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Quality of massively parallel sequencing/nextgeneration sequencing (MPS/NGS) methods - Qualità dei metodi di sequenziamento massivo parallelo/sequenziamento di nuova generazione (MPS/NGS)
Abstract
ISO's concern about the neglected quality of MPS/NGS is
more than well-founded. Laboratory operations are considered analogous to a manufacturing process where the industrial process is replaced by the measurement process, the output of which is the results of examinations. Process control is therefore an essential element of the quality management system. The complexity of the MPS/NGS process is obviously daunting for some medical laboratories. The expression of the final result in nominal format is misleading. The ISO 20397-2 document provides many indicators and useful examples for doing laboratory estimation of result uncertainty for MPS/NGS.
... Molti dettagli sul tema specifico si trovano nel documento ISO 20397-2:2021 e in letteratura. [46][47][48] CLSI MM07 (per ibridazione in situ a fluorescenza, FISH) consiglia il conteggio di cellule con un segnale anormale in un vetrino di riferimento. 49 CLSI MM20 per esami genetici molecolari 50 prevede controlli con sequenze del genoma umano o controlli sintetici positivo (a livello basso, vicino al LoD, o medio) e negativo, i cui risultati sono analizzati con grafici e statistiche. ...
La norma ISO 15189 revisionata è stata pubblicata il 6 dicembre 2022, quasi un anno dopo il termine previsto. SIPMeL, dopo aver partecipato attivamente al processo di revisione, mette a disposizione una serie di documenti di Raccomandazioni, con i principali contenuti della norma ISO in lingua italiana, qualche riflessione critica, e le indicazioni per applicare i requisiti della norma. Le Raccomandazioni SIPMeL Q19 concernono i requisiti di UNI EN ISO 15189:2023 su validità dei risultati degli esami, con il controllo di qualità interno (CQI), la valutazione esterna della qualità (VEQ) e la comparabilità dei risultati degli esami. I requisiti di ISO 15189 per il CQI non scendono in dettagli, ma le Raccomandazioni Q19 richiamano almeno ISO 15198 (compiti dei produttori) e CLSI C24 (statistica del CQI), oltre al Vocabolario Internazionale di Metrologia (VIM). Pubblicando il documento ISO 20914, è stato stabilito che il CQI non serve solo per il monitoraggio dei risultati, ma fornisce la base per la stima dell’incertezza. Il documento Q19 raccoglie elementi da vari documenti CLSI, come le guide per l’immunometria, quelle per la microbiologia, quelle per i metodi molecolari e quelle per gli esami POCT. CLSI poi fornisce indicazioni sulle alternative per i materiali di controllo. Q19 introduce infine la tematica del monitoraggio dei risultati qualitativi. ISO 15189 fornisce più dettagli sulla partecipazione a VEQ, ma la Raccomandazione Q19 aggiunge i riferimenti a guide CLSI come QMS24 e MM14. I requisiti per VEQ di ISO 15189 hanno grande importanza per l’esito delle verifiche di accreditamento. ISO 15189 affianca infine nella stessa clausola CQI, VEQ e comparabilità dei risultati degli esami. Le Raccomandazioni Q19 correggono la terminologia in base al VIM, richiamano le guide CLSI EP31 e CLSI EP09, nonché le proprie Raccomandazioni sulla stessa materia del 2016. Secondo Q19, le procedure di comparazione hanno caratteristiche più vicine alle attività di validazione e verifica dei metodi, diverse da CQI e VEQ, da cui dovrebbero essere distinte. Q19 propone ai laboratori un approccio semplice, poco oneroso e con statistiche non troppo sofisticate.
----- The revised ISO 15189 standard was published on 6 December 2022, almost one year after the deadline. SIPMeL, having actively participated in the revision process, provides a series of Recommendations documents, with the main contents of the ISO standard in Italian language, some critical reflections, and indications for applying the requirements of the standard. The SIPMeL Recommendations Q19 concern the requirements of UNI EN ISO 15189:2023 on validity of examination results, with internal quality assurance (IQC), external quality assessment (EQA) and comparability of examination results. The requirements of ISO 15189 for CQI do not go into detail, but Recommendation Q19 at least recalls ISO 15198 (manufacturers' tasks) and CLSI C24 (CQI statistics), as well as the International Vocabulary of Metrology (VIM). By publishing ISO 20914, it was established that the IQC not only serves for monitoring results, but also provides the basis for estimating uncertainty.
