eCerto-versatile software for interlaboratory data evaluation and documentation during reference material production.

The statistical tool eCerto was developed for the evaluation of measurement data to assign property values and associated uncertainties of reference materials. The analysis is based on collaborative studies of expert laboratories and was implemented using the R software environment. Emphasis was put on comparability of eCerto with SoftCRM, a statistical tool based on the certification strategy of the former Community Bureau of Reference. Additionally, special attention was directed towards easy usability from data collection through processing, archiving, and reporting. While the effects of outlier removal can be flexibly explored, eCerto always retains the original data set and any manipulation such as outlier removal is (graphically and tabularly) documented adequately in the report. As a major reference materials producer, the Bundesanstalt für Materialforschung und -prüfung (BAM) developed and will maintain a tool to meet the needs of modern data processing, documentation requirements, and emerging fields of RM activity. The main features of eCerto are discussed using previously certified reference materials.

Comparison of reproducibility precision on mass fraction in some interlaboratory studies of methods of food analysis.

The relationship between reproducibility standard deviation and mass fraction in food analysis has been studied in compilations of statistics from collaborative trials and from proficiency tests. There was a broad tendency for both categories of statistics to follow the Horwitz function although systematic deviations from it were easily detected at both extremes of the mass fraction range (below 10-7 and above 10-2). The two compilations were found to have very similar properties over the whole range of mass fractions, that is from about 10-10 (0.1 ppb) upwards. This similarity has implications for the determination of detection limit.

Serum HE4 and diagnosis of ovarian cancer in postmenopausal women with adnexal masses.

BACKGROUND: Transvaginal ultrasound and serum CA125 are routinely used for differential diagnosis of pelvic adnexal mass. Use of human epididymis 4 was approved in the United States in 2011. However, there is scarcity of studies evaluating the additional value of human epididymis 4. OBJECTIVE: The objective of the study was to evaluate the performance characteristics of transvaginal ultrasound, CA125, and human epididymis 4 for differential diagnosis of ovarian cancer in postmenopausal women with adnexal masses. STUDY DESIGN: This was a cohort study nested within the screen arms of the multicenter randomized controlled trial, United Kingdom Collaborative Trial of Ovarian Cancer Screening, based in England, Wales, and Northern Ireland. In United Kingdom Collaborative Trial of Ovarian Cancer Screening, 48,230 women randomized to transvaginal ultrasound screening and 50,078 to multimodal screening (serum CA125 interpreted by Risk of Ovarian Cancer Algorithm with second line transvaginal ultrasound) underwent the first (prevalence) screen. Women with adnexal lesions and/or persistently elevated risk were clinically assessed and underwent surgery or follow-up for a median of 10.9 years. Banked samples taken within 6 months of transvaginal ultrasound from all clinically assessed women were assayed for human epididymis 4 and CA125. Area under the curve and sensitivity for diagnosing ovarian cancer of multiple penalized logistic regression models incorporating logCA125, log human epididymis 4, age, and simple ultrasound features of the adnexal mass were compared. RESULTS: Of 1590 (158 multimodal, 1432 ultrasound) women with adnexal masses, 78 were diagnosed with ovarian cancer (48 invasive epithelial ovarian, 14 type I, 34 type II; 24 borderline epithelial; 6 nonepithelial) within 1 year of scan. The area under the curve (0.893 vs 0.896; P = .453) and sensitivity (74.4% vs 75.6% ;P = .564) at fixed specificity of 90% of the model incorporating age, ultrasound, and CA125 were similar to that also including human epididymis 4. Both models had high sensitivity for invasive epithelial ovarian (89.6%) and type II (>91%) cancers. CONCLUSION: Our population cohort study suggests that human epididymis 4 adds little value to concurrent use of CA125 and transvaginal ultrasound in the differential diagnosis of adnexal masses in postmenopausal women.

Expanded coverage of non-targeted LC-HRMS using atmospheric pressure chemical ionization: a case study with ENTACT mixtures.

Non-targeted analysis (NTA) is a rapidly evolving analytical technique with numerous opportunities to improve and expand instrumental and data analysis methods. In this work, NTA was performed on eight synthetic mixtures containing 1264 unique chemical substances from the U.S. Environmental Protection Agency's Non-Targeted Analysis Collaborative Trial (ENTACT). These mixtures were analyzed by atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) using both positive and negative polarities for a total of four modes. Out of the 1264 ENTACT chemical substances, 1116 were detected in at least one ionization mode, 185 chemicals were detected using all four ionization modes, whereas 148 were not detected. Forty-four chemicals were detected only by APCI, and 181 were detected only by ESI. Molecular descriptors and physicochemical properties were used to assess which ionization type was preferred for a given compound. One ToxPrint substructure (naphthalene group) was found to be enriched in compounds only detected using APCI, and eight ToxPrints (e.g., several alcohol moieties) were enriched in compounds only detected using ESI. Examination of physicochemical parameters for ENTACT chemicals suggests that those with higher aqueous solubility preferentially ionized by ESI-. While ESI typically detects a larger number of compounds, APCI offers chromatograms with less background, fewer co-elutions, and additional chemical space coverage, suggesting both should be considered for broader coverage in future NTA research. Graphical abstract.

The strength in numbers: comprehensive characterization of house dust using complementary mass spectrometric techniques.

Untargeted analysis of a composite house dust sample has been performed as part of a collaborative effort to evaluate the progress in the field of suspect and nontarget screening and build an extensive database of organic indoor environment contaminants. Twenty-one participants reported results that were curated by the organizers of the collaborative trial. In total, nearly 2350 compounds were identified (18%) or tentatively identified (25% at confidence level 2 and 58% at confidence level 3), making the collaborative trial a success. However, a relatively small share (37%) of all compounds were reported by more than one participant, which shows that there is plenty of room for improvement in the field of suspect and nontarget screening. An even a smaller share (5%) of the total number of compounds were detected using both liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Thus, the two MS techniques are highly complementary. Most of the compounds were detected using LC with electrospray ionization (ESI) MS and comprehensive 2D GC (GC×GC) with atmospheric pressure chemical ionization (APCI) and electron ionization (EI), respectively. Collectively, the three techniques accounted for more than 75% of the reported compounds. Glycols, pharmaceuticals, pesticides, and various biogenic compounds dominated among the compounds reported by LC-MS participants, while hydrocarbons, hydrocarbon derivatives, and chlorinated paraffins and chlorinated biphenyls were primarily reported by GC-MS participants. Plastics additives, flavor and fragrances, and personal care products were reported by both LC-MS and GC-MS participants. It was concluded that the use of multiple analytical techniques was required for a comprehensive characterization of house dust contaminants. Further, several recommendations are given for improved suspect and nontarget screening of house dust and other indoor environment samples, including the use of open-source data processing tools. One of the tools allowed provisional identification of almost 500 compounds that had not been reported by participants.

Bench Test for the Detection of Bacterial Contamination in Platelet Concentrates Using Rapid and Cultural Detection Methods with a Standardized Proficiency Panel.

BACKGROUND: The most frequent infectious complication in transfusion therapy in developed countries is related to the bacterial contamination of platelet concentrates (PCs). Rapid and cultural screening methods for bacterial detection in platelets are available, but external performance evaluation, especially of rapid methods, has been difficult to realize so far. Here we summarize the results of three individual collaborative trials using an external quality assessment program (EQAP) for the application of current rapid and cultural screening methods. METHODS: Three different modules were available for the detection of bacterial contamination: module 1: rapid methods, module 2: culture methods, module 3: bacterial identification methods. The sample set-up included up to six different bacterial strains, 1-2 negative samples and 4-6 positive samples with stabilized bacterial cell counts (approximately 10(3)/10(4)/10(5) CFU/ml). Time schedule for testing was limited (module 1: 6 h, module 2 and 3: 7 days). RESULTS: Samples of module 1 were analyzed with two different rapid methods (BactiFlow, NAT). The results of the three individual collaborative trials showed that all participants detected the negative samples with both assays correctly. Samples spiked with 10(4) to 10(5) CFU/ml of bacteria obtained positive results with both rapid screening methods, whereas samples spiked with only 10(3) CFU/ml disclosed a lower number of correctly identified positive results by NAT (86.6-93.8% sensitivity) compared to BactiFlow (100% sensitivity). The results for modules 2 and 3 revealed a 100% diagnostic sensitivity and specificity in all three collaborative trials. CONCLUSION: This proficiency panel facilitates the verification of the analytical sensitivity of rapid and cultural bacterial detection systems under controlled routine conditions. The concept of samples provided in this EQAP has three main advantages: i) samples can be examined by both rapid and culture methods, ii) the provided material is matrix-equivalent, and iii) the sample material is ready-to-use.

Establishment, Maintenance, and Performance of the Cooperative Osteosarcoma Study Group (COSS).

INTRODUCTION: Osteosarcoma treatment has benefitted greatly from collaborative research. This paper describes the history and accomplishments of the Cooperative Osteosarcoma Study Group (COSS), mainly dedicated to clinical questions, as well as remaining challenges. MATERIALS AND METHODS: Narrative review of over four decades of uninterrupted collaboration within the multi-national German-Austrian-Swiss COSS group. RESULTS: Since its very first prospective osteosarcoma trial starting in 1977, COSS has continuously been able to provide high-level evidence on various tumor- and treatment-related questions. This includes both the cohort of patients enrolled into prospective trials as well as those patients excluded from them for various reasons, followed in a prospective registry. Well over one hundred disease-related publications attest to the group's impact on the field. Despite these accomplishments, challenging problems remain. DISCUSSION: Collaborative research within a multi-national study group resulted in better definitions of important aspects of the most common bone tumor, osteosarcoma, and its treatments. Important challenges continue to persist.

