Interlaboratory Evaluation Research Articles

New statistical framework for interlaboratory evaluation of anti-doping testing results by WADA

The World Anti-doping Agency (WADA) International Standard for Laboratories (ISL), developed as part of the World Anti-Doping Program, requires satisfactory laboratory performance in the WADA External Quality Assessment Scheme (EQAS) in order to obtain and maintain WADA accreditation. Under this mandate, WADA regularly distributes urine and blood test samples to anti-doping laboratories to continuously monitor their proficiency. Over the years, WADA has employed classical, generic statistical methods, in accordance to ISO 13528, to evaluate quantitative EQAS results. Here, we set out the rationale for a modern statistical approach that recognizes and addresses the particular features of the measurement results typically obtained in such tests and present an approach involving Bayesian measurement models and statistical data analysis that is tailored specifically to anti-doping testing.

Interlaboratory evaluation of LC-MS-based biomarker assays.

Validation of biomarker assays is crucial for effective drug development and clinical applications. Interlaboratory reproducibility is vital for reliable comparison and combination of data from different centers. This review summarizes interlaboratory studies of quantitative LC-MS-based biomarker assays using reference standards for calibration curves. The following points are discussed: trends in reports, reference and internal standards, evaluation of analytical validation parameters, study sample analysis and normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studies is limited, necessitating further research. Some reports suggest methods to address variations in biomarker assay data among laboratories, facilitating organized studies and data combination. Method validation across laboratories is crucial for reducing interlaboratory differences and reflecting target biomarker responses.

Interlaboratory Comparison of SpermX™ and Conventional Differential Extractions Indicated High Male DNA Recovery by the SpermX Method

The National Institute of Justice reported that current methods for processing sexual assault samples have a high failure rate, with 60 to 80 percent of tested kits unable to produce usable DNA profiles. Even when samples test positive for male DNA, 34 percent of sexual assault kits (SAKs) do not yield recovered male DNA after differential extraction. Less than 30% recovery of available sperm DNA contributes to this low success rate. The SpermX™ method (SX) has been shown to recover 80 percent or more of sperm DNA from sexual assault samples. An interlaboratory evaluation compared SX to standard differential extraction (DE) protocols. Mock samples with known ratios of female epithelial cells and sperm cells were processed using both methods. Results revealed that SX consistently provided CODIS up-loadable DNA profiles, even with as few as 25 sperm cells, whereas DE failed to produce usable results. On average, SX yielded a seven-fold increase in the recovery of unshared male alleles compared to DE. In conclusion, SX outperformed DE in recovering higher quantities of male DNA with minimal female carryover in sexual assault-type samples. This improved success rate in obtaining usable DNA profiles can significantly aid in solving sexual assault cases.

Improvement of interlaboratory evaluation method of olfactometry in Japan

Improvement of interlaboratory evaluation method of olfactometry in Japan

Interlaboratory evaluation of quality control methods for circulating cell-free DNA extraction

Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is significantly impacted by sample quality and standardised approaches for assessing the quality of ccfDNA are not yet established. In this study we evaluated the application of nucleic acid “spike-in” control materials to aid quality control (QC) and standardisation of cfDNA isolation for use in in vitro diagnostic assays. We describe an approach for the design and characterisation of in-process QC materials, illustrating it with a spike-in material containing an exogenous Arabidopsis sequence and DNA fragments approximating to ccfDNA and genomic DNA lengths. Protocols for inclusion of the spike-in material in plasma ccfDNA extraction and quantification of its recovery by digital PCR (dPCR) were assessed for their suitability for process QC in an inter-laboratory study between five expert laboratories, using a range of blood collection devices and ccfDNA extraction methods. The results successfully demonstrated that spiking plasmid-derived material into plasma did not deleteriously interfere with endogenous ccfDNA recovery. The approach performed consistently across a range of commonly-used extraction protocols and was able to highlight differences in efficiency and variability between the methods, with the dPCR quantification assay performing with good repeatability (generally CV <5%). We conclude that initial findings demonstrate that this approach appears “fit for purpose” and spike-in recovery can be combined with other extraction QC metrics for monitoring the performance of a process over time, or in the context of external quality assessment.