Document Q19 collects elements from various CLSI documents, such as the guides for immunometry, those for microbiology, those for molecular methods and those for POCT examinations. CLSI then provides guidance on alternatives for control materials. Q19 finally introduces the topic of monitoring quality results. ISO 15189 provides more detail on participation in VEQ, but Recommendation Q19 adds references to CLSI guides such as QMS24 and MM14. The requirements for VEQ in ISO 15189 are of great importance for the outcome of accreditation audits. ISO 15189 finally combines IQC, VEQ and comparability of examination results in the same clause. The Q19 Recommendations correct the terminology according to VIM, recall the CLSI EP31 and CLSI EP09 guides as well as its own 2016 Recommendations on the same subject. According to Q19, comparison procedures have characteristics that are closer to method validation and verification activities, different from IQC and VEQ, from which they should be distinguished. Q19 proposes to the laboratories a simple, inexpensive approach with not too sophisticated statistics.
SIPMeL actively participates in the standardisation work carried out in the Technical Committee UNI 044 'Biomedical and Diagnostic Technologies' and through this in at least two international technical committees, one in Europe (CEN/TC 140) and one worldwide (ISO/TC 212). The work of the European committee is intensive and of great relevance to medical laboratories. The standards developed by CEN/TC 140 have supported the uniform application of the requirements of the European Directive 98/79/EC on 'In vitro diagnostic medical devices' and are about to do the same for the IVD Regulation 2017/746 on 'In vitro diagnostic medical devices'. In this note, the points of the CEN standards on NGS methods that are of particular relevance for ISO accreditation visits and the preparation of related evidence of conformity to requirements are highlighted. The study of the standards allows the distillation of some general considerations and the identification of some critical points that deserve attention and resolution. NGS methods demolish the traditional classification of laboratory tests based simplistically on results: numerical (quantitative), nominal (qualitative), ordinal. The complexity of the multi-stage process is reflected in the complexity of the presentation of results. The glossary of standards on NGS methods contains many terms that would also be useful in other standards documents, including those at ISO level. However, in the CEN standards, confusions between clinical, metrological, numerical and nominal results are noted, even with internal contradictions. In addition to the revision of these specific documents, specific standardisation tools aimed at resolving conflicts between industry glossaries and basic and general standards, such as the International Metrology Vocabulary and standards on statistical concepts applied to laboratory methods, could be useful. --- SIPMeL partecipa attivamente al lavoro normativo svolto nella Commissione tecnica UNI 044 “Tecnologie biomediche e diagnostiche” e attraverso questa ad almeno due comitati tecnici internazionali, uno in ambito europeo (CEN/TC 140) ed uno mondiale (ISO/TC 212). Il lavoro del comitato europeo è intenso e di grande rilevanza per i laboratori medici. Le norme sviluppate dal CEN/TC 140 hanno sostenuto l’applicazione uniforme dei requisiti della Direttiva europea 98/79/CE sui "Dispositivi medico-diagnostici in vitro" e si apprestano a fare altrettanto per il Regolamento IVD 2017/746 sui "Dispositivi medico-diagnostici in vitro". In questa nota sono stati evidenziati i punti delle norme CEN sui metodi NGS particolarmente significativi per le visite di accreditamento ISO e la predisposizione delle relative evidenze di conformità ai requisiti. Lo studio degli standard consente la distillazione di alcune considerazioni di carattere generale e l’individuazione di alcune criticità, meritevoli di attenzione e risoluzione. I metodi NGS demoliscono la tradizionale classificazione degli esami di laboratorio basata semplicisticamente sui risultati: numerici (quantitativi), nominali (qualitativi), ordinali. La complessità del processo, articolato in diverse fasi, si riflette nella complessità della presentazione dei risultati. Il glossario degli standard sui metodi NGS contiene molti termini che sarebbero utili anche in altri documenti normativi, compreso quelli di livello ISO. Tuttavia, negli standard CEN si notano confusioni tra ambito clinico, metrologico, risultati numerici e risultati nominali, anche con contraddizioni interne. Oltre alla revisione di questi specifici documenti, potrebbero essere utili strumenti normativi specifici finalizzati a risolvere i contrasti tra i glossari di settore e le norme di base e generali, come il Vocabolario Internazionale di Metrologia e gli standard sui concetti di statistica applicata ai metodi di laboratorio.
