RESUMO
This study aimed to investigate whether the inhibition of the TLR4/NF-κB pathway can promote lipopolysaccharide (LPS)-induced microglial polarization from the M1 to M2 phenotype, and thus exert neuroprotection. LPS-induced microglia were used as a model for inflammation in vitro. TLR4-specific inhibitor resatorvid (TAK-242) and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) were used to verify the effect of the TLR4/NF-κB pathway on microglia activation and polarization. Cell proliferation was measured by cell counting, and nitric oxide (NO) and reactive oxygen species (ROS) release was measured using the Griess reagent and ROS kit, respectively. Immunofluorescence and RT-qPCR analyses were used to detect the expression of microglial activation markers, phenotypic markers, related pathway molecules, and inflammatory factors. TLR4 specific inhibitor TAK-242 and NF-κB inhibitor PDTC alleviated LPS-induced microglia over-activation by inhibiting the TLR4/NF-κB pathway, and reduced LPS-stimulated cell proliferation and the release of NO, ROS, TNF-a, and IL-6 and IL-1ß. Meanwhile, TAK-242 and PDTC promoted LPS-induced polarization of microglia from M1 to M2 phenotype, decreased the expression of microglial activation marker Iba1 and M1 phenotypic markers (TNF-a and CD86), and increased the expression of M2 phenotypic markers (Arg-1 and CD206). The mechanism may be related to inhibiting the TLR4/NF-κB pathway. The inhibition of the TLR4/NF-κB pathway can promote LPS-induced polarization of BV2 microglia from M1 phenotype to M2 phenotype.
Assuntos
Lipopolissacarídeos , NF-kappa B , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Microglia , Receptor 4 Toll-Like/metabolismo , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , FenótipoRESUMO
Introduction: Metagenomic next-generation sequencing (mNGS) assay for detecting infectious agents is now in the stage of being translated into clinical practice. With no approved approaches or guidelines available, laboratories adopt customized mNGS assays to detect clinical samples. However, the accuracy, reliability, and problems of these routinely implemented assays are not clear. Objectives: To evaluate the performance of 90 mNGS laboratories under routine testing conditions through analyzing identical samples. Methods: Eleven microbial communities were generated using 15 quantitative microbial suspensions. They were used as reference materials to evaluate the false negatives and false positives of participating mNGS protocols, as well as the ability to distinguish genetically similar organisms and to identify true pathogens from other microbes based on fictitious case reports. Results: High interlaboratory variability was found in the identification and the quantitative reads per million reads (RPM) values of each microbe in the samples, especially when testing microbes present at low concentrations (1 × 103 cell/ml or less). 42.2% (38/90) of the laboratories reported unexpected microbes (i.e. false positive problem). Only 56.7% (51/90) to 83.3% (75/90) of the laboratories showed a sufficient ability to obtain clear etiological diagnoses for three simulated cases combined with patient information. The analysis of the performance of mNGS in distinguishing genetically similar organisms in three samples revealed that only 56.6% to 63.0% of the laboratories recovered RPM ratios (RPM S. aureus /RPM S. epidermidis ) within the range of a 2-fold change of the initial input ratios (indicating a relatively low level of bias). Conclusion: The high interlaboratory variability found in both identifying microbes and distinguishing true pathogens emphasizes the urgent need for improving the accuracy and comparability of the results generated across different mNGS laboratories, especially in the detection of low-microbial-biomass samples.
Assuntos
Metagenômica , Staphylococcus aureus , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metagenoma , Metagenômica/métodos , Reprodutibilidade dos TestesRESUMO
Flocculation is one of the commonly used sludge conditioning methods in water supply plants, which can improve the sludge dewatering performance by reducing the specific resistance of sludge (SRF), decreasing the amount of sludge, and finally lowering the transportation cost and subsequent disposal cost of sludge. Therefore, it is particularly important to develop new and efficient flocculants. In this paper, the template copolymer of acryloxy trimethylammonium chloride (DAC) and acrylamide (AM) was successfully synthesized by microwave-template copolymerization (MV-TP) using sodium polyacrylate (NaPAA) as template. The template copolymer was analyzed by infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance hydrogen spectroscopy (1H NMR), and scanning electron microscopy (SEM). It was found that this template copolymer had obvious cationic microblock structure. In addition, the test results of association constant (KM) and polymerization kinetics showed that the MW-TP was assigned to free radical initiated polymerization and the polymerization mechanism was I Zip-up (ZIP). It confirmed the formation of cation fragment structure again. Due to its dense positive charges in this new cationic microblock structure, it greatly improved the functions of electric neutralization, electrical patching, and adsorption bridging. The cationic fragment structure in the template copolymer could help to generate large and dense floc structure and form stable drainage channels. Under external pressure, these large and compact floc structures had greater compressive resistance, which avoided deformation and blockage of drainage channels and voids. It was beneficial to reduce SRF and evidently enhanced sludge dewatering performance.
