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BACKGROUND: Programmed cell death receptors and ligands in cancer tissue samples are established companion diagnostics for immune checkpoint inhibitor (ICI) therapies. OBJECTIVE: To investigate the relevance of soluble PD-1, PD-L1 and PD-L2 for estimating therapy response and prognosis in non-small cell lung cancer patients (NSCLC) undergoing platin-based combination chemotherapies. METHODS: In a biomarker substudy of a prospective, multicentric clinical trial (CEPAC-TDM) on advanced NSCLC patients, soluble PD-1, PD-L1 and PD-L2 were assessed in serial serum samples by highly sensitive enzyme-linked immunosorbent assays and correlated with radiological response after two cycles of chemotherapy and with overall survival (OS). RESULTS: Among 243 NSCLC patients, 185 achieved response (partial remission and stable disease) and 58 non-response (progression). The distribution of PD-1, PD-L1 and PD-L2 at baseline (C1), prior to staging (C3) and the relative changes (C3/C1) greatly overlapped between the patient groups with response and non-response, thus hindering the discrimination between the two groups. None of the PD markers had prognostic value regarding OS. CONCLUSIONS: Neither soluble PD-1, PD-L1 nor PD-L2 did provide clinical utility for predicting response to chemotherapy and prognosis. Studies on the relevance of PD markers in ICI therapies are warranted. [ABSTRACT FROM AUTHOR]

BACKGROUND: Protein tumor markers are released in high amounts into the blood in advanced non-small cell lung cancer (NSCLC). OBJECTIVE: To investigate the relevance of serum tumor markers (STM) for prognosis, prediction and monitoring of therapy response in NSCLC patients receiving chemotherapy. METHODS: In a biomarker substudy of a prospective, multicentric clinical trial (CEPAC-TDM) on 261 advanced NSCLC patients, CYFRA 21-1, CEA, SCC, NSE, ProGRP, CA125, CA15-3 and HE4 were assessed in serial serum samples and correlated with radiological response after two cycles of chemotherapy and overall (OS) and progression-free survival (PFS). RESULTS: While pretherapeutic STM levels at staging did not discriminate between progressive and non-progressive patients, CYFRA 21-1, CA125, NSE and SCC at time of staging did, and yielded AUCs of 0.75, 0.70, 0.69 and 0.67 in ROC curves, respectively. High pretherapeutic CA15-3 and CA125 as well as high CYFRA 21-1, SCC, CA125 and CA15-3 levels at staging were prognostic for shorter PFS and OS –also when clinical variables were added to the models. CONCLUSIONS: STM at the time of first radiological staging and pretherapeutic CA15-3, CA125 are predictive for first-line treatment response and highly prognostic in patients with advanced NSCLC. [ABSTRACT FROM AUTHOR]

Background: Severe coronavirus disease 2019 (COVID-19) disease courses are characterized by immuno-inflammatory, thrombotic, and parenchymal alterations. Prediction of individual COVID-19 disease courses to guide targeted prevention remains challenging. We hypothesized that a distinct serologic signature precedes surges of IL-6/D-dimers in severely affected COVID-19 patients. Methods: We performed longitudinal plasma profiling, including proteome, metabolome, and routine biochemistry, on seven seropositive, well-phenotyped patients with severe COVID-19 referred to the Intensive Care Unit at the German Heart Center. Patient characteristics were: 65 ± 8 years, 29% female, median CRP 285 ± 127 mg/dL, IL-6 367 ± 231 ng/L, D-dimers 7 ± 10 mg/L, and NT-proBNP 2616 ± 3465 ng/L. Results: Based on time-series analyses of patient sera, a prediction model employing feature selection and dimensionality reduction through least absolute shrinkage and selection operator (LASSO) revealed a number of candidate proteins preceding hyperinflammatory immune response (denoted ΔIL-6) and COVID-19 coagulopathy (denoted ΔD-dimers) by 24–48 h. These candidates are involved in biological pathways such as oxidative stress/inflammation (e.g., IL-1alpha, IL-13, MMP9, C-C motif chemokine 23), coagulation/thrombosis/immunoadhesion (e.g., P- and E-selectin), tissue repair (e.g., hepatocyte growth factor), and growth factor response/regulatory pathways (e.g., tyrosine-protein kinase receptor UFO and low-density lipoprotein receptor (LDLR)). The latter are host- or co-receptors that promote SARS-CoV-2 entry into cells in the absence of ACE2. Conclusions: Our novel prediction model identified biological and regulatory candidate networks preceding hyperinflammation and coagulopathy, with the most promising group being the proteins that explain changes in D-dimers. These biomarkers need validation. If causal, our work may help predict disease courses and guide personalized treatment for COVID-19. [ABSTRACT FROM AUTHOR]

Background: Throughout the COVID-19 pandemic, veterinary diagnostic laboratories have tested diagnostic samples for SARS-CoV-2 both in animals and over 6 million human samples. An evaluation of the performance of those laboratories is needed using blinded test samples to ensure that laboratories report reliable data to the public. This interlaboratory comparison exercise (ILC3) builds on 2 prior exercises to assess whether veterinary diagnostic laboratories can detect Delta and Omicron variants spiked in canine nasal matrix or viral transport medium. Methods: The ILC organizer was an independent laboratory that prepared inactivated Delta variant at levels of 25 to 1000 copies per 50 µL of nasal matrix for blinded analysis. Omicron variant at 1000 copies per 50 µL of transport medium was also included. Feline infectious peritonitis virus (FIPV) RNA was used as a confounder for specificity assessment. Fourteen test samples were prepared for each participant. Participants used their routine diagnostic procedures for RNA extraction and real-time reverse transcriptase-PCR. Results were analyzed according to International Organization for Standardization (ISO) 16140–2:2016. Results: Overall, laboratories demonstrated 93% detection for Delta and 97% for Omicron at 1000 copies per 50 µL. Specificity was 97% for blank samples and 100% for blank samples with FIPV. No differences in Cycle Threshold (Ct) values were significant for samples with the same virus levels between N1 and N2 markers, nor between the 2 variants. Conclusions: The results indicated that all ILC3 participants were able to detect both Delta and Omicron variants. The canine nasal matrix did not significantly affect SARS-CoV-2 detection. [ABSTRACT FROM AUTHOR]

Food fraud, even when not in the news, is ubiquitous and demands the development of innovative strategies to combat it. A new non-targeted method (NTM) for distinguishing spelt and wheat is described, which aids in food fraud detection and authenticity testing. A highly resolved fingerprint in the form of spectra is obtained for several cultivars of spelt and wheat using liquid chromatography coupled high-resolution mass spectrometry (LC-HRMS). Convolutional neural network (CNN) models are built using a nested cross validation (NCV) approach by appropriately training them using a calibration set comprising duplicate measurements of eleven cultivars of wheat and spelt, each. The results reveal that the CNNs automatically learn patterns and representations to best discriminate tested samples into spelt or wheat. This is further investigated using an external validation set comprising artificially mixed spectra, samples for processed goods (spelt bread and flour), eleven untypical spelt, and six old wheat cultivars. These cultivars were not part of model building. We introduce a metric called the D score to quantitatively evaluate and compare the classification decisions. Our results demonstrate that NTMs based on NCV and CNNs trained using appropriately chosen spectral data can be reliable enough to be used on a wider range of cultivars and their mixes. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans. [ABSTRACT FROM AUTHOR]

Featured Application: Droplet digital PCR (ddPCR) in combination with propidium monoazide (PMA) treatment provides a method that is accurate, efficient, and reliable and that can be easily and quickly applied in routine use to quantify Campylobacter jejuni and coli in chicken meat rinses from slaughterhouse and retail. The EU commission established Regulation (2017/1495) in 2017 to reduce Campylobacter on chicken skin and to decrease the number of human cases of campylobacteriosis attributable to the consumption of poultry meat. A Process Hygiene Criterion based on colony-forming unit data was set to a maximum of 1000 CFU Campylobacter spp. per gram chicken neck skin at slaughterhouses. Confronted with stressors, including cold, oxidative stress or antibiotic treatment, live cells may enter into a viable but non-cultivable state (VBNC) and lose the ability to grow, in reference to the plate count ISO 10272-2:2017 method, but still possess the potential to recover and cause infections under favorable conditions. In this study, a droplet digital PCR combined with the intercalating dye propidium monoazide (PMA) was established for quantification of C. coli and C. jejuni in chicken meat rinses. The PMA was used to inactivate DNA from dead cells in this technique. This method was successfully validated against the reference method according to ISO 16140-2:2016 for accuracy and relative trueness. Additionally, it presented a 100% selectivity for Campylobacter jejuni and C. coli. Moreover, the technical measurement uncertainty was determined according to ISO 19036:2019, and the applicability of ddPCR for quantifying C. coli and C. jejuni in chicken meat rinses was investigated on naturally contaminated samples from slaughterhouses and supermarkets. Results obtained from this study demonstrated a strong correlation to qPCR as well as the classical microbiological reference method. [ABSTRACT FROM AUTHOR]

Background: Despite the significance of colonoscopy for early diagnosis of colorectal adenocarcinoma (CRC), population-wide screening remains challenging, mainly because of low acceptance rates. Herein, exosomal (exo-miR) and free circulating microRNA (c-miR) may be used as liquid biopsies in CRC to identify individuals at risk. Direct comparison of both compartments has shown inconclusive results, which is why we directly compared a panel of 10 microRNAs in this entity. Methods: Exo-miR and c-miR levels were measured using real-time quantitative PCR after isolation from serum specimens in a cohort of 69 patients. Furthermore, results were compared to established tumor markers CEA and CA 19-9. Results: Direct comparison of exo- and c-miR biopsy results showed significantly higher microRNA levels in the exosomal compartment (p < 0.001). Exo-Let7, exo-miR-16 and exo-miR-23 significantly differed between CRC and healthy controls (all p < 0.05), while no c-miR showed this potential. Sensitivity and specificity can be further enhanced using combinations of multiple exosomal miRNAs. Conclusions: Exosomal microRNA should be considered as a promising biomarker in CRC for future studies. Nonetheless, results may show interference with common comorbidities, which must be taken into account in future studies. [ABSTRACT FROM AUTHOR]

Meat species authentication in food is most commonly based on the detection of genetic variations. Official food control laboratories frequently apply single and multiplex real-time polymerase chain reaction (PCR) assays and/or DNA arrays. However, in the near future, DNA metabarcoding, the generation of PCR products for DNA barcodes, followed by massively parallel sequencing by next generation sequencing (NGS) technologies, could be an attractive alternative. DNA metabarcoding is superior to well-established methodologies since it allows simultaneous identification of a wide variety of species not only in individual foodstuffs but even in complex mixtures. We have recently published a DNA metabarcoding assay for the identification and differentiation of 15 mammalian species and six poultry species. With the aim to harmonize analytical methods for food authentication across EU Member States, the DNA metabarcoding assay has been tested in an interlaboratory ring trial including 15 laboratories. Each laboratory analyzed 16 anonymously labelled samples (eight samples, two subsamples each), comprising six DNA extract mixtures, one DNA extract from a model sausage, and one DNA extract from maize (negative control). Evaluation of data on repeatability, reproducibility, robustness, and measurement uncertainty indicated that the DNA metabarcoding method is applicable for meat species authentication in routine analysis. [ABSTRACT FROM AUTHOR]

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results. [ABSTRACT FROM AUTHOR]

Background: Fish and fish products are one of the most important food sources of high commercial interest. The global food trade and the associated risks are constantly presenting new challenges to consumer protection and public authorities, which, among other things, demand state-of-the-art analytical methods to ensure food authenticity. Objective: The establishment of MS-based strategies plays a decisive role alongside the (further) development of ELISAor DNA-oriented methods. Methods: In the present work, therefore, the development and in-house validation of an LC-MS and LC-MS/MS-based assay for authenticity testing of certain fish species is described. Results: Based on the execution of a validated bottom-up LC-electrospray-MS and MS/MS assay and multivariate analysis, the commercially available species Lutjanus malabaricus (red snapper) and Sebastes spp. (redfish) are distinguished from each other, whereas an additional 68 samples [nine additional marine species such as pangasius (Pangasianodon hypophthalmus), salmon (Salmo salar), turbot (Scophthalmus maximus), plaice (Pleuronectes platessa), sole (Solea solea), lemon sole (Glyptocephalus cynoglossus), halibut (Reinhardtius hypoglossoides), red salmon (Oncorhynchus nerka), and great scallop (Pecten jacobaeus)] served as blinded negative controls to ensure the specificity of the assay. Conclusions and Highlights: A promising LC-MS and LC-MSMS based assay has been developed that could enable the detection of fish fraud at the protein level in the future. [ABSTRACT FROM AUTHOR]