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69 % of spiked compounds using LC-HRMS and 20-60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants' own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from -9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

Analysis of four polycyclic aromatic hydrocarbons in plant-based food supplements-results of a collaborative study.

The determination of carcinogenic polycyclic aromatic hydrocarbons (PAHs) in food supplements is challenging, especially due to the presence of other e.g. heterogeneous PAH-like compounds in the matrix. A collaborative study with 12 participants was conducted in order to assess performance characteristics of a fast method intended to analyse the four regulated PAHs (PAH 4) benzo[b]fluoranthene [BbF], benz[a]anthracene [BaA], chrysene [CHR] and benzo[a]pyrene [BaP] in five different plant-based food supplements in the form of capsules, powder, and tablets. The principle of the method includes the extraction of PAHs with ethyl acetate: cyclohexane followed by a two-step SPE cleanup and final analysis by GC-MS or LC-FLD. The regulated maximum level for BaP is 10 µg/kg and, for the PAH 4, 50 µg/kg. Accordingly, the method was validated for the regulated PAH 4 analytically challenging concentration range from 2.5 µg/kg to 6.9 µg/kg. The performance criteria for the method set in European Regulation No 333/2007 for the overall repeatability, reproducibility (HorRat values below 2), and recovery (range 50-120%) were fulfilled. Based on the statistical evaluation of the results, it was concluded that the method is a suitable alternative to existing methods and should be studied for additional matrices.

Determining MOSH and MOAH with High Sensitivity in Vegetable Oil─A New, Reliable, and Comparable Approach Using Online LC-GC-FID─Evaluation of Method Precision Data.

A method for the analysis of saturated mineral oil hydrocarbons (MOSH) and aromatic mineral oil hydrocarbons (MOAH) has been developed to improve interlaboratory precisions especially for amounts below 10 mg/kg. This approach was adopted as the standard method DGF C-VI 22 (20) of the German Society of Fat Sciences. Therefore, this method was evaluated on different edible oils containing a variety of interfering biogenic substances. The precision data were determined in an interlaboratory trial with an international group of 14 laboratories from Germany, Austria, and Italy. Good reproducible relative standard deviations for total MOSH (12.5-20.7%) and total MOAH (12.4-39.5%) as well as HorRat values ranging between 1.1 and 2.3 for total MOSH and between 0.9 and 2.6 for total MOAH have been achieved. As some matrices showed residual interferences in the MOAH fraction, these substances were further analyzed by online high-performance liquid chromatography-comprehensive two dimensional gas chromatography with time of flight mass detection.

Validation by collaborative trial of a method for the determination by GC-MS and LC-MS/MS of boar taint marker compounds in pork tissue.

Meat from male pigs may develop an off-flavour, commonly known as boar taint. Castration of male piglets prevents the potential formation of off-flavour. In the suggested method, three marker compounds for boar taint (skatole, androstenone and indole) are quantified in pork fat by isotope dilution gas chromatography mass spectrometry (GC-MS) or by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). This method was validated by collaborative trial according to ISO 5725-2:1994. The studied concentration ranges included sensorial thresholds. The repeatability relative standard deviation (RSDr) ranges from 3% to 10% and the reproducibility relative standard deviation (RSDR) from 10% to about 30%. The method has proven to be robust and free from matrix interferences. The method performance characteristics are compliant with requirements for official control methods in the area of food contaminants; therefore, the method is regarded as fit for its intended purpose.

Interlaboratory validation of an LC-MS/MS method for the determination of melamine and cyanuric acid in animal feed.

Melamine and cyanuric acid have been mixed illegally into food and feed to increase the nitrogen content, which results in deceptively high protein contents. As a consequence, a maximum level for melamine of 2.5 mg kg-1 feed was established by the European Union under Directive 2002/32/EC. The Technical Committee (TC) 327 of the European Committee for Standardisation (CEN) commissioned the standardisation of a method for the analysis of melamine and cyanuric acid in animal feed. One main task in the standardisation process is the performance of a full international collaborative trial, which is described in this paper. After performing a pre-trial study, in the main study eight different feed samples with different concentration levels of melamine and/or cyanuric acid were distributed as double-blind samples to 13 participants. The minimum criterion of eight laboratories submitting results per sample is fulfilled for melamine but only partly for cyanuric acid. The evaluation showed for both analytes a Horwitz ratio (HorRat) well below 2, and meets the requirements stated in the appropriate international protocols. The results demonstrated that the method seems to be suitable for the analysis of melamine and cyanuric acid in animal feed.