Next-generation sequencing (NGS) of tumor cell-derived DNA/RNA to screen for targetable genomic alterations is now widely available and has become part of routine practice in oncology. NGS testing strategies depend on cancer type, disease stage and the impact of results on treatment selection. The European Society for Medical Oncology (ESMO) has recently published recommendations for the use of NGS in patients with advanced cancer. We complement the ESMO recommendations with a practical review of how oncologists should read and interpret NGS reports. A concise and straightforward NGS report contains details of the tumor sample, the technology used and highlights not only the most important and potentially actionable results, but also other pathogenic alterations detected. Variants of unknown significance should also be listed. Interpretation of NGS reports should be a joint effort between molecular pathologists, tumor biologists and clinicians. Rather than relying and acting on the information provided by the NGS report, oncologists need to obtain a basic level of understanding to read and interpret NGS results. Comprehensive annotated databases are available for clinicians to review the information detailed in the NGS report. Molecular tumor boards do not only stimulate debate and exchange, but may also help to interpret challenging reports and to ensure continuing medical education.
Background: The performance assessment of tests that express qualitative results in the medical laboratory is of primary importance in characterization, diagnosis, follow-up, and screening. An important contribution to this type of assessment may be the publication of the Eurachem AQA 2021 guide. The text intends to principally discuss the consistency of the subclauses of this guide with ISO 15189 and CLSI EP12-A2. Methods: The study involves a literature review within the scope of qualitative tests. Results: Tables are used for crossing AQA. with ISO 15189 and CLSI EP12-A2. Conclusions: Consistency with ISO 15189 and CLSI EP12-A2 is demonstrated in the study. Introducing “uncertainty of proportion” reflects the necessity of assessing uncertainties when dealing with qualitative results.
In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.
Introduction
In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing (“NGS first”) as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate.
Methods
Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled.
Results
Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication.
Conclusions
Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.
Next Generation Sequencing (NGS) is increasingly used in diagnostic centers for the assessment of genomic alterations to select patients for precision oncology. The Italian Society of Anatomic Pathology and Diagnostic Cytopathology (SIAPEC) through the Molecular Pathology and Predictive Medicine Study Group (PMMP) has been following the progressive development of centers that have adopted NGS technology in diagnostics over time. In July 2017, a study network on massive parallel sequencing was activated in Italy and recognized as the NGS SIAPeC National Network by the SIAPeC Scientific Society Board. Since then, activities have been implemented within the network that provide for alignment of laboratories through diagnostic concordance analysis and monitoring of centers adhering to the Network. Recently, considering the growing need for extended genomic analyses, the PMMP distributed a national survey to assess activities related to the use of genomic diagnostics in oncology within the NGS SIAPEC National Network.
Thirty centers participated in the survey. Eighty percent of the centers are laboratories within Pathology Departments. The distribution of laboratories in the country, the diagnostic laboratory/population ratio, the staff dedicated, the type and number of sequencing and mechatronics platforms available, the genomic panels utilized, and the type and number of diagnostic tests carried out in the last year in each center, are reported.
The centers were also asked whether they participated in a multidisciplinary Molecular Tumor Board (MTB) for management of patients. Thirty percent of the centers had a MTB that was ratified by regional decree. The professionals most frequently involved in the core team of the MTB are the pathologist, oncologist, molecular biologist, geneticist, pharmacologist, and bioinformatician.
