In-house Standards Research Articles

Saccharide dosage content of meningococcal polysaccharide conjugate vaccines determined using WHO International Standards for serogroup A, C, W, Y and X polysaccharides

Potency of meningococcal polysaccharide-protein conjugate vaccines relies on the polysaccharide content to prevent meningitis. NIBSC, as the official national control laboratory in UK, analysed ten different mono- and multi-meningococcal conjugate vaccines, using established International Standards for meningococcal serogroups A, C, W, Y and X, by resorcinol or HPAEC-PAD assay. Most saccharide contents were within ±20% of their claimed content for licensure with taking different O-acetylation levels into consideration, with only MenC content in two vaccines below (by 60% and 54%) the labelled value, however, previous study showed different dosage was not necessarily correlated to the immunogenicity of those vaccines. This study demonstrated the use of International Standards to quantify saccharide content in polysaccharide-based vaccines with different percentage of O-acetylation. These International Standards are suitable to serve as either quantitative standard or calibrator of in-house standards, with supplied stability data.

Xenometabolite signatures in the UC Davis type 2 diabetes mellitus rat model revealed using a metabolomics platform enriched with microbe-derived metabolites.

The gut microbiome has the potential to create or modify xenometabolites (i.e., nonhost-derived metabolites) through de novo synthesis or modification of exogenous and endogenous compounds. While there are isolated examples of xenometabolites influencing host health and disease, wide-scale characterization of these metabolites remains limited. We developed a metabolomics platform ("XenoScan") using liquid chromatography-mass spectrometry to characterize a range of known and suspected xenometabolites and their derivatives. This assay currently applies authentic standards for 190 molecules, enriched for metabolites of microbial origin. As a proof-of-principle, we characterized the cecal content xenometabolomics profile in adult male lean Sprague-Dawley (LSD) and University of California, Davis type 2 diabetes mellitus (UCD-T2DM) rats at different stages of diabetes. These results were correlated to specific bacterial species generated via shotgun metagenomic sequencing. UCD-T2DM rats had a unique xenometabolite profile compared with LSD rats, regardless of diabetes status, suggesting that at least some of the variation is associated with host genetics. Furthermore, modeling approaches revealed that several xenometabolites discriminated UCD-T2DM rats at early stages of diabetes versus those at 3 mo postdiabetes onset. Several xenometabolite hubs correlated with specific bacterial species in both LSD and UCD-T2DM rats. For example, indole-3-propionic acid negatively correlated with species within the Oscillibacter genus in UCD-T2DM rats considered to be prediabetic or recently diagnosed diabetic, in contrast to gluconic acid and trimethylamine, which were positively correlated with Oscillibacter species. The application of a xenometabolite-enriched metabolomics assay in relevant milieus will enable rapid identification of a wide variety of gut-derived metabolites, their derivatives, and their potential biochemical origins of xenometabolites in relationship to host gastrointestinal microbial ecology.NEW & NOTEWORTHY We debut a liquid chromatography-mass spectrometry (LC/MS) platform called the XenoScan, which is a metabolomics platform for xenometabolites (nonself-originating metabolites). This assay has 190 in-house standards with the majority enriched for microbe-derived metabolites. As a proof-of-principle, we used the XenoScan to discriminate genetic differences from cecal samples associated with different rat lineages, in addition to characterizing diabetes progression in rat model of type 2 diabetes. Complementing microbial sequencing data with xenometabolites uncovered novel microbial metabolism in targeted organisms.

Intestinal, Hepatic, Muscle, and Renal Xenometabolite Release and Uptake in Healthy Conscious Pigs

Abstract Objectives To characterize the intestinal, hepatic, muscle, and renal release and uptake of xenometabolites (non-host derived metabolites) and endogenously modified xenometabolites by measuring their across-organ net release and uptake in a post-absorptive conscious pig model. Methods Twelve 2–3 months old female pigs (25.6 ± 2.2 kg) had surgically-implanted catheters across portal drained viscera (PDV), splanchnic area (SPL), liver, kidney, and hindquarter muscle. Ten days after catheter implantation, overnight fasted arterial and venous plasma was collected in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Xenometabolites were identified by retention time and MS2 spectra from in-house authentic standards and expressed as peak areas. Plasma flow measurements determined with para-aminohippuric acid dilution technology. Net organ balance rate (peak area/kg body weight/min) was calculated. Significant (P < 0.05) release or uptake was determined if tissue net balance differed from zero, using the Wilcoxon Signed Rank Test. Results Forty-eight xenometabolites were structurally identified in this model. Of these, 31 had at least one tissue with a significant net release or uptake. Metabolites suggestive of intestinal origin (i.e., significant PDV net release) included 2-hydroxybenzoic acid, 4-hydroxyphenyllactic acid, acetic acid, atrolactic acid, dodecanedioic acid, hydroxycinnamic acid, p-cresol sulfate, p-cresol glucuronide, stercobilin, and several bile acids and indole derivatives. Of these, atrolactic acid, hydrocinnamic acid, indole-3-acetic acid, stercobilin, and all of the bile acids were taken up by the liver; p-cresol glucuronide was taken up by the kidneys. No xenometabolite released from the gut was taken up by muscle, but 4-hydroxyphenyllactic acid, dodecanedioic acid, and indole-3-carboxaldehyde were also released by muscle tissue. Conclusions Our study confirm gastrointestinal origins for a number of known xenometabolites. Liver and kidney are key organs for xenometabolite uptake. Other putative xenometabolites appear to have either non-gut origins or released by other tissues. Funding Sources Funding provided by the US Department of Agriculture-Agricultural Research Service.

Chemical Separation of Uranium and Precise Measurement of 234U/238U and 235U/238U Ratios in Soil Samples Using Multi Collector Inductively Coupled Plasma Mass Spectrometry

A new chemical separation has been developed to isolate uranium (U) using two UTEVA columns to minimize iron and thorium interferences from high background area soil samples containing minerals like monazites and ilmenite. The separation method was successfully verified in some certified reference materials (CRMs), for example, JSd-2, JLk-1, JB-1 and JB-3. The same method was applied for purification of U in Fukushima soil samples affected by the Fukushima dai-ichi nuclear power station (FDNPS) accident. Precise and accurate measurement of 234U/238U and 235U/238U isotope ratios in chemically separated U were carried out using a multi-collector inductively coupled plasma mass spectrometer (MC-ICP-MS). In this mass spectrometric method, an array of two Faraday cups (1011 Ω, 1012 Ω resistor) and a Daly detector were simultaneously employed. The precision of U isotope ratios in an in-house standard was evaluated by replicate measurement. Relative standard deviation (RSD) of 234U/238U and 235U/238U were found to be 0.094% (2σ) and 0.590% (2σ), respectively. This method has been validated using a standard reference material SRM 4350B, sediment sample. The replicate measurements of 234U/238U in SRM shows 0.7% (RSD). This developed method is suitable for separation of U and its isotope ratio measurement in environmental samples.

DELUSIONAL SEVERITY IN A COGNITIVELY MIXED SAMPLE OF OLDER ADULTS IS ASSOCIATED WITH ABNORMALITIES IN WHITE MATTER TEXTURE