Interlaboratory comparison study of a chemical profiling method for methylphosphonic dichloride, a nerve agent precursor

An interlaboratory evaluation of a chemical profiling method for a nerve agent precursor was performed by eight chemical analytical laboratories with a wide global distribution. A set of chemical attribution signatures (i.e. impurities) present in methylphosphonic dichloride (DC) were analysed by gas chromatography-mass spectrometry (GC/MS). The GC/MS analyses were performed with the individual instrumentation and analytical methods used by the laboratories in proficiency tests organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Two batches of DC, produced by two different production routes, were dispatched together with one reference sample. By the use of a targeted MS-library, sixteen chemical attribution signatures were analysed in the DC samples. Retention indices, mass spectra and relative peak areas of the GC/MS data were evaluated. When the within batch-data of the eight laboratories were compared, similarity values of 0.720–0.995 were obtained. In addition, the two different batches had clearly unique chemical attribution profiles, as indicated by a large between batch-distance similarity values of 0.509–0.576. Retention indices showed less than ± 14 retention index unit variation for the 16 chemical attribution signatures. This study showed the potential of getting consistent chemical profiling data over multiple laboratories by the use of retention indices for alignment of GC/MS-data. The results indicate that the GC/MS instrumentation and methods used at most OPCW designated laboratories are valid tools for the acquisition of chemical profiling data of importance for the attribution analysis to study suspected production or use of chemical warfare agents.

Interlaboratory prevalidation of a new in vitro transcriptional activation assay for the screening of (anti-)androgenic activity of chemicals using the UALH-hAR cell line

We report an interlaboratory evaluation of a recently developed androgen receptor (AR) transactivation assay using the UALH-hAR reporter cell line that stably expresses the luciferase gene under the transcriptional control of androgen receptor elements (AREs) with no glucocorticoid receptor (GR) crosstalk. Herein, a two-step prevalidation study involving three laboratories was conducted to assess performance criteria of the method such as transferability as well as robustness, sensitivity, and specificity. The first step consisted in the validation of the transfer of the cell line to participant laboratories through the testing of three reference chemicals: the AR agonist dihydrotestosterone, the AR antagonist hydroxyflutamide and the glucocorticoid dexamethasone. Secondly, a blinded study was conducted by screening a selection of ten chemicals, including four AR agonists, five AR antagonists, and one non-active chemical. All test compounds yielded the same activity profiles in all laboratories. The logEC50 (agonist assay) or logIC50 (antagonist assay) were in the same range, with intra-laboratory coefficients of variation (CVs) of 0.1–3.4% and interlaboratory CVs of 1–4%, indicating very good within- and between-laboratory reproducibility. Our results were consistent with literature and regulatory data (OECD TG458). Overall, this interlaboratory study demonstrated that the UALH-hAR assay is transferable, produces reliable, accurate and specific (anti)androgenic activity of chemicals, and can be considered for further regulatory validation.

Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer.

Native ion mobility-mass spectrometry (IM-MS) has emerged as an information-rich technique for gas phase protein structure characterization; however, IM resolution is currently insufficient for the detection of subtle structural differences in large biomolecules. This challenge has spurred the development of collision-induced unfolding (CIU) which utilizes incremental gas phase activation to unfold a protein in order to expand the number of measurable descriptors available for native protein ions. Although CIU is now routinely used in native mass spectrometry studies, the interlaboratory reproducibility of CIU has not been established. Here we evaluate the reproducibility of the CIU data produced across three laboratories (University of Michigan, Texas A&M University, and Vanderbilt University). CIU data were collected for a variety of protein ions ranging from 8.6-66 kDa. Within the same laboratory, the CIU fingerprints were found to be repeatable with root mean square deviation (RMSD) values of less than 5%. Collision cross section (CCS) values of the CIU intermediates were consistent across the laboratories, with most features exhibiting an interlaboratory reproducibility of better than 1%. In contrast, the activation potentials required to induce protein CIU transitions varied between the three laboratories. To address these differences, three source assemblies were constructed with an updated ion activation hardware design utilizing higher mechanical tolerance specifications. The production-grade assemblies were found to produce highly consistent CIU data for intact antibodies, exhibiting high precision ion CCS and CIU transition values, thus opening the door to establishing databases of CIU fingerprints to support future biomolecular classification efforts.

Electrodialysis with Bipolar Membranes for the Generation of NaOH and HCl Solutions from Brines: An Inter-Laboratory Evaluation of Thin and Ultrathin Non-Woven Cloth-Based Ion-Exchange Membranes.

The SEArcularMINE project aims to recover critical raw materials (CRMs) from brines from saltworks, thus facing a CRM shortage within Europe. To promote a fully circular scheme, the project valorises concentrated brines using electrodialysis with bipolar membranes (EDBM) to generate the required amounts of reactants (i.e., acids and bases). Regarding the performances of new non-woven cloth ion-exchange membranes (Suez): (i) an ultra-thin non-woven polyester cloth and (ii) a thin polypropylene cloth acting as the support structures were assessed. Additionally, the anion layer includes a catalyst to promote the water dissociation reaction. The effect of current density (100, 200, and 300 A m-2) on the performance of two combinations of membranes in an inter-laboratory exercise using 2 M NaCl was evaluated. According to statistical analysis ANOVA, there was an agreement on the results obtained in both laboratories. NaOH/HCl solutions up to 0.8 M were generated working at 300 A m-2 using both combinations of membranes. Regarding the performance parameters, stack set-ups incorporating thin polypropylene membranes showed lower specific energy consumption (SEC) and higher specific productivity (SP) than ultra-thin polypropylene ones. Hence, for ultra-thin polypropylene membranes, SEC was reported to be between 2.18 and 1.69 kWh kg-1NaOH and SP between 974 and 314 kg m-2 y-1.

Interlaboratory Evaluation of Enterococcus faecium NRRL B-2354 as a Salmonella Surrogate for Validating Thermal Treatment of Multiple Low-Moisture Foods

This multi-institutional study assessed the efficacy of Enterococcus faecium NRRL B-2354 as a nonpathogenic Salmonella surrogate for thermal processing of nonfat dry milk powder, peanut butter, almond meal, wheat flour, ground black pepper, and date paste. Each product was analyzed by two laboratories (five independent laboratories total), with the lead laboratory inoculating (E. faecium or a five-strain Salmonella enterica serovar cocktail of Agona, Reading, Tennessee, Mbandaka, and Montevideo) and equilibrating the product to the target water activity before shipping. Both laboratories subjected samples to three isothermal treatments (between 65 and 100°C). A log-linear and Bigelow model was fit to survivor data via one-step regression. On the basis of D80°C values estimated from the combined model, E. faecium was more thermally resistant (P < 0.05) than Salmonella in nonfat dry milk powder (DEf-80°C, 100.2 ± 5.8 min; DSal-80°C, 28.9 ± 1.0 min), peanut butter (DEf-80°C, 133.5 ± 3.1 min; DSal-80°C, 57.6 ± 1.5 min), almond meal (DEf-80°C, 34.2 ± 0.4 min; DSal-80°C, 26.1 ± 0.2 min), ground black pepper (DEf-80°C, 3.2 ± 0.8 min; DSal-80°C, 1.5 ± 0.1 min), and date paste (DEf-80°C, 1.5 ± 0.0 min; DSal-80°C, 0.5 ± 0.0 min). Although the combined laboratory D80°C for E. faecium was lower (P < 0.05) than for Salmonella in wheat flour (DEf-80°C, 9.4 ± 0.1 min; DSal-80°C, 10.1 ± 0.2 min), the difference was ∼7%. The zT values for Salmonella in all products and for E. faecium in milk powder, almond meal, and date paste were not different (P > 0.05) between laboratories. Therefore, this study demonstrated the impact of standardized methodologies on repeatability of microbial inactivation results. Overall, E. faecium NRRL B-2354 was more thermally resistant than Salmonella, which provides support for utilizing E. faecium as a surrogate for validating thermal processing of multiple low-moisture products. However, product composition should always be considered before making that decision.