Assuntos
Esgotos , Eliminação de Resíduos Líquidos , Resinas Acrílicas , Cátions , Floculação , Espectroscopia de Infravermelho com Transformada de Fourier , ÁguaRESUMO
BACKGROUND: BRCA1/2 gene mutation testing, based on next-generation sequencing (NGS), has been gradually applied in the clinic to serve as preventive early screening for predisposed individuals or to provide treatment options for patients with hereditary breast or ovarian cancers. Here, we evaluated the accuracy of NGS-based mutation detection in BRCA1/2 and the consistency in variant interpretation among clinical laboratories to find the possible reasons underlying inaccurate results and discrepant variant interpretation. METHODS: Laboratories were asked to use their routine procedures to detect six mimetic DNA samples with different BRCA1/2 germline variants. The results of variant detection were required to be submitted via a web-based evaluation system and were automatically scored, according to predefined criteria. The variant interpretation report, including the detailed clinical evidence, was summarized and analyzed for reasons underlying inconsistent results. RESULTS: Overall, only 55.2% (16/29) of laboratories, whose detection score was higher than 90 points, was found to be an acceptable detection capability level. 82.9% (29/35) of the errors were genotype errors. The variant classification results were generally consistent, and 77.8% (7/9) of the variants were given the consistent classification answer. Only two single nucleotide variants (SNVs) had a discrepant classification opinion across laboratories. CONCLUSIONS: The BRCA1/2 variant detection performance should be further improved, especially in reporting the correct genome coordinates. Inconsistent variant classification may be a result of the different clinical pieces of evidence collected by the laboratories. However, discordant clinical evidence also appeared within the same classification results. Therefore, our study provided clear clinical evidence assessment strategies for BRCA1/2 variants, which was aimed at obtaining a consistent variant classification strategy for providing accurate clinical reports to the clinicians.
Assuntos
Neoplasias da Mama/diagnóstico , Proteína BRCA1 , Proteína BRCA2 , Neoplasias da Mama/genética , Feminino , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MasculinoRESUMO
BACKGROUND: Astrovirus (AstV), Sapovirus (SaV) and Poliovirus (PV) are important pathogens that cause infections in children under five years of age. It is a very important task to systematically monitor and evaluate the diagnostic performance of these viruses in clinical laboratories. METHODS: In our study, we performed a multicenter evaluation study among 21 laboratories across China using simulated stool samples spiked with self-designed AstV, SaV and PV pseudoviral particles. RESULTS: The testing capability of 80.0% (16/20, AstV), 52.6% (10/19, SaV), and 25.0% (2/8, PV) of the participating laboratories were found to be "competent" in reporting correct results for all samples. The main type of errors were false negatives. None of the laboratories identified the subtypes of AstV and SaV, and six laboratories specifically identified the subtypes of PV. Lacking of well-trained personnel and adequate funding were the main challenges. From the questionnaire results, 55.6% laboratories (10/18) believe that training personnel could improve the laboratory testing performance. CONCLUSIONS: The laboratories showed a competent diagnostic performance for AstV, but inferior diagnostic performances for SaV and PV. Sensitivity of detection and the ability for virus typing should be improved clinically. Professional and standardized personnel training is urgently needed to further improve laboratory performance.
