SI-traceable characterisation of the first reference material for nanoparticle number concentration in suspension to support regulatory compliance.

Reference materials (RMs) are increasingly needed to support number-based characterisation of nanomaterials (NM) in a regulatory context for the purpose of method development, validation and measurement quality control. To date, RMs for number concentration in suspension with a directly assigned value that is SI traceable have been rather scarce, being the LGCQC5050 the only material commercialised so far. This could be attributed to the limited availability of metrologically validated measurement methods and stability challenges associated with long-term storage of NM suspensions.

Development of an SI traceable value assigned amino acid matrix-matched material to underpin European external quality assessment.

Plasma amino acid analysis plays a vital role in monitoring inherited metabolic diseases (IMDs), such as phenylketonuria (PKU), where dietary therapy is the cornerstone of management and patients are regularly monitored using blood or plasma phenylalanine throughout life. Recent findings from external quality assessment (EQA) schemes demonstrate large variability between results from different clinical laboratories. EQA schemes are hampered by factors including suboptimal sample type and frequency of distributions.

Simple steps to achieve harmonisation and standardisation of dried blood spot phenylalanine measurements and facilitate consistent management of patients with phenylketonuria.

Objectives: Management of phenylketonuria (PKU) relies upon life-long monitoring of phenylalanine (Phe) in dried blood spots (DBS), thus comparability of measurements is important. The lack of harmonisation and standardisation between laboratories, combined with the variable quality of patient-collected DBS specimens, are currently preventing this from being achieved. A traceable, matrix-matched Phe certified reference material, common methodology and means to ensure patient collected DBS specimens are of consistent quality would improve comparability between laboratories.

The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing.

Background: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available.

Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease.

Diagnostic tests were heralded as crucial during the Coronavirus disease (COVID-19) pandemic with most of the key methods using bioanalytical approaches that detected larger molecules (RNA, protein antigens or antibodies) rather than conventional clinical biochemical techniques. Nucleic Acid Amplification Tests (NAATs), like the Polymerase Chain Reaction (PCR), and other molecular methods, like sequencing (that often work in combination with NAATs), were essential to the diagnosis and management during COVID-19. This was exemplified both early in the pandemic but also later on, following the emergence of new genetic SARS-CoV-2 variants.

Clinically and industrially relevant incurred reference materials to improve analysis of food allergens, milk, egg, almond, hazelnut and walnut.

Measurement of food allergen protein concentrations against thresholds can improve allergen risk management and precautionary allergen labelling. Such measurement suffers well known problems which could be ameliorated by well characterised reference materials (RMs) providing meaningful information for risk assessors. We investigated the preparation and characterisation of the first consensus informed industrially and clinically relevant multi-allergen matrix RM kit for five priority allergens.

Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.

The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material.

Improving Harmonization and Standardization of Expanded Newborn Screening Results by Optimization of the Legacy Flow Injection Analysis Tandem Mass Spectrometry Methods and Application of a Standardized Calibration Approach.

Background: Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors.

Applicability of Control Materials To Support Gene Promoter Characterization and Expression in Engineered Cells Using Digital PCR.

The use of standardized components and processes in engineering underpins the design-build-test model, and the engineering of biological systems is no different. Substantial efforts to standardize both the components and the methods to validate the engineered biological systems is ongoing. This study has developed a panel of control materials encoding the commonly used reporter genes GFP and RFP as DNA or RNA molecules.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load.

The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing.

Methods: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed.

On-column internal standardisation as an alternative calibration strategy for speciation analysis: feasibility demonstration through analysis of inorganic As in rice.

Species-specific isotope dilution mass spectrometry (SS-IDMS) has been the calibration method of choice for high accuracy speciation analysis because it can correct for detector sensitivity drifts, matrix effects, and analyte loss during sample preparation and analysis. However, in many cases SS-IDMS calibration is either not applicable (e.g.

Evaluation of a Common Internal Standard Material to Reduce Inter-Laboratory Variation and Ensure the Quality, Safety and Efficacy of Expanded Newborn Screening Results When Using Flow Injection Analysis Tandem Mass Spectrometry with Internal Calibration.

In 2015, the newborn screening (NBS) programmes in England and Wales were expanded to include four additional disorders: Classical Homocystinuria, Isovaleric Acidemia, Glutaric Aciduria Type 1 and Maple Syrup Urine Disease, bringing the total number of analytes quantified to eight: phenylalanine, tyrosine, leucine, methionine, isovalerylcarnitine, glutarylcarnitine, octanoylcarnitine and decanoylcarnitine. Post-implementation, population data monitoring showed that inter-laboratory variation was greater than expected, with 90th centiles varying from 17 to 59%. We evaluated the effect of stable isotope internal standard (IS) used for quantitation on inter-laboratory variation.

Mass Spectrometry Characterization of Higher Order Structural Changes Associated with the Fc-glycan Structure of the NISTmAb Reference Material, RM 8761.

As monoclonal antibodies (mAbs) rapidly emerge as a dominant class of therapeutics, so does the need for suitable analytical technologies to monitor for changes in protein higher order structure (HOS) of these biomolecules. Reference materials (RM) serve a key analytical purpose of benchmarking the suitability and robustness of both established and emerging analytical procedures for both drug producers and regulators. Here, two simple enzymatic protocols for generating Fc-glycan variants from the NISTmAb RM are described and both global and localized changes in HOS between the RM and these Fc-glycan variants are characterized using hydrogen deuterium exchange-mass spectrometry (HDX-MS) and ion mobility spectrometry-mass spectrometry (IMS-MS) measurements.

Lessons learned from inter-laboratory studies of carbon isotope analysis of honey.

Forensic application of carbon isotope ratio measurements of honey and honey protein to investigate the degree of adulteration with high fructose corn syrup or other C plant sugars is well established. These measurements must use methods that exhibit suitable performance criteria, particularly with regard to measurement uncertainty and traceability - low levels of adulteration can only be detected by methods that result in suitably small measurement uncertainties such that differences of 1‰ or less can be reliably detected. Inter-laboratory exercises are invaluable to assess the state-of-the art of measurement capabilities of laboratories necessary to achieve such performance criteria.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

Background: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use.

Feedback from the European Bioanalysis Forum: focus workshop on current analysis of immunogenicity: best practices and regulatory hurdles.

European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.

A generic standard additions based method to determine endogenous analyte concentrations by immunoassays to overcome complex biological matrix interference.

We describe a novel generic method to derive the unknown endogenous concentrations of analyte within complex biological matrices (e.g. serum or plasma) based upon the relationship between the immunoassay signal response of a biological test sample spiked with known analyte concentrations and the log transformed estimated total concentration.

Almond or Mahaleb? Orthogonal Allergen Analysis During a Live Incident Investigation by ELISA, Molecular Biology, and Protein Mass Spectrometry.

It is now well known that an incident investigated in the United Kingdom in 2015 of cumin alleged to be contaminated with almond, a risk for people with almond allergy, was caused by the Prunus species, Prunus mahaleb. In the United Kingdom, the Government Chemist offers a route of technical appeal from official findings in the food control system. Findings of almond in two official samples, cumin and paprika, which had prompted action to exclude the consignments from the food chain, were so referred.

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

Background: Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1 (%BCR-ABL1) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples.