External quality assurance of the carcinoembryonic antigen (CEA) assay: main findings in six years' experience.

In 1984 we initiated a national external quality assessment (EQA) program (supported by the Italian National Research Council, CNR) for the CEA assay; at present, about 200 Italian laboratories are participating in the program. The laboratories assayed the quality control (QC) samples according to their routine procedures and returned the results together with the name of the method/kit they used. The collected results were computer-processed and reports were sent back to the participants. A significant reduction of the CVt (mean between-laboratory agreement) of the CEA assay was observed throughout the EQA survey (from 35% in 1985 to 20-25% in the last cycles). In order to better clarify the differences in variability observed in the first QC cycles against the last ones, we used the ANOVA technique to evaluate the components of variability. The improvement in between-laboratory agreement was mainly due to the reduction of the between-kit component (from 30.5% to 15.2%), rather than to the smaller decrease observed for the within-kit variability (from 18.4% to 14.0%). The results reported for QC samples from different materials showed differences in the between-lab variability and substantial changes of the kit biases, thus suggesting a different specificity of the antibodies used in the various method/kits against different families of CEA molecules. Considerable uncertainty was also encountered in the clinical classification of low pathological samples, which seems mainly due to the variability in cut-off values used by the laboratories for the clinical assessment of the same analytical results. Our data indicate a progressive increase in the reliability of CEA determination during our study and confirm that EQA has improved the reliability of analysis carried out by the participating laboratories, thus stimulating the kit manufacturers to provide more reliable products.     

AIAP: A Quality Control and Integrative Analysis Package to Improve ATAC-seq Data Analysis

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis. Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data; however, key quality control (QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics for ATAC-seq data, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package (AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity (20%–60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and it can be executed by one command line to generate a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed qATACViewer for the user-friendly interaction with the QC report. The software, source code, and documentation of AIAP are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.     

Estimation of the Optimal Statistical Quality Control Sampling Time Intervals Using a Residual Risk Measure

Background

An open problem in clinical chemistry is the estimation of the optimal sampling time intervals for the application of statistical quality control (QC) procedures that are based on the measurement of control materials. This is a probabilistic risk assessment problem that requires reliability analysis of the analytical system, and the estimation of the risk caused by the measurement error.

Methodology/Principal Findings

Assuming that the states of the analytical system are the reliability state, the maintenance state, the critical-failure modes and their combinations, we can define risk functions based on the mean time of the states, their measurement error and the medically acceptable measurement error. Consequently, a residual risk measure rr can be defined for each sampling time interval. The rr depends on the state probability vectors of the analytical system, the state transition probability matrices before and after each application of the QC procedure and the state mean time matrices. As optimal sampling time intervals can be defined those minimizing a QC related cost measure while the rr is acceptable. I developed an algorithm that estimates the rr for any QC sampling time interval of a QC procedure applied to analytical systems with an arbitrary number of critical-failure modes, assuming any failure time and measurement error probability density function for each mode. Furthermore, given the acceptable rr, it can estimate the optimal QC sampling time intervals.

Conclusions/Significance

It is possible to rationally estimate the optimal QC sampling time intervals of an analytical system to sustain an acceptable residual risk with the minimum QC related cost. For the optimization the reliability analysis of the analytical system and the risk analysis of the measurement error are needed.     

Multicenter cell processing for cardiovascular regenerative medicine applications: the Cardiovascular Cell Therapy Research Network (CCTRN) experience

Background aimsMulticenter cellular therapy clinical trials require the establishment and implementation of standardized cell-processing protocols and associated quality control (QC) mechanisms. The aims here were to develop such an infrastructure in support of the Cardiovascular Cell Therapy Research Network (CCTRN) and to report on the results of processing for the first 60 patients.MethodsStandardized cell preparations, consisting of autologous bone marrow (BM) mononuclear cells, prepared using a Sepax device, were manufactured at each of the five processing facilities that supported the clinical treatment centers. Processing staff underwent centralized training that included proficiency evaluation. Quality was subsequently monitored by a central QC program that included product evaluation by the CCTRN biorepositories.ResultsData from the first 60 procedures demonstrated that uniform products, that met all release criteria, could be manufactured at all five sites within 7 h of receipt of BM. Uniformity was facilitated by use of automated systems (the Sepax for processing and the Endosafe device for endotoxin testing), standardized procedures and centralized QC.ConclusionsComplex multicenter cell therapy and regenerative medicine protocols can, where necessary, successfully utilize local processing facilities once an effective infrastructure is in place to provide training and QC.     

A two-stage, multilevel quality control system for serological assays in anthrax vaccine clinical trials

A two-stage, multilevel assay quality control (QC) system was designed and implemented for two high stringency QC anthrax serological assays; a quantitative anti-PA IgG enzyme-linked immunosorbent assay (ELISA) and an anthrax lethal toxin neutralization activity (TNA) assay. The QC system and the assays were applied for the congressionally mandated Centers for Disease Control and Prevention (CDC) Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax). A total of 57,284 human serum samples were evaluated by anti-PA enzyme-linked immunosorbent assay (ELISA) and 11,685 samples by anthrax lethal toxin neutralization activity (TNA) assay. The QC system demonstrated overall sample acceptance rates of 86% for ELISA and 90% for the TNA assays respectively. Monitoring of multiple assay and test sample variables showed no significant long term trends or degradation in any of the critical assay reagents or reportable values for both assays. Assay quality control data establish the functionality of the quality control system and demonstrates the reliability of the serological data generated using these assays.     

Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K₂EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2^nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.     

Standards for Immunohistochemical Imaging: A Protein Reference Device for Biomarker Quantitation

We are developing a reference device to be used in the validation of immunohistochemical imaging of biomarkers by microscopy. The prototype device consists of p53 protein immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with assays that incorporate commercially available anti-p53 antibodies. p53 protein was characterized by mass spectrometry and covalently immobilized through amide linkage to the (3-aminopropyl)trietoxysilane-modified glass surface. This procedure is reproducible and provides a chemically stable product in high yield. The surface-bound protein was shown to be immunoreactive by its specific interaction with anti-p53 antibody (Ab) and detection by absorbance and fluorescence spectroscopy. Also, comparison was made with microscopic images of Ab-stained tissue samples, known to stain positive for p53. Further development will be required to establish accurate surface protein concentrations in the range required for specific clinical applications. (J Histochem Cytochem 58:1005–1014, 2010)     

A 3-year experience of quality control and quality assurance in the multisite delivery of a lymphocyte-based cellular therapy for renal cell carcinoma

Autolymphocyte therapy (ALT) is outpatient-based adoptive immunotherapy using ex vivo-activated memory T-cells. To support the safe and reproducible delivery of ALT at three cell processing facilities (Boston, MA; Atlanta, GA; Orange, CA) we created a comprehensive quality assurance/quality control program compliant with recent FDA guidance relevant to activated lymphocytes and somatic cell therapies. Each facility performed extensive QC testing to ensure sterility, viability, and proper cell yield. Additonally, several QC tests were performed at Cellocr's centralized reference laboratory to monitor cell potency and identity of the ex vivo-processed lymphocytes. We report here the successful implementation of this QA/QC program for ALT which has resulted in the safe preparation and delivery of cell infusion products amounting to over 3600 treatments at seven clinical sites nationwide. We believe this program will serve as a model for other cellular therapies.     

Alignment of practices for data harmonization across multi-center cell therapy trials: a report from the Consortium for Pediatric Cellular Immunotherapy

Immune effector cell (IEC) therapies have revolutionized our approach to relapsed B-cell malignancies, and interest in the investigational use of IECs is rapidly expanding into other diseases. Current challenges in the analysis of IEC therapies include small sample sizes, limited access to clinical trials and a paucity of predictive biomarkers of efficacy and toxicity associated with IEC therapies. Retrospective and prospective multi-center cell therapy trials can assist in overcoming these barriers through harmonization of clinical endpoints and correlative assays for immune monitoring, allowing additional cross-trial analysis to identify biomarkers of failure and success. The Consortium for Pediatric Cellular Immunotherapy (CPCI) offers a unique platform to address the aforementioned challenges by delivering cutting-edge cell and gene therapies for children through multi-center clinical trials. Here the authors discuss some of the important pre-analytic variables, such as biospecimen collection and initial processing procedures, that affect biomarker assays commonly used in IEC trials across participating CPCI sites. The authors review the recent literature and provide data to support recommendations for alignment and standardization of practices that can affect flow cytometry assays measuring immune effector function as well as interpretation of cytokine/chemokine data. The authors also identify critical gaps that often make parallel comparisons between trials difficult or impossible.     

Quality assurance and quality control processes: summary of a metabolomics community questionnaire

Introduction

The Metabolomics Society Data Quality Task Group (DQTG) developed a questionnaire regarding quality assurance (QA) and quality control (QC) to provide baseline information about current QA and QC practices applied in the international metabolomics community.

Objectives

The DQTG has a long-term goal of promoting robust QA and QC in the metabolomics community through increased awareness via communication, outreach and education, and through the promotion of best working practices. An assessment of current QA and QC practices will serve as a foundation for future activities and development of appropriate guidelines.

Method

QA was defined as the set of procedures that are performed in advance of analysis of samples and that are used to improve data quality. QC was defined as the set of activities that a laboratory does during or immediately after analysis that are applied to demonstrate the quality of project data. A questionnaire was developed that included 70 questions covering demographic information, QA approaches and QC approaches and allowed all respondents to answer a subset or all of the questions.

Result

The DQTG questionnaire received 97 individual responses from 84 institutions in all fields of metabolomics covering NMR, LC-MS, GC-MS, and other analytical technologies.

Conclusion

There was a vast range of responses concerning the use of QA and QC approaches that indicated the limited availability of suitable training, lack of Standard Operating Procedures (SOPs) to review and make decisions on quality, and limited use of standard reference materials (SRMs) as QC materials. The DQTG QA/QC questionnaire has for the first time demonstrated that QA and QC usage is not uniform across metabolomics laboratories. Here we present recommendations on how to address the issues concerning QA and QC measurements and reporting in metabolomics.

     

A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays

RNA interference (RNAi) high-throughput screening (HTS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high-quality RNAi HTS assays. To obtain high-quality HTS assays, there is a strong need for an easily interpretable and theoretically based quality control (QC) metric. Signal-to-noise ratio (S/N), signal-to-background ratio (S/B), and Z-factor have been adopted as QC metrics in HTS assays. In this paper, I proposed a pair of new parameters, strictly standardized mean difference (SSMD) and coefficient of variability in difference (CVD), as QC metrics in RNAi HTS assays. Compared to S/B and S/N, SSMD and CVD capture the variabilities in both compared populations. Compared to Z-factor, SSMD and CVD have a clear probability interpretation and a solid statistical basis. Accordingly, the cutoff criteria of using SSMD or CVD as a QC metric in HTS assays are fully theoretically based. In addition, I discuss the relationship between the SSMD-based criterion and the popular Z-factor-based criterion and elucidate why p-value from t-test of testing mean difference fails to serve as a QC metric.     

The Italian External Quality Assessment scheme for fragile x syndrome: the results of a 5-year survey

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.     

Five-year experience of quality control for a 3D LSO-based whole-body PET scanner: Results and considerations

PET scanners require routine monitoring and quality control (QC) to ensure proper scanner performance. QC helps to ensure that PET equipment performs as specified by the manufacturer and that there have not been significant changes in the system response since acceptance. In this work we describe the maintenance history and we report on the results obtained from the PET system QC testing program over 5 years at two centers, both utilizing a Siemens Biograph 16 HiRez PET/CT system. QC testing programs were based on international standards and included the manufacturer’s daily QC, monthly uniformity and sensitivity, quarterly cross-calibration and annual resolution and image quality.For the Winnipeg and Novara sites, two and one PET detector blocks have been replaced, respectively. Neither system has had other significant PET system related hardware replacements. The manufacturer’s suggested daily QC was sensitive to detecting problems in the function of PET detector elements. The same test was not sensitive for detecting long term drifts in the systems: the Novara system observed a significant deterioration over five years of testing in the sensitivity which exhibited a decrease of 16% as compared to its initial value measured at system installation. The measure of the energy spectrum, showed that the 511 keV photopeak had shifted to a position of 468 keV. This shift was corrected by having service personnel perform a complete system calibration and detector block setup.We recommend including tests of system energy response and of sensitivity as part of a QC program since they can provide useful information on the actual performance of the scanner. A modification of the daily QC test by the manufacturer is suggested to monitor the long term stability of the system. Image quality and spatial resolution tests have proven to be of limited value for monitoring the system over time.     

Quality control issues in point of care testing

* Quality Control (QC) in Point of Care Testing (PoCT) is often thought of as a complex issue; however intelligent system analysis can simplify matters and greatly increase the chances of a well controlled system. What we want to achieve is a QC program which adequately controls the PoCT system, but does not excessively contribute to the operating costs or complexity of maintaining a PoCT instrument, or network of instruments. * Don't neglect effective pre-analytical work: good documentation, operator training, monitoring, and analyser maintenance programs are essential, as for any analyser. * Look closely at your analyser: Is it a "laboratory type" instrument or cartridge or strip based? Can it perform multiple test types or a single test only? How is it calibrated? Does it have built in self-check capabilities or an electronic check cartridge? Is the sample in contact with the instrument? What are the cartridge/strip/reagent storage requirements? * Establish where the analysis is taking place and which system component is involved. * Tailor your QC program to target this component, but still check the system as a whole. * A common approach is to check cartridges/strips on delivery and run a QA sample at least monthly to check storage conditions and operator performance. If there is no independent electronic instrument check, daily QC checks are also recommended. * Don't be afraid to stray beyond conventional QC models if necessary. Some PoCT systems are not adequately controlled by the application of conventional QC alone.     

Meta-QC-Chain: Comprehensive and Fast Quality Control Method for Metagenomic Data

Next-generation sequencing(NGS) technology has revolutionized and significantly impacted metagenomic research.However,the NGS data usually contains sequencing artifacts such as low-quality reads and contaminating reads,which will significantly compromise downstream analysis.Many quality control(QC) tools have been proposed,however,few of them have been verified to be suitable or efficient for metagenomic data,which are composed of multiple genomes and are more complex than other kinds of NGS data.Here we present a metagenomic data QC method named Meta-QC-Chain.Meta-QC-Chain combines multiple QC functions:technical tests describe input data status and identify potential errors,quality trimming filters poor sequencing-quality bases and reads,and contamination screening identifies higher eukaryotic species,which are considered as contamination for metagenomic data.Most computing processes are optimized based on parallel programming.Testing on an 8-GB real dataset showed that Meta-QC-Chain trimmed low sequencing-quality reads and contaminating reads,and the whole quality control procedure was completed within 20 min.Therefore,Meta-QC-Chain provides a comprehensive,useful and high-performance QC tool for metagenomic data.Meta-QC-Chain is publicly available for free at:http://computationalbioenergy.org/meta-qc-chain.html.     

High-throughput sample preparation procedures for the quantitation of a new bone integrin alpha(nu)beta(3) antagonist in human plasma and urine using liquid chromatography-tandem mass spectrometry

High throughput LC-MS/MS assays to quantitate a new alpha(nu)beta(3) bone integrin antagonist (I) in human plasma and urine have been developed using instruments programmed to automate sample preparation procedures. Packard liquid handling system-MultiPROBE II EX was programmed for preparing calibration standards in control plasma and urine, acidifying all standards, quality control (QC), and clinical samples with necessary dilutions, and adding the internal standard to the acidified samples. TOMTEC Quadra 96 was programmed to perform the solid phase extraction (SPE) process on a 3M 96-well mixed phase cation standard density (MPC-SD) plate to isolate the analytes from the sample matrix. The extract collected from both types of matrices was directly injected into reversed-phase LC-MS/MS system with a Turbo Ion Spray (TIS) interface in the positive ionization mode. The plasma and urine assays have the calibration range of 0.5-1500 and 2-6000 ng/mL, respectively. Validation of the automated and the manual plasma assays showed that application of MultiPROBE II to sample preparation gave comparable accuracy and precision. Overall, the automated approaches with minimum manual intervention enhanced the throughput of sample preparation.     

Tissue microarray study for classification of breast tumors

Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erbB2 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors.     

Detection of an East European wolf haplotype puzzles mitochondrial DNA monomorphism of the Italian wolf population

Southern European wolves suffered from reiterated population declines during glacial periods and historically due to human persecution. Differently from other European wolf populations, a single mitochondrial DNA (mtDNA) control region haplotype (W14) has been so far described in the Italian wolves, although no intensive genetic sampling has ever been conducted in historical source populations from central and southern Italy. Using non-invasive genetic techniques, we report the occurrence of an unexpected mtDNA haplotype (W16) in the wolf population of the Abruzzo, Lazio and Molise National Park (PNALM), central Italy. This haplotype, detected in three out of 90 faecal samples from the PNALM, was previously reported in wolves from the North Carpathians, Slovakia and the Balkans only. Microsatellite analysis and molecular sex determination confirmed that the W16 samples belonged to three distinct wolves. Although alternative explanations can be formulated for the origin of this mtDNA haplotype in the otherwise monomorphic Italian wolf population, assignment procedures indicated the likely admixed ancestry of one W16 sample with East European wolves. Anthropogenic introgression with dogs has been detected in the Italian wolf population using nuclear DNA microsatellites, but no population-wide genetic survey had previously reported a mtDNA control region variant in Italian wolves. Our findings strongly suggest that, in addition to wolf × dog hybridization, captive-released wolves or wolf × dog hybrids may successfully interbreed with wolves in the wild, and that human-mediated introgression may occur even in well established protected areas.