The level of anti-D antibodies in human immunoglobulin products for intravenous administration (IVIG) is controlled by the direct haemagglutination method prescribed by the European Pharmacopoeia (Ph. Eur.) that requires 2 control reference reagents. The World Health Organization (WHO) positive control International Reference Reagent (IRR; 02/228) with a nominal titre of 8 defines the highest acceptable titre, while the negative control preparation (02/226) has a nominal titre of <2. Working reference preparations (04/132 and 04/140) were subsequently established as Biological Reference Preparations (BRPs) for the Ph. Eur., and for distribution by the United States Food and Drug Administration (US FDA) and the National Institute for Biological Standards and Control (NIBSC). Due to diminishing stocks of these working reference preparations across the 3 institutions, a joint international study was organised to establish harmonised replacement batches. Sixteen laboratories contributed data to the study to evaluate positive and negative candidate replacement batches (13/148 and 12/300, respectively) against the WHO positive and negative control IRRs and the current working reference preparations (BRPs). The results show that the candidate reference preparations (13/148 and 12/300) are indistinguishable from the corresponding IRRs and current BRPs. The candidate preparations 13/148 and 12/300 were adopted by the Ph. Eur. Commission as Immunoglobulin (anti-D antibodies test) BRP batch 2 and Immunoglobulin (anti-D antibodies test negative control) BRP batch 2 with nominal haemagglutination titres of 8 and <2, respectively. The same materials were also adopted as NIBSC and US FDA reference preparations, thus ensuring full harmonisation.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test\'s analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.

A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the N-glycan structures and proportions including their loci and occupancy of ricin CRM-LS-1. The glycan profile was compared with ricin from different preparations and other cultivars and isoforms. A total of 15 different oligomannosidic or paucimannosidic structures were identified in CRM-LS-1. Paucimannose was mainly found within the A-chain and oligomannose constituted the major glycan type of the B-chain. Furthermore, the novel primary structure variants E138 and D138 and four different C-termini of the A-chain as well as two B-chain variants V250 and F250 were elucidated. While the glycan proportions and loci were similar among all variants in CRM-LS-1 and ricin isoforms D and E of all cultivars analysed, a different stoichiometry for isoforms D and E and the amino acid variants were found. This detailed physicochemical characterization of ricin regarding the glycan profile and amino acid sequence variations yields unprecedented insight into the molecular features of this protein toxin. The variable attributes discovered within different cultivars present signature motifs and may allow discrimination of the biotoxin\'s origin that are important in molecular forensic profiling. In conclusion, our data of in-depth CRM-LS-1 characterization combined with the analysis of other cultivars is representative for known ricin variants.

Spinach (Spinacia oleracea) is one of the most famous vegetables worldwide, rich in essential metabolites for various health benefits. It is a valuable plant source that has the potential to be a nutraceutical. This study aimed to evaluate the single characteristic marker compound to establish the validation of HPLC-DAD methods applied to the development of a nutraceutical using spinach samples. Six metabolites (1-6) were identified from the spinach samples such as freeze-dried spinach (FDS) and spinach extract concentrate (SEC) by LC-Q-TOF/MS analysis. Among the six metabolites, 3\',4\',5-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4\'-glucuronide (TMG) was selected as a marker compound due to its highest abundance and high selectivity. The specificity, accuracy, linearity, precision, repeatability, limit of detection (LOD), and limit of quantification (LOQ) of TMG in the spinach samples (FDS and SEC) were validated according to AOAC international guideline. The specificity was confirmed by monitoring the well separation of the marker compound from other compounds of spinach samples in the base peak intensity (BPI) and ultraviolet (UV) chromatogram. The calibration curve of TMG (15.625~500 μg/mL) had reasonable linearity (R2 = 0.999) considered with LOD and LOQ values, respectively. Recovery rate of TMG was 93-101% for FDS and 90-95% for SEC. The precision was less than 3 and 6% in the intraday and interday. As a result, the HPLC-DAD validation method of TMG in the spinach samples (FDS and SEC) was first established with AOAC and KFDA regulations for approving functional ingredients in functional foods.