The data from this survey indicate that NGS diagnostics in Italy is still heterogeneous in terms of geographical distribution and the characteristics of laboratories and diagnostic test performed. The implementation of activities that favors harmonization, the logistics and the convergence of biological material in reference centers for molecular analyses is a priority for the development of a functional laboratory network.
The aim of this study was to evaluate the performance of a novel NGS-based assay to monitor mixed chimerism (MC) and compare its technical capacity to established techniques for chimerism analysis. Artificial and clinical samples with increasing amounts of patient DNA were compared using real-time PCR detection of indels and SNP, fragment analysis of short-tandem repeats (STR) and NGS analysis of indels. Real-time PCR displayed excellent sensitivity (>0,01%) but poor accuracy (>20 CV% at MC > 20%), while fragment analysis exhibited good accuracy (<5 CV% at MC > 20%) with limited sensitivity (>2,5%). In contrast, NGS chimerism demonstrated a sensitivity (>0,1%) equal to real-time PCR and an accuracy equal or better than STR analysis throughout an extensive range of mixed chimerism (0,1 – 100%). To evaluate performance of the separate techniques for chimerism determination, 75 retrospective patient monitoring samples (3–7 weeks post-HSCT) with low (<5%), intermediate (5–20%) or high mixed chimerism (>20%) were analyzed. The between run precision for the NGS assay varied from 0,72% (>20% MC) to 7,38% (MC < 5%). In conclusion, NGS displayed a combination of high sensitivity with good accuracy in both artificial and clinical chimerism samples.
Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendations on clinical WGS analytical validation for the diagnosis of individuals with suspected germline disease with a focus on test development, upfront considerations for test design, test validation practices, and metrics to monitor test performance. This work also provides insight into the current state of WGS testing at each member institution, including the utilization of reference and other standards across sites. Importantly, members of this initiative strongly believe that clinical WGS is an appropriate first-tier test for patients with rare genetic disorders, and at minimum is ready to replace chromosomal microarray analysis and whole-exome sequencing. The recommendations presented here should reduce the burden on laboratories introducing WGS into clinical practice, and support safe and effective WGS testing for diagnosis of germline disease.
Purpose
The purpose of this document is to provide guidance for the use of next-generation sequencing (NGS, also known as massively parallel sequencing or MPS) in Canadian clinical genetic laboratories for detection of genetic variants in genomic DNA and mitochondrial DNA for inherited disorders, as well as somatic variants in tumour DNA for acquired cancers. They are intended for Canadian clinical laboratories engaged in developing, validating and using NGS methods.
Methods of statement development
The document was drafted by the Canadian College of Medical Geneticists (CCMG) Ad Hoc Working Group on NGS Guidelines to make recommendations relevant to NGS. The statement was circulated for comment to the CCMG Laboratory Practice and Clinical Practice committees, and to the CCMG membership. Following incorporation of feedback, the document was approved by the CCMG Board of Directors.
Disclaimer
The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada and should not be considered to be inclusive of all information laboratories should consider in the validation and use of NGS for a clinical laboratory service.
Next-generation sequencing (NGS) methods for cancer testing have been rapidly adopted by clinical laboratories. To establish analytical validation best practice guidelines for NGS gene panel testing of somatic variants, a working group was convened by the Association of Molecular Pathology with liaison representation from the College of American Pathologists. These joint consensus recommendations address NGS test development, optimization, and validation, including recommendations on panel content selection and rationale for optimization and familiarization phase conducted before test validation; utilization of reference cell lines and reference materials for evaluation of assay performance; determining of positive percentage agreement and positive predictive value for each variant type; and requirements for minimal depth of coverage and minimum number of samples that should be used to establish test performance characteristics. The recommendations emphasize the role of laboratory director in using an error-based approach that identifies potential sources of errors that may occur throughout the analytical process and addressing these potential errors through test design, method validation, or quality controls so that no harm comes to the patient. The recommendations contained herein are intended to assist clinical laboratories with the validation and ongoing monitoring of NGS testing for detection of somatic variants and to ensure high quality of sequencing results.
