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Owing to highly conductive solid ionic conductors, all-solid-state batteries attract significant attention as promising next-generation energy storage devices. A lot of research is invested in the search and optimization of solid electrolytes with higher ionic conductivity. However, a systematic study of an interlaboratory reproducibility of measured ionic conductivities and activation energies is missing, making the comparison of absolute values in literature challenging. In this study, we perform an uncertainty evaluation via a Round Robin approach using different Li-argyrodites exhibiting orders of magnitude different ionic conductivities as reference materials. Identical samples are distributed to different research laboratories and the conductivities and activation barriers are measured by impedance spectroscopy. The results show large ranges of up to 4.5 mScm-1 in the measured total ionic conductivity (1.3 – 5.8 mScm-1 for the highest conducting sample, relative standard deviation 35 – 50% across all samples) and up to 128 meV for the activation barriers (198 – 326 meV, relative standard deviation 5 – 15%, across all samples), presenting the necessity of a more rigorous methodology including further collaborations within the community and multiplicate measurements.

Background and objective: Although the endogenous thrombin potential (ETP)-based activated protein C (APC) resistance is recommended for the development of steroid contraceptive agents, one of the main limitations of this technique was its lack of standardization, which hampered study-to-study comparison. A validated methodology that meets all the regulatory requirements in terms of analytical performances has been developed recently. To ensure a wide implementation of this test, the assessment of the interlaboratory variability was needed.Method: The assay was implemented in three testing laboratories. First, dose-response curves were performed to locally define APC concentration leading to 90% of ETP inhibition on healthy donors. Intra- and inter-run repeatability were assessed on a reference plasma and three quality controls. To investigate the variability in results among the different testing units, 60 donor samples were analyzed at each site.Results: The APC concentration leading to 90% of ETP inhibition was defined at 1.21 µg/ml and 1.14 µg/ml in the two receiving units. Intra- and inter-run repeatability showed standard deviation below 3%. Analyses of the 60 donor samples showed no statistically significant difference. The sensitivity of the test in the different laboratories was maintained and subgroup analyses still reported significant differences depending on hormonal status of donors.Conclusion: This study is the first reporting the interlaboratory variability of the ETP-based APC resistance assay. Data revealed excellent intra- and interlaboratory reproducibility. These results support the concept that this blood coagulation test provides an appropriate sensitivity irrespective of the laboratory in which analyses are performed.

Aim: Although the fit-for-purpose approach has been proposed for validation procedures and acceptance criteria for biomarker assays, practical biomarker assays to facilitate clinical application and regulatory documents on biomarker assays remain limited. Materials & methods: We assigned six independent laboratories and selected three lysophosphatidylcholines (LPCs): LPC(16:0), LPC(18:0) and LPC(18:1) as model biomarkers. Using LC–MS, the following key validation parameters were evaluated: calibration curve, carryover, parallelism, precision and relative accuracy and these values were similar among all laboratories. Further, we determined LPC levels in six lots of rat plasma at unknown concentrations and compared them among the laboratories. Conclusion: Our multilaboratory validation and reproducibility data are useful for the development of future biomarker assay validation procedures, as well as regulatory documents.

Ten years ago we issued, in conjunction with the Journal of Chemical Information and Modeling, an open prediction challenge to the cheminformatics community. Would they be able to predict the intrinsic solubilities of 32 druglike compounds using only a high-precision set of 100 compounds as a training set? The "Solubility Challenge" was a widely recognized success and spurred many discussions about the prediction methods and quality of data. Regardless of the obvious limitations of the challenge, the conclusions were somewhat unexpected. Despite contestants employing the entire spectrum of approaches available then to predict aqueous solubility and disposing of an extremely tight data set, it was not possible to identify the best methods at predicting aqueous solubility, a variety of methods and combinations all performed equally well (or badly). Several authors have suggested since then that it is not the poor quality of the solubility data which limits the accuracy of the predictions, but the deficient methods used. Now, ten years after the original Solubility Challenge, we revisit it and challenge the community to a new test with a much larger database with estimates of interlaboratory reproducibility.

