WorldWideScience

Sample records for flow cytometer assay

  1. Hyperspectral imaging flow cytometer

    Science.gov (United States)

    Sinclair, Michael B.; Jones, Howland D. T.

    2017-10-25

    A hyperspectral imaging flow cytometer can acquire high-resolution hyperspectral images of particles, such as biological cells, flowing through a microfluidic system. The hyperspectral imaging flow cytometer can provide detailed spatial maps of multiple emitting species, cell morphology information, and state of health. An optimized system can image about 20 cells per second. The hyperspectral imaging flow cytometer enables many thousands of cells to be characterized in a single session.

  2. Phase-sensitive flow cytometer

    Energy Technology Data Exchange (ETDEWEB)

    Steinkamp, J.A.

    1992-12-31

    This report describes phase-sensitive flow cytometer (FCM) which provides additional FCM capability to use the fluorescence lifetime of one or more fluorochromes bound to single cells to provide additional information regarding the cells. The resulting fluorescence emission can be resolved into individual fluorescence signals if two fluorochromes are present or can be converted directly to a decay lifetime from a single fluorochrome. The excitation light for the fluorochromes is modulated to produce an amplitude modulated fluorescence pulse as the fluorochrome is excited in the FCM. The modulation signal also forms a reference signal that is phase-shifted a selected amount for subsequent mixing with the output modulated fluorescence intensity signal in phase-sensitive detection circuitry. The output from the phase-sensitive circuitry is then an individual resolved fluorochrome signal or a single fluorochrome decay lifetime, depending on the applied phase shifts.

  3. Microfluidic Multichannel Flow Cytometer, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed innovation is a "Microfluidic Multichannel Flow Cytometer." Several novel concepts are integrated to produce the final design, which is compatible with...

  4. White Blood Cell Differentiation Using a Solid State Flow Cytometer

    NARCIS (Netherlands)

    Doornbos, R.M.P.; Doornbos, R.M.P.; Hennink, E.J.; Putman, C.A.J.; Putman, C.A.J.; de Grooth, B.G.; Greve, Jan

    1993-01-01

    A flow cytometer using a solid state light source and detector was designed and built. For illumination of the sample stream two types of diode lasers (670 nm and 780 nm) were tested in a set-up designed to differentiate human leukocytes by means of light scattering. The detector is an avalanche

  5. Soluble Protein Analysis using a Compact Bench-top Flow Cytometer

    Science.gov (United States)

    Pappas, Dimitri; Kao, Shib-Hsin; Cyr, Johnathan

    2004-01-01

    Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health (blood cell count, leukocyte differential, etc.) and a wide array of biochemistry and immunology assays. research settings, the application of this technique to soluble protein analysis is also possible. Proteomic beads using fluorescent dyes for optical encoding were used to monitor six cytokines simultaneously in cell medium of cell cultures in stationary and rotating cell culture systems. The results of this work demonstrate that a compact flow cytometer, such as a system proposed for space flight, can detect a variety of soluble proteins for crew health and biotechnology experiments during long-term missions.

  6. Microchip Flow Cytometer with Integrated Polymer Optical Elements for Measurement of Scattered Light

    DEFF Research Database (Denmark)

    Wang, Zhenyu; El-Ali, Jamil; Perch-Nielsen, Ivan Ryberg

    2004-01-01

    channels to form a complete microchip flow cytometer. All the optical elements, the microfluidic system, and the fiber-to-waveguide couplers were defined in one layer of polymer (SU-8, negative photoresist) by standard photolithography. With only one single mask procedure, all the fabrication and packaging...... processes can be finished in one day. Polystyrene beads were measured in the microchip flow cytometer, and three signals (forward scattering, large angle scattering and extinction) were measured simultaneously for each bead. The average intensities of the forward Scattered light and the incident light...

  7. Reconfigurable acquisition system with integrated optics for a portable flow cytometer

    Energy Technology Data Exchange (ETDEWEB)

    Kirleis, Matthew A., E-mail: matthew.kirleis@nrl.navy.mil; Mathews, Scott A.; Verbarg, Jasenka; Erickson, Jeffrey S.; Piqué, Alberto [Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375 (United States)

    2013-11-15

    Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-color excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument.