Introduction Background: Delusions occur in older patients and may be a harbinger of later cognitive impairment. Studies to date have suggested a correlation with white matter lesion burden [2] [3] but the correlation between delusions and white matter texture (WMT) is not well understood. White matter texture refers to tissue organization in normal-appearing brain matter (NABM) related to the overall diffusion capabilities of brain tissue. There is growing interest in analyzing these regions of the brain since they may contain early signs of disease [4]. Whether delusion severity is correlated with WMT is not so clear. Knowing the relationship between delusional severity and WMT may provide insight into potential mechanisms. Objective: Using neuroimaging biomarkers measured from 212 FLAIR MRI volumes, we investigated the relationship between delusional level and texture of the NABM in a cognitively mixed cohort extracted from the Alzheimer's Disease Neuroimaging (ADNI) data set. Methods Method: We extracted a cognitively diverse sample of patients with delusions based on NPI-Q scores from the ADNI data set. The delusion group was organized according to severity (1, 2, 3) based on Neuropsychiatric Inventory-Quick (NPI-Q) scores, with 1 being the lowest severity, and FLAIR MRI volumes for each subject was downloaded. There were 32 subjects (130 volumes) in group 1, 11 subjects (49 volumes) in group 2 and 9 subjects (33 volumes) in group 3 for analysis. Using validated, in-house brain extraction and intensity standardization algorithms [5][6][7], the normal-appearing brain matter (NABM) of FLAIR images were extracted. Texture analysis was performed on the NABM with a 2D texture feature that measures spatial correlation among pixel intensities [1]. This 2D spatial correlation metric was extracted on a per pixel basis, using 3mm x 3mm windows to find local, microstructural texture differences. The spatial feature maps were averaged over all slices to find a volume representation and a slight erosion was applied to remove outliers caused by the large brain-background edge. From the volume's correlation histogram, the bottom 2% and top 98% of the features are removed to further improve robustness to outliers. The mean and median of these feature maps were statistically compared between delusion groups (1, 2, 3). High spatial correlation indicates neighboring pixels have similar intensities (and is more homogeneous). Results Results: Boxplots for the NABM texture measures are shown for groups 1, 2 and 3 in Figure 1. Subjects with high delusional severity (group 3) had the most noticeable change in WMT when compared to lowest delusional severity (group 1). Group 3 had a higher spatial correlation value of 6.0947 e+03 +/- 592.6178 than group 1 with values of 5.7630 e+03 +/- 832.5040. A higher value indicates more spatial correlation among pixels (i.e. local homogeneity since closer pixels are more likely to have similar intensity values). This suggests that there are larger regions that are smooth in Group 3 compared to Group 1, which could indicate less WM “structure” in the more severe delusions group. This effect is shown in Figure 2. T-test results in Table 1 shows the highest difference is between groups 1 and 3 when examining the mean and median values with p-values both Conclusions Discussion: This study provides the first evidence that severity of delusions in a cognitively diverse sample of older adults is correlated with abnormalities in white matter texture. This finding suggests that alterations in white matter texture may contribute to the development of delusions in older patients. There is a notable difference between an individual with mild symptoms and severe symptoms based on their white matter texture as shown. Due to the subjective nature of an individual being scored either NPI-Q 1 or 2, or, 2 or 3, differentiating between mild and moderate, or moderate and severe disease is often ill-defined. This may contribute to challenges in differentiating between these groups. Further study comparing our results to patients without delusions and controlling for severity of cognitive impairment is required to further validate our findings. This research was funded by: St. Michael's Hospital Foundation, Heather and Eric Donnelly endowment

Risk factors associated with progressive lacunar strokes and benefit from dual antiplatelet therapy

Early neurological deterioration (END) occurs in 20%-30% of patients with lacunar stroke and challenges their clinical management. This retrospective cohort study analyzed clinical and neuroimaging risk factors predicting the occurrence of END, the functional outcome after END and potential benefit from dual antiplatelet therapy (DAPT) in patients with lacunar strokes. Factors associated with END and benefit from DAPT were retrospectively analyzed in 308 patients with lacunar stroke symptoms and detected lacunar infarction by magnetic resonance imaging. END was defined by deterioration of ≥3 total National Institutes of Health Stroke Scale (NIHSS) points, ≥2 NIHSS points for limb paresis or documented deterioration within 5 days after admission. Patients were treated with DAPT according to in-house standards. The primary efficacy end-point for functional outcome was fulfilled if NIHSS at discharge improved after END at least to the score at admission. Male gender [odds ratio (OR)2.08; 95% confidence interval (CI) 1.09-4.00], higher age (OR=1.65 per 10years; 95% CI 1.18-2.31), motor paresis (OR=18.89, 95% CI 4.66-76.57) and infarction of the internal capsule or basal ganglia (OR=3.58, 95% CI 1.26-10.14) were associated with an increased risk for END. A larger diameter of infarction (OR=0.85, 95% CI 0.76-0.95), more microangiopathic lesions (OR=0.75, 95% CI 0.57-0.99) and pontine localization (OR=0.29, 95% CI 0.12-0.65) were factors associated with unfavorable functional outcome after END occurred. Localization in the internal capsule or basal ganglia was identified as a significant predictive factor for a benefit from DAPT after END. Identified clinical and neuroimaging factors predicting END occurrence, functional outcome after END and potential benefit from DAPT might improve the clinical management of patients with lacunar strokes.