Background Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool. Methods In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at −80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing. Findings The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at −80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at −80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p

A proficiency test (PT) for the determination of the tropane alkaloids (TAs) atropine and scopolaminein buckwheat flour and maize flour was organised by the European Union Reference Laboratory for mycotoxins & plant toxins (EURLMP) between August and October 2020. This PT was carried out by Wageningen Food Safety Research (WFSR) in accordance with ISO/IEC 17043 (R013). The measurandlevels were targeted to provide insight on the measurement capabilities of the EU Member States’National Reference Laboratories (NRLs) at concentrations corresponding to the levels of TAs in processed cereal-based foods and baby foods for infants and young children, containing millet, sorghum, buckwheat or their derived products as regulated by Commission Regulation (EU) 2016/239. In addition to this food product, a maize sample containing a higher level of TAs was included in this PT since an amendment to the legislation is foreseen.The participants were asked to quantify atropine and scopolamine in two materials and to report the compounds individually as well as the sum value. The participants performance was assessed as z-score in both materials for the individual TAs (maximum score 4 out of 4) and for the sum of the two TAs in one sample (maximum score 2 out of 2).Thirty-eight participants, of which 29 NRLs for mycotoxins and/or plant toxins in food and feed (from 22 EU Member States plus Iceland and Norway) and 9 Official Laboratories (8 from 4 EU Member States and Switzerland) participated in the PT.Two materials, buckwheat flour (material A) and maize flour (material B), were prepared containing atropine and scopolamine. Levels were artificially increased by spiking with atropine and scopolamine standard solutions. Both materials were sufficiently homogeneous and stable during the PT. Each participant received one test sample of each material.For the identification and quantification of atropine and scopolamine 34 participants used liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS), one participant used LC single quadrupole MS, two participants used LC high resolution mass spectrometry (HRMS) and one participant provided no information. In this PT the robust mean was used as consensus value. The consensus value based on the participants’ results was used as the assigned value. The assigned values of atropine and scopolamine in material A were, respectively, 1.15 and 1.16 μg/kg and in material B, respectively, 15.3 and 52.7 μg/kg. Obtained interlaboratory reproducibility (RSDR) ranged from 14% to 26%. The RSDR were for all TAs below the target standard deviation, except for scopolamine (26%) in material A. For the sum of TAs (atropine and scopolamine) the RSDR was 20% and 17% for material A and B, respectively. The proficiency of the participants was assessed through z-scores, calculated using the assigned values and a relative target standard deviation of 25%. All participants submitted results for atropine and scopolamine. One participant analysed only material A. For both materials (A and B) 87% of the results for atropine and scopolamine were rated with satisfactory z-scores (|z|≤ 2), 6% of the results fell into the questionable range with 2

Circular RNAs (circRNAs) have recently gained substantial attention as potential biomarkers in several human diseases, most prominently in cancer. However, the detection and quantification of circRNA biomarkers pose specific challenges owing to their circular nature. In particular, the use of enzymes, such as reverse transcriptases, may lead to impaired quantification, poor interlaboratory reproducibility and even false-positive results. Therefore, the development of methods for accurate quantification of circRNA biomarkers is a high priority. An enzyme-free and digital quantification method, named NanoString nCounter, was recently adapted for circRNA detection. In this review, I describe the advantages of using this technology for circRNA quantification as well as methodological considerations, potential pitfalls and disadvantages.

The larva of the greater wax moth Galleria mellonella is an increasingly popular model for assessing the virulence of bacterial pathogens and the effectiveness of antimicrobial agents. In this review, we discuss details of the components of the G. mellonella larval immune system that underpin its use as an alternative infection model, and provide an updated overview of the state of the art of research with G. mellonella infection models to study bacterial virulence, and in the evaluation of antimicrobial efficacy. Emphasis is given to virulence studies with relevant human and veterinary pathogens, especially Escherichia coli and bacteria of the ESKAPE group. In addition, we make practical recommendations for larval rearing and testing, and overcoming potential limitations of the use of the model, which facilitate intra- and interlaboratory reproducibility.

BackgroundNew and fully validated tests need to be brought into clinical practice to improve the estimation of recurrence risk in patients with colon cancer. The aim of this study was to assess the analytical performances of the Immunoscore (IS) and show its contribution to prognosis prediction.MethodsImmunohistochemical staining of CD3+ and CD8+ T cells on adjacent sections of colon cancer tissues were quantified in the core of the tumor and its invasive margin with dedicated IS modules integrated into digital pathology software. Staining intensity across samples collected between 1989 and 2016 (n=595) was measured. The accuracy of the IS workflow was established by comparing optical and automatic counts. Analytical precision of the IS was evaluated within individual tumor block on distant sections and between eligible blocks. The IS interlaboratory reproducibility (n=100) and overall assay precision were assessed (n=3). Contribution of the IS to prediction of recurrence based on clinical and molecular parameters was determined (n=538).ResultsOptical and automatic counts for CD3+ or CD8+ were strongly correlated (r=0.94, pConclusionIS is a robust and validated clinical assay leveraging immune scoring to predict recurrence risk of patient with localized colon cancer. The strong and independent prognostic value of IS should pave the way for it use in clinical practice.