Assuntos
Gastroenterite , Poliovirus , Sapovirus , Criança , Pré-Escolar , China , Fezes , Humanos , Patologia Molecular , Sapovirus/genéticaRESUMO
INTRODUCTION: Microbiome research based on high-throughput sequencing has grown exponentially in recent years, but methodological variations can easily undermine the reproducibility across studies. OBJECTIVES: To systematically evaluate the comparability of sequencing results of 16S rRNA gene sequencing (16Ss)- and shotgun metagenomic sequencing (SMs)-based microbial community profiling in laboratories under routine conditions. METHODS: We designed a multicenter study across 35 participating laboratories in China using designed mock communities and homogenized fecal samples. RESULTS: A wide range of practices and approaches was reported by the participating laboratories. The observed microbial compositions of the mock communities in 46.2% (12/26) of the 16Ss and 82.6% (19/23) of the SMs laboratories had significant correlations with the expected result (Spearman r>0.59, P <0.05). The results from laboratories with near-identical protocols showed slight interlaboratory deviations. However, a high degree of interlaboratory deviation was found in the observed abundances of specific taxa, such as Bacteroides spp. (range: 0.3%-53.5%), Enterococci spp. (range: 0.8%-43.9%) and Fusobacterium spp. (range: 0.1%-39.8%). SMs performed better than 16Ss in detecting low-abundance bacteria (B. bifidum). The differences in DNA extraction methods, amplified regions and bioinformatics analysis tools (taxonomic classifiers and database) were important factors causing interlaboratory deviations. Addressing laboratory contamination is an urgent task because various sources of unexpected microbes were found in negative control samples. CONCLUSIONS: Well-defined control samples, such as the mock communities in this study, should be routinely used in microbiome research for monitoring potential biases. The findings in this study will provide guidance in the choice of more reasonable operating procedures to minimize potential methodological biases in revealing human microbiota composition.
RESUMO
The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.
Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Rearranjo Gênico , Ensaio de Proficiência Laboratorial/métodos , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Melhoria de Qualidade , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , China/epidemiologia , Feminino , Células HEK293 , Humanos , Hibridização in Situ Fluorescente/métodos , Camundongos , Camundongos Nus , Medicina de Precisão/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: Success of multiple-gene mutation tests by next-generation sequencing (NGS), associated with molecular targeting therapies for cancers, depending on the accuracy and consistency of interpreting variants. Here, we summarized reports from clinical laboratories for cases with non-small cell lung cancer (NSCLC) and discussed conflicting interpretations of somatic variants. Methods: Three mimetic DNA samples, containing six somatic mutations, were prepared based on three clinical case reports of NSCLC. Clinical reports and genetic testing questionnaires were collected from 67 laboratories enrolled in this investigation. Results: Thirty-four laboratories with correct variant results identified two variants, based on FDA approval of targeted drugs for the same tumor, consistently, with strong clinical significance, whereas the other variants were classified with conflicting interpretations. Discordant interpretations were reported for ERBB2 with three different classifications, including strong clinical significance (53.0%, 18/34), potential clinical significance (38.2%, 13/34), and unknown significance (8.8%, 3/34). In the variant therapeutic drug recommendation section, 32.4% of the laboratories (11/34) did not recommend all the available therapeutic drugs designated by the National Comprehensive Cancer Network (NCCN). In the remaining group of 33 laboratories with incorrect variant results, less correct classifications were acquired for the variants with strong clinical significance. Conclusions: Owing to numerous reasons, the interpretation of variants differed greatly, which might in turn lead to the inappropriate clinical care of patients with NSCLC. By analyzing the limitations of different databases used by laboratories, we integrated various types of databases with different levels of evidence to form a comprehensive and detailed variant interpretation pipeline, aiming to standardize the variant classification and provide accurate and sufficient therapeutic drug recommendation to clinicians for minimal-inappropriate therapeutic options.