Stable Isotope Analysis of Intact Oxyanions Using Electrospray Quadrupole-Orbitrap Mass Spectrometry.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1-2‰ for sulfate δ34S and δ18O and nitrate δ15N and δ18O, based on in-house and international standards. Less abundant variants such as δ17O, δ33S, and δ36S, and the 34S-18O "clumped" sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine 18O/34S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

Development and validation of a HPLC method for simultaneous quantitative determination of adapalene and benzoyl peroxide in gel products

Adapalene (AP) and Benzoyl peroxide (BO) have been used to treat acnes very efficient for many years, especially in gel products. But nowaday, that gel products still don't have in-house standard. And furthermore, the most difficult thing to build in-house standard is to assay simultaneously. The aim of this research is to build a method to assay two of AP and BO simultaneously by HPLC method. Subjects: AP (from India) and BO (from China). Solvents get the quality standard to use in liquid chromatography. Sample: gel product (AP 0.1% and BO 2.5%) prepared at Laboratory of School of Medicine, National University of HCMC. Chromatographic system: HPLC Shimadzu system with column C18 RP (250 x 4.6 mm; 5 mm), injection volumn 20 mL, detector at 270 nm, flow rate 1 mL/min, temperature 30oC, mobile phase with acetonitril - tetrahydrofuran - acid formic 0.1% in water 42 : 32 : 26. Validations: system suitability, specificity, linearity, accuracy, precision adapt to ICH. Results: The HPLC method for simultaneous quantitative determination of adapalene and benzoyl peroxide in gel products was built successfully. The method was achieved all validations term adapt to ICH: system suitability, specificity, linearity, accuracy, precision. Furthermore, that method is used to assay the concentration of two API (AP and BO) in the product (prepared at Laboratory of School of Medicine, National University of HCMC) with result: the concentration of AP is 101.38 0.87% and that of BO is 95.56 0.04 %, get the standard of USP 41.

Computer aided design of knitted and woven fabrics and virtual garment simulation

Pattern cutting, sizing and fit are major issues for the clothing brands. All fashion companies are interested on how the product will fit their target customers and this involves making samples that eventually will not look or fit as per the desired design. 3D technology solutions are truly the best to deal with the existing needs of clothing manufacturers in order to diminish the costs and time of the sampling process, to improve the quality and reduce the rejects. In this study, the authors used automation techniques like computer pattern design, computer aided fabric production and 3D Simulation software. Two different fabrics (woven and knitted) were designed and produced. Then their material properties which have to be known by the 3D simulation were determined by official and in-house standards. Finally, 3D visualizations of dresses were created by using pattern and material data of the fabrics. By this study we were able to explain the product development route, from fabric design to garment prototype by using computers, for the companies willing to benefit from the advantages of computer aided design and manufacture.

Novel Method of Extraction for Radiocarbon Measurements of Atmospheric Carbon dioxide

ABSTRACTIn this paper, we present the first data from an alternative extraction method for atmospheric 14CO2 analysis, based on the direct trapping of whole air samples onto a molecular sieve zeolite (13X) trap, incorporated into a commercially available automated graphitization system. Results are presented for both inter-laboratory comparison samples and an in-house reference standard. The in-house reference was used to calculate the standard deviation of measurements (2.0‰). This newly developed method will facilitate faster sample processing and therefore lower cost per analysis, critical for scaling up such studies.

Development of Reference Control Material for the Evaluation of the Cepheid Prototype * Xpert® BCR-ABL P190 Ultra Assay

Background: Patients with Philadelphia+ Acute Lymphoid Leukemia (Ph+ ALL) respond to the same tyrosine kinase inhibitors (TKIs) that work against Chronic Myeloid Leukemia (CML). However, the dominant BCR-ABL breakpoint is the e1a2 (p190) minor transcript with the e13a2/e14a2 (p210) major transcripts, accounting for 20-30% of the ALL patients, together with <5% of the CML patients. Currently, unlike the p210 major transcripts for CML, there are no International Standards available for the quantitation of the e1a2 p190 transcript for both CML and ALL. This poses a challenge when developing a quantitative assay to monitor the e1a2 p190 transcript levels. Objectives: Implementation of a certified plasmid DNA (pDNA) reference material through copy number (CN) calibration was used to develop a primary control pDNA at Cepheid. The reference pDNA contains ABL and BCR-ABL b3a2-p210 (IRMM_ERM-AD623 BCR-ABL Calibrator) for standardization of ABL and BCR-ABL e1a2-p190 quantifications. This process allows generation of in-house secondary RNA control panel, for calibration and standardization of the Xpert BCR-ABL p190 Ultra assay to the WHO IS (NIBSC-09/138). Method: Titration studies were performed with IRMM's pDNA to derive a CN calibration curve of the ABL control gene relative to ABL Cycle Threshold (Ct), to assign CN to Cepheid's pDNA containing e1a2 target gene and ABL control gene in a 1:1 ratio. The obtained CN information for Cepheid's pDNA was then used to generate corresponding ABL Ct and e1a2 Ct in a simulated biological matrix, followed by the assignment of CN to the in-house RNA control material. A 5-log concentration range of a malignant BCR-ABL e1a2 positive ALL cell line, SUP-B15, is used as an interim in-house control material with an assigned % BCR-ABL e1a2/ABL value to evaluate the assay's linearity/dynamic range performance. Results: Through the ABL calibration curve generated from IRMM reference material, CN of Cepheid's e1a2:ABL pDNA were assigned. The ABL Ct was generated using IRMM reference material with a 6-log dynamic range of ABL CN, slope of -1.41, R2=0.9924 (Figure 1). Using the ABL Ct obtained from the Cepheid's pDNA construct, allowed us to link Cepheid's pDNA ABL CN to IRMM's, thus linking e1a2 CN to ABL CN, demonstrated in a 5-log dynamic range of ABL and e1a2 CN. Similar slopes of -1.491, R2=0.9972 and -1.464, R2=0.9966 were obtained from Cepheid's pDNA between input CN and output Ct for e1a2 and ABL, respectively (Figure 2). Finally, a 5-log dynamic range of ∆Ct (e1a2-ABL) was generated from SUP-B15 cells, yielding slope of -1.427, R2=0.9917 (Figure 3), allowing assignment of %BCR-ABL e1a2/ABL value. Conclusion: In conclusion, implementing a certified pDNA reference material (IRMM) allowed linkage of known ABL CN to Cepheid's pDNA. Preliminary data from SUP-B15 cells suggested that we achieved the goal of generating a control material with assigned e1a2 CN. * Prototype test. Not for use in diagnostic procedures. Not reviewed by any regulatory body. Disclosures No relevant conflicts of interest to declare.

Toward Improved Accuracy in Chlorine Isotope Analysis: Synthesis Routes for In-House Standards and Characterization via Complementary Mass Spectrometry Methods.

Increasing applications of compound-specific chlorine isotope analysis (CSIA) emphasize the need for chlorine isotope standards that bracket a wider range of isotope values in order to ensure accurate results. With one exception (USGS38), however, all international chlorine isotope reference materials (chloride and perchlorate salts) fall within the narrow range of one per mille. Furthermore, compound-specific working standards are required for chlorine CSIA but are not available for most organic substances. We took advantage of isotope effects in chemical dehalogenation reactions to generate (i) silver chloride (CT16) depleted in 37Cl/35Cl and (ii) compound-specific standards of the herbicides acetochlor and S-metolachlor (Aceto2, Metola2) enriched in 37Cl/35Cl. Calibration against the international reference standards USGS38 (-87.90 ‰) and ISL-354 (+0.05 ‰) by complementary methods (gas chromatography-isotope ratio mass spectrometry, GC-IRMS, versus gas chromatography-multicollector inductively coupled plasma mass spectrometry, GC-MC-ICPMS) gave a consensus value of δ37ClCT16 = -26.82 ± 0.18 ‰. Preliminary GC-MC-ICPMS characterization of commercial Aceto1 and Metola1 versus Aceto2 and Metola2 resulted in tentative values of δ37ClAceto1 = 0.29 ± 0.29 ‰, δ37ClAceto2 = 18.54 ± 0.20 ‰, δ37ClMetola1 = -4.28 ± 0.17 ‰ and δ37ClMetola2 = 5.12 ± 0.27 ‰. The possibility to generate chlorine isotope in-house standards with pronounced shifts in isotope values offers a much-needed basis for accurate chlorine CSIA.

Chemodiversity of Calophyllum inophyllum L. oil bioactive components related to their specific geographical distribution in the South Pacific region.

