Alison S. Devonshire

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

AbstractCirculating 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. FigureComparison of single and multiple reference gene normalisation for quantification of plasma cell free DNA

Rat Primary Hepatocytes Show Enhanced Performance and Sensitivity to Acetaminophen During Three-Dimensional Culture on a Polystyrene Scaffold Designed for Routine Use

The in vitro evaluation of hepatotoxicity is an essential stage in the research and development of new pharmaceuticals as the liver is one of the most commonly impacted organs during preclinical toxicity studies. Fresh primary hepatocytes in monolayer culture are the most commonly used in vitro model of the liver but often exhibit limited viability and/or reduction or loss of important liver-specific functions. These limitations could potentially be overcome using three-dimensional (3D) culture systems, but their experimental nature and limited use in liver toxicity screening and drug metabolism has impaired their uptake into commercial screening programs. In this study we use a commercially available polystyrene scaffold developed for routine 3D cell culture to maintain primary rat hepatocytes for use in metabolism and toxicity studies over 72 h. We show that primary hepatocytes retain their natural cuboidal morphology with significantly higher viability (>74%) than cells grown in monolayer culture (maximum of 57%). Hepatocytes in the 3D scaffolds exhibit differential expression of genes associated with phase I, II, and III drug metabolism under basal conditions compared with monolayer culture and can be induced to stably express significantly higher levels of the cytochrome-P450 enzymes 1A2, 2B1, and 3A2 over 48 h. In toxicity studies the hepatocytes in the 3D scaffolds also show increased sensitivity to the model toxicant acetaminophen. These improvements over monolayer culture and the availability of this new easy to use 3D scaffold system could facilitate the uptake of 3D technologies into routine drug screening programs.

Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis.

Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed.

Highly Reproducible Absolute Quantification of Mycobacterium tuberculosis Complex by Digital PCR

Digital PCR (dPCR) offers absolute quantification through the limiting dilution of template nucleic acid molecules and has the potential to offer high reproducibility. However, the robustness of dPCR has yet to be evaluated using complex genomes to compare different dPCR methods and platforms. We used DNA templates from the pathogen Mycobacterium tuberculosis to evaluate the impact of template type, master mixes, primer pairs and, crucially, extraction methods on dPCR performance. Performance was compared between the chip (BioMark) and droplet (QX100) formats. In the absence of any external calibration, dPCR measurements were generally consistent within ∼2-fold between different master mixes and primers. Template DNA integrity could influence dPCR performance: high molecular weight gDNA resulted in underperformance of one master mix, while restriction digestion of a low molecular weight sample also caused underestimation. Good concordance (≤1.5-fold difference) was observed between chip and droplet formats. Platform precision was in agreement with predicted Poisson error based on partition number, but this was a minor component (<10%) of the total variance when extraction was included. dPCR offers a robust reproducible method for DNA measurement; however, as a predominant source of error, the process of DNA extraction will need to be controlled with suitable calibrators to maximize agreement between laboratories.

Applicability of RNA standards for evaluating RT-qPCR assays and platforms

The availability of diverse RT-qPCR assay formats and technologies hinder comparability of data between platforms. Reference standards to facilitate platform evaluation and comparability are needed. We have explored using universal RNA standards for comparing the performance of a novel qPCR platform (Fluidigm® BioMark™) against the widely used ABI 7900HT system. Our results show that such standards may form part of a toolkit to evaluate the key performance characteristics of platforms.

Validation of reference gene stability for APAP hepatotoxicity studies in different in vitro systems and identification of novel potential toxicity biomarkers

Liver cell lines and primary hepatocytes are becoming increasingly valuable for in vitro toxicogenomic studies, with RT-qPCR enabling the analysis of gene expression profiles following exposure to potential hepatotoxicants. Supporting the accurate normalisation of RT-qPCR data requires the identification of reference genes which have stable expression during in vitro toxicology studies. Therefore, we performed a comprehensive analysis of reference gene stability in two routinely used cell types, (HepG2 cells and primary rat hepatocytes), and two in vitro culture systems, (2D monolayer and 3D scaffolds). A robust reference gene validation strategy was performed, consisting of geNorm analysis, to test for pair wise variation in gene expression, and statistical analysis using analysis of variance. This strategy identified stable reference genes with respect to acetaminophen treatment and time in HepG2 cells (GAPDH and PPIA), and with respect to acetaminophen treatment and culture condition in primary hepatocytes (18S rRNA and α-tubulin). Following the selection of reference genes, the novel target genes E2F7 and IL-11RA were identified as potential toxicity biomarkers for acetaminophen treatment. We conclude that accurate quantification of gene expression requires the use of a validated normalisation strategy for each species and experimental system employed.

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

ABSTRACT Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.

Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

BackgroundDNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS). The techniques were evaluated for linearity, accuracy and precision.ResultsMethyLight qPCR displayed the best linearity across the range of tested samples. Observed methylation measured by MethyLight- and MSRE/MDRE-qPCR and -dPCR were not significantly different to expected values whilst bisulfite amplicon NGS analysis over-estimated methylation content. Bisulfite amplicon NGS showed good precision, whilst the lower precision of qPCR and dPCR analysis precluded discrimination of differences of < 25% in methylation status. A novel dPCR MethyLight assay is also described as a potential method for absolute quantification that simultaneously measures both sense and antisense DNA strands following bisulfite treatment.ConclusionsOur findings comprise a comprehensive benchmark for the quantitative accuracy of key methods for methylation analysis and demonstrate their applicability to the quantification of circulating tumour DNA biomarkers by using sample concentrations that are representative of typical clinical isolates.

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.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

BackgroundReal-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring.MethodsTo assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories.ResultsdPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude.ConclusionsTB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.