RESUMO
Genome-edited human cell lines are important resources for producing quality control materials for clinical molecular genetic testing. Generating cell lines with defined mutations through homology-directed repair-based methods are inefficient and can lead to unwanted insertions and deletions in the target loci. Nonhomologous end joining in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system was harnessed to generate genome-engineered cell lines harboring target mutations. Donor plasmids containing target sites for the single guide RNA (sgRNA) and homologous DNA fragments harboring important cancer gene mutations were cotransfected with the Cas9/sgRNA vector into wild-type human cells. The introduced mutations were validated in-house and in 44 laboratories using various techniques, including next-generation sequencing. Exogenous sequences containing the target mutations were efficiently integrated into the ALK receptor tyrosine kinase (ALK) locus in HEK293T and A549 cells. Successful introduction of artificial mutations was confirmed via both Sanger sequencing and the amplification refractory mutation system. Results of external pilot testing revealed that the DNA samples derived from genome-edited cell lines were widely applicable across multiple platforms and laboratories. This study demonstrates that CRISPR/Cas9-induced nonhomologous end joining is a valuable and novel method for generating artificial mutants for use in quality control applications in clinical molecular genetics.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Testes Genéticos/métodos , Sequência de Bases , Edição de Genes , Técnicas de Introdução de Genes , Genoma Humano , Células HEK293 , Humanos , Mutação/genética , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
This study evaluated the ability of laboratories in the Chinese mainland to conduct molecular detection of seasonal A(H1N1), A(H1N1)pdm09, A(H3N2), A(H5N1), A(H7N9), A(H9N2), B(Victoria), and B(Yamagata). Based on a genetically engineered system of virus-like particles (VLPs), the National Center for Clinical Laboratories of China (NCCLs) developed an external quality assessment (EQA) panel. The panel was distributed to 35 laboratories in mainland China to investigate the proficiency of the 16 assays for influenza molecular detection. Using genetic engineering technology, VLPs encapsulating the 37 target genes of 8 influenza viruses were generated. After verification and quantification, 26 influenza virus surrogates with different concentrations were prepared for EQA. Among the 35 participating laboratories, 319 datasets were returned to the NCCLs. Overall, 95.6% (305/319) of datasets correctly reported all 30â¯samples, while 2.2% (7/319) of datasets with more than one incorrect result were considered as "improvable". A total of 16 misdiagnosed and 18 undiagnosed results were reported. The data analyzed in this study showed good reproducibility in China, but improvements are needed to decrease misdiagnosed and undiagnosed cases, particularly for the A(H9N2) NA gene. Moreover, VLPs are a good alternative specimen type for assay training and proficiency testing purposes.
Assuntos
Influenza Humana/diagnóstico , Ensaio de Proficiência Laboratorial/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Orthomyxoviridae/isolamento & purificação , China , Humanos , Virossomos/genética , Virossomos/isolamento & purificaçãoRESUMO
BACKGROUND: KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. METHODS: We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. RESULTS: We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. CONCLUSION: The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.
Assuntos
Sistemas CRISPR-Cas/genética , Glicina/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Valina/genética , Linhagem Celular , Análise Mutacional de DNA , Células HEK293 , Humanos , TransfecçãoRESUMO
BACKGROUND: Due to the significant risk of developing Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), the use of carbamazepine is not recommended in patients carrying the human leukocyte antigen B (HLA-B) *15:02 allele. In an effort to guarantee reliable community-based HLA-B*15:02 testing throughout China, a HLA-B*15:02 genotyping external quality assessment (EQA) program was set up. METHODS: In 2016, 10 genomic DNA samples with known HLA-B*15:02 allele status were sent to 37 laboratories from 16 provinces with a request for routine HLA-B*15:02 screening. The samples were validated using Sanger sequencing by a reference laboratory. Both genotyping results and clinical written reports were evaluated. RESULTS: Thirty-six of the participating laboratories correctly identified the HLA-B*15:02 allele status for all EQA samples. However, one lab failed to identify any positive challenges. The overall analytical sensitivity was 97.3% (180/185 challenges; 95% confidence interval: 93.8%-99.1%) and the analytic specificity was 100% (185/185; 95% confidence interval: 98.0%-100%). A review of the written reports showed that the clinical reporting for HLA-B*15:02 detection should be improved. Some essential information was missing, most notably laboratory information/contact, therapeutic recommendations, and methodology. CONCLUSION: External quality assessment is valuable in assessing and improving the quality of laboratory testing of HLA-B*15:02 allele.