Leaching processes are responsible for the release of biocides from treated materials into the environment. Adequate modeling of emission processes is required in order to predict emission values in the framework of the risk assessment of biocidal products intended for long-term service life. Regression approaches have been applied to data obtained from the long-term monitoring of biocide emissions in experiments involving semi-field conditions. Due to the complex interaction of different underlying mechanisms such as water and biocide diffusion and desorption, however, these attempts have proven to be of limited usefulness – at least, for the available biocide emission data. It seems that the behavior of the biocide emission curve depends to a considerable extent on whichever underlying mechanism is slowest at a given point in time, thus limiting the amount of biocide available for release. Building on results obtained in the past few years, the authors propose a criterion for determining which mechanism controls the leaching process at a given point in time based on the slope of the log-log emission curve. In addition, a first-order approximation of this slope value is presented which displays advantages both in terms of computability and interpretability. Finally, an algorithm for the determination of breakpoints in the slope of the log-log emission curve is presented for the demarcation of phases within which one mechanism acts as a limiting factor. Image 1 • The usefulness of regression approaches to model leaching processes is limited. • Controlling mechanisms are identified via slope values of log-log emission curves. • A promising expression for these slope values is derived. • Breakpoints in emission curves can be identified by maximum likelihood estimation. [ABSTRACT FROM AUTHOR]

Various standardised test methods are used by laboratories to determine the hazardous properties and safety characteristics of chemical substances and mixtures, e.g. test methods published by the European Union (2008) and by the United Nations (2015). The test results are the basis (amongst others) for the correct classification and labelling of chemicals as hazardous according to GHS/CLP or as dangerous goods according to Transport Regulations. The REACH Regulation obliges all manufacturers and importers of chemicals to identify and manage risks associated with the substances they manufacture and market. Via the eChemPortal (www.echemportal.org) many data from the REACH registration dossiers are available. However, the quality and correctness of the information remains in the responsibility of the data submitter. Unfortunately, the quality of information on physicochemical properties is not always adequate, so the quality and adequacy of the data submitted shall be improved. This paper focusses on the quality of test results obtained by laboratories and the validation of the test methods used e.g. for classification of dangerous goods and indicates recent developments as well as steps to be taken for further improvement. In particular, the importance of interlaboratory tests and the need to validate test methods are presented in this publication. Interlaboratory tests play a decisive role in assessing the reliability of laboratory test results. Participation in interlaboratory tests is not only a crucial element of the quality assurance of laboratories; as such it is explicitly recommended in ISO/IEC 17025, ISO (2017). In addition, interlaboratory tests are also used to improve and validate test methods and can be used for the determination of the measurement uncertainty, Hässelbarth (2004), ISO (2004). This paper specially addresses interlaboratory tests for the validation of test methods performed by CEQATDGHS (Centre for quality assurance for testing of dangerous goods and hazardous substances, www.ceqatdghs. bam.de) and outlines typical results and general conclusions and steps to be taken to guarantee that laboratory test results are fit for the purpose and of high quality. [ABSTRACT FROM AUTHOR]

ABSTRACT A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Multiple factors contribute to the development and progression of breast cancer. Markers of tumor growth and invasion, cell death, immune activation, and angiogenesis can be assessed in parallel by a novel multiplex immunoassay panel. The diagnostic performance of a multiplex cancer biomarker magnetic bead panel comprising 24 tumor associated parameters was evaluated in sera of 154 women including 77 patients with breast cancer, 10 with precancerous lesions, 31 with benign breast diseases, and 36 healthy controls. Marker levels were log-transformed for variance stabilization. Significance testing was done using t-test or Wilcoxon rank-sum test with correction of p values for multiple testing. Furthermore, receiver operating characteristic analyses were performed. Serum levels of several biomarkers were significantly (p = 0.001) higher in cancer patients than in healthy controls, particularly alpha-fetoprotein, cancer antigen 15-3, cancer antigen 19-9, migration inhibitory factor, carcinoembryonic antigen, cancer antigen 125, hepatocyte growth factor, soluble Fas, tumor necrosis factor-a, stem cell factor, and osteopontin. As most markers were also elevated in benign breast diseases, only cancer antigen 15-3 showed significant differences to cancer patients (p = 0.001). The resulting areas under the curve in receiver operating characteristic curves for discrimination between benign and malignant breast diseases achieved 0.71 with a sensitivity of 33.8% at 95% specificity. Multiplexing enables parallel analysis of different biomarker classes for cancer detection. Established cancer antigen 15-3 proved to be most relevant for differential diagnosis. [ABSTRACT FROM AUTHOR]

