of tumors such as brain tumors and neuroblastoma. These tumors are characterized by the Warburg effect. Because renal cell carcinoma (RCC) presents similar energetic features as neuroblastoma, KD might also be effective in the treatment of RCC. To test this, we established xenografts with RCC 786-O cells in CD-1 nu/nu mice and then randomized them to a control diet or to KDs with different triglyceride contents. Although the KDs tended to reduce tumor growth, mouse survival was dramatically reduced due to massive weight loss. A possible explanation comes from observations of human RCC patients, who often experience secondary non-metastatic hepatic dysfunction due to secretion of high levels of inflammatory cytokines by the RCCs. Measurement of the mRNA levels of tumor necrosis factor alpha (TNFα) and interleukin-6 revealed high expression in the RCC xenografts compared to the original 786-O cells. The expression of TNFα, interleukin-6 and C-reactive protein were all increased in the livers of tumor-bearing mice, and KD significantly boosted their expression. KDs did not cause weight loss or liver inflammation in healthy mice, suggesting that KDs are per se safe, but might be contraindicated in the treatment of RCC patients presenting with Stauffer’s syndrome, because they potentially worsen the associated hepatic dysfunction.

We report on a preanalytical issue we encountered during routine clinical chemistry analyses, potentially leading to deviated analysis results and believe that it might help other laboratories to overcome similar problems. In a heparin-gel tube we measured an implausible glucose value of 0.06 mmol/L. Re-measurement of the same sample resulted in a glucose value of 5.4 mmol/L. After excluding an analytical error, we inspected the sample closer and found a white material as well as fatty droplets floating on the surface of the plasma tube. Evaluation of these structures revealed that the white particulate matter (WPM) consisted of fibrinogen, platelets and leukocytes and the fatty droplets most probably originated from the separator gel. We concluded that these structures formed a temporary clot in the instruments pipetting needle thereby altering the sampling volume and subsequently the measured glucose value. The formation of WPM might be attributable to high speed centrifugation, high cholesterol levels, the gel formulation or a combination of several issues such as temperature, heparin concentration, pH and patient-specific factors. The gel droplets were most probably caused by an aberrant gel formulation in combination with an improper storage of the empty tubes on the wards prior to phlebotomy. After adding an additional instrument cleansing cycle and changing to another batch of heparin tubes the problems could be significantly reduced.

The chroman-like chalcone Xanthohumol C, originally found in hops, was demonstrated to be a potent neuroregenerative and neuroprotective natural product and therefore constitutes a strong candidate for further pharmaceutical research. The bottleneck for in vivo experiments is the low water solubility of this chalcone. Consequently, we developed and validated a suitable formulation enabling in vivo administration. Cyclodextrins were used as water-soluble and nontoxic complexing agents, and the complex of Xanthohumol C and 2-hydroxypropyl-β-cyclodextrin was characterized using HPLC, HPLC-MS, NMR, and differential scanning calorimetry. The water solubility of Xanthohumol C increases with increasing concentrations of cyclodextrin. Using 50 mM 2-hydroxypropyl-β-cyclodextrin, solubility was increased 650-fold. Furthermore, in vitro bioactivity of Xanthohumol C in free and complexed form did not significantly differ, suggesting the release of Xanthohumol C from 2-hydroxypropyl-β-cyclodextrin. Finally, a small-scaled in vivo experiment in a rat model showed that after i. p. administration of the complex, Xanthohumol C can be detected in serum, the brain, and the cerebrospinal fluid at 1 and 6 h post-administration. Mean (± SD) Xanthohumol C serum concentrations after 1, 6, and 12 h were determined as 463.5 (± 120.9), 61.9 (± 13.4), and 9.3 (± 0.8) ng/mL upon i. v., and 294.3 (± 22.4), 45.5 (± 0.7), and 13 (± 1.0) ng/mL after i. p. application, respectively. Accordingly, the formulation of Xanthohumol C/2-hydroxypropyl-β-cyclodextrin is suitable for further in vivo experiments and further pharmaceutical research aiming for the determination of its neuroregenerative potential in animal disease models.

Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call lipid droplet-accumulating microglia (LAM), are defective in phagocytosis, produce high levels of reactive oxygen species, and secrete pro-inflammatory cytokines. RNA sequencing analysis of LAM revealed a transcriptional profile driven by innate inflammation distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin, are causes of autosomal dominant forms of human neurodegenerative diseases. We thus propose that LAM contribute to age-related and genetic forms of neurodegeneration.

BackgroundMarinobufagenin (MBG) is an endogenous cardiotonic steroid (CTS) that inhibits the Na+/K+-ATPase. Human MBG is significantly increased in end-stage renal disease and immunization against MBG attenuates cardiovascular fibrosis in a rat model of uremic cardiomyopathy. Mineralocorticoid antagonists (MRA) block MBG binding sites and decrease proteinuria in chronic kidney disease (CKD) patients. We therefore aimed to investigate the association of MBG and albuminuria, as a marker of renal damage, as well as MBG and decline of glomerular filtration rate (GFR).MethodsThe Graz endocrine causes of hypertension (GECOH) study is a single center study of adults routinely referred for screening of endocrine hypertension. Plasma MBG was measured by an enzyme-linked immunoassay, and in a post-hoc analysis, follow-up creatinine levels were obtained. Patients with proteinuria >3.5g/day at baseline were excluded from further evaluation.ResultsWe measured MBG concentrations in 40 hypertensive subjects and excluded one patient due to pre-existing proteinuria. Plasma MBG was significantly correlated with albuminuria (Spearman ρ = .357; p = .028) and proteinuria (ρ = .336; p = .039). In linear regression analysis, the association remained significant after adjustment for age, sex, and BMI (β = .306; p = .036), and for mean systolic blood pressure (β = .352; p = .034). In follow-up analyses (N = 30), MBG was significantly associated with decline in GFR after adjustment for time-to-follow-up (β = -.374; p = .042).ConclusionThe findings suggest that MBG plasma concentrations were associated with albuminuria as well as decline in kidney function. Whether MBG predicts hard renal endpoints warrants further investigations.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17 kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17 kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (P = .0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.

As a small proportion of obese individuals do not develop metabolic complications and non-alcoholic fatty liver disease (NAFLD), this study aimed to provide a comprehensive clinical, metabolic and genetic description of obese subjects with healthy livers.A total of 183 subjects were stratified, according to BMI, presence of metabolic syndrome, biochemical liver tests and hepatic steatosis on ultrasound, into: (i) lean controls (n = 69); (ii) obese healthy (n = 50); and (iii)obese NAFLD (n = 62) groups. Detailed clinical, genetic and metabolic evaluations were then performed.Obese healthy subjects did not differ in glucose parameters from lean controls, and had a lower rate of minor TM6SF2 gene variants compared with obese NAFLD (2/49 vs. 11/60, respectively; P = 0.035) and lean controls (13/64; P = 0.035), but significantly higher leptin concentrations than lean controls (P 0.001); they also higher adiponectin concentrations (P 0.001), and lower TNF-α and IL-6 concentrations (P = 0.01 and P 0.001, respectively), than obese NAFLD subjects. Also, metabolomic studies identified ether- and ester-containing phospholipids [PC ae C44:6, PC ae C42:5, PC aa C40:4; P 0.001, corrected by the false discovery rate (FDR) method] and found that the amino-acids lysine, glycine and isoleucine (FDR 0.001) differed between the two obese groups, but not between lean controls and obese healthy subjects.Obese people with healthy livers are characterized by intact glucose homoeostasis, lower pro-inflammatory cytokine levels, and higher adiponectin and leptin concentrations compared with obese people with NAFLD. In addition, the major allele of TM6SF2, a set of phosphatidylcholines and several amino acids are associated with healthy livers in obesity.

Introduction: Although centrifugation is performed in almost every blood sample, recommendations on duration and g-force are heterogeneo-us and mostly based on expert opinions. In order to unify this step in a fully automated laboratory, we aimed to evaluate different centrifugation settings and their influence on the results of routine clinical chemistry analytes. Materials and methods: We collected blood from 41 healthy volunteers into BD Vacutainer PST II-heparin-gel- (LiHepGel), BD Vacutainer SST II-serum-, and BD Vacutainer Barricor heparin-tubes with a mechanical separator (LiHepBar). Tubes were centrifuged at 2000xg for 10 minutes and 3000xg for 7 and 5 minutes, respectively. Subsequently 60 and 21 clinical chemistry analytes were measured in plasma and serum samples, respec-tively, using a Roche COBAS instrument. Results: High sensitive Troponin T, pregnancy-associated plasma protein A, ß human chorionic gonadotropin and rheumatoid factor had to be excluded from statistical evaluation as many of the respective results were below the measuring range. Except of free haemoglobin (fHb) measure-ments, no analyte result was altered by the use of shorter centrifugation times at higher g-forces. Comparing LiHepBar to LiHepGel tubes at different centrifugation setting, we found higher lactate-dehydrogenase (LD) (P = 0.003 to < 0.001) and lower bicarbonate values (P = 0.049 to 0.008) in the lat ter. Conclusions: Serum and heparin samples may be centrifuged at higher speed (3000xg) for a shorter amount of time (5 minutes) without alteration of the analytes tested in this study. When using LiHepBar tubes for blood collection, a separate LD reference value might be needed.

Background Inappropriate utilization of laboratory resources is an increasing concern especially in high-throughput facilities. Until now, no reliable information has been published addressing to which extent laboratory results are actually used for clinical decision-making. Therefore, we aimed to close this gap using a novel retrospective approach including a survey of clinicians and nurses. Methods We retrospectively evaluated the number of re-orders for potassium (K), lactate dehydrogenase (LD), aspartate-aminotransferase (AST), activated partial thromboplastin-time (APTT) and prothrombin-time/INR (PT/INR), after the initial order had to be cancelled due to preanalytical non-conformities. We analyzed subgroups regarding time to re-order, ward and sample priority (urgent vs. routine). Subsequently, we surveyed clinicians and nurses, asking for their estimate of the amount of failed re-orders as well as for possible reasons. Results From initially cancelled tests, only ~20% of K, LD, AST and ~30% of APTT and PT/INR tests were re-ordered within 24 h. 70% of the investigated clinical chemistry and 60% of coagulation tests were re-ordered one week after cancellation or not at all. Survey participants quite accurately estimated these numbers. Routine laboratory panels, short stay of out-patients, obsolete test results and avoiding additional phlebotomies were the main reasons for not re-ordering cancelled tests. Conclusions Overall, 60–70% of test results in the investigated assays ordered in a high throughput laboratory are potentially inappropriate or of doubtful clinically importance. Although clinicians and nurses are aware of this situation, it is the duty of laboratory specialists to overcome overutilization in close collaboration with all involved healthcare workers.