Assuntos
Carbamazepina/efeitos adversos , Antígeno HLA-B15/genética , Síndrome de Stevens-Johnson , Carbamazepina/uso terapêutico , Contraindicações de Medicamentos , Epilepsia/tratamento farmacológico , Predisposição Genética para Doença/genética , Humanos , Fatores de Risco , Sensibilidade e Especificidade , Síndrome de Stevens-Johnson/epidemiologia , Síndrome de Stevens-Johnson/etiologia , Síndrome de Stevens-Johnson/genéticaRESUMO
BACKGROUND: Reports serve as a bridge between laboratories and clinicians, help synthesize an overwhelming amount of raw data into evidence-based medicine, and play a significant role in designing clinical treatments. In an effort to guarantee high-quality epidermal growth factor receptor (EGFR) gene mutation testing and reporting performance, the National Center for Clinical Laboratories launched a proficiency testing (PT) scheme reflecting clinical practices in China since 2014. This study focuses on the quality assessment of gene mutation reports. MATERIALS AND METHODS: Fifty-three laboratories that submitted reports in both 2014 and 2016 EGFR gene mutation PT schemes were selected for report analysis and comparison according to predefined evaluation criteria. RESULTS: The average score for reports from 2014 was 14 out of 30 points. The overall scores for reports from 2016 improved substantially, yielding an average score of 20 out of 30 points. Among the evaluation criteria, general items were well documented in the reports. However, items specific to molecular diagnosis were far from satisfactory, and some items were even missing. CONCLUSION: The quality assessment of clinical written reports from 2014 and 2016 demonstrates that substantial improvements have been made in overall reporting performance. However, not all statements pertaining to important elements met expectations. To continue education, repeated PT schemes need to be executed in a timely fashion to expose and address existing shortcomings in clinical reports. There remains ample room for improvement towards generating concise, comprehensive, and readable reports. IMPLICATIONS FOR PRACTICE: This article compares the quality of clinical gene mutation reports submitted in 2014 to those submitted in 2016 epidermal growth factor receptor proficiency testing schemes, exposes the existing shortcomings, and discusses ways to communicate results more effectively in the future. The findings demonstrate that notable progress was observed in the overall reporting performance. However, key points specific to molecular diagnosis were far from expectation, and some items were even missing. Standardization needs to be emphasized to improve the report format and content. This article provides a reference that laboratories can use to write concise, comprehensive, and readily accessible clinical reports.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Ensaio de Proficiência Laboratorial/normas , Patologia Molecular/normas , Carcinoma Pulmonar de Células não Pequenas/patologia , China , Receptores ErbB/genética , Feminino , Humanos , Mutação , Garantia da Qualidade dos Cuidados de SaúdeRESUMO
BACKGROUND: Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS: We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories. RESULTS: The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFR T790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2. CONCLUSIONS: The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.
Assuntos
Biomarcadores Tumorais/sangue , Biópsia/métodos , DNA de Neoplasias/sangue , Biomarcadores Tumorais/genética , Biópsia/normas , DNA de Neoplasias/genética , Variação Genética , Células HEK293 , Humanos , Variações Dependentes do ObservadorRESUMO
Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.
Assuntos
Neoplasias da Mama/genética , Ácidos Nucleicos Livres/genética , Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The early detection of type 1 diabetes (T1D) largely depends on a reliable approach to monitor ß cell loss. An effective way to evaluate the decline of ß cell mass would allow early preventative intervention to preserve insulin secretion. MAIN BODY: Recent progress in the development of novel biomarkers, based on tissue-specific methylation patterns, has inspired relevant studies in T1D. In this review, we focus on the application of circulating ß cell-derived unmethylated insulin (INS) DNA. Circulating ß cell-derived unmethylated INS DNA has a potential clinical value for the early detection of T1D, surveillance of islet transplantation rejection, and evaluation of response to therapy. Utilizing differentiated methylation patterns in different organs and employing a wide variety of molecular technologies also provide insights into the interrogation of biomarkers in other diseases with massive tissue-specific cell loss. CONCLUSION: Circulating unmethylated INS DNA is a promising molecular biomarker for the early detection of T1D.
Assuntos
Diabetes Mellitus Tipo 1/diagnóstico , Células Secretoras de Insulina/fisiologia , Insulina/genética , Biomarcadores/sangue , Morte Celular , Metilação de DNA , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Diagnóstico Precoce , Humanos , Insulina/sangue , Especificidade de ÓrgãosRESUMO
BACKGROUND: The emergence of Zika virus demands accurate laboratory diagnostics. Nucleic acid testing is currently the definitive method for diagnosis of Zika infection. In 2016, an external quality assurance (EQA) for assessing the quality of molecular testing of Zika virus was carried out in China. METHODS: A single armored RNA encapsulating a 4942-nucleotides (nt) long specific RNA sequence of Zika virus was prepared and used as positive samples. A pre-tested EQA panel, consisting of 4 negative and 6 positive samples with different concentrations of armored RNA, was distributed to 38 laboratories that perform molecular detection of Zika virus. RESULTS: A total of 39 data sets (1 laboratory used two test kits in parallel), produced by using commercial (n=38) or laboratory developed (n=1) quantitative reverse-transcriptase PCR (qRT-PCR) kits, were received. Of these, 35 (89.7%) had correct results for all 10 samples, and 4 (10.3%) reported at least 1 error (11 in total). The testing errors were all false-negatives, highlighting the need of improvements in detecting sensitivity. CONCLUSIONS: The EQA reveals that the majority of participating laboratories are proficient in molecular testing of Zika virus.