DNA sequencing continues to evolve quickly even after > 30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data.
We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients' representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a 'rating system' for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.European Journal of Human Genetics advance online publication, 28 October 2015; doi:10.1038/ejhg.2015.226.
Science is defined in part by an honest exposition of the uncertainties that arise in measurements and propagate through calculations and inferences, so that the reliabilities of its conclusions are made apparent. The recent rapid development of high-throughput DNA sequencing technologies has dramatically increased the number of measurements made at the biochemical and molecular level. These data come from many different DNA-sequencing technologies, each with their own platform-specific errors and biases, which vary widely. Several statistical studies have tried to measure error rates for basic determinations, but there are no general schemes to project these uncertainties so as to assess the surety of the conclusions drawn about genetic, epigenetic, and more general biological questions. We review here the state of uncertainty quantification in DNA sequencing applications, describe sources of error, and propose methods that can be used for accounting and propagating these errors and their uncertainties through subsequent calculations.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Despite the routine nature of comparing sequence variations identified during clinical testing to database records, few databases meet quality requirements for clinical diagnostics. To address this issue, The Royal College of Pathologists of Australasia (RCPA) in collaboration with the Human Genetics Society of Australasia (HGSA), and the Human Variome Project (HVP) is developing standards for DNA sequence variation databases intended for use in the Australian clinical environment. The outputs of this project will be promoted to other health systems and accreditation bodies by the Human Variome Project to support the development of similar frameworks in other jurisdictions.
Advances in next-generation sequencing (NGS) technologies have rapidly improved sequencing fidelity and substantially decreased sequencing error rates. However, given that there are billions of nucleotides in a human genome, even low experimental error rates yield many errors in variant calls. Erroneous variants can mimic true somatic and rare variants, thus requiring costly confirmatory experiments to minimize the number of false positives. Here, we discuss sources of experimental errors in NGS and how replicates can be used to abate such errors.
Next-generation sequencing technologies have been and continue to be deployed in clinical laboratories, enabling rapid transformations in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude, and continuous advances are being made. It is now feasible to analyze an individual’s near-complete exome or genome to assist in the diagnosis of a wide array of clinical scenarios. Next-generation sequencing technologies are also facilitating further advances in therapeutic decision making and disease prediction for at-risk patients. However, with rapid advances come additional challenges involving the clinical validation and use of these constantly evolving technologies and platforms in clinical laboratories. To assist clinical laboratories with the validation of next-generation sequencing methods and platforms, the ongoing monitoring of next-generation sequencing testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics has developed the following professional standards and guidelines.
Genet Med15 9, 733–747.
This publication was a collaborative effort with expertise from industry, academia, regulatory experts and CLIA lab directors, AMP members, CAP members to provide guidance for the implementation, validation and proficiency testing of NGS procedures in clinical settings.
To the Editor
Most test results in medical laboratories are numbers, known as “quantitative results” if they have linearity (a ratio or interval scale) and are related to the international/national reference standard. Results consisting of nouns or adjectives (known as “qualitative results”) are less frequent but no less important to service quality.
The Eurachem/Cooperation on International Traceability in Analytical Chemistry (CITAC) guide “Evaluating Performance and Uncertainty in Qualitative Chemical Analysis” has recently been published (1). However, the Eurachem/CITAC guide states that it does not address all available tools for evaluating the performance of qualitative methods and the uncertainty of qualitative results. It is limited to comparison of results to a reference and does not consider, for example, concordance between qualitative methods or the classification on ordinal scales. In fact, a number of criticisms of the guide have recently been reported, including the lack of the repeatability and reproducibility components for the accuracy of the methods (2).
ISO published the draft for final approval of the revision of ISO 15189 standard. Following ISO directives, ISO 15189 must be aligned with ISO/IEC 17025:2017 and should be less prescriptive. Draft ISO/DIS 15189 deviates in some points from ISO 17025 and the ISO indications to limit prescriptiveness: equipment, uncertainty, quality control. This do not seem to be justified by medical specificities and could complicate the understanding of the new requirements in medical laboratories.
Background
Accurate human leucocyte antigen (HLA) typing is essential for solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). Next-generation sequencing (NGS) is increasingly adopted by clinical laboratories performing HLA typing. To ensure reliable NGS-based HLA typing throughout China, a HLA-NGS proficiency testing (PT) scheme was set up.
Methods
A panel of 10 DNA reference materials for 11 HLA loci were distributed to 24 participants. They were asked to submit NGS typing results for at least six loci including HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1. Information regarding individual laboratory’s sample preparation method, NGS platform, and genotyping software was also collected.
Results
Thirteen participants correctly identified HLA alleles in all samples. The total concordance rate for all HLA alleles typed was 99.2% (3390/3420). The overall discordance was 0.2% (3/1440) for Class I, and 1.4% (27/1980) for Class II loci. Further investigation of the typing mistakes pointed to poor sequencing coverage and wrong allele calls. The errors identified reflect the deficient aspect of NGS in HLA typing, which may have implications for quality improvement for relevant laboratories.
Conclusions
The results of this PT underscore the necessity of regular participation in external quality assessment for clinical laboratories that perform HLA-NGS typing.
Background and aims
Hereditary anemia (HA) encloses a wide group of rare inherited disorders with clinical and hematologic overlaps that complicate diagnosis.
Materials and methods
A 48-gene panel was developed to diagnose HA by Next Generation Sequencing (NGS) in a large cohort of 165 patients from 160 unrelated families.
Results
Patients were divided in: A) patients who had a suspicion of a specific type of HA (n=109), and B) patients who had a suspicion of HA but with no clear type (n=56). Diagnostic performance was 83.5% in group A and a change of the initial diagnosis occurred in 11% of these patients. In group B, 35.7% of patients achieved a genetic diagnosis. NGS identified 6 cases of xerocytosis, 6 of pyruvate kinase (PK) deficiency, 4 of G6PD, and 1 case of phytosterolemia with no initial suspicion of these pathologies, which is clinically relevant since they have specific treatment. Five patients were found to carry variants associated to two different pathologies (4 of them combining a metabolic deficiency and a membrane defect), and 44 new variants were identified in 41 patients.
Conclusion
The use of NGS is a sensitive technique to diagnose HA and it shows better performance when patients are better characterized.
Membranopathies are a group of inherited blood disorders where the diagnosis could form a challenge due to phenotype‐genotype heterogeneity. In this review, the usage and limitations of diagnostic methods for membranopathies in Asian countries were evaluated. A systematic review was done using articles from PubMed, Google Scholar, and EBSCO from 2000 to 2020. Thirty‐six studies conducted in seven Asian countries had used different diagnostic methods to confirm membranopathies. In 58.3% of studies, full blood count (FBC), reticulocyte count, and peripheral blood smear (PBS) were used in preliminary diagnosis. The combination of the above three with osmotic fragility (OF) test was used in 38.8%. The flowcytometric osmotic fragility (FC‐OF) test was used in 27.7% where it showed high sensitivity (92%–100%) and specificity (96%–98%). The eosin‐5‐maleimide (EMA) assay was used in 68.1% with high sensitivity (95%–100%) and specificity (93%–99.6%). About 36.1% of studies had used sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as a further diagnostic method to detect defective proteins. Genetic analysis to identify mutations was done using Sanger sequencing, next‐generation sequencing (NGS), and whole‐exome sequencing (WES) in 33.3%, 22.2%, and 13.8% of studies, respectively. The diagnostic yield of NGS ranged from 63% to 100%. Proteomics was used in 5.5% of studies to support the diagnosis of membranopathies. A single method could not diagnose all membranopathies. Next‐generation sequencing, Sanger sequencing, and proteomics will supplement the well‐established screening and confirmatory methods, but not replace them in hereditary hemolytic anemia assessment.
Background:
Metagenomic next-generation sequencing (mNGS) for pathogen detection is becoming increasingly available as a method to identify pathogens in cases of suspected infection. mNGS analyzes the nucleic acid content of patient samples with high-throughput sequencing technologies to detect and characterize microorganism DNA and/or RNA. This unbiased approach to organism detection enables diagnosis of a broad spectrum of infection types and can identify more potential pathogens than any single conventional test. This can lead to improved ability to diagnose patients, although there remains concern regarding contamination and detection of nonclinically significant organisms.
Content:
We describe the laboratory approach to mNGS testing and highlight multiple considerations that affect diagnostic performance. We also summarize recent literature investigating the diagnostic performance of mNGS assays for a variety of infection types and recommend further studies to evaluate the improvement in clinical outcomes and cost-effectiveness of mNGS testing.
Summary:
The majority of studies demonstrate that mNGS has sensitivity similar to specific PCR assays and will identify more potential pathogens than conventional methods. While many of these additional organism detections correlate with the expected pathogen spectrum based on patient presentations, there are relatively few formal studies demonstrating whether these are true-positive infections and benefits to clinical outcomes. Reduced specificity due to contamination and clinically nonsignificant organism detections remains a major concern, emphasizing the importance of careful interpretation of the organism pathogenicity and potential association with the clinical syndrome. Further research is needed to determine the possible improvement in clinical outcomes and cost-effectiveness of mNGS testing.
The new European regulations, and in particular that on in vitro diagnostics, have made the principles already developed by ISO and CEN mandatory obligations. In particular, in post-market surveillance laboratories and manufacturers are now obliged to share important information throughout the life cycle of devices. Both serious accidents with the relative corrective actions, and in general the performance and safety, user reports, the frequency and severity of events, the drifts over time. Documents such as a surveillance plan and periodic safety reports are expected. The manufacturer collects from laboratories and proactively evaluates performance data and relevant scientific data related to the use of a device that bears the CE marking, with the aim of confirming safety, performance and scientific validity for life expected device. This means concretely starting from the results of external quality assessments (VEQ) and from those of internal quality control (CQI), arriving at the reports of clinicians and patients up to real complaints.
ISO/TR 20416 for its part covers the post-market phase of medical devices.
Close collaboration between laboratories and manufacturers is already present in ISO 15189 for accreditation, but it is clearly explicit at least in ISO 22367 for risk management and ISO 15198 for internal quality control, not to mention operator safety standards ISO 15190 and ISO 35001. Manufacturers and laboratories cannot fail to undertake to develop this collaboration in the narrow period of time that separates us from the full application of the European Regulation.
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https://www.minervamedica.it/en/journals/medicina-laboratorio/article.php?cod=R54Y2020N04A0309
ISO, the International Organization for Standardization, is preparing document ISO/TS 17822-2 on the quality control of nucleic acid amplification techniques (NAAT). ISO 17822-2 takes into consideration many aspects that affect quality. ISO 17822 prescribes the use of control materials and describes different types of them. ISO 17822-2 states that the laboratory shall decide the appropriate type and frequency of use of internal and external controls, but says however that the results must remain within the pre-established limits, without saying how the limits should be fixed. Worldwide quality control in medical laboratories is often not performed correctly. Quality costs represent 22% of the laboratory’s direct costs. The SIPMeL Quality and Accreditation Commission has therefore produced its recommendations on quality control, with the transposition of ISO 15198: 2004, reconfirmed also in 2018. Diagnostic system manufacturers have a primary role in this activity. The guidelines for quality control are in the document Clinical Laboratory Standards Institute (CLSI) C24.
Comprehensive next-generation sequencing (NGS) tests are increasingly used as first-line tests in the evaluation of patients with suspected heritable disease. Despite major technical simplifications, these assays still pose significant challenges for molecular testing laboratories. Existing professional guidelines and recommendations provide a framework for laboratories implementing such tests, but in-depth, concrete guidance is generally not provided. Consequently, there is variability in how laboratories interpret and subsequently implement these regulatory frameworks. To address the need for more detailed guidance, the College of American Pathologists with representation from the Association for Molecular Pathologists assembled a working group to create a practical resource for clinical laboratories. This initial work is focused on variant detection in the setting of inherited disease and provides structured worksheets that guide the user through the entire life cycle of an NGS test, including design, optimization, validation, and quality management with additional guidance for clinical bioinformatics. This resource is designed to be a living document that is publicly available and will be updated with user and expert feedback as the wet bench and bioinformatic landscapes continue to evolve. It is intended to facilitate the standardization of NGS testing across laboratories and therefore to improve patient care. © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology
Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical advisement for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.
Diverse microbial communities of bacteria, archaea, viruses and single-celled eukaryotes have crucial roles in the environment and in human health. However, microbes are frequently difficult to culture in the laboratory, which can confound cataloging of members and understanding of how communities function. High-throughput sequencing technologies and a suite of computational pipelines have been combined into shotgun metagenomics methods that have transformed microbiology. Still, computational approaches to overcome the challenges that affect both assembly-based and mapping-based metagenomic profiling, particularly of high-complexity samples or environments containing organisms with limited similarity to sequenced genomes, are needed. Understanding the functions and characterizing specific strains of these communities offers biotechnological promise in therapeutic discovery and innovative ways to synthesize products using microbial factories and can pinpoint the contributions of microorganisms to planetary, animal and human health.
The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.
Context:
The higher throughput and lower per-base cost of next-generation sequencing (NGS) as compared to Sanger sequencing has led to its rapid adoption in clinical testing. The number of laboratories offering NGS-based tests has also grown considerably in the past few years, despite the fact that specific Clinical Laboratory Improvement Amendments of 1988/College of American Pathologists (CAP) laboratory standards had not yet been developed to regulate this technology.
Objective:
To develop a checklist for clinical testing using NGS technology that sets standards for the analytic wet bench process and for bioinformatics or "dry bench" analyses. As NGS-based clinical tests are new to diagnostic testing and are of much greater complexity than traditional Sanger sequencing-based tests, there is an urgent need to develop new regulatory standards for laboratories offering these tests.
Design:
To develop the necessary regulatory framework for NGS and to facilitate appropriate adoption of this technology for clinical testing, CAP formed a committee in 2011, the NGS Work Group, to deliberate upon the contents to be included in the checklist. Results . -A total of 18 laboratory accreditation checklist requirements for the analytic wet bench process and bioinformatics analysis processes have been included within CAP's molecular pathology checklist (MOL).
Conclusions:
This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality.
Next-generation sequencing (also known as massively parallel sequencing) technologies are revolutionising our ability to characterise cancers at the genomic, transcriptomic and epigenetic levels. Cataloguing all mutations, copy number aberrations and somatic rearrangements in an entire cancer genome at base pair resolution can now be performed in a matter of weeks. Furthermore, massively parallel sequencing can be used as a means for unbiased transcriptomic analysis of mRNAs, small RNAs and noncoding RNAs, genome-wide methylation assays and high-throughput chromatin immunoprecipitation assays. Here, I discuss the potential impact of this technology on breast cancer research and the challenges that come with this technological breakthrough.
A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced
is first subjected to limited attack by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide
a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely
a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone
library has been prepared, the speed of this approach (>1000 nucleotides of randomly selected sequence per day) is limited
mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount
of new sequence information per clone is substantially diminished as the sequence nears completion.
Next Generation Sequencing (NGS)
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Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists
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a Nationwide Signal-Finding Clinical Trial: Molecular Analysis for Therapy Choice Clinical Trial
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Q16 - Raccomandazioni per la stima dell'incertezza di misura nei laboratori medici
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Eurachem/CITAC guide: assessment of performance and uncertainty in qualitative chemical analysis. Prima Edizione. Teddington: Eurachem
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Exigences Particulieres Pour L'accreditation Des Laboratoires Medicaux Qui Realisent Des Analyses De Tumeurs Hematologiques Et Solides Selon La Methode Next Generation Sequencing (Ngs)
- Belac. De Belgische Accreditatie-Instelling