Carbonate clumped isotope (Δ ) thermometry examines the temperature-dependent excess abundance of the C- O bond in the carbonate lattice. Inconsistent temperature calibrations and standard values have been reported among laboratories, which has led to use of equilibrated gases and carbonate standards for standardization. Furthermore, different acid fractionation factors and isotopic parameter sets have been proposed to improve inter-laboratory data comparability. However, few long-term datasets have been generated to explore the effects of these factors on the long-term reproducibility of Δ data within a laboratory. Four standards (ISTB-1, NBS-19, GBWO4416, and GB04417) were analyzed as unknowns from 2015 to 2019. Δ values were calibrated using the ETH standards. We investigated the Assonov, Brand, and Gonfiantini isotope parameter sets for carbon and oxygen isotopes, as well as two correction schemes of equilibrated gas and carbonate standardization, using the same sample measurements to determine which procedures enhanced reproducibility. ISTB-1 (calcite) and ZK312-346W (dolomite) were measured to determine the 90 C acid fractionation factor. The corrected 90 C acid fractionation factors are 0.076±0.008‰ for ISTB-1 and 0.077±0.009‰ for ZK312-346W. The choice of isotope parameter set had no significant influence on final Δ values in this study. However, using the Assonov parameters to calculate Δ values improved the reproducibility of results. The use of carbonate standards improved reproducibility through time compared to use of equilibrated gases for standardization. At 90 C, the acid fractionation factors of calcite and dolomite are statistically indistinguishable. We find an insignificant effect from changing the isotope parameter set, suggesting that the choice of isotope parameter set among laboratories is not a major factor affecting interlaboratory reproducibility. We find that using carbonate standards improved the reproducibility of results, suggesting that the use of carbonate standards may help achieve interlaboratory comparability of results in future studies.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioflm.2019.100010., The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays., This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska – Curie grant agreement No 722467, as part of the Print-Aid consortium. The information and views set out in this article are those of the authors and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein. This work received additional financial support by: project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

Background Viability testing is a common practice in laboratories. The goal of this study was to ascertain current laboratory practices internationally for performing viability testing for cryopreserved cord blood (CB) products and glean information about how to standardize the method to improve interlaboratory reproducibility. Study design and methods A survey to evaluate current laboratory practices for viability testing was designed and distributed internationally. The question topics included sampling and testing methods, responses to unexpected results, and the rating of the reliability of the CB quality tests, together with expectations for standardization. Results There were 32 respondents to the survey, of whom 28 responded to the more detailed questionnaire about viability methods. Overall, responses indicated that various stains were used among the laboratories, and when multiple sites used the same viability stain the methods differed. The majority of the respondents were in favor of standardizing the viability testing methods. A wide variety of preferences were communicated about how to standardize the method, but a majority did advocate the use of 7-aminoactinomycin D (7-AAD) with flow cytometry. Conclusions The survey results revealed a variety of tests and inconsistent interlaboratory practices for performing the viability assay. Flow cytometry with a 7-AAD dye was suggested as a first step toward standardization.

The iQ-Check Salmonella II Real-Time PCR test kit utilizes Salmonella-specific oligonucleotide probes and primers for the rapid and specific detection of Salmonella species in select food types. The alternative method was evaluated by using 375 g test portions in an unpaired study design for two matrices, milk chocolate and dry dog food. Each matrix was compared with the U.S. Food and Drug Administration Chapter 5 Salmonella reference method. Fourteen technicians from 12 laboratories, including academia and industry, located within the United States and Canada participated in the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2–2 CFU/test portion), and a high inoculum level (2–5 CFU/test portion). The statistical analysis was conducted according to the Probability of Detection (POD) statistical model. The results obtained for the low inoculum level test portions produced a difference in the candidate presumptive and confirmatory results (dLPOD) value with a 95% confidence interval of −0.05, (−0.15, 0.06) for the milk chocolate and 0.10, (−0.01, 0.21) for the dry dog food. The dLPOD results indicate an equivalence between the candidate method and reference method for the matrices evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for each matrix and produce values of