Assuntos
RNA Viral/normas , Infecção por Zika virus/diagnóstico , Zika virus/isolamento & purificação , China , Técnicas de Laboratório Clínico , Reações Falso-Negativas , Humanos , Variações Dependentes do Observador , Controle de Qualidade , RNA Viral/síntese química , Kit de Reagentes para Diagnóstico/normasRESUMO
Tamoxifen has been successfully used for treating breast cancer and preventing cancer recurrence. Cytochrome P450 2D6 (CYP2D6) plays a key role in the process of metabolizing tamoxifen to its active moiety, endoxifen. Patients with variants of the CYP2D6 gene may not receive the full benefit of tamoxifen treatment. The CYP2D6*10 variant (the most common variant in Asians) was analyzed to optimize the prescription of tamoxifen in China. To ensure referring clinicians have accurate information for genotype-guided tamoxifen treatment, the Chinese National Center for Clinical Laboratories (NCCL) organized a national proficiency testing (PT) to evaluate the performance of laboratories providing CYP2D6*10 genotyping. Ten genomic DNA samples with CYP2D6 wild-type or CYP2D6*10 variants were validated by PCR-sequencing and sent to 28 participant laboratories. The genotyping results and pharmacogenomic test reports were submitted and evaluated by NCCL experts. Additional information regarding the number of samples tested, the accreditation/certification status, and detecting technology was also requested. Thirty-one data sets were received, with a corresponding analytical sensitivity of 98.2% (548/558 challenges; 95% confidence interval: 96.7-99.1%) and an analytic specificity of 96.5% (675/682; 95% confidence interval: 97.9-99.5%). Overall, 25/28 participants correctly identified CYP2D6*10 status in 10 samples; however, two laboratories made serious genotyping errors. Most of the essential information was included in the 20 submitted CYP2D6*10 test reports. The majority of Chinese laboratories are reliable for detecting the CYP2D6*10 variant; however, several issues revealed in this study underline the importance of PT schemes in continued external assessment and provision of guidelines.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocromo P-450 CYP2D6/genética , Técnicas de Genotipagem , Ensaio de Proficiência Laboratorial , Tamoxifeno/uso terapêutico , Neoplasias da Mama/enzimologia , Linhagem Celular , Quimioterapia Adjuvante , China , Feminino , Humanos , Reprodutibilidade dos Testes , Relatório de PesquisaRESUMO
BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is as a target gene for trastuzumab in patients with breast cancer. Accurate determination of HER2 status and strict quality control are necessary to ensure reproducibility and accuracy of the techniques used for the determination of HER2 status. METHODS: We used three different types of samples: formalin-fixed and paraffin-embedded (FFPE) samples prepared from cell lines, agarose gel samples using cell lines, and xenograft tumor samples. One cell line for FFPE or xenografts did not overexpress HER2, while the others showed different levels of HER2 overexpression. We compared the morphology, HER2 gene amplification status, and HER2 protein expression status of these samples with those of clinical specimens. RESULTS: We successfully produced three kinds of samples for quality control. Cells from the cell line-sample sections were dispersed while those from the agarose gel-sample sections and xenograft tumor sample sections (prepared from the both cell lines) were concentrated in one area. The FISH results for all three kinds of samples were as expected. The IHC results of the cell line samples and xenograft tumor samples were as expected, but the staining level of the agarose gel samples, using HER2-overexpressed cell lines was weak which might be regarded as a false negative result. CONCLUSIONS: Xenograft tumor samples might be used as an additional option for quality control in FISH and IHC. However, it might not replace the clinical specimen quality controls directly.