BackgroundDifferent parts of the tree Calophyllum inophyllum L. (nuts, leaves, roots, bark, fruits, nut oil and resin) are used as traditional medicines and cosmetics in most of the Pacific Islands. The oil efficiency as a natural cure and in traditional cosmetics has been largely described throughout the South Pacific, which led us to investigate C. inophyllum’s chemical and genetic diversity. A correlative study of the nut resin and leaf DNA from three distinct archipelagos in the South Pacific was carried out in order to identify diversity patterns in C. inophyllum across the South Pacific.MethodsCalophyllum inophyllum plants were sampled from French Polynesia, New Caledonia and Fiji. We extracted tamanu oil (nut oil) resin for chemo-diversity studies and sampled leaf tissues for genetic studies. We applied an analysis method designed for small quantities (at a microscale level), and used High Performance Liquid Chromatography (HPLC) to establish the chemo-diversity of tamanu oil resin. In-house standards were co-eluted for qualitative determination. Genetic diversity was assessed using chloroplast barcoding markers (the Acetyl-CoA carboxylase (accD) gene and the psaA-ycf3 intergenic spacer region).ResultsOur HPLC analysis revealed 11 previously known tamanu oil constituents, with variability among plant samples. We also isolated and characterized two new neoflavonoids from tamanu oil resin namely, tamanolide E1 and E2 which are diastereoisomers. Although genetic analysis revealed low genetic variation, our multivariate analysis (PCA) of the tamanu oil resin chemical profiles revealed differentiation among geographic regions.ConclusionWe showed here that chromatographic analysis using formalized in-house standards of oil resin compounds for co-elution studies against oil resin samples could identify patterns of variation among samples of C. inophyllum, and discriminate samples from different geographical origins.

Autoantibodies in Pandemrix®-induced narcolepsy: Nine candidate autoantigens fail the conformational autoantibody test

Study objectives: Narcolepsy type 1 (NT1) is a chronic sleep disorder characterized by loss of hypocretin-producing neurons. Increased NT1 incidence was observed in Sweden following mass-vaccination with Pandemrix®. Genetic association to HLA DQB1*06:02 implies an autoimmune origin, but target autoantigen remains unknown. Candidate autoantigens for NT1 have previously been identified in solid-phase immunoassays, while autoantibodies against conformation-dependent epitopes are better detected in radiobinding assays. The aims are to determine autoantibody levels against nine candidate autoantigens representing (1) proteins of the hypocretin transmitter system; Preprohypocretin (ppHypocretin), Hypocretin peptides 1 and 2 (HCRT1 and HCRT2) and Hypocretin receptor 2 (HCRTR2); (2) proteins previously associated with NT1; Tribbles homologue 2 (TRIB2), Pro-opiomelanocortin/alpha-melanocyte-stimulating-hormone (POMC/α-MSH) and Prostaglandin D2 Receptor DP1 (DP1); (3) proteins suggested as autoantigens for multiple sclerosis (another HLA DQB1*06:02-associated neurological disease); ATP-dependent Inwardly Rectifying Potassium Channel Kir4.1 (KIR4.1) and Calcium-activated chloride channel Anoctamin 2 (ANO2).Methods: Serum from post-Pandemrix® NT1 patients (n = 31) and their healthy first-degree relatives (n = 66) were tested for autoantibody levels in radiobinding assays separating autoantibody bound from free labelled antigen with Protein A-Sepharose. 125I-labelled HCRT1 and HCRT2 were commercially available while 35S-methionine-labelled ppHypocretin, HCRTR2, TRIB2, α-MSH/POMC, DP1, KIR4.1 or ANO2 was prepared by in vitro transcription translation of respective cDNA. In-house standards were used to express data in arbitrary Units/ml (U/ml).Results: All radiolabelled autoantigens were detected in a concentration-dependent manner by respective standard sera. Levels of autoantibodies in the NT1 patients did not differ from healthy first-degree relatives in any of the nine candidate autoantigens.Conclusions: None of the nine labelled proteins proposed to be autoantigens were detected in the radiobinding assays for conformation-dependent autoantibodies. The results emphasise the need of further studies to identify autoantigen(s) and clarify the mechanisms in Pandemrix®-induced NT1.

