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. Each panel contained up to six samples with increasingly small copy number differences between the two reporter genes that ranged from 1- to 2-fold differences. These copy number differences represent the magnitude of changes that may need to be measured to validate an engineered system. Using digital PCR (dPCR), we demonstrated that it is possible to quantify changes in both gene and gene transcript numbers both within and between samples down to 1.05-fold. We corroborated these findings using a simple gene circuit within a bacterial model to demonstrate that dPCR was able to precisely identify small changes in gene expression of two transcripts in response to promoter stimulation. Finally, we used our findings to highlight sources of error that can contributed to the measurement uncertainty in the measurement of small ratios in biological systems. Together, the development of a panel of control materials and validation of a high accuracy method for the measurement of small changes in gene expression, this study can contribute to the engineering biology "toolkit" of methods and materials to support the current standardization efforts.

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. METHODS: Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis-tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches. RESULTS: Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 µL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1-46.0) to 5.4% (range 3.0-8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1-25.0) to 7.1% (range 4.1-11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer's stated values of ≥98%. CONCLUSIONS: For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.

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. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

Guidance for characterization of in-house reference materials for light element stable isotope analysis.

RATIONALE: Preparation of in-house reference materials (RMs) is an important aspect of light element stable isotope analysis. While some relevant information is available, there is as yet no clear set of guidelines available covering all aspects of in-house production and characterization of RMs. METHODS: To address this need, the experience of production of certified reference materials under accreditation to ISO 17034:2016 and ISO/IEC 17025:2017 has been distilled into guidance for production of in-house RMs that are fit-for-purpose. RESULTS: The guidance provided covers five areas: (i) planning; (ii) material considerations including preparation, packaging, and storage; (iii) measurements and assessments; (iv) value and uncertainty assignment; and (v) monitoring and use. CONCLUSIONS: In-house RMs prepared by following this guidance can be used to provide traceability to measurement results when used for normalization or for quality control and/or assurance purposes.

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 C4 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. National and designated metrology institutes from a number of countries recently participated in an inter-laboratory assessment (CCQM-K140) of stable carbon isotope ratio determination of bulk honey. The same sample material was distributed to a number of forensic isotope analysis laboratories that could not participate directly in the metrological comparison. The results from these studies have demonstrated that the majority of participants provided isotope delta values with acceptable performance metrics; that all participants ensured traceability of their results; and that where measurement uncertainties were reported; these were fit-for-purpose. A number of the forensic laboratories only reported precision rather than full estimates of measurement uncertainty and this was the major cause of the few instances of questionable performance metrics. Reporting of standard deviations in place of measurement uncertainties is common practice outside metrology institutes and the implications for interpretations of small differences in isotopic compositions are discussed. The results have also highlighted a number of considerations that are useful for organisers of similar inter-laboratory studies in the future.

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. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

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. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

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-ABL1IS (%BCR-ABL1IS) 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. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS: BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS: Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS: RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.

Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS.

BACKGROUND: Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. METHOD: Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. RESULTS: Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ionization efficiency studies and structural analog analysis proved the method to be robust against interferences. Through analysis of 34 CRMs (83-764 nmol/L), expanded measurement uncertainty was calculated to be 5% (95% CI). The mean bias between the measured and target CRM concentrations was statistically insignificant at -0.08%. CONCLUSIONS: The accuracy and low measurement uncertainty of this method qualify it as a CRM procedure. Metrological traceability has been achieved through the analysis of higher-order CRMs. This method could be used to underpin serum cortisol EQA schemes to provide samples with a traceable target value, enabling participating laboratories to determine the accuracy and measurement uncertainty of their assays.

Considerations for digital PCR as an accurate molecular diagnostic tool.

BACKGROUND: Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT: This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY: In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp circulating nucleic acid (CNA) kit, NucleoSpin Plasma XS (NS) kit and FitAmp plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity.

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

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. The 100 day mission to respond to future pandemic threats highlights the need for effective diagnostics, therapeutics and vaccines. Of the three, diagnostics represents the first opportunity to manage infectious diseases while also being the most poorly supported in terms of the infrastructure needed to demonstrate effectiveness. Where performance targets exist, they are not well served by consensus on how to demonstrate they are being met; this includes analytical factors such as limit of detection (LOD) false positive results as well as how to approach clinical evaluation. The selection of gold standards or use of epidemiological factors such as predictive value, reference ranges or clinical thresholds are seldom correctly considered. The attention placed on molecular diagnostic tests during COVID-19 illustrates important considerations and assumptions on the use of these methods for infectious disease diagnosis and beyond. In this manuscript, we discuss state-of-the-art approaches to diagnostic evaluation and explore how they may be better tailored to diagnostic techniques like NAATs to maximise the impact of these highly versatile bioanalytical tools, both generally and during future outbreaks.

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. It is a medium analytical difficulty processed food chocolate paste matrix (a) devoid of allergens, and (b) incurred with five allergens at the clinically relevant concentration of 10 mg kg-1 expressed as protein. The allergen raw materials: hens' egg white powder, skimmed cows' milk powder, almond powder (full fat), hazelnut powder (partially defatted), and walnut powder (partially defatted), are also available as RMs. The preparation, gravimetric traceability to the SI, homogeneity, and stability were found to be fit-for-purpose and the RMs are now available to the analytical community.

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. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 106 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed.

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. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Evaluation of digital PCR for absolute DNA quantification.

The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.

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. for monoisotopic elements) or not feasible (e.g. limited by the cost and availability of like-for-like isotopically enriched species). The work presented here demonstrates the potential of a novel on-column species-specific internal calibration approach, which is based on the chromatographic injection of the same species of the analyte as the internal standard (IS), after the sample injection. It can compensate for on-column analyte losses and signal drift and can be applied with any detector capable of recording time-resolved data, provided that enough species resolution can be achieved. The feasibility of this novel calibration strategy for accurate quantitative elemental speciation in complex matrices is demonstrated here through the analysis of inorganic arsenic in rice. An expanded uncertainty (k = 2) of <10% was obtained for a mass fraction range of 60 to 300 µg kg-1 inorganic-As (i-As) in dry rice products. The method is currently used for the certification of i-As in baby food matrices to support Commission Regulation (EU) 2015/1006 in regard to the maximum levels of i-As in foodstuffs.