Interlaboratory validation of a real-time PCR detection method for bovine- and ovine-derived material.

In this work we performed interlaboratory validation of a Taqman real-time PCR method for the identification of bovine and ovine material. The Bos taurus beta-actin gene (ACTB) and Ovis aries prolactin receptor gene (PRLR) were selected as the bovine and ovine species-specific amplifying target sequences, and primers and TaqMan probes were designed accordingly. The precision, efficiency, false positive rate, limit of detection (LOD95%) and probability of detection (POD) were determined, and the results demonstrated that both bovine and ovine detection methods performed well. The high homogeneity of the results indicates that the detection methods are suitable for a wide range of applications, and the tools developed herein could be applied by official and third-party detection institution to maintain quality in the food and feedstuff industries.

Searching for new biomarkers in ovarian cancer patients: Rationale and design of a retrospective study under the Mermaid III project.

Ovarian cancer is a silent killer and, due to late diagnosis, the primary cause of death amongst gynecological cancers, killing approximately 376 women annually in Denmark. The discovery of a specific and sensitive biomarker for ovarian cancer could improve early diagnosis, but also treatment, by predicting which patients will benefit from specific treatment strategies. The Mermaid III project is consisting of 3 parts including "Early detection, screening and long-term survival," "Biomarkers and/or prognostic markers" and "The infection theory." The present paper gives an overview of the part regarding biomarkers and/or prognostic markers, with a focus on rationale and design. The study described has 3 major branches: microRNAs, epigenetics and Next Generation Sequencing. Tissue and blood from ovarian cancer patients, already enrolled in the prospective ongoing pelvic mass cohort, will be examined. Relevant microRNAs and DNA methylation patterns will be investigated using array technology. Patient exomes will be fully sequenced, and identified genetic variations will be validated with Next Generation Sequencing. In all cases, data will be correlated with clinical information on the patient, in order to identify possible biomarkers. A thorough investigation of biomarkers in ovarian cancer, including large numbers of different markers, has never been done before. Besides from improving diagnosis and treatment, other outcomes could be markers for screening, knowledge of the molecular aspects of cancer and the discovery of new drugs. Moreover, biomarkers are a prerequisite for the development of precision medicine. This study will attack the ovarian cancer problem from several angles, thereby increasing the chance of successfully contributing to saving lives.

Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.

A collaborative trial was conducted to determine the performance characteristics of an analytical method for the quantification of inorganic arsenic (iAs) in food. The method is based on (i) solubilisation of the protein matrix with concentrated hydrochloric acid to denature proteins and allow the release of all arsenic species into solution, and (ii) subsequent extraction of the inorganic arsenic present in the acid medium using chloroform followed by back-extraction to acidic medium. The final detection and quantification is done by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS). The seven test items used in this exercise were reference materials covering a broad range of matrices: mussels, cabbage, seaweed (hijiki), fish protein, rice, wheat, mushrooms, with concentrations ranging from 0.074 to 7.55mgkg(-1). The relative standard deviation for repeatability (RSDr) ranged from 4.1 to 10.3%, while the relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 22.8%.

Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella species and Entero-invasive Escherichia coli in the Netherlands.

An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coli (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic methods and a sample panel, consisting of DNA-extracts and spiked stool samples with different concentrations of Shigella flexneri, was provided to each MML. The results of the trial showed an enormous variety in culture-dependent and molecular diagnostic techniques currently used among MMLs. Despite the various molecular procedures, 15 out of 16 MMLs were able to detect Shigella species or EIEC in all the samples provided, showing that the diversity of methods has no effect on the qualitative detection of Shigella flexneri. In contrast to semi quantitative analysis, the minimum and maximum values per sample differed by approximately five threshold cycles (Ct-value) between the MMLs included in the study. This indicates that defining a uniform Ct-value cut-off for notification to health authorities is not advisable.

Selected reaction monitoring method to determine the species origin of blood-based binding agents in meats: a collaborative study.

Binding products or food 'glues' are used throughout the food industry to increase the meat use rate or to augment economic efficiency. Some of these binders contain thrombin from bovine and porcine blood. The European parliament has recently banned thrombin-based additives and labelling legislation governs their use in the US. A mass spectrometry screening method is available to detect the addition of thrombin agents to foods as there is a need to protect consumers and to avoid misleading trade practices. We report the details of an inter-laboratory trial to determine the transferability of this method to operators in various food testing laboratories, each using a different triple quadrupole mass spectrometer design. The trial was successful with the species origin of the binding agent contained in each of the 43 test materials being correctly reported by the participants. This is consistent with a false positive and false negative rate of 0%. This is the first collaborative study, as far as we are aware, which involves a liquid chromatography mass spectrometry (LC-MS/MS) application to approach a food authenticity issue.