The BD FACSVia™ system is a novel flow cytometer with improved workflow efficiencies. To evaluate the HLA-B27 application developed on the BD FACSVia system utilizing the BD™ HLA-B27 kit, we conducted a concordance study at three centers to compare with the BD FACSCalibur™ system. Prepared donor samples (n = 594) were analyzed on both the BD FACSVia and BD FACSCalibur for the HLA-B27 assay. Adjudication of HLA-B27 discordant results was performed using the reverse sequence-specific oligonucleotide (rSSO) DNA typing method (LABType® SSO, One Lambda). On the BD FACSVia system 80 B27 positive, 499 B27 negative and 15 "Inconclusive" samples were observed. The corresponding BD FACSCalibur results were 73 B27 positive, 502 B27 negative and 19 "gray zone" samples. The overall concordance of HLA-B27 determination was 98% between the two systems with seven more positives identified on BD FACSVia as compared to BD FACSCalibur. The equivocal zone between positive and negative on BD FACSVia (named "Inconclusive") and on BD FACSCalibur (named "gray zone") is due to antibody cross reactivity of HLA-B27 clone GS145.2. One negative sample verified with the rSSO DNA method was reported as HLA-B27 positive by the BD FACSVia system leading to a false positive result. Our study demonstrated concordance results between the BD FACSVia system and BD FACSCalibur. Intersite reproducibility of BD HLA-B27 assay remained within the limits of acceptability. © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Carbonate clumped isotopes offer a potentially transformational tool to interpret Earth's history, but the proxy is still limited by poor interlaboratory reproducibility. Here, we focus on the uncertainties that result from the analysis of only a few replicate measurements to understand the extent to which unconstrained errors affect calibration relationships and paleoclimate reconstructions. We find that highly precise data can be routinely obtained with multiple replicate analyses, but this is not always done in many laboratories. For instance, using published estimates of external reproducibilities we find that typical clumped isotope measurements (three replicate analyses) have margins of error at the 95% confidence level (CL) that are too large for many applications. These errors, however, can be systematically reduced with more replicate measurements. Second, using a Monte Carlo-type simulation we demonstrate that the degree of disagreement on published calibration slopes is about what we should expect considering the precision of Δ47 data, the number of samples and replicate analyses, and the temperature range covered in published calibrations. Finally, we show that the way errors are typically reported in clumped isotope data can be problematic and lead to the impression that data are more precise than warranted. We recommend that uncertainties in Δ47 data should no longer be reported as the standard error of a few replicate measurements. Instead, uncertainties should be reported as margins of error at a specified confidence level (e.g., 68% or 95% CL). These error bars are a more realistic indication of the reliability of a measurement.

Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-coded Staphylococcus aureus DNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of >75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods.

Background Recently a lipidomics approach was able to identify perturbed fatty acyl chain (FAC) and sphingolipid moieties that could stratify patients according to the severity of coronary calcification, a form of subclinical atherosclerosis. Nevertheless, these findings have not yet been reproduced before generalising their application. The aim of this study was to evaluate the reproducibility of lipidomics approaches by replicating previous lipidomic findings in groups of patients with calcific coronary artery disease (CCAD). Methods Patients were separated into the following groups based on their calcium score (CS); no calcification (CS: 0; n = 26), mild calcification (CS: 1–250; n = 27) and severe calcification (CS: > 250; n = 17). Two serum samples were collected from each patient and used for comparative analyses by 2 different laboratories, in different countries and time points using liquid chromatography coupled to mass spectrometry untargeted lipidomics methods. Results Six identical metabolites differentiated patients with severe coronary artery calcification from those with no calcification were found by both laboratories independently. Additionally, relative intensities from the two analyses demonstrated high correlation coefficients. Phosphatidylcholine moieties with 18-carbon FAC were identified in lower intensities and 20:4 FAC in higher intensities in the serum of diseased group. Moreover, 3 common sphingomyelins were detected. Conclusion This is the first interlaboratory reproducibility study utilising lipidomics applications in general and specifically in patients with CCAD. Lipid profiling applications in patients with CCAD are very reproducible in highly specialised and experienced laboratories and could be applied in clinical practice in order to spare patients diagnostic radiation.

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N-nitroso-N-ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay.

Following a previous study which aimed to determine the interlaboratory reproducibility of biorelevant dissolution testing in the USP 2 apparatus for commercial formulations of two weak acids (ibuprofen and zafirlukast), this study attempts to determine the interlaboratory reproducibility using a similar protocol for a commercially available formulation of a weak base, indinavir. Fourteen partners including twelve industrial and two academic partners participated in this study. To ensure uniformity, all partners were provided with a standardized protocol to perform (i) a single medium dissolution test in fasted state simulated gastric and intestinal fluids (FaSSGF and FaSSIF, respectively) and (ii) a two-stage dissolution experiment simulating gastrointestinal transfer. Optionally, partners could run a single-stage dissolution test in fed state simulated intestinal fluid (FeSSIF). For each dissolution test, one Crixivan® capsule (containing 400 mg indinavir as its sulfate salt) was added as dose of interest. For the single medium dissolution test in FaSSIF, all partners observed rapid release of indinavir resulting in supersaturated concentrations, followed by precipitation to equilibrium solubility. The degree and period of supersaturation varied among the participating laboratories. Average dissolution profiles in FeSSIF appeared to be highly reproducible with dissolved concentrations remaining lower than the thermodynamic solubility of indinavir in FeSSIF. For the two-stage dissolution test, most partners observed supersaturated concentrations in the intestinal compartment; two partners observed no supersaturation due to immediate precipitation. Given the fact that a high interlaboratory but low intralaboratory variability was observed when supersaturation/precipitation occurred, an undefined factor was hypothesized as a potential cause of the variability in precipitation. Hence, the impact of several experimental factors on the supersaturation and precipitation behavior of indinavir was investigated in a next step. The investigation indicated that variability is likely attributable to a combination of factors, especially, the time elapsed between sampling and dilution of the sample with the mobile phase. Therefore, when designing a test in which supersaturation and precipitation is anticipated, stringent control of the test methodology, especially regarding sampling and dilution, is needed. ispartof: EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS vol:140 pages:141-148 ispartof: location:Netherlands status: published