  8. Reconfigurable acquisition system with integrated optics for a portable flow cytometer

    International Nuclear Information System (INIS)

    Kirleis, Matthew A.; Mathews, Scott A.; Verbarg, Jasenka; Erickson, Jeffrey S.; Piqué, Alberto

    2013-01-01

    Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-color excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument

  9. Reconfigurable acquisition system with integrated optics for a portable flow cytometer.

    Science.gov (United States)

    Kirleis, Matthew A; Mathews, Scott A; Verbarg, Jasenka; Erickson, Jeffrey S; Piqué, Alberto

    2013-11-01

    Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-color excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument.

  10. Evaluation and purchase of an analytical flow cytometer: some of the numerous factors to consider.

    Science.gov (United States)

    Zucker, Robert M; Fisher, Nancy C

    2013-01-01

    When purchasing a flow cytometer, the decision of which brand, model, specifications, and accessories may be challenging. The decisions should initially be guided by the specific applications intended for the instrument. However, many other factors need to be considered, which include hardware, software, quality assurance, support, service, and price and recommendations from colleagues. These issues are discussed to help guide the purchasing process.

  11. Parallel imaging microfluidic cytometer.

    Science.gov (United States)

    Ehrlich, Daniel J; McKenna, Brian K; Evans, James G; Belkina, Anna C; Denis, Gerald V; Sherr, David H; Cheung, Man Ching

    2011-01-01

    By adding an additional degree of freedom from multichannel flow, the parallel microfluidic cytometer (PMC) combines some of the best features of fluorescence-activated flow cytometry (FCM) and microscope-based high-content screening (HCS). The PMC (i) lends itself to fast processing of large numbers of samples, (ii) adds a 1D imaging capability for intracellular localization assays (HCS), (iii) has a high rare-cell sensitivity, and (iv) has an unusual capability for time-synchronized sampling. An inability to practically handle large sample numbers has restricted applications of conventional flow cytometers and microscopes in combinatorial cell assays, network biology, and drug discovery. The PMC promises to relieve a bottleneck in these previously constrained applications. The PMC may also be a powerful tool for finding rare primary cells in the clinic. The multichannel architecture of current PMC prototypes allows 384 unique samples for a cell-based screen to be read out in ∼6-10 min, about 30 times the speed of most current FCM systems. In 1D intracellular imaging, the PMC can obtain protein localization using HCS marker strategies at many times for the sample throughput of charge-coupled device (CCD)-based microscopes or CCD-based single-channel flow cytometers. The PMC also permits the signal integration time to be varied over a larger range than is practical in conventional flow cytometers. The signal-to-noise advantages are useful, for example, in counting rare positive cells in the most difficult early stages of genome-wide screening. We review the status of parallel microfluidic cytometry and discuss some of the directions the new technology may take. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Hansen, Ole; Petersen, Peter Kalsen

    2006-01-01

    Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring...... of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DER The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell...

  13. Design of portable ultraminiature flow cytometers for medical diagnostics

    Science.gov (United States)

    Leary, James F.

    2018-02-01

    Design of portable microfluidic flow/image cytometry devices for measurements in the field (e.g. initial medical diagnostics) requires careful design in terms of power requirements and weight to allow for realistic portability. True portability with high-throughput microfluidic systems also requires sampling systems without the need for sheath hydrodynamic focusing both to avoid the need for sheath fluid and to enable higher volumes of actual sample, rather than sheath/sample combinations. Weight/power requirements dictate use of super-bright LEDs with top-hat excitation beam architectures and very small silicon photodiodes or nanophotonic sensors that can both be powered by small batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses. Microfluidic cytometry also requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically in less than 15 minutes) initial medical decisions for patients in the field. This is not something conventional cytometry traditionally worries about, but is very important for development of small, portable microfluidic devices with small-volume throughputs. It also provides a more reasonable alternative to conventional tubes of blood when sampling geriatric and newborn patients for whom a conventional peripheral blood draw can be problematical. Instead one or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the doctor's office or field.

  14. High-speed cell recognition algorithm for ultrafast flow cytometer imaging system

    Science.gov (United States)

    Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

    2018-04-01

    An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform.

  15. Functional analysis and classification of phytoplankton based on data from an automated flow cytometer.

    Science.gov (United States)

    Malkassian, Anthony; Nerini, David; van Dijk, Mark A; Thyssen, Melilotus; Mante, Claude; Gregori, Gerald

    2011-04-01

    Analytical flow cytometry (FCM) is well suited for the analysis of phytoplankton communities in fresh and sea waters. The measurement of light scatter and autofluorescence properties of particles by FCM provides optical fingerprints, which enables different phytoplankton groups to be separated. A submersible version of the CytoSense flow cytometer (the CytoSub) has been designed for in situ autonomous sampling and analysis, making it possible to monitor phytoplankton at a short temporal scale and obtain accurate information about its dynamics. For data analysis, a manual clustering is usually performed a posteriori: data are displayed on histograms and scatterplots, and group discrimination is made by drawing and combining regions (gating). The purpose of this study is to provide greater objectivity in the data analysis by applying a nonmanual and consistent method to automatically discriminate clusters of particles. In other words, we seek for partitioning methods based on the optical fingerprints of each particle. As the CytoSense is able to record the full pulse shape for each variable, it quickly generates a large and complex dataset to analyze. The shape, length, and area of each curve were chosen as descriptors for the analysis. To test the developed method, numerical experiments were performed on simulated curves. Then, the method was applied and validated on phytoplankton cultures data. Promising results have been obtained with a mixture of various species whose optical fingerprints overlapped considerably and could not be accurately separated using manual gating. Copyright © 2011 International Society for Advancement of Cytometry.

  16. Phytoplankton cell counts from a moored submersible flow cytometer at Martha's Vineyard (Massachusetts) Coastal Observatory, May 10 - December 15, 2003 (NODC Accession 0002299)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Phytoplankton cell counts data were collected using a moored submersible flow cytometer from a Coastal Observatory at the Martha's Vineyard in Masschutsetts from 10...

  17. Phytoplankton cell counts from a moored submersible flow cytometer at Martha's Vineyard (Massachusetts) Coastal Observatory, May - December 2006 (NODC Accession 0036656)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Phytoplankton cell counts were collected from using a moored submersible flow cytometer from the Martha's Vineyard Coastal Observatory in the Northwest Atlantic...

  18. Phytoplankton cell counts from a moored submersible flow cytometer at Martha's Vineyard Coastal Observatory, Massachusetts, May - September 2004 (NODC Accession 0002722)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Phytoplankton cell counts were collected from using a moored submersible flow cytometer from the Martha's Vineyard Coastal Observatory in the Northwest Atlantic...

  19. Simplified Monolithic Flow Cytometer Chip With Three-Dimensional Hydrodyanmic Focusing And Integrated Fiber-Free Optics

    DEFF Research Database (Denmark)

    Motosuke, Masahiro; Jensen, Thomas Glasdam; Zhuang, Guisheng

    2011-01-01

    A miniaturized flow cytometry incorporating both fluidic and optical systems has a great possibility for portable biochemical sensing or point-of-care diagnostics. This paper presents a simple microfluidic flow cytometer combining reliable 3D hydrodynamic focusing and optical detection without...... optical fibers in a monolithic architecture fabricated by a single photolithographic process. The vertical flow focusing is achieved by the optimized inlet geometry in a PDMS lid onto the substrate with detection channel and integrated optics. The simplified approach indicates the possibility...

  20. A Raman Flow Cytometer: An Innovative Microfluidic Approach for Continuous Label-Free Analysis of Cells via Raman Spectroscopy

    KAUST Repository

    De Grazia, Antonio

    2015-05-05

    In this work a Raman flow cytometer is presented. It is a whole new microfluidic device that takes advantage of basic principles of Raman spectroscopy and fluorescent flow cytometry mixed together in a system of particularly shaped channels. These are indeed composed by specific shape and sizes – thanks to which cells can flow one-by-one – and a trap by means of which cells are trapped in order to perform Raman analysis on single ones in a constant and passive way. In this sense the microfluidic device promotes a fast method to look for single cells in a whole multicellular sample. It is a label-free analysis and this means that, on the contrary of what happens with fluorescent flow cytometry, the sample does not need to undergo any particular time-consuming pretreatment before being analyzed. Moreover it gives a complete information about the biochemical content of the sample thanks to the involvement of Raman spectroscopy as method of analysis. Many thought about a device like this, but eventually it is the first one being designed, fabricated and tested. The materials involved in the production of the Raman flow cytometer are chosen wisely. In particular the chip – the most important component of the device – is multilayered, being composed by a slide of calcium fluoride (which gives a negligible signal in Raman analyses), a photosensitive resist containing a pattern with channels and another slide of calcium fluoride in order for the channels to be sealed on both sides. The chip is, in turn, connected to gaskets and external frames. Several fabrication processes are followed to ultimately get the complete Raman flow cytometer and experiments on red blood cells demonstrate its validity in this field.

  1. Experimental and numerical investigation into micro-flow cytometer with 3-D hydrodynamic focusing effect and micro-weir structure.

    Science.gov (United States)

    Hou, Hui-Hsiung; Tsai, Chien-Hsiung; Fu, Lung-Ming; Yang, Ruey-Jen

    2009-07-01

    This study presents a novel 3-D hydrodynamic focusing technique for micro-flow cytometers. In the proposed approach, the sample stream is compressed initially in the horizontal direction by a set of sheath flows such that it is constrained to the central region of the microchannel and is then focused in the vertical direction by a second pair of sheath flows. Thereafter, the focused sample stream passes over a micro-weir structure positioned directly beneath an optical detection system to capture polystyrene beads fluorescent signal. The microchannel configuration and operational parameters are optimized by performing a series of numerical simulations. An experimental investigation is then performed using a micro-flow cytometer fabricated using conventional micro-electro-mechanical systems techniques and an isotropic wet etching method. The results indicate that the two sets of sheath flows successfully constrain the sample stream within a narrow, well-defined region of the microchannel. Furthermore, the micro-weir structure prompts the separation of a mixed sample of 5 and 10 microm polystyrene beads in the vertical direction and ensures that the beads flow through the detection region of the microchannel in a sequential fashion and can therefore be reliably detected and counted.

  2. COPASutils: an R package for reading, processing, and visualizing data from COPAS large-particle flow cytometers.

    Directory of Open Access Journals (Sweden)

    Tyler C Shimko

    Full Text Available The R package COPASutils provides a logical workflow for the reading, processing, and visualization of data obtained from the Union Biometrica Complex Object Parametric Analyzer and Sorter (COPAS or the BioSorter large-particle flow cytometers. Data obtained from these powerful experimental platforms can be unwieldy, leading to difficulties in the ability to process and visualize the data using existing tools. Researchers studying small organisms, such as Caenorhabditis elegans, Anopheles gambiae, and Danio rerio, and using these devices will benefit from this streamlined and extensible R package. COPASutils offers a powerful suite of functions for the rapid processing and analysis of large high-throughput screening data sets.

  3. Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.

    Science.gov (United States)

    Glier, Hana; Heijnen, Ingmar; Hauwel, Mathieu; Dirks, Jan; Quarroz, Stéphane; Lehmann, Thomas; Rovo, Alicia; Arn, Kornelius; Matthes, Thomas; Hogan, Cassandra; Keller, Peter; Dudkiewicz, Ewa; Stüssi, Georg; Fernandez, Paula

    2017-07-29

    The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria

  4. A novel all-fiber optic flow cytometer technology for Point-of Care and Remote Environments

    Science.gov (United States)

    Mermut, Ozzy

    Traditional flow cytometry designs tend to be bulky systems with a complex optical-fluidic sub-system and often require trained personnel for operation. This makes them difficult to readily translate to remote site testing applications. A new compact and portable fiber-optic flow cell (FOFC) technology has been developed at INO. We designed and engineered a specialty optical fiber through which a square hole is transversally bored by laser micromachining. A capillary is fitted into that hole to flow analyte within the fiber square cross-section for detection and counting. With demonstrated performance benchmarks potentially comparable to commercial flow cytometers, our FOFC provides several advantages compared to classic free-space con-figurations, e.g., sheathless flow, low cost, reduced number of optical components, no need for alignment (occurring in the fabrication process only), ease-of-use, miniaturization, portability, and robustness. This sheathless configuration, based on a fiber optic flow module, renders this cytometer amenable to space-grade microgravity environments. We present our recent results for an all-fiber approach to achieve a miniature FOFC to translate flow cytometry from bench to a portable, point-of-care device for deployment in remote settings. Our unique fiber approach provides the capability to illuminate a large surface with a uniform intensity distri-bution, independently of the initial shape originating from the light source, and without loss of optical power. The CVs and sensitivities are measured and compared to industry benchmarks. Finally, integration of LEDs enable several advantages in cost, compactness, and wavelength availability.

  5. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  6. Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.

    Science.gov (United States)

    Dubelaar, G B; Gerritzen, P L; Beeker, A E; Jonker, R R; Tangen, K

    1999-12-01

    The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring. Copyright 1999 Wiley-Liss, Inc.

  7. Seston Data from Flow Cytometers and Microscope Environmental Data from Sondes

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Seston data with phytoplankton and size fractioned non-living particles counted by flow cytomter from Penebscot River, Maine in April, May, and June of 2015. High...