An in vitro functional assay to measure the biological activity of TB vaccine candidate H4-IC31

Potency assays for vaccine products are an important regulatory requirement, and are used to assess product quality and consistency prior to lot release for clinical testing. Ideally they measure an established correlate of efficacy or protection. In cases where there is no known correlate of protection, however, a functional assay that measures a biological response to a vaccine can be applied as a potency assay. Here we describe an in vitro assay which quantitatively measures human T cell activation as a biological response to the TB vaccine candidate H4-IC31. The Cytokine Secretion Assay (CSA) is based on the ability of peripheral blood mononuclear cells (PBMCs) from a Bacillus Calmette-Guérin (BCG)-vaccinated human donor to process and respond to H4-IC31. The ability of H4-IC31 to stimulate a cellular immune response is measured through the quantification of secreted IFNγ and is reported as relative stimulatory activity (RSA) compared to an in-house reference standard. The CSA is specific to the H4-IC31 vaccine, determines the RSA of H4-IC31 in the range of 50% to 150% of the reference standard, and is stability indicating as it detects differences in RSA between intact and heat treated H4-IC31. Although the CSA does not provide a link to clinical efficacy, it fulfills the critical requirements for a biological potency test to assess TB vaccine candidates and can be used along with biochemical and immunochemical assays to define a product profile during clinical development, while eliminating the use of animals for product testing.

Dual Antiplatelet Therapy Improves Functional Outcome in Patients With Progressive Lacunar Strokes.

Background and Purpose- In 20% to 30% of patients with lacunar strokes, early neurological deterioration (END) occurs within the first days after stroke onset. However, effective treatment strategies are still missing for these patients. The purpose of this study was to analyze efficacy of dual antiplatelet therapy (DAPT) in patients presenting with END. Methods- Four hundred fifty-eight patients with lacunar strokes and corresponding neuroimaging evidence of lacunar ischemia were retrospectively screened for END, which was defined by deterioration of ≥3 total National Institutes of Health Stroke Scale points, ≥2 National Institutes of Health Stroke Scale points for limb paresis, or documented clinical deterioration within 5 days after admission. Patients with END were treated with DAPT according to in-house standards. Primary efficacy end point was fulfilled if National Institutes of Health Stroke Scale score at discharge improved at least to the score at admission. Secondary end points were Rankin Scale score, further clinical fluctuation, and symptomatic bleeding complications. Results- END occurred in 130 (28%) of 458 patients with lacunar strokes. Ninety-seven (75%) of these patients were treated with DAPT after END, mostly for 5 days. DAPT was associated with improved functional outcome. The primary end point was met in 68% (66) of patients with DAPT compared with 36% (12) of patients with standard treatment ( P=0.0019). Further clinical fluctuations were absent in 79% (77) of patients with DAPT versus 33% (11) of patients without DAPT ( P<0.001). Symptomatic bleeding complications were not observed in any patient. Conclusions- The results demonstrated potential positive effects of DAPT in patients with progressive lacunar strokes.

Effect of ultrafilter pretreatment, acid strength and decalcification duration on archaeological bone collagen yield

Recent attempts to date batches of poorly preserved archaeological bone prompted a re-evaluation of existing protocols for bone sample preparation and ultrafilter cleaning methods at the Keck-Carbon Cycle Accelerator Mass Spectrometer (KCCAMS) facility. The emphasis of the study was to optimize decalcification time and ultrafiltration retention efficiency to improve overall collagen yields, especially for poorly preserved bone. Decalcification time and acid strength tests were done on four in-house bone standards of varying states of preservation. Through systematic testing, we found that 200 mg bone chip samples decalcified in 4 mL of 1.0 N HCl over 16 h showed increases of collagen yield across all samples relative to our existing protocol. Ultrafilters precleaned with weak acid (0.01 N HCl) showed greater retention efficiencies than those cleaned by sonication in ultrapure Milli-Q (MQ) water. Collagen produced from each sample were analyzed via AMS dating to check the quality of the collagen.