BACKGROUND: The exploration of cancer vaccines has yielded a multitude of studies, resulting in a diverse collection of information. The heterogeneity of cancer vaccine data significantly impedes effective integration and analysis. While CanVaxKB serves as a pioneering database for over 670 manually annotated cancer vaccines, it is important to distinguish that a database, on its own, does not offer the structured relationships and standardized definitions found in an ontology. Recognizing this, we expanded the Vaccine Ontology (VO) to include those cancer vaccines present in CanVaxKB that were not initially covered, enhancing VO\'s capacity to systematically define and interrelate cancer vaccines.
RESULTS: An ontology design pattern (ODP) was first developed and applied to semantically represent various cancer vaccines, capturing their associated entities and relations. By applying the ODP, we generated a cancer vaccine template in a tabular format and converted it into the RDF/OWL format for generation of cancer vaccine terms in the VO. \'12MP vaccine\' was used as an example of cancer vaccines to demonstrate the application of the ODP. VO also reuses reference ontology terms to represent entities such as cancer diseases and vaccine hosts. Description Logic (DL) and SPARQL query scripts were developed and used to query for cancer vaccines based on different vaccine\'s features and to demonstrate the versatility of the VO representation. Additionally, ontological modeling was applied to illustrate cancer vaccine related concepts and studies for in-depth cancer vaccine analysis. A cancer vaccine-specific VO view, referred to as \"CVO,\" was generated, and it contains 928 classes including 704 cancer vaccines. The CVO OWL file is publicly available on: http://purl.obolibrary.org/obo/vo/cvo.owl , for sharing and applications.
CONCLUSIONS: To facilitate the standardization, integration, and analysis of cancer vaccine data, we expanded the Vaccine Ontology (VO) to systematically model and represent cancer vaccines. We also developed a pipeline to automate the inclusion of cancer vaccines and associated terms in the VO. This not only enriches the data\'s standardization and integration, but also leverages ontological modeling to deepen the analysis of cancer vaccine information, maximizing benefits for researchers and clinicians.
BACKGROUND: The VO-cancer GitHub website is: https://github.com/vaccineontology/VO/tree/master/CVO .

OBJECTIVE: To assess the adequacy of linear function of calibration according to GOST R ISO 11095-2007 for ethanol mass concentration measurement using internal reference materials (RMs).
METHODS: An experiment on calibration in accordance with the GOST R ISO 11095-2007 National standard of the RF was carried out using internal RMs, namely aqueous solutions of ethanol at different concentrations. Measurements were performed for two subbands of ethanol concentrations at RMs: 0.15-1.05 and 1.0-7.0 mg/ml - according to the certified methodology.
RESULTS: The graphs of the calibration\'s functions based on experimental data are consistent with the assumption of the calibration function\'s linearity, as well as the assumption of the standard deviation\'s constance of residues is equitable for two subbands of RMs.
CONCLUSIONS: Proven linear models in the calibration experiment may be recommended for use in the ethanol mass concentration measurement.
UNASSIGNED: Оценка адекватности линейной функции калибровки по ГОСТ Р ИСО 11095-2007 для измерения массовой концентрации этанола с применением внутренних образцов сравнения (Reference Material, RM).
UNASSIGNED: Проведен эксперимент по калибровке в соответствии с Национальным стандартом РФ ГОСТ Р ИСО 11095-2007 с применением внутренних RM — водных растворов этанола в разных концентрациях. Измерения выполняли для двух поддиапазонов концентраций этанола на RM: 0,15—1,05 и 1,0—7,0 мг/мл — в соответствии с аттестованной методикой.
UNASSIGNED: Графики функций калибровки, построенные по экспериментальным данным, согласуются с предположением о линейности функции калибровки, а также предположение о постоянстве стандартного отклонения остатков является справедливым для двух поддиапазонов RM.
UNASSIGNED: Подтвержденные линейные модели в ходе эксперимента по калибровке могут быть рекомендованы к применению на практике для измерения массовой концентрации этанола.