Nine cooperating laboratories, distributed throughout the United States, determined the interlaboratory reproducibility of a sensitive, selective method for isolation of Campylobacter jejuni and Campylobacter coli from foods, and determined the prevalence and distribution of the organism in retail meats. A double-blind inoculated/recovery experiment demonstrated the ability to detect two cells of C. jejuni and C. coli per g of meat at a rate of 96% among the cooperating laboratories. However, a 7.5% false-positive rate for the presumptive detection of the organism was also reported. Samples of ground beef, beef flank steak, lamb stew meat, broiler chicken, pork sausage (without antimicrobials), and pork chops were selected to assess the presence of campylobacters. Each cooperator purchased five of each of the above samples from the refrigerated case of two retail outlets at quarterly intervals throughout the year. A total of 2,160 retail samples were analyzed for the presence of C. jejuni and C. coli . Results indicated that about 30% of the 360 chickens sampled yielded the organism. Analysis of 1,800 red meat products yielded campylobacters at a rate of about 5.1%. Pork samples yielded C. coli and other meats yielded C. jejuni . Higher numbers of isolations from the red meats were made during June and September (8.6%) as compared with December and March (4.2%). These results provide a baseline, for the prevalence of campylobacters in these selected foods, and also support epidemiologic data associating mishandled foods of animal origin as a potential vehicle in human gastroenteritis.

Summary Background Sporicidal surface disinfection is recommended to control transmission of Clostridium difficile in healthcare facilities. EN 17126 provides a method to determine the sporicidal activity in suspension and has been approved as a European standard. In addition, a sporicidal surface test has been proposed. Aim To determine the interlaboratory reproducibility of a test method for evaluating the susceptibility of a C. difficile spore preparation to a biocidal formulation following the 4-field test (EN 16615 methodology). Methods Nine laboratories participated. C. difficile NCTC 13366 spores were used. Glutaraldehyde (1% and 6%; 15 min) and peracetic acid (PAA; 0.01% and 0.04%; 15 min) were used to determine the spores' susceptibility in suspension in triplicate. Findings One-percent glutaraldehyde revealed a mean decimal log10 reduction of 1.03 with variable results in the nine laboratories (0.37–1.49) and a reproducibility of 0.38. The effect of 6% glutaraldehyde was stronger (mean: 2.05; range: 0.96–4.29; reproducibility: 0.86). PAA revealed similar results. An exemplary biocidal formulation based on 5% PAA was used at 0.5% (non-effective concentration) and 4% (effective concentration) to determine the sporicidal efficacy (4-field test) under clean conditions in triplicate with a contact time of 15 min. When used at 0.5% it demonstrated an overall log10 reduction of 2.68 (range: 2.35–3.57) and at 4% of 4.61 (range: 3.82–5.71). The residual contamination on the three primarily uncontaminated test fields was Conclusion The interlaboratory reproducibility seems to be robust.

Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.

Label-free quantification (LFQ) and isobaric labeling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT SPS/MS3 10-plex workflow to a label free single shot data-independent acquisition (DIA) workflow on a controlled sample set. The sample set consisted of ten samples derived from 10 biological replicates of mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. For a fair comparison, we matched the instrument time for the two workflows. The LC-MS data were acquired at two facilities to assess interlaboratory reproducibility. Both methods resulted in a high proteome coverage (>5000 proteins) with low missing values on protein level (

In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real-time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST-/RQ- by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined "borderline" (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST-/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ-. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next-generation sequencing have the potential to overcome the current limitations.

Biorelevant media are increasingly being employed as dissolution media in drug development, including in smaller volumes than 900ml and under non-sink conditions. The objectives were to assess interday repeatability, interanalyst repeatability, and interlaboratory reproducibility of dissolution profiles from biorelevant media, as well as to assess the impacts of biorelevant media production method and biorelevant medium volume on dissolution profiles. Ibuprofen and ketoconazole tablets were subjected to dissolution testing in 500ml, 300ml, and 40ml of fasted state simulated gastric fluid (FaSSGF), fed state simulated gastric fluid (FeSSGF), fasted state simulated intestinal fluid version 2 (FaSSIF-V2), and fed state simulated intestinal fluid version 2 (FeSSIF-V2). f2 was used to assess repeatability and reproducibility of dissolution profiles. Results indicate favorable interday repeatability (83 of 88 comparisons were similar), favorable interanalyst repeatability (19 of 21 comparisons were similar), and favorable interlaboratory reproducibility (10 of 14 comparisons were similar) of dissolution profiles from biorelevant media, with commercial media showing greater interlaboratory reproducibility than 'from scratch' media. However, biorelevant medium production had low impact on profiles when one analyst conducted all medium preparations and study procedures at one location. Additionally, biorelevant media detected differences when products were not similar. Overall, biorelevant media showed favorable repeatability and reproducibility performance.

Inadequate and (or) inconsistent soil sample preparation techniques (SPT) contribute to excessive variance, difficulties in soil test interpretation, and incorrect lime and fertilizer recommendations. The objective of this study was to evaluate the effect of SPT of five laboratories in Quebec (Canada) on chemical parameters measurement reliability. Samples of fine (G1), medium (G2), and coarse (G3) textured soils were collected from the surface layer. Three 500 g portions of each soil were sent to each laboratory for preparation. In addition, all samples were analyzed by the same laboratory for routine analyses. Nested ANOVA in a hierarchical model were performed with components of SPT interlaboratory reproducibility, SPT intralaboratory replicability, and intralaboratory soil analysis repeatability. Before samples were analyzed, we observed an important interlaboratory heterogeneity of particle size distributions for the same samples; due to sample preparation techniques, this can affect results of the analyses. Of all variables analyzed, the only significant, outside acceptable variations due to SPT were (1) pHwater in G1; (2) PM-III, AlM-III, and (P/Al)M-III in G1 and G2; (3) KM-III, CaM-III, MgM-III and organic matter in G3; and (4) MnM-III and CuM-III in G1, G2, and G3. The steps in SPT, mostly drying and crushing, require standardization to reduce the variance of the entire soil testing process.

This study examines formally the proposition that validation of analytical procedures can be best undertaken by interlaboratory study. It approaches the task by considering the fundamental nature of validation and the meaning of ‘procedure’ in relation to uncertainty. The contention is that (a) only procedures as such can be validated, (b) the results of a procedure are empirical, (c) empirical procedures are unbiased, and (d) the interlaboratory reproducibility standard deviation (SDR) associated with an unbiased procedure is an unbiased estimate of the uncertainty. The definitive interlaboratory study for estimating SDR is the collaborative trial, but the case is made that, with due caution, data from proficiency tests can also provide useful estimates of SDR and thence estimates of uncertainty.

Temperature dependence of gas chromatography retention indices should be considered one of the main sources of their interlaboratory irreproducibility. The regularities of this dependence for packed and capillary columns or individual compounds allow revealing and characterizing other factors affecting reproducibility of the indices. These include the ratio of amounts of target analytes and reference compounds, as well as their polarity. Considering the anomalies in temperature dependences of retention indices of polar compounds on nonpolar phases allows assuming that it can be one of the causes of the asymmetry of the sets of reference data.

The quality control of culture media used for Legionella spp. isolation and enumeration is paramount to achieve a satisfactory degree of comparability among water testing results from different laboratories. Here, we report on a comparative assessment of the sensitivity and selectivity of MWY and BCYE&alpha, media supplied by two different manufacturers (i.e., Xebios Diagnostics GmbH and Oxoid Ltd) for the detection of Legionella spp. from environmental water samples. Even though our analysis showed an excellent agreement between the recovery rates of the four media tested (90.5%), the quantitative recovery of Legionella spp. colonies using Xebios media was significantly greater than that achieved by Oxoid media (P = 0.0054). Furthermore, the sensitivity of detection was significantly higher when samples were plated on MWY Xebios agar (P = 0.0442), while the selectivity of MWY appeared to be the same regardless of the manufacturer. Furthermore, MWYXebios agar favored the growth of much larger colonies compared to those observed on MWYOxoid agar. Finally, MWYXebios medium enhanced the recovery of non-pneumophila Legionella species. Collectively, our findings demonstrate that quality control is crucial to ensure high selectivity and sensitivity of the culture media used for the detection and enumeration of Legionella spp. from environmental water resources. As water remediation measures strictly depend on Legionella spp. recovery, culture protocol standardization, as well as quality control of the culture media, is essential to achieve intra- and interlaboratory reproducibility and accuracy.