Potassium isotopic compositions of international geological reference materials

There is a renewed interest in K isotope geochemistry driven by the advances in analytical precision and it is emerging as a useful tracer in a variety of disciplines ranging from Earth sciences to biology. However, high-quality K isotopic data for reference materials are still limited but highly needed. Here, we report high-precision stable K isotopic compositions (δ41K) for 23 commercially available international reference materials, including igneous, sedimentary, and metamorphic rocks, as well as an in-house seawater standard. Potassium in digested samples was separated by cation-exchange chromatography with Bio-Rad AG50W-X8 (200–400 mesh) resin in 0.5 N HNO3 media. Potassium isotopes were measured on a Nu Plasma II high-resolution MC-ICPMS. The reproducibility of K isotopic analysis, based on over one year of measurements of pure K solutions and rock standards, was ≤0.06‰ (95% confidence interval). Synthetic solutions made by mixing single element standards to represent various rock matrices confirmed the accuracy of our methods. The 23 reference materials have δ41K values ranging from −0.562‰ to −0.253‰ and the seawater standard has a much higher δ41K value of 0.143‰. The comprehensive dataset presented here provides a reference for quality control and inter-laboratory comparison of high-precision K isotopic analyses for future studies.

The Middle East and Africa Code of Promotional Practices in the Pharmaceutical Industry

The pharmaceutical industry is known for investing heavily in promotions targeted at healthcare professionals (HCPs). Governments around the world try to regulate unwanted promotional practices in different ways. Where binding laws are in place in the U.S.A., European governments favor self-regulation. The purpose of this research is the evaluation of the Middle East and Africa Code of Promotional Practices (MEACPP) as a preliminary draft and its implications. Our paper fills a research gap by looking into the perceptions of the parties involved, analyzing their interests, and predicting possible outcomes. We used a mixed-method approach. Interviews were conducted with pharmaceutical companies and associations; while a questionnaire was administered to HCPs. Our findings suggest that all parties are in favor of more transparency. However, when it comes to disclosing the received financial support, the HCPs are hesitant. An estimated 20% would be willing to fully disclose their received benefits, which is in line with their European colleagues. Multinational pharmaceutical companies follow their own in-house standards and fear being at a competitive disadvantage when local companies can promote their drugs without any strings attached. MEA pharmaceutical companies do not see the potential benefits of analyzing the publicly available data to identify key opinion leaders (KOLs). The limitation of our research is the fact that the MEACPP has not been implemented yet and survey results are therefore based on expectations rather than real events.

Analysis and options for optimization of preoperative assessment for anesthesia at auniversity hospital

Risk assessment prior to elective surgery is an important tool in the context of perioperative patient care; however, only a few studies have been carried out to address the processes and problems during preoperative assessment for anesthesia. Over aperiod of several weeks all preoperative anesthesia evaluations prior to elective surgery were prospectively recorded in order to generate adata pool with a view to identifying options for process optimization. All preoperative evaluations over aperiod of 38working days at the University Medical Center Regensburg were recorded and analyzed with respect to waiting time for the patient and the duration of the preoperative consultation on medication. Also documented were the patient age, ASA score, the faculty carrying out the operation, type and risk of surgery, planned time of surgery, professional experience of the anesthesiologist and the approval status for surgery. In addition, all problems which occurred during the preoperative anesthesia evaluation were documented using a questionnaire. Overall 2233 preoperative assessments for anesthesia were recorded and analyzed. The number of patients attending the preoperative assessment clinic differed markedly in the course of aday and was lower at the end of the week. Approval for surgery with no reservations was given more frequently by anesthesiologists with more than 5 years professional experience and consultants compared to younger colleagues. The main reason for approval with reservations or no approval was the lack of patient records and test results, which should have been presented according to the in-house standard for preoperative assessment for anesthesia. The mean waiting time was 58.6± 30.3 min, the mean duration of the patient documentation review and physician-patient consultation together was 33.6± 16.3 min. Anesthesiologists with 2-5years professional experience needed significantly less time for patient documentation reviews and physician-patient consultations than younger and more experienced colleagues. The duration of the preoperative assessment for anesthesia correlated with the ASA score and risks of surgery. The analysis of processes and problems in the context of preoperative assessment for anesthesia revealed several options for optimization. Major efforts should be the implementation of an appointment system for the preoperative assessment clinic in order to generate ahomogeneous distribution of patients during the course of aday. Furthermore, surgeons and case managers should be requested to refer patients to the preoperative assessment clinic only with complete records and test results according to the in-house standard.