Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification that can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.
Abstract
Accurate and comparable quantification of somatic mutations is essential for precision oncology, as clinical decision-making increasingly relies on the quantification of molecular biomarkers. Despite major technological advances, inter-laboratory variability and the lack of metrological traceability remain significant barriers to harmonization and confidence in mutation testing results. Reference Measurement Procedures (RMPs) represent a critical framework to address these challenges by anchoring molecular measurements to common quantitative standards. Here, we describe the development and validation of a candidate RMP for the detection and quantification of the clinically relevant NRAS p.Q61R mutation using digital PCR (dPCR). The assay was systematically optimized to maximize specificity and minimize cross-reactivity between wild-type and mutant alleles. Analytical characterization demonstrated excellent linearity across a broad range of variant allele frequencies (vAF), with a limit of detection of 0.1 %. Precision studies performed on commercially available circulating tumour DNA reference materials (RM) showed good repeatability and intermediate precision, while a full measurement uncertainty budget confirmed the robustness of the approach. Comparison with a commercial dPCR assay provided independent support for assay comparability and consistent vAF estimates across the investigated range. Preliminary inter-laboratory assessment supported transferability of the candidate RMP and comparability of the resulting measurements. Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification. Its implementation can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.
Some of the recent work on dPCR-based RMPs are described and how this can be applied to improve nucleic acid analysis measurements on a global scale in EQA schemes, clinical laboratories and harmonization studies for infectious disease diagnostics are discussed.
S. Falak, D. O'Sullivan, Megan H. Cleveland et al.· 150th anniversary of the Met...· 0 citations
A new generation of molecular tests has revolutionized laboratory medicine through allowing for quick, sensitive, and precise identification of the pathogen; genetic mutation or alteration; and disease-related bio-markers. Of all the molecular techniques that exist today, PCR is still the most versatile, and clinically applicable technique available. This paper reviews the development of PCR technology from traditional amplification to advanced platforms including qPCR, dPCR, RT-PCR, and multiplex PCR. Each of the advanced platforms reviewed will be examined for their contributions to both the accurate detection, and quantification of nucleic acids along with the ability to perform high throughput analyses. Additionally, this paper also discusses how PCR technology can be integrated with additional technologies such as NGS, CRISPR-based diagnostic systems, microarrays, and epigenetic profiling to provide a larger view of disease states than was previously possible. Specifically, the focus of this paper will include many clinically relevant biomarkers and molecular targets used in cancer research including EGFR, KRAS, BRAF, BCR-ABL1, circulating tumor DNA (ctDNA), and methylated SEPT9. These biomarkers and molecular targets have greatly enhanced early diagnosis, prognostication, treatment monitoring and precision medicine in cancer research and other areas. The use of PCR-based technologies in diagnosing infectious diseases, tracking AMR, detecting heritable disorders, studying microbial communities, and MRD monitoring were also discussed. Further discussion included current trends in integrating multiple omics data types into a single platform using AI assisted data analysis tools. Liquid biopsy technologies and point-of-care molecular test platforms were also discussed. Although there are continuing challenges related to costs associated with developing these technologies, establishing standardized protocols for each technology type developed, having skilled technicians develop and validate these technologies, and managing large amounts of data generated when using these technologies; continued advances are providing greater access to these technologies to clinicians and ultimately patients.
M. Saleem, Taimoor Riaz, Fatima Saleem et al.· Clinica chimica acta; intern...· 0 citations
This review sets out the analytical validation, standardization, and quality assurance requirements that would allow a research-grade catalytic protease assay to become a laboratory-developed test or an approved in vitro diagnostic and proposes a staged minimum validation-and-reporting framework.