Development and inter-laboratory evaluation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA

Responding to the growing scientific and practical interest in forensic DNA phenotyping, the VISible Attributes through GEnomics (VISAGE) Consortium was founded in 2017 with the main goal of developing and validating new and reliable molecular and statistical tools to predict appearance, ancestry and age from DNA. Here, we describe the development and inter-laboratory evaluation and validation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA. The VISAGE Enhanced Tool for Appearance and Ancestry is the first forensic-driven genetic laboratory tool that comprises well-established markers for eye, hair and skin color with more recently discovered DNA markers for eyebrow color, freckling, hair shape and male pattern baldness and bio-geographic ancestry informative DNA markers. The bio-geographic ancestry markers include autosomal SNPs (bi- and tri-allelic SNPs), X-SNPs, Y-SNPs and autosomal Microhaplotypes. In total, primers targeting 524 SNPs (representing a 97.6% assay conversion rate) were successfully designed using AmpliSeq into a single primer pool (i.e., one multiplex assay) and sequenced with the Ion S5. In a collaborative framework, five VISAGE laboratories tested the VISAGE Enhanced Tool for Appearance and Ancestry on reproducibility, sensitivity, genotyping concordance, mixtures, species specificity and performance in relevant forensic conditions, including inhibitor-spiked, mock casework and artificially degraded samples. Based on our results, the VISAGE Enhanced Tool for Appearance and Ancestry is a robust, reproducible, and – for the large SNP number - fairly sensitive MPS assay with high concordance rates. With the VISAGE Enhanced Tool for Appearance and Ancestry introduced here, the VISAGE Consortium delivers the first single DNA-test for combined appearance prediction based on seven traits together with bio-geographic ancestry inference based on major continental regions for separated bi-parental and paternal ancestry, which represents the most comprehensive validated laboratory tool currently available for Forensic DNA Phenotyping.

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa=0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation=0.04). The ICC was 0.83 (95% confidence interval=0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.

Multicentre interlaboratory analysis of routine susceptibility testing with a challenge panel of resistant strains

In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation.

Interlaboratory Evaluation of the Analytical Performance of Size Exclusion Chromatography for Small Aggregates in Biopharmaceuticals and Considerations on Development of Analytical Procedures

Interlaboratory Evaluation of the Analytical Performance of Size Exclusion Chromatography for Small Aggregates in Biopharmaceuticals and Considerations on Development of Analytical Procedures

Inter-laboratory validation of an ISO test method for measuring enzyme activities in soil samples using colorimetric substrates.

The evaluation of soil quality requires the use of robust methods to assess biologically based indicators. Among them, enzyme activities are used for several decades, but there is a clear need to update their measurement methods for routine use, in combining feasibility, accuracy, and reliability. To this end, the platform Biochem-Env optimized a miniaturized method to measure enzyme activities in soils using colorimetric substrates in micro-well plates. The standardization of the method was carried out within the framework of ISO/TC 190/SC 4/WG 4 "Soil quality - Biological methods" workgroup, recommending an inter-laboratory evaluation for the publication of a full ISO standard. That evaluation, managed by the platform, was based on the measurement, in six soils of contrasted physicochemical properties, of the ten soil enzyme activities described in the standard. Eight laboratories were involved in the validation study. Only 2.7% of outliers were identified from the analyses of the whole dataset. The repeatability and reproducibility of the method were determined by computing, respectively, the intra-laboratory (CVr,) and inter-laboratory (CVR) coefficients of variation for each soil and enzyme. The mean CVr ranged from 4.5% (unbuffered phosphatase) to 9.9% (α-glucosidase), illustrating a reduced variability of enzyme activities within laboratories. The mean CVR ranged from 13.8% (alkaline phosphatase) to 30.9% (unbuffered phosphatase). Despite this large CVR noticed for unbuffered phosphatase, the method was repeatable, reproducible, and sensitive. It also proved to be applicable for measuring enzyme activities in different types of soils. These results have been found successful by ISO/TC 190/SC4 and resulted in the publication of ISO 20130:2018 standard.

