Detection of cortisol in serum using quantitative resonance Raman spectroscopy

Measurement of cortisol in serum is used commonly as an indicator of stress and disease. Conventional analytical techniques have limited utility given that they remain largely laboratory based, they do not directly measure the deemed biologically active free cortisol, and there is no robust correlation between the free cortisol measurements within serum and saliva. It would therefore be desirable to measure both the free and total cortisol readily within the same matrix in a portable device in the field or at the bedside. This paper demonstrates the utility of a portable Raman approach to measure both the biological active free cortisol as well as total cortisol in human serum, compared to a laboratory-based chemiluminescence analysis technique. This alternative portable Raman method produced results that were consistent with results obtained from previous methods, which has the potential for further miniaturisation for point of test applications

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection; however, the use of different qPCR components, calibration materials and DNA extraction methods reduces the comparability between clinics, which could result in false diagnosis and discrepancies in patient care. The establishment of a metrological framework for nucleic-acid tests is expected to improve the degree of standardisation of pathogen detection and quantification methods applied in a clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) allows quantification of nucleic acids and has already been used for a myriad of applications, including pathogen quantification. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessments on its actual performance characteristics should be collected before it can be implemented in a metrological framework and to allow an adequate estimation of the measurement uncertainty. Here, high repeatability and reproducibility of dPCR for quantification of DNA from human cytomegalovirus were demonstrated. Using extracted DNA and whole-virus material, each of five dPCR platforms from four laboratories demonstrated high intermediate precision between three consecutive experiments. Furthermore, discrepancies in estimated mean DNA copy-number concentrations between different laboratories were less than two-fold, with DNA extraction recognised as the main source of variability. Our results demonstrate dPCR-based methods can be very repeatable and reproducible for quantification of viral DNA, and should be considered as potent reference method candidates for implementation in a metrological framework.JRC.F.6-Reference Material

A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer's disease (AD). However, the in-fluence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap (R) DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements

A flexible synthesis of C-6 and N-1 analogues of a 4-amino-1,3-dihydroimidazo[4,5-c]pyridin-2-one core

A flexible route which enables access to derivatives of 4-amino-1,3-dihydroimidazo[4,5-c]pyridin-2-ones is described. Issues of selectivity, reaction safety, and low yields in original routes are overcome with the key improvements to the route, including a Negishi cross-coupling and use of a carbamate as a protecting group and intrinsic carbonyl source. The new route enables variation of C-6 and N-1 substituents