BACKGROUND: Chlorinated paraffins (CPs) are industrial chemicals categorised as persistent organic pollutants because of their toxicity, persistency and tendency to long-range transport, bioaccumulation and biomagnification. Despite having been the subject of environmental attention for decades, analytical methods for CPs still struggle reaching a sufficient degree of accuracy. Among the issues negatively impacting the quantification of CPs, the unavailability of well-characterised standards, both as pure substances and as matrix (certified) reference materials (CRMs), has played a major role. The focus of this study was to provide a matrix CRM as quality control tool to improve the comparability of CPs measurement results.
RESULTS: We present the process of certification of ERM®-CE100, the first fish reference material assigned with certified values for the mass fraction of short-chain and medium-chain chlorinated paraffins (SCCPs and MCCPs, respectively). The certification was performed in accordance with ISO 17034:2016 and ISO Guide 35:2017, with the value assignment step carried out via an intercomparison of laboratories of demonstrated competence in CPs analysis and applying procedures based on different analytical principles. After confirmation of the homogeneity and stability of the CRM, two certified values were assigned for SCCPs, depending on the calibrants used: 31 ± 9 μg kg-1 and 23 ± 7 μg kg-1. The MCCPs certified value was established as 44 ± 17 μg kg-1. All assigned values are relative to wet weight in the CRM that was produced as a fish paste to enhance similarity to routine biota samples.
UNASSIGNED: The fish tissue ERM-CE100 is the first matrix CRM commercially available for the analysis of CPs, enabling analytical laboratories to improve the accuracy and the metrological traceability of their measurements. The certified CPs values are based on results obtained by both gas and liquid chromatography coupled with various mass spectrometric techniques, offering thus a broad validity to laboratories employing different analytical methods and equipment.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

BACKGROUND: Estimation of prevalence and diagnostic test accuracy in tuberculosis (TB) prevalence surveys suffer from reference standard and verification biases. The former is attributed to the imperfect reference test used to bacteriologically confirm TB disease. The latter occurs when only the participants screening positive for any TB-compatible symptom or chest X-ray abnormality are selected for bacteriological testing (verification). Bayesian latent class analysis (LCA) alleviates the reference standard bias but suffers verification bias in TB prevalence surveys. This work aims to identify best-practice approaches to simultaneously alleviate the reference standard and verification biases in the estimates of pulmonary TB prevalence and diagnostic test performance in TB prevalence surveys.
METHODS: We performed a secondary analysis of 9869 participants aged ≥15 years from a community-based multimorbidity screening study in a rural district of KwaZulu-Natal, South Africa (Vukuzazi study). Participants were eligible for bacteriological testing using Xpert Ultra and culture if they reported any cardinal TB symptom or had an abnormal chest X-ray finding. We conducted Bayesian LCA in five ways to handle the unverified individuals: (i) complete-case analysis, (ii) analysis assuming the unverified individuals would be negative if bacteriologically tested, (iii) analysis of multiply-imputed datasets with imputation of the missing bacteriological test results for the unverified individuals using multivariate imputation via chained equations (MICE), and simultaneous imputation of the missing bacteriological test results in the analysis model assuming the missing bacteriological test results were (iv) missing at random (MAR), and (v) missing not at random (MNAR). We compared the results of (i)-(iii) to the analysis based on a composite reference standard (CRS) of Xpert Ultra and culture. Through simulation with an overall true prevalence of 2.0%, we evaluated the ability of the models to alleviate both biases simultaneously.
RESULTS: Based on simulation, Bayesian LCA with simultaneous imputation of the missing bacteriological test results under the assumption that the missing data are MAR and MNAR alleviate the reference standard and verification biases. CRS-based analysis and Bayesian LCA assuming the unverified are negative for TB alleviate the biases only when the true overall prevalence is <3.0%. Complete-case analysis produced biased estimates. In the Vukuzazi study, Bayesian LCA with simultaneous imputation of the missing bacteriological test results under the MAR and MNAR assumptions produced overall PTB prevalence of 0.9% (95% Credible Interval (CrI): 0.6-1.9) and 0.7% (95% CrI: 0.5-1.1) respectively alongside realistic estimates of overall diagnostic test sensitivity and specificity with substantially overlapping 95% CrI. The CRS-based analysis and Bayesian LCA assuming the unverified were negative for TB produced 0.7% (95% CrI: 0.5-0.9) and 0.7% (95% CrI: 0.5-1.2) overall PTB prevalence respectively with realistic estimates of overall diagnostic test sensitivity and specificity. Unlike CRS-based analysis, Bayesian LCA of multiply-imputed data using MICE mitigates both biases.
CONCLUSIONS: The findings demonstrate the efficacy of these advanced techniques in alleviating the reference standard and verification biases, enhancing the robustness of community-based screening programs. Imputing missing values as negative for bacteriological tests is plausible under realistic assumptions.

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients.