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OBJECTIVES: The quality control of serological assays remains controversial. The aim of this project was to describe the problems associated with a working model for controlling these assays and solutions, including using a source of well-defined targets and acceptable limits, a process to identify lot-to-lot reagent variation and an interpretation of the result that accounted for the clinical situation. False-negative results are problematic but can be reduced by identifying and comparing reagent lot variation with previous results. METHODS: The components of the Quality Assurance strategy are the following: Lot-to-lot reagent and calibrator variation assessment; dynamic, big-data approach to determine accurate targets and acceptable limits for manufacturer-provided QC material; negative QC monitoring process; use of commutable EQA with a sufficient method subgroup size to assess bias; clinical assessment of any statistically flagged error; and provision of support to the clinician for the interpretation of results. RESULTS: The model described has been used for twelve months, and acceptable variation has been maintained. CONCLUSIONS: The paper presents a solution that emphasizes the early detection of reagent lot variation and patient risk rather than instrument control. Reducing the risk of a false result to patients requires optimal assay quality control and an effective mechanism to support the clinician's use of these results in diagnosis and monitoring. The problems of serological assays are well-known, but there remain few integrated solutions in the literature.
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Control de Calidad , Pruebas Serológicas , Humanos , Pruebas Serológicas/normasRESUMEN
Microplastic (MP) research faces challenges due to costly, time-consuming, and error-prone analysis techniques. Additionally, the variability in data quality across studies limits their comparability. This study addresses the critical need for reliable and cost-effective MP analysis methods through validation of a semi-automated workflow, where environmentally relevant MP were spiked into and recovered from marine fish gastrointestinal tracts (GITs) and blue mussel tissue, using Nile red staining and machine learning automated analysis of different polymers. Parameters validated include trueness, precision, uncertainty, limit of quantification, specificity, sensitivity, selectivity, and method robustness. For fish GITs a 95 ± 9 % recovery rate was achieved, and 87 ± 11 % for mussels. Polymer identification accuracies were 76 ± 8 % for fish GITs and 80 ± 13 % for mussels. Polyethylene terephthalate fragments showed more variability with lower accuracies. The proposed validation parameters offer a step towards quality management guidelines, as such aiding future researchers and fostering cross-study comparability.
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Traditionally, for indoor radon testing, predominantly passive measurements have been used, typically applying the solid-state alpha track-etch method for long-term and the charcoal method for short-term measurements. However, increasingly, affordable consumer-grade active monitors have become available in the last few years, which can generate a concentration time series of an almost arbitrary duration. Firstly, we argue that consumer-grade monitors can well be used for quality-assured indoor radon assessment and consequent reliable decisions. Secondly, we discuss the requirements of quality assurance, which actually allow for reliable decision-making. In particular, as part of a rational strategy, we discuss how to interpret measurement results from low-cost active monitors with high and low sensitivity with respect to deciding on conformity with reference levels that are the annual average concentration of indoor radon. Rigorous analysis shows that temporal variations in radon are a major component of the uncertainty in decision-making, the reliability of which is practically independent of monitor sensitivity. Manufacturers of low-cost radon monitors already provide sufficient reliability and quality of calibration for their devices, which can be used by both professional inspectors and the general public. Therefore, within the suggested measurement strategy and metrologically assured criteria, we only propose to clarify the set and values of the key metrological characteristics of radon monitors as well as to upgrade user-friendly online tools. By implementing clear metrological requirements as well as the rational measurement strategy for the reliable conformity assessment of a room (building) with radon safety requirements, we anticipate significant reductions in testing costs, increased accessibility, and enhanced quality assurance and control (QA/QC) in indoor radon measurements.
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Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.
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Mitocondrias , Mitofagia , Animales , Mitocondrias/metabolismo , Genes Reporteros , Drosophila/metabolismo , Microscopía Fluorescente/métodos , Drosophila melanogaster/metabolismo , Lisosomas/metabolismo , Autofagia/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genéticaRESUMEN
We outline our approach for studying the selective autophagy of peroxisomes (pexophagy), using fluorescence microscopy in tissue cell culture models. Ratiometric reporters, which specifically localize to peroxisomes, allow a quantitative assessment of pexophagy in fixed and live cells, as well as whole organisms. We discuss chemical and physiological inducers of pexophagy and any overlap with the induction of mitophagy.
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Microscopía Fluorescente , Peroxisomas , Peroxisomas/metabolismo , Microscopía Fluorescente/métodos , Humanos , Animales , Autofagia/fisiología , MitofagiaRESUMEN
Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.
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Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.
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INTRODUCTION: During the Metabolomics 2023 conference, the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) presented a QA/QC workshop for LC-MS-based untargeted metabolomics. OBJECTIVES: The Best Practices Working Group disseminated recent findings from community forums and discussed aspects to include in a living guidance document. METHODS: Presentations focused on reference materials, data quality review, metabolite identification/annotation and quality assurance. RESULTS: Live polling results and follow-up discussions offered a broad international perspective on QA/QC practices. CONCLUSIONS: Community input gathered from this workshop series is being used to shape the living guidance document, a continually evolving QA/QC best practices resource for metabolomics researchers.
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Espectrometría de Masas , Metabolómica , Control de Calidad , Metabolómica/métodos , Metabolómica/normas , Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Espectrometría de Masas/métodos , Humanos , Consenso , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography-mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.
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Proteínas Recombinantes de Fusión , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Flujo de Trabajo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/análisis , Glucagón/análisis , Glucagón/química , Humanos , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Patient-based real-time QC (PBRTQC) uses patient-derived data to assess assay performance. PBRTQC algorithms have advanced in parallel with developments in computer science and the increased availability of more powerful computers. The uptake of Artificial Intelligence in PBRTQC has been rapid, with many stated advantages over conventional approaches. However, until this review, there has been no critical comparison of these. The PBRTQC algorithms based on moving averages, regression-adjusted real-time QC, neural networks and anomaly detection are described and contrasted. As Artificial Intelligence tools become more available to laboratories, user-friendly and computationally efficient, the major disadvantages, such as complexity and the need for high computing resources, are reduced and become attractive to implement in PBRTQC applications.
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Algoritmos , Control de Calidad , Humanos , Redes Neurales de la Computación , Inteligencia Artificial , Laboratorios Clínicos/normasRESUMEN
Background: Breast cancer is a major global cancer, for which radiation and chemotherapy are the main treatments. Natural remedies are being studied to reduce the side effects. Etoposide (ETO), a chemo-drug, and quercetin (QC), a phytochemical, are considered potential factors for adaptation to conventional treatments. Objectives: The anticancer effect of the synergy between ETO and Quercetin-loaded solid lipid nanoparticles (QC-SLNs), was investigated in MDA-MB-231 cells. Methods: We developed QC-SLNs for efficient cellular delivery, characterizing their morphology, particle size, and zeta potential. We assessed the cytotoxicity of QC-SLNs and ETO on breast cancer cells via the MTT assay. Effects on apoptosis intensity in MDA-MB-231 cells have been detected utilizing annexin V-FITC, PI, and caspase activities. Real-time PCR assessed Bax gene and Bcl-2 gene fold change expression, while Western blot analysis determined p53 and p21 protein levels. Results: Spherical, negatively charged QC-SLNs, when combined with ETO, significantly enhanced inhibition of MDA-MB-231 cell proliferation compared to ETO or QC-SLNs alone. The combined treatment also notably increased the apoptosis pathway. QC-SLNs + ETO increased the Bax/Bcl-2 gene ratio, elevated p53 and p21 proteins, and activated caspase 3 and 9 enzymes. These results indicate the potential for QC-SLNs + ETO as a strategy for breast cancer treatment, potentially overcoming ETO-resistant breast cancer chemoresistance. Conclusion: These results suggest that QC-SLN has the potential to have a substantial impact on the breast cancer cure by improving the efficacy of ETO. This enhancement could potentially help overcome chemoresistance observed in ETO-resistant breast cancer.
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PURPOSE: Quality Control (QC) of the Positron Emission Tomography-Computed Tomography (PET-CT) system must be performed prior to the PET-CT acquisition to ensure the reproducibility as per the manufacturer recommendation. In this study we have evaluated the performance of daily PET QC test by utilising lutetium yttrium oxyorthosilicate (LYSO) scintillation crystal natural radioactivity of 176Lu as a source of radiation to perform the PET uCare.iQC with uMI550 digital PET-CT system. This was also compared with existing radioactive external source-based QC test with other manufacturer PET-CT systems. METHOD: This radioactive source free daily QC study was performed on uMI550 digital PET-CT system. The daily QC data report was captured and interpreted. This PET-CT system has unique feature that utilises the inherent property of LYSO crystal that is 176Lu with natural radioactivity abundance of 2.6%. The Lutetium-176 (176Lu) radioactivity is used to perform the daily QC in PET in place of external radioactive source of Germanium-68 (68Ge). This feature work automatically in preschedule manner to complete the daily QC at preset time in the morning and system get ready after the QC test. RESULTS: Over 120 automatic PET daily uCare.iQC test were performed. The daily PET QC test was prescheduling setup for 6:00 am in every morning. No failure on daily QC test were observed. The QC parameters and system parameters consistency was observed. CONCLUSION: It was concluded that the daily PET QC can be performed by utilising LYSO crystal inherent natural radioactivity of 176Lu as a source of radiation to perform the test as replacement of external 68Ge radioactive source. IMPLICATION FOR PRACTICE: PET-CT daily QC by utilizing the 176Lu radioactivity of LYSO crystal results in reducing the radiation exposure to operation staff and reducing operational cost by elimination 68Ge shield source Phantom.
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Lutecio , Tomografía Computarizada por Tomografía de Emisión de Positrones , Control de Calidad , Radioisótopos , Reproducibilidad de los Resultados , Silicatos , HumanosRESUMEN
In recent years, plastic pollution in the environment has also increased due to the increasing production and consumption of plastics worldwide. The presence of microplastics (MPs) in the environment from different sources is observed almost everywhere, especially in aquatic environments. A standard method for sampling, identification, and quantification of MPs in wastewater has not yet been established. In this study, it was aimed to determine the MPs and their characteristics in the effluent of an advanced biological domestic wastewater treatment plant. The seasonal changes of MPs in a year were revealed. Pre-treatments suitable for the studied wastewater were developed for visual determination of MPs. Fibers are the dominant type of MPs, with numbers ranging between 32.0 and 95.5 particle/L. MPs in five different polymer structures were determined by FTIR analysis. These are Polyethylene, Polypropylene, Polyester, Polyurethane and Polyethylene terephthalate. The results were evaluated according to QA/QC and determined to meet the standards.
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Monitoreo del Ambiente , Microplásticos , Eliminación de Residuos Líquidos , Aguas Residuales , Contaminantes Químicos del Agua , Aguas Residuales/química , Microplásticos/análisis , Contaminantes Químicos del Agua/análisis , Monitoreo del Ambiente/métodos , Plásticos/análisisRESUMEN
The Westgard quality control (QC) rules are often applied in infectious diseases serology to validate the quality of results, but this requires a reasonable tradeoff between maximum sensitivity to errors and minimum false rejections. This article, in addition to illustrate the six sigma methodology in the QC management of the (anti-HCV Architect®) test, it discusses the main influencing factors on sigma value. Data from low positive and in-kit control materials spreading over 6 months and using four reagent kits, were used to calculate the precision of the test. The difference between the control material reactivity and the cut-off defined the error budget. Sigma values were > 6, which indicates that the method produces four erroneous results per million tests. The application of the six sigma concept made it possible to argue the choice of the new QC strategy (use of 13S rule with one positive control) and to relax the existing QC rules. This work provides a framework for infectious diseases serology laboratories to evaluate tests performances against a quality requirement and design an optimal QC strategy.
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Hepatitis C , Control de Calidad , Pruebas Serológicas , Gestión de la Calidad Total , Humanos , Hepatitis C/sangre , Hepatitis C/diagnóstico , Gestión de la Calidad Total/normas , Pruebas Serológicas/normas , Pruebas Serológicas/métodos , Anticuerpos contra la Hepatitis C/sangre , Anticuerpos contra la Hepatitis C/análisis , Hepacivirus/aislamiento & purificación , Hepacivirus/inmunología , Sensibilidad y Especificidad , Juego de Reactivos para Diagnóstico/normas , Reproducibilidad de los Resultados , Garantía de la Calidad de Atención de Salud/normas , Garantía de la Calidad de Atención de Salud/métodos , Laboratorios Clínicos/normasRESUMEN
Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10 mg kg-1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48 h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. KEY POINTS: There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles.
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Ratones Transgénicos , Mitofagia , Músculo Esquelético , Sepsis , Animales , Sepsis/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Ratones , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Masculino , Mitocondrias Musculares/metabolismo , Autofagia/fisiologíaRESUMEN
Advanced therapy medicinal products (ATMP) are complex medicines based on gene therapy, somatic cell therapy, and tissue engineering. These products are rapidly arising as novel and promising therapies for a wide range of different clinical applications. The process for the development of well-established ATMPs is challenging. Many issues must be considered from raw material, manufacturing, safety, and pricing to assure the quality of ATMPs and their implementation as innovative therapeutic tools. Among ATMPs, cell-based ATMPs are drugs altogether. As for standard drugs, technologies for quality control, and non-invasive isolation and production of cell-based ATMPs are then needed to ensure their rapidly expanding applications and ameliorate safety and standardization of cell production. In this review, emerging approaches and technologies for quality control of innovative cell-based ATMPs are described. Among new techniques, microfluid-based systems show advantages related to their miniaturization, easy implementation in analytical process and automation which allow for the standardization of the final product.
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Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Genética , Ingeniería de Tejidos , Animales , Humanos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Terapia Genética/métodos , Terapia Genética/normas , Control de Calidad , Ingeniería de Tejidos/métodos , Ingeniería de Tejidos/normasRESUMEN
BACKGROUND: Both functional brain imaging studies and autopsy reports have indicated the presence of synaptic loss in the brains of depressed patients. The activated microglia may dysfunctionally engulf neuronal synapses, leading to synaptic loss and behavioral impairments in depression. However, the mechanisms of microglial-synaptic interaction under depressive conditions remain unclear. METHODS: We utilized lipopolysaccharide (LPS) to induce a mouse model of depression, examining the effects of LPS on behaviors, synapses, microglia, microglial phagocytosis of synapses, and the C1q/C3-CR3 complement signaling pathway. Additionally, a C1q neutralizing antibody was employed to inhibit the C1q/C3-CR3 signaling pathway and assess its impact on microglial phagocytosis of synapses and behaviors in the mice. RESULTS: LPS administration resulted in depressive and anxiety-like behaviors, synaptic loss, and abnormal microglial phagocytosis of synapses in the hippocampal dentate gyrus (DG) of mice. We found that the C1q/C3-CR3 signaling pathway plays a crucial role in this abnormal microglial activity. Treatment with the C1q neutralizing antibody moderated the C1q/C3-CR3 pathway, leading to a decrease in abnormal microglial phagocytosis, reduced synaptic loss, and improved behavioral impairments in the mice. CONCLUSIONS: The study suggests that the C1q/C3-CR3 complement signaling pathway, which mediates abnormal microglial phagocytosis of synapses, presents a novel potential therapeutic target for depression treatment.
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Complemento C1q , Complemento C3 , Depresión , Modelos Animales de Enfermedad , Microglía , Fagocitosis , Transducción de Señal , Sinapsis , Animales , Complemento C1q/metabolismo , Microglía/metabolismo , Sinapsis/metabolismo , Ratones , Transducción de Señal/fisiología , Depresión/metabolismo , Fagocitosis/fisiología , Complemento C3/metabolismo , Masculino , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BLRESUMEN
Objective: A quality control (QC) system based on the electronic portal imaging device (EPID) system was used to realize the Multi-Leaf Collimator (MLC) position verification and dose verification functions on Primus and VenusX accelerators. Methods: The MLC positions were calculated by the maximum gradient method of gray values to evaluate the deviation. The dose of images acquired by EPID were reconstructed using the algorithm combining dose calibration and dose calculation. The dose data obtained by EPID and two-dimensional matrix (MapCheck/PTW) were compared with the dose calculated by Pinnacle/TiGRT TPS for γ passing rate analysis. Results: The position error of VenusX MLC was less than 1 mm. The position error of Primus MLC was significantly reduced after being recalibrated under the instructions of EPID. For the dose reconstructed by EPID, the average γ passing rates of Primus were 98.86% and 91.39% under the criteria of 3%/3 mm, 10% threshold and 2%/2 mm, 10% threshold, respectively. The average γ passing rates of VenusX were 98.49% and 91.11%, respectively. Conclusion: The EPID-based accelerator quality control system can improve the efficiency of accelerator quality control and reduce the workload of physicists.
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Planificación de la Radioterapia Asistida por Computador , Radioterapia de Intensidad Modulada , Planificación de la Radioterapia Asistida por Computador/métodos , Dosificación Radioterapéutica , Algoritmos , Calibración , Electrónica , Radioterapia de Intensidad Modulada/métodos , Radiometría/métodosRESUMEN
PURPOSE: Indian Council of Medical Research (ICMR) initiated an Inter-Laboratory Quality Control testing (ILQC) program for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) testing. Under this program, SARS-CoV-2 testing laboratories across the country submit specimens to the assigned State Quality Control (SQCs) laboratories for ILQC testing. This study aimed to investigate the performance of public and private SARS-CoV-2 testing laboratories in Delhi and highlights the country's effort in ramping up testing facility with close monitoring of the quality of Covid-19 testing results. METHODS: In the present study, two-years of SARS-CoV-2 testing data is included. During July 2020 through February 2022, a total of 1791 anonymised specimens were received from 56 public and private laboratories. These specimens were processed by reverse transcriptase - polymerase chain reaction (RT-PCR) tests as per National Institute of Virology (NIV) protocol and the results were uploaded on the ICMR quality control/quality assurance (QC/QA) portal without directly conveying the results to respective participating laboratories. This portal generated a final report stating concordance and intimate results to individual laboratories. RESULTS: Among the 1791 specimens, 25 were rejected and the remaining 1766 were tested. Among these specimens 1691 (95.75%) revealed concordance, and 75 (4.24%) were discordant. A total of 29 laboratories had 100% concordance, 21 laboratories had over 90% concordance and six laboratories had over 80% concordance. CONCLUSIONS: The study demonstrates that the establishment of an inter-laboratory comparison program for SARS-CoV-2 testing helped in monitoring quality of SARS-CoV-2 testing in the country.
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Prueba de COVID-19 , COVID-19 , SARS-CoV-2 , Humanos , India , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Prueba de COVID-19/métodos , Prueba de COVID-19/normas , Control de Calidad , Garantía de la Calidad de Atención de Salud , Laboratorios/normas , Laboratorios Clínicos/normas , PandemiasRESUMEN
Liquid chromatography-mass spectrometry (LC-MS)-based peptidomics methods allow for the detection and identification of many peptides in a complex biological mixture in an untargeted manner. Quantitative peptidomics approaches allow for comparisons of peptide abundance between different samples, allowing one to draw conclusions about peptide differences as a function of experimental treatment or physiology. While stable isotope labeling is a powerful approach for quantitative proteomics and peptidomics, advances in mass spectrometry instrumentation and analysis tools have allowed label-free methods to gain popularity in recent years. In a general label-free quantitative peptidomics experiment, peak intensity information for each peptide is compared across multiple LC-MS runs. Here, we outline a general approach for label-free quantitative peptidomics experiments, including steps for sample preparation, LC-MS data acquisition, data processing, and statistical analysis. Special attention is paid to address run-to-run variability, which can lead to several major problems in label-free experiments. Overall, our method provides researchers with a framework for the development of their own quantitative peptidomics workflows applicable to quantitation of peptides from a wide variety of different biological sources.