Innovative approaches to thermochromic materials for adaptive building envelopes

Thermochromic (TC) materials are characterized by a change of their optical response at a specific temperature. They can work based on both, the alteration of solar reflection by temperature, or the change of photoluminescence intensity. In building applications, this type of smart materials enhances the rejection of solar heat for high temperatures to favour cooling of the envelopes and reduces this rejection for low temperatures to improve surface heating. This adaptive optical response improves energy efficiency and reduces environmental impact of urban areas. Most of the current advances in this area are related to TC glazing based on Vanadium oxide, while opaque TC materials have been developed as based on Leuco dyes. The main drawback of these last materials is their significant aging in outdoor applications due to a photo-degradation process. The present work shows the recent results of a multidisciplinary and multinational consortium for research on innovative approaches to thermochromic materials for adaptive building envelopes. Next steps will be focused on building simulation to evaluate material choices across different performance aspects, while physical prototypes will be used for inter-laboratory evaluation of such performance and material durability.

Development and Interlaboratory Evaluation of an LC-MS/MS Method for the Quantification of Lysozyme in Wine Across Independent Instrument Platforms.

Various processing aids and fining agents are used in winemaking to help improve sensory characteristics. Some of these materials may contain or be derived from allergenic foods, such as eggs. In order to ensure food safety and that products meet regulatory compliance, it is essential to have robust and effective analytical methods to verify the removal of allergenic proteins following their use. Current methods include ELISA and MS methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. MS methods offer the potential to test for multiple proteins within a single assay to improve cost and efficiency, whereas ELISA methods typically analyze for a single protein per assay. This study focuses on the development of a LC-tandem MS (MS/MS) quantitative method for lysozyme in white wine and compares performance across two laboratories utilizing two different instrument platforms. Lysozyme target peptides were selected by conducting bottom-up discovery proteomics. Candidate targets were evaluated using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) LC-MS/MS, depending on the instrument in each laboratory. Quantification of lysozyme was conducted using internal, stable isotope-labeled synthetic peptide standards. Three of eight candidate target peptides showed performance suitable for the final quantitative method. White wine spiked with 0.1 and 0.5 ppm lysozyme demonstrated quantitative recovery of 70-120%. While the PRM method delivered better repeatability, the SRM method gave higher quantitative recovery values. A targeted LC-MS/MS method for quantification of lysozyme in white wine has been developed and deployed on two different MS instrument platforms in two laboratories. Both SRM and PRM targeted LC-MS/MS methodologies can be used for quantification of lysozyme in white wine. This study is among the first to evaluate an MS method for food allergen quantification in multiple laboratories.

Performance of quantum dot nanobeads-based immunoassay in diagnosis of toxoplasmosis

Background: Diagnosis of toxoplasmosis relies mainly on determination of IgG and IgM using ELISA.However, inter-laboratory evaluation of performance of many commercial kits gave unsatisfactoryagreements. Merging the innovative use of quantum dots nanobeads (QDNBs) immunoassay may yieldhigher sensitivity and specificity than the conventional used methods.Objective: The present study aims to evaluate the performance of QDNBs based immunoassay inserodiagnosis of human toxoplasmosis.Subjects and Methods: The study included 175 subjects divided into three categories; 122 patientswith clinical manifestations suggestive of toxoplasmosis; 32 patients proved positive for other parasiticinfections, and 21 healthy control subjects. In comparison to Sabin Feldman dye test (SFDT), as a goldstandard test, serum samples were examined in an immunoassay utilizing QDNBs for detection of T. gondiispecific IgG and IgM antibodies.Results: Compared to SFDT, combined QDNBs-IgG/IgM immunoassay exhibited sensitivity of 95.4%,specificity of 98.9%, positive predictive value (PPV) of 98.8%, and negative predictive value (NPV) of95.6%, with relative agreement, validity, and accuracy of almost 97%.Conclusion: It was concluded that QDNBs-based immunoassay improves the performance of ELISAparticularly sensitivity as well as shorten the assay time.

Interlaboratory evaluation of plasma N-glycan antennary fucosylation as a clinical biomarker for HNF1A-MODY using liquid chromatography methods

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels.