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Peter E Barker

Publications and source records attributed to Peter E Barker.

18 recordsLinked to original sources
PubMed2006

Standards for plasma and serum proteomics in early cancer detection: a needs assessment report from the national institute of standards and technology--National Cancer Institute Standards, Methods, Assays, Reagents and Technologies Workshop, August 18-19, 2005.

NIST and the National Cancer Institute cosponsored a workshop on August 18-19, 2005, to examine needs for reference materials for early cancer detection. This meeting focused on standards, methods, assays, reagents, and technologies. Needs for plasma and serum proteomics, DNA methylation, and specimen reference collections were discussed, and recommendations from participants were solicited. This report summarizes the discussion and recommendations for proteomics reference materials.

Biomarkers, Tumor↗
PubMed2006

Quantum dots--a versatile tool in plant science?

An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system. In contrast, detection of QDs in in situ hybridisation of several plant chromosomes, using several high-copy sequences, was less sensitive than Alexa 488. Thus, semiconductor nanocrystal fluorophores are more suitable for immunostaining but not for in situ hybridisation of plant chromosomes.

Journal Article↗
PubMed2005

Preparation and characterization of candidate reference materials for telomerase assays.

BACKGROUND: Telomerase has been measured in body fluids of cancer patients, and clinical tests for telomerase may have utility as noninvasive, cost-effective methods for the early detection of cancer. However, telomerase activity measured by common methods such as the telomerase repeat amplification protocol (TRAP) and telomerase reverse transcriptase catalytic subunit (hTERT) mRNA by reverse transcription-PCR (RT-PCR) varies among laboratories. METHODS: We prepared a CHAPS buffer lysate from cultured A549 cells and stored it at -80 degrees C. Telomerase activity was measured by TRAP/PCR and real-time TRAP/PCR in conjunction with RT-PCR measurements of hTERT mRNA. Activity measured with use of the robot-assisted TRAP (RApidTRAP) multicapillary electrophoresis system was compared with single-capillary and slab-gel measurements in the range 10 to 10 000 cell equivalents. RESULTS: Preparations made after flash freezing and sonication of cells were approximately 3-fold more active. Although the slab-gel and capillary instruments detected telomerase activity, the multicapillary instrument was better suited for high-throughput studies. Measurements of telomerase by TRAP/real-time PCR and hTERT mRNA/RT-PCR yielded reproducible titrations in the range 10 to 10 000 cell equivalents (CVs, 1%-8% and 1%-3%, respectively). CONCLUSIONS: We have prepared and characterized a candidate reference material that appears to be suitable for use in a wide range of assays of telomerase activity and expression.

Biomarkers, Tumor↗
PubMed2005

Mitochondrial DNA as a cancer biomarker.

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Adult↗
PubMed2004

Inter-rater agreement on chromosome 5 breakage in FISH-based mutagen sensitivity assays (MSAs).

In chromosome breakage assays, validated, universal criteria for selection of cells and classification of chromosome aberrations may enhance their utility for cancer susceptibility screening. To standardize a fluorescence in situ hybridization (FISH) modification of the mutagen sensitivity assay (MSA), scoring criteria were evaluated by web-based validation. Two hundred digital FISH images were assigned random identification numbers. With this set of images, criteria for inclusion of cells and measurement of the frequency of abnormal cells were evaluated by eight observers, all of whom had five or more years of experience. Observers included doctoral and MS/BS level cytogeneticists, and were drawn from a randomized pool of 54 volunteers. Questions addressed were: (1) how uniformly were criteria applied to analysis of a standard digital FISH image set and (2) did concordance vary with educational level? These data suggest inter-rater agreement within a factor of 2 for average breakage frequency, but revealed greater variability in cell selection. These results aid in estimating the components of assay variance due to definitions, technical parameters and biological variables.

Chromosome Aberrations↗
PubMed2004

Semiconductor nanocrystal probes for human metaphase chromosomes.

To improve signal stability and quantitation, an optically stable, novel class of fluorophore for hybridization analysis of human metaphase chromosomes is demonstrated. Detection of hybridization sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide [(CdSe)ZnS]. Fluorescence of nanocrystal fluorophores was significantly brighter and more photostable than organic fluorophores Texas Red and fluorescein. Thus, semiconductor nanocrystal fluorophores offer a more stable and quantitative mode of fluorescence in situ hybridization (FISH) for research and clinical applications.

Biotinylation↗
PubMed2004

Standards requirements for systems biology approaches to health care: mitochondrial proteomics.

A review of the standards needs of the mitochondrial proteomics communities is presented based on the presentations and discussions at National Institute of Standards and Technology (NIST) workshop, Systems Biology Approaches to Health Care: Mitochondrial Proteomics, held on September 17-18, 2002. The mitochondrial proteomics areas addressed for standards needs are model systems, methods and data. This review outlines the challenges in the field, proposes standards efforts that the community would like to see pursued to meet those challenges, and is followed by a summary and NIST's planned efforts to address these standards requirements.

Journal Article↗
PubMed2004

Data standards for proteomics: mitochondrial two-dimensional polyacrylamide gel electrophoresis data as a model system.

Proteomics has emerged as a major discipline that led to a re-examination of the need for consensus and a nationally sanctioned set of proteomics technology standards. Such standards for databases and data reporting may be applied to two-dimensional polyacrylamide gel electrophoresis (2D PAGE) technology as a pilot project for assessing global and national needs in proteomics, and the role of the National Institute of Standards and Technology (NIST) and other similar standards and measurement organizations. The experience of harmonizing the heterogeneous data included in the Protein Data Bank (PDB) provides a paradigm for technology in an area where significant heterogeneity in technical detail and data storage has evolved. Here we propose an approach toward standardizing mitochondrial 2D PAGE data in support of a globally relevant proteomics consensus.

Journal Article↗
PubMed2004

Analytical validation of telomerase activity for cancer early detection: TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells.

Activation of telomerase plays a critical role in unlimited proliferation and immortalization of cells. Telomerase activity has been shown to correlate with tumor progression, indicating that tumors expressing this enzyme possess aggressive clinical behavior and that telomerase activity may be a useful biomarker for early detection of cancer. However, measurements of telomerase activity by current methods such as telomeric repeat amplification protocol (TRAP)/polymerase chain reaction (PCR) or antibody-based radioimmunoassay (RIA) are low-throughput and not robust enough to easily accommodate the required statistical analysis to determine whether telomerase activity is a practical biomarker. As part of the National Cancer Institute Early Detection Research Network of analytical validation, we have developed a robot assisted TRAP assay (RApidTRAP) of telomerase, a potential biomarker for cancer early detection. Measurements of human telomerase reverse transcriptase catalytic subunit (hTERT) mRNA were performed in concert with measurement of telomerase activity. For this purpose we determined hTERT mRNA concentration and telomerase activity in human normal (RPE-28) and cancer (A549) cell lines as well as in human serum (SRM 1951A). Telomerase activity measurements were made using the TRAP/PCR capillary electrophoresis (CE) method on (50 to 1000) cells/reaction isolated from cell extracts. Measurement of hTERT mRNA was made using specific primers and probes on a LightCycler in the range of (10 to 7000) cells/reaction. Comparison of high-throughput telomerase activity measurements using the robot and those performed manually were consistent in sensitivity and reproducibility. Using this combination of telomerase activity and hTERT mRNA measurements, the automated system improved efficiency over traditional TRAP/PCR methods.

Biomarkers↗
PubMed2004

Measurement reproducibility in the early stages of biomarker development.

Biomarker discovery and development requires measurement reproducibility studies in addition to case-control studies. Parallel pursuit of reproducibility studies is especially important for emerging technologies such as protein biomarkers based on time-of-flight mass spectrometry, the case considered in this paper. For parallel studies, a way to improve reproducibility prior to identification of protein species is necessary. One approach is use of functional principal components analysis (PCA) as the basis for assessing measurement reproducibility. Reproducibility studies involve repeated measurement of a reference material such as a human serum standard. Measurement in our example is by SELDI-TOF (surface-enhanced laser desorption and ionization time-of-flight) mass spectrometry. Reproducibility is defined in reference to a source of variation, which in our example is associated with a type of commercially available protein biochip. We obtained spectra for 8 spots on each 11 chips. Two spectra are generally more alike when obtained from the same chip rather than different chips. Thus, our experiment indicates potential improvements from reducing variation in chip manufacture and chip handling during measurement. Our analysis involves careful registration of the spectra and characterization of the spectral differences. As shown by our example, a metrological analysis may enhance case-control studies by guiding optimization of the measurements underlying the biomarker.

Biomarkers↗
PubMed2003

Cancer biomarker validation: standards and process: roles for the National Institute of Standards and Technology (NIST).

Rigorous validation of biomarkers for early detection of cancer differs at the National Institute of Standards and Technology (NIST) from similar processes common among research laboratories. As a newly discovered biomarker assay makes the transition from a research setting to the clinical diagnostic laboratory, it should progress through defined stages of assay confirmation. The first task of a validation laboratory is evaluation of research assay technology, performance, and specifications (analytical validation). However, the ultimate goal is initial validation of the test to identify early stage cancer (clinical validation). Upon technical and clinical confirmation, assays are moved systematically toward a standardized, reproducible, high-throughput format for clinical diagnostic implementation. With laboratory performance rigorously established, the clinical variables can subsequently be analyzed to define limitations, applications, and clinical utility. The role of NIST in technology evaluation for early cancer testing is described in the context of similar programs and prior experience at NIST. Here we conceptualize the validation steps of cancer test development and examine how NIST activities impact health care through institutional focus on measurement, technology, and standards development programs.

Biomarkers, Tumor↗
PubMed2002

NIST physical standards for DNA-based medical testing.

As DNA and RNA become major targets for clinical laboratory analysis, benchmark reagents will play an increasingly important role in standardization. Reliable national and international nucleic acid standards promote automation and third-party reimbursement for clinical testing. Furthermore, nucleic acid standards provide materials for quality assurance and quality control (QA/QC), and proficiency testing. Standard methods and training initially evolved from consensus guidelines endorsed by professional societies and governmental agencies. The National Institute of Standards and Technology (NIST), a nonregulatory agency of the U.S. Department of Commerce, develops and certifies physical and chemical standards in support of national commerce, manufacturing, and science. In its role supporting U.S. science and industry, the NIST responds to specific standards needs, most recently for medically and biologically important analytes. Broad-based consensus developed through interdisciplinary NIST workshops initiated development of NIST-certified DNA standards. Such materials serve the diagnostic community and help manufacturers benchmark a variety of DNA diagnostic testing platforms. Here we summarize the NIST experience and programs for development of national standards for DNA-based medical diagnostic testing.

Clinical Laboratory Techniques↗
PubMed2002

Cytonectin expression in Alzheimer disease.

Cytonectin is a novel 35,000 molecular weight protein that displays remarkable ion-independent adherence properties. This consigns it to a family of well-known adherence molecules essential for cell communication and the development of 3-dimensional tissue structures. Cytonectin is expressed in a variety of organs and tissues, being evolutionarily conserved from human to avian species. It is hypothesized to serve as a key structural component of the body, and as a "do not attack" signal molecule that prevents tissue destruction by cells of monocyte lineage. This paper describes the properties of cytonectin and its proposed role in normal and disease states. The protein is overexpressed in Alzheimer disease entorhinal cortex as compared to normal age-matched controls. It is also detected in tissues from patients with Down syndrome and leukemia. Its presence in all 3 of these related conditions may prove important to their etiopathogenesis.

Aged↗
PubMed2002

Nanotechnology: Emerging Developments and Early Detection of Cancer. A two-day workshop sponsored by the National Cancer Institute and the National Institute of Standards and Technology, August 30-31 2001, on the National Institute of Standards and Technology Campus, Gaithersburg, MD, USA.

A recent meeting jointly sponsored by the National Cancer Institute (NCI) and National Institute of Standards and Technology (NIST) brought together researchers active in nanotechnology and cancer molecular biology to discuss and evaluate the interface between disciplines. Emerging areas where nanotechnologies may impact cancer prevention and early cancer detection were elaborated by key researchers who catalyzed interdisciplinary dialogue aimed at fostering cross-discipline communications and future collaboration.

Biomarkers, Tumor↗
PubMedDate not supplied

Measurement of DNA biomarkers for the safety of tissue-engineered medical products, using artificial skin as a model.

To test the hypothesis that the process of tissue engineering introduces genetic damage to tissue-engineered medical products, we employed the use of five state-of-the-art measurement technologies to measure a series of DNA biomarkers in commercially available tissue-engineered skin as a model. DNA was extracted from the skin and compared with DNA from cultured human neonatal control cells (dermal fibroblasts and epidermal keratinocytes) and adult human fibroblasts from a 55-year-old donor and a 96-year-old donor. To determine whether tissue engineering caused oxidative DNA damage, gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry were used to measure six oxidatively modified DNA bases as biomarkers. Normal endogenous levels of the modified DNA biomarkers were not elevated in tissue-engineered skin when compared with control cells. Next, denaturing high-performance liquid chromatography and capillary electrophoresis-single strand conformation polymorphism were used to measure genetic mutations. Specifically, the TP53 tumor suppressor gene was screened for mutations, because it is the most commonly mutated gene in skin cancer. The tissue-engineered skin was found to be free of TP53 mutations at the level of sensitivity of these measurement technologies. Lastly, fluorescence in situ hybridization was employed to measure the loss of Y chromosome, which is associated with excessive cell passage and aging. Loss of Y chromosome was not detected in the tissue-engineered skin and cultured neonatal cells used as controls. In this study, we have demonstrated that tissue engineering (for TestSkin II) does not introduce genetic damage above the limits of detection of the state-of-the-art technologies used. This work explores the standard for measuring genetic damage that could be introduced during production of novel tissue-engineered products. More importantly, this exploratory work addresses technological considerations that need to be addressed in order to expedite accurate and useful international reference standards for the emerging tissue-engineering industry.

Biomarkers↗