Background: Hemolytic specimens are among the most challenging preanalytical issues in laboratory diagnostics. The type of blood collection tube in use is claimed to influence in vitro hemolysis. We aimed to examine this hypothesis and estimate the respective financial impact, evaluating routine blood samples from the past 4 years. Methods: A total of 47,820 hemolysis index (HI) values from five different time intervals (IV1–IV5) were compared against each other, representing the following tubes: IV1-Sarstedt Monovette; IV2-8 mL/16×100 mm Greiner BioOne (GBO) Vacuette; IV3/IV4-5 mL/16×100 mm GBO Vacuette; IV5-4.5 mL/13×75 mm GBO Vacuette. For estimation of the economic impact, material, personnel and analytical costs were calculated. Results: HI mean values in time interval IV2 were significantly higher than in all other intervals, while mean values amongst all other intervals were comparable. The number of moderately and severely hemolyzed samples increased with incrementing vacuum. Overall comparable costs between intervals IV1 and IV5 were €11,370, €14,045, €12,710, €11,213 and €8138 per 10,000 samples, respectively. Conclusions: Aspiration tubes and low vacuum tubes show comparable hemolysis rates. Increasing vacuum levels are associated with higher hemolysis rates. By decreasing in vitro hemolysis, financial savings up to €5907 per 10,000 samples could be gained.

BACKGROUND Carotid endarterectomy is widely performed under regional anaesthesia. Ultrasound guidance is increasingly used in many regional anaesthetic procedures to improve safety and efficacy, and because it can reduce the amount of local anaesthetic required. Despite this, an ideal approach and dosing regimen for cervical plexus block remain elusive. OBJECTIVE The aim of this study was to compare two different concentrations of ropivacaine in terms of analgesic adequacy, haemodynamic effects and plasma concentration using an ultrasound-guided triple approach for intermediate cervical plexus blockade. DESIGN A randomised, placebo-controlled, blinded study. SETTING University Clinic Salzburg, Department of Anaesthesiology, Perioperative Medicine and Intensive Care, Paracelsus Medical University, Salzburg, Austria, from 16 November 2012 to 17 September 2013. PATIENTS Forty-six patients prospectively randomised to receive ultrasound-guided intermediate cervical block with either 20 ml ropivacaine 0.75% or 20 ml ropivacaine 0.375% each with 20 ml prilocaine 1%. INTERVENTION After subcutaneous infiltration, blocks were performed using ultrasound-guided infiltration below the sternocleidomastoid muscle, and ultrasound-guided infiltration of the carotid sheath. Ropivacaine and prilocaine plasma concentrations were measured at intervals. MAIN OUTCOME The primary study endpoint was the volume of supplementary lidocaine 1% required to achieve adequate surgical anaesthesia. Perioperative haemodynamic variables and pain scores were recorded. RESULTS There was no statistical difference in the volume of supplementary lidocaine given: 5.0 (±3.63) ml in the ropivacaine 0.375% group and 5.17 (±2.76) ml in the ropivacaine 0.75% group (P = 0.846). Pain scores were similarly low across both groups. Measured concentrations of ropivacaine and prilocaine did not reach toxic levels in either group. Levels of ropivacaine were approximately two-fold higher in the 0.75% group [mean area under the curve (AUC) 10 531.11 (±2912.84) vs. 5264.34 ng (±1594.69), P

Epidermolysis bullosa simplex (EBS) is a rare genetic, blistering skin disease for which there is no cure. Treatments that address the pathophysiology of EBS are needed.Compare the impact of 1% diacerein cream with placebo in reducing the number of blisters in EBS.In a randomized, placebo-controlled, phase 2/3 trial we used a 1% diacerein topical formulation to treat defined skin areas in 17 patients. In a 2-period crossover trial, patients were randomized to either placebo or diacerein for a 4-week treatment and a 3-month follow-up in period 1. After a washout, patients were crossed over during period 2. The prespecified primary end point was the proportion of patients with a reduction of number of blisters by more than 40% from baseline in selected areas over the treatment episode.Of the patients receiving diacerein, 86% in episode 1 and 37.5% in episode 2 met the primary end point (vs 14% and 17% with placebo, respectively). This effect was still significant after the follow-up. Changes in absolute blister numbers were significant for the diacerein group only. No adverse effects were observed.Low patient numbers and no invasive data acquisition because of clinical burden in children.This trial provides evidence of the impact of 1% diacerein cream in the treatment of EBS.