Background: This meta-analysis evaluated whether pretherapy serum levels of carcinoembryonic antigen (CEA) and cytokeratin-19 fragments (CYFRA 21-1) are predictive of response to therapy in non-small cell lung cancer (NSCLC) and whether changes in these markers during vs pretherapy are indicative of response.Methods: Original peer-reviewed studies enrolling adults with untreated advanced NSCLC were identified using PubMed. Two reviewers independently extracted data from eligible studies and assessed study heterogeneity and the risk of study bias.Results: Fourteen studies were eligible; 11 had objective response as an end point and three evaluated clinical benefit (i.e., response and stable disease). Study bias was relatively low. Both markers showed comparable modest predictive value across studies, with baseline CYFRA 21-1 numerically better in predicting treatment benefit. A good performance in identifying objective response during treatment was seen (AUC 0.724 (95% CI 0.667-0.785) for CYFRA 21-1 and 0.728 (95% CI, 0.599-0.871) for CEA). A decline in CYFRA 21-1 levels during treatment was highly indicative for objective response (sensitivity 79.1% (95% CI 71.5-85.1)).Conclusions: Comprehensive analysis of study heterogeneity and bias provides a high level of evidence for the clinical utility of CEA and CYFRA 21-1 for the prediction and monitoring of response in NSCLC. [ABSTRACT FROM AUTHOR]

Supplemental digital content is available in the text. Objectives: The aim of this study was to determine whether the Risk of Ovarian Malignancy Algorithm (ROMA) is more accurate than the human epididymis 4 (HE4) or carbohydrate antigen 125 (CA125) biomarkers with respect to the differential diagnosis of women with a pelvic mass. The secondary objective is to assess the performance of ROMA in early-stage ovarian cancer (OC) and late-stage OC, as well as premenopausal and postmenopausal patient populations. Methods/Materials: The PubMed and Google Scholar databases were searched for relevant clinical studies. Eligibility criteria included comparison of ROMA with both HE4 and CA125 levels in OC (unspecified, epithelial, and borderline ovarian tumors), use of only validated ROMA assays, presentation of area under the curve and sensitivity/specificity data, and results from early-stage OC, late-stage OC and premenopausal and postmenopausal women. Area under the curve (AUC), sensitivity/specificity, and the diagnostic odds ratio (DOR) results were summarized. Results: Five studies were selected comprising 1975 patients (premenopausal, n = 1033; postmenopausal, n = 925; benign, n = 1387; early stage, n = 192; and late stage, n = 313). On the basis of the AUC (95% confidence interval) data for all patients, ROMA (0.921 [0.855–0.960]) had a numerically greater diagnostic performance than CA125 (0.883 [0.771–0.950]) and HE4 (0.899 [0.835–0.943]). This was also observed in each of the subgroup populations, in particular, the postmenopausal patients and patients with early OC. The sensitivity and specificity (95% confidence interval) results showed ROMA (sensitivity, 0.873 [0.752–0.940]; specificity, 0.855 [0.719–0.932]) to be numerically superior to CA125 (sensitivity, 0.796 [0.663–0.885]; specificity, 0.825 [0.662–0.919]) and HE4 (sensitivity, 0.817 [0.683–0.902]; specificity, 0.851 [0.716–0.928]) in all patients and for the early- and late-stage OC subgroups. Finally, the ROMA log DOR results were better than HE4 and CA125 log DOR results especially for the early-stage patient group. Conclusions: The results presented support the use of ROMA to improve clinical decision making, most notably in patients with early OC. [ABSTRACT FROM AUTHOR]