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Determination of somatic mutations in human erythrocytes by cytometry

Description: Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab.
Date: June 21, 1985
Creator: Jensen, R.H.; Langlois, R.G. & Bigbee, W.L.
Partner: UNT Libraries Government Documents Department

Somatic cell genotoxicity at the glycophorin A locus in humans

Description: We have developed an assay for detecting variant erythrocytes that occur as a result of in vivo allele loss at the glycophorin A (GPA) locus on chromosome 4 in humans. This gene codes for an erythroid- specific cell surface glycoprotein, and with our assay we are able to detect rare variant erythrocytes that have lost expression of one of the two GPA alleles. Two distinctly different variant cell types are detected with this assay. One variant cell type (called N{O}) is hemizygous. Our assay also detects homozygous variant erythrocytes that have lost expression of the GPA(M) allele and express the GPA(N) allele at twice the heterozygous level. The results of this assay are an enumeration of the frequency of N{O} and NN variant cell types for each individual analyzed. These variant cell frequencies provide a measure of the amount of somatic cell genotoxicity that has occurred at the GPA locus. Such genotoxicity could be the result of (1) reactions of toxic chemicals to which the individual has been exposed, or (2) high energy radiation effects on erythroid precursor cells, or (3) errors in DNA replication or repair in these cells of the bone marrow. Thus, the GPA-based variant cell frequency can serve as a biodosimeter that indicates the amount of genotoxic exposure each individual has received. Because two very different kinds of variant cells are enumerated, different kinds of genotoxicity should be distinguishable. Results of the GPA somatic genotoxicity assay may also provide valuable information for cancer-risk estimation on each individual. 16 refs.
Date: December 28, 1990
Creator: Jensen, R.H.; Grant, S.G.; Langlois, R.G. & Bigbee, W.L.
Partner: UNT Libraries Government Documents Department

The glycophorin A assay for somatic cell mutations in humans

Description: In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs.
Date: August 18, 1989
Creator: Langlois, R.G.; Bigbee, W.L. & Jensen, R.H.
Partner: UNT Libraries Government Documents Department

Pre-symptomatic Prediction of Illness in Mice Inoculated with Cowpox

Description: We describe here research directed towards early (presyndromic) diagnosis of infection. By using a mouse model and a multi-component blood protein diagnostic tool we detected cowpox infection several days in advance of overt symptoms with high accuracy. We provide details of the experimental design and measurement technique and elaborate on the long-range implication of these results.
Date: April 19, 2007
Creator: Kercher, J R; Colston, Jr., B W; Langlois, R G; Lyons, C R & Milanovich, F P
Partner: UNT Libraries Government Documents Department

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

Description: Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.
Date: May 5, 2000
Creator: Venkateswaran, K.S.; Nasarabadi, S. & Langlois, R.G.
Partner: UNT Libraries Government Documents Department

Pathomics: Final Report

Description: Pathomics is a research project to explore the feasibility for developing biosignatures for early infectious disease detection in humans, particularly those that represent a threat from bioterrorism. Our goal is to use a science-based approach to better understand the underlying molecular basis of disease and to find sensitive, robust, and specific combinations of biological molecules (biosignatures) in the host that will indicate the presence of developing infection prior to overt symptoms (pre-syndromic). The ultimate goal is develop a national surveillance system for monitoring for the release and managing the consequences of a biothreat agent or an emerging disease. Developing the science for a more comprehensive understanding of the molecular basis of infectious disease and the development of biosignature-based diagnostics could help detect both emerging and engineered treats to humans.
Date: December 8, 2006
Creator: Turteltaub, K W; Ascher, M; Langlois, R; Fodor, I; Kercher, J; Laughlin, K M et al.
Partner: UNT Libraries Government Documents Department

Autonomous system for pathogen detection and identification

Description: This purpose of this project is to build a prototype instrument that will, running unattended, detect, identify, and quantify BW agents. In order to accomplish this, we have chosen to start with the world´┐Ż s leading, proven, assays for pathogens: surface-molecular recognition assays, such as antibody-based assays, implemented on a high-performance, identification (ID)-capable flow cytometer, and the polymerase chain reaction (PCR) for nucleic-acid based assays. With these assays, we must integrate the capability to: l collect samples from aerosols, water, or surfaces; l perform sample preparation prior to the assays; l incubate the prepared samples, if necessary, for a period of time; l transport the prepared, incubated samples to the assays; l perform the assays; l interpret and report the results of the assays. Issues such as reliability, sensitivity and accuracy, quantity of consumables, maintenance schedule, etc. must be addressed satisfactorily to the end user. The highest possible sensitivity and specificity of the assay must be combined with no false alarms. Today, we have assays that can, in under 30 minutes, detect and identify simulants for BW agents at concentrations of a few hundred colony-forming units per ml of solution. If the bio-aerosol sampler of this system collects 1000 Ymin and concentrates the respirable particles into 1 ml of solution with 70% processing efficiency over a period of 5 minutes, then this translates to a detection/ID capability of under 0.1 agent-containing particle/liter of air.
Date: September 24, 1998
Creator: Belgrader, P; Benett, W; Langlois, R; Long, G; Mariella, R; Milanovich, F et al.
Partner: UNT Libraries Government Documents Department

Laser-based blood cell measurements for the biological reconstruction of ionizing radiation doses using a novel technique for cytometry/biochemical reaction analysis

Description: A novel biodosimetry method based on the red blood cells analysis by means of laser flow cytometry and a specially designed monoclonal antibodies is used for reconstructing ionizing radiation doses for Chernobyl Accident victims. In addition to analysis of the patient`s blood samples, we are evaluating both spectroscopy procedures and laser-based flow techniques. It was shown that resonance energy transfer processes can take place among the two different dyes links to the spherical cell surface. This can affect the accuracy of rare mutant cell scoring. Also a new technique is described to provide conventional flow cytometric analysis and kinetic measurements of the elementary stages of biochemical reactions are described.
Date: January 1, 1994
Creator: Pleshanov, P. G.; Langlois, R. G.; Azarov, L. A.; Budayev, D. A.; Ossipenkov, A. L. & Shamayev, D. M.
Partner: UNT Libraries Government Documents Department

Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

Description: The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.
Date: April 20, 2001
Creator: Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R. et al.
Partner: UNT Libraries Government Documents Department

Laser-based flow cytometric analysis of genotoxicity of humans exposed to ionizing radiation during the Chernobyl accident

Description: An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accident at Chernobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ionizing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure. 17 refs., 5 figs.
Date: September 12, 1990
Creator: Jensen, R.H.; Bigbee, W.L.; Langlois, R.G.; Grant, S.G. (Lawrence Livermore National Lab., CA (USA)); Pleshanov, P.G. (Ministerstvo Zdravookhraneniya SSSR, Moscow (USSR)); Chirkov, A.A. (Institut Biofiziki, Moscow (USSR)) et al.
Partner: UNT Libraries Government Documents Department