A. Lesner· Analytical and Bioanalytical...· 0 citations
BackgroundProstate-specific antigen (PSA) remains the most widely used biomarker for prostate cancer screening, diagnosis, and monitoring. However, despite decades of standardization efforts, significant inter-assay variability persists, with important consequences for clinical interpretation and decision-making.ObjectiveThis review aims to evaluate the impact of PSA calibration and harmonization on reference intervals, clinical thresholds, and population-based screening strategies in contemporary clinical practice.MethodsA literature-based analysis was conducted, examining studies on PSA assay standardization, analytical variability, and evidence from population screening trials, including considerations from a Health Technology Assessment perspective.ResultsThe introduction of the World Health Organization (WHO) International Standard 96/670 improved comparability among PSA assays, yet clinically relevant differences between platforms remain. This variability is driven by differences in calibration, antibody specificity, epitope recognition, and assay design. As a result, PSA values are not directly interchangeable across assays, and assay-specific cut-offs may be necessary to maintain diagnostic performance. Historically established thresholds, such as the 4 μg/L cut-off and the "gray zone," were derived using specific assay systems and are influenced by methodological limitations. PSA-derived metrics, including PSA density, improve specificity but are still affected by inter-assay variability. Evidence from large randomized trials supports a PSA cut-off of 3.0 μg/L for population screening, showing a reduction in prostate cancer mortality when implemented within structured programs; however, this threshold is intrinsically linked to the analytical characteristics of the assays used. PSA also shows relevant intra-individual biological variability beyond analytical variation, with within-subject variation of 6-13%.ConclusionsPSA standardization remains incomplete, and inter-assay variability and biological variability continues to influence clinical interpretation. The implementation of PSA-based screening programs must explicitly consider the assay-specific nature of evidence-derived thresholds, including the 3.0 μg/L cut-off, to ensure consistent, effective, and safe clinical decision-making.
X. Filella· Tumour biology : the journal...· 0 citations
BACKGROUND
Accurate identification of actionable somatic variants is essential for therapeutic stratification in colorectal cancer (CRC). While next-generation sequencing (NGS) enables comprehensive genomic profiling, targeted approaches may provide faster and more practical alternatives for routine diagnostics.
METHODS
This study evaluated the analytical performance of the Agena Bioscience iPLEX® High Sensitivity (HS) Colon Panel using MassARRAY MALDI-TOF technology for detection of hotspot variants in KRAS, NRAS, BRAF, and PIK3CA from formalin-fixed paraffin-embedded (FFPE) specimens. A total of 60 unique clinical and reference samples were analyzed, targeting 26 single-nucleotide variants and compared with an orthogonal targeted NGS assay. Limit of detection (LOD) was assessed using serial dilutions of reference materials, and intra- and inter-run reproducibility was evaluated across multiple runs. Analytical performance metrics including positive and negative percent agreement, predictive values, and error rates were calculated.
RESULTS
The assay demonstrated complete concordance with NGS across all evaluated variants, requiring only 20 ng of DNA input, compared to 80-120 ng for a successful NGS run. LOD studies showed reliable detection of multiple variants at approximately 5% variant allele frequency, with BRAF p.V600E detectable near 1%. Intra- and inter-run analyses achieved 100% concordance, confirming assay reproducibility. Aggregated performance metrics demonstrated high sensitivity and specificity across a heterogeneous sample set.
CONCLUSIONS
These findings establish the iPLEX® HS Colon Panel as a reliable platform for rapid detection of clinically actionable hotspot mutations. This study represents analytical validation using mixed FFPE tumor specimens; evaluation in larger colorectal cancer cohort, is warranted.
Vishakha Vashisht, A. Vashisht, A. Mondal et al.· Frontiers in Bioscience· 0 citations
This work sought to systematically apply CAP laboratory-developed test validation requirements for next-generating sequencing to targeted CFTR gene sequencing on an Illumina iSeq100 Sequencing System and to address assay-specific translational guidance gaps.
Muhammad Fareeduddin, Muhammad Usama, Shakir Hussain et al.· Laboratoriums Medizin· 0 citations
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