The original version of this article has been corrected to include authors middle initials in the author list. The details given in this correction are correct.

Introduction Cerebrospinal fluid α-synuclein level is increased in sporadic Creutzfeldt-Jakob disease cases. However, the clinical value of this biomarker remains to be established. In this study, we have addressed the clinical validation parameters and the interlaboratory reproducibility by using an electrochemiluminescent assay. Methods Cerebrospinal fluid α-synuclein was quantified in a total of 188 sporadic Creutzfeldt-Jakob disease and non–Creutzfeldt-Jakob-disease cases to determine sensitivity and specificity values and lot-to-lot variability. Two round robin tests with 70 additional cases were performed in six independent laboratories. Results A sensitivity of 93% and a specificity of 96% were achieved in discriminating sporadic Creutzfeldt-Jakob disease. No differences were detected between lots. The mean interlaboratory coefficient of variation was 23%, and the intralaboratory coefficient of variations ranged 2.70%–11.39%. Overall, 97% of samples were correctly diagnosed. Discussion The herein validated α-synuclein assay is robust, accurate, and reproducible in identifying Creutzfeldt-Jakob disease cases. Thus, it is ready for implementation in the clinical practice to support the diagnosis of Creutzfeldt-Jakob disease., Highlights • A new cerebrospinal fluid α-synuclein assay is validated for the diagnosis of sCJD. • High diagnostic accuracy allows 97% of correct diagnoses. • Two round robin tests unveil high interlaboratory reproducibility. • Clinical parameters are superior to those of other established biomarkers.

Increasing the interlaboratory reproducibility of gas chromatographic retention indices requires avoiding measurements distorted by overloading effects. Several criteria of evaluating the limits of the mass overloading of gas chromatographic systems are compared and reconsidered. The criteria mostly appropriate for practical purposes are based on (i) the dependences of factors of peak broadening (ratio of peak height and its width) vs. amount of analyte injected into the chromatographic column and (ii) the dependence of parameters characterizing the peak distortion (asymmetry factor) vs. the amount of analyte. Both these criteria provide mutually comparable evaluations of the overloading limits for analytes of different polarity. At the same time, the dependence of retention indices vs. amounts of analyte injected in the chromatographic column cannot be recommended for overloading control, because the parameters of the corresponding linear regressions indicate temperature dependence. The interpretation of certain gas chromatographic anomalies requires the correct evaluation of overloading limits. For example, the unusual temperature dependence of retention indices of polar analytes on non-polar stationary phases and the dependence of retention indices on ratio of amounts of target analytes and reference compounds.

The accurate prediction of solubility of drug-like molecules is difficult, and perhaps a satisfactory general model is not yet available. The most cited challenge to good prediction has been the lack of enough high-quality and drug-relevant solubility data that adequately cover the chemical space of drugs. This review addresses data quality in solubility measurement. Specifically, the “gold standard” shake-flask and related methods used to measure equilibrium solubility of ionizable drug-like compounds as a function of pH were reviewed. Over 800 publications were examined. Many factors affecting the quality of the measurement were recognized, and a number of suggestions are offered to improve the experimental methodology. Some of the suggestions focus on improving methods for future measurements, and some refer to improvements in data mining, i.e., to ways of extracting more reliable information from existing data. By normalizing data for pH (i.e., deriving intrinsic solubility, S 0 ) and for temperature (by transforming measurements performed in the range 20 – 40 °C to 25 °C), it is suggested that the 0.6-0.7 log unit currently expected interlaboratory reproducibility can be reduced to less than 0.15. It is the aim of the review that the improvements in data quality would lead to better predictions of drug solubility using in silico methods.

Differential counting of peripheral blood cells is an important diagnostic tool. However, manual morphological analysis using the microscope is time-consuming and requires highly trained personnel. The digital microscope is capable of performing an automated peripheral blood cell differential, which is as reliable as manual classification by experienced laboratory technicians. To date, information concerning the interlaboratory variation and quality of cell classification by independently operated digital microscopy systems is limited. We compared four independently operated digital microscope systems for their ability in classifying the five main peripheral blood cell classes and detection of blast cells in 200 randomly selected samples. Set against the averaged results, the R(2) values for neutrophils ranged between 0.90 and 0.96, for lymphocytes between 0.83 and 0.94, for monocytes between 0.77 and 0.82, for eosinophils between 0.70 and 0.78, and for blast cells between 0.94 and 0.99. The R(2) values for the basophils were between 0.28 and 0.34. This study shows that independently operated digital microscopy systems yield reproducible preclassification results when determining the percentages of neutrophils, eosinophils, lymphocytes, monocytes, and blast cells in a peripheral blood smear. Detection of basophils was hampered by the low incidence of this cell class in the samples.

The high number of poorly water-soluble compounds in drug development has increased the need for enabling formulations to improve oral bioavailability. One frequently applied approach is to induce supersaturation at the absorptive site, e.g., the small intestine, increasing the amount of dissolved compound available for absorption. However, due to the stochastic nature of nucleation, supersaturating drug delivery systems may lead to inter- and intrapersonal variability. The ability to define a feasible range with respect to the supersaturation level is a crucial factor for a successful formulation. Therefore, an in vitro method is needed, from where the ability of a compound to supersaturate can be defined in a reproducible way. Hence, this study investigates the reproducibility of an in vitro small scale standardized supersaturation and precipitation method (SSPM). First an intralaboratory reproducibility study of felodipine was conducted, after which seven partners contributed with data for three model compounds; aprepitant, felodipine, and fenofibrate, to determine the interlaboratory reproducibility of the SSPM. The first part of the SSPM determines the apparent degrees of supersaturation (aDS) to investigate for each compound. Each partner independently determined the maximum possible aDS and induced 100, 87.5, 75, and 50% of their determined maximum possible aDS in the SSPM. The concentration-time profile of the supersaturation and following precipitation was obtained in order to determine the induction time (t

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

The increasing request for hair ethyl glucuronide (HEtG) in alcohol consumption monitoring according to cut-off levels set by the Society of Hair Testing (SoHT) has triggered a proficiency testing program based on interlaboratory comparisons (ILC). Here, the outcome of nine consecutive ILC rounds organised by the SoHT on the determination of HEtG between 2011 and 2017 is summarised regarding interlaboratory reproducibility and the influence of procedural variants. Test samples prepared from cut hair (1mm) with authentic (in-vivo incorporated) and soaked (in-vitro incorporated) HEtG concentrations up to 80pg/mg were provided for 27-35 participating laboratories. Laboratory results were evaluated according to ISO 5725-5 and provided robust averages and relative reproducibility standard deviations typically between 20 and 35% in reasonable accordance with the prediction of the Horwitz model. Evaluation of results regarding the analytical techniques revealed no significant differences between gas and liquid chromatographic methods In contrast, a detailed evaluation of different sample preparations revealed significantly higher average values in case when pulverised hair is tested compared to cut hair. This observation was reinforced over the different ILC rounds and can be attributed to the increased acceptance and routine of hair pulverisation among laboratories. Further, the reproducibility standard deviations among laboratories performing pulverisation were on average in very good agreement with the prediction of the Horwitz model. Use of sonication showed no effect on the HEtG extraction yield.

Dissolution testing with biorelevant media has become widespread in the pharmaceutical industry as a means of better understanding how drugs and formulations behave in the gastrointestinal tract. Until now, however, there have been few attempts to gauge the reproducibility of results obtained with these methods. The aim of this study was to determine the interlaboratory reproducibility of biorelevant dissolution testing, using the paddle apparatus (USP 2). Thirteen industrial and three academic laboratories participated in this study. All laboratories were provided with standard protocols for running the tests: dissolution in FaSSGF to simulate release in the stomach, dissolution in a single intestinal medium, FaSSIF, to simulate release in the small intestine, and a "transfer" (two-stage) protocol to simulate the concentration profile when conditions are changed from the gastric to the intestinal environment. The test products chosen were commercially available ibuprofen tablets and zafirlukast tablets. The biorelevant dissolution tests showed a high degree of reproducibility among the participating laboratories, even though several different batches of the commercially available medium preparation powder were used. Likewise, results were almost identicalbetween the commercial biorelevant media and those produced in-house. Comparing results to previous ring studies, including those performed with USP calibrator tablets or commercially available pharmaceutical products in a single medium, the results for the biorelevant studies were highly reproducible on an interlaboratory basis. Interlaboratory reproducibility with the two-stage test was also acceptable, although the variability was somewhat greater than with the single medium tests. Biorelevant dissolution testing is highly reproducible among laboratories and can be relied upon for cross-laboratory comparisons.

LabCyte EPI-MODEL24 is a commercially available reconstructed human cultured epidermal model produced by Japan Tissue Engineering Co. Ltd., Japan. It consists of normal human epidermal keratinocytes whose biological origin is neonatal foreskin. The several validation studies of the in vitro skin irritation test method using LabCyte EPI-MODEL24 (LabCyte EPI-MODEL24 SIT) were conducted at three laboratories to assess both intra- and interlaboratory reproducibility as well as predictive capacity of this test method using set of coded test chemicals.