  8. Miniaturized flow cytometer with 3D hydrodynamic particle focusing and integrated optical elements applying silicon photodiodes

    NARCIS (Netherlands)

    Rosenauer, M.; Buchegger, W.; Finoulst, I.; Verhaert, P.D.E.M.; Vellekoop, M.

    2010-01-01

    In this study, the design, realization and measurement results of a novel optofluidic system capable of performing absorbance-based flow cytometric analysis is presented. This miniaturized laboratory platform, fabricated using SU-8 on a silicon substrate, comprises integrated polymer-based

  9. Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

    Science.gov (United States)

    Bae, Candice; Sharpe, Julia Z.; Bishara, Andrew M.; Nelson, Emily S.; Weaver, Aaron S.; Brown, Daniel; McKay, Terri L.; Griffin, DeVon; Chan, Eugene Y.

    2014-01-01

    Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described. PMID:25490614

  10. Strategies for enumeration of circulating microvesicles on a conventional flow cytometer: Counting beads and scatter parameters.

    Science.gov (United States)

    Alkhatatbeh, Mohammad J; Enjeti, Anoop K; Baqar, Sara; Ekinci, Elif I; Liu, Dorothy; Thorne, Rick F; Lincz, Lisa F

    2018-01-01

    Enumeration of circulating microvesicles (MVs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: MV/μl = (MV count/bead count) × final bead concentration. We sought to optimize each variable in the equation by determining the best parameters for detecting 'MV count' and examining the effects of different bead preparations and concentrations on the final calculation. Three commercially available bead preparations (TruCount, Flow-Count and CountBright) were tested, and MV detection on a BD FACSCanto was optimized for gating by either forward scatter (FSC) or side scatter (SSC); the results were compared by calculating different subsets of MV on a series of 74 typical patient plasma samples. The relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations ( R 2 ≥ 0.997). However, TruCount beads produced the most consistent (concentration variation = 3.8%) calculated numbers of plasma CD41 + /Annexin V + MV, which were significantly higher from that calculated using either Flow-Count or CountBright ( p beads by FSC and 0.16 μm beads by SSC, but there were significantly more background events using SSC compared with FSC (3113 vs. 470; p = 0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV ( p beads provided linear results at concentrations ranging from 6 beads/μl to 100 beads/μl, but TruCount was the most consistent. Using SSC to gate MV events produced high background which negatively affected counting bead enumeration and overall MV calculations. Strategies to reduce SSC background should be employed in order to reliably use this technique.

  11. Improved Diffuse Fluorescence Flow Cytometer Prototype for High Sensitivity Detection of Rare Circulating Cells In Vivo

    Science.gov (United States)

    Pestana, Noah Benjamin

    Accurate quantification of circulating cell populations is important in many areas of pre-clinical and clinical biomedical research, for example, in the study of cancer metastasis or the immune response following tissue and organ transplants. Normally this is done "ex-vivo" by drawing and purifying a small volume of blood and then analyzing it with flow cytometry, hemocytometry or microfludic devices, but the sensitivity of these techniques are poor and the process of handling samples has been shown to affect cell viability and behavior. More recently "in vivo flow cytometry" (IVFC) techniques have been developed where fluorescently-labeled cells flowing in a small blood vessel in the ear or retina are analyzed, but the sensitivity is generally poor due to the small sampling volume. To address this, our group recently developed a method known as "Diffuse Fluorescence Flow Cytometry" (DFFC) that allows detection and counting of rare circulating cells with diffuse photons, offering extremely high single cell counting sensitivity. In this thesis, an improved DFFC prototype was designed and validated. The chief improvements were three-fold, i) improved optical collection efficiency, ii) improved detection electronics, and iii) development of a method to mitigate motion artifacts during in vivo measurements. In combination, these improvements yielded an overall instrument detection sensitivity better than 1 cell/mL in vivo, which is the most sensitive IVFC system reported to date. Second, development and validation of a low-cost microfluidic device reader for analysis of ocular fluids is described. We demonstrate that this device has equivalent or better sensitivity and accuracy compared a fluorescence microscope, but at an order-of-magnitude reduced cost with simplified operation. Future improvements to both instruments are also discussed.

  12. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    International Nuclear Information System (INIS)

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer

  13. Determination of blood leukocyte concentration with constant volume acquisition on a flow cytometer is comparable to individualized single platform testing with beads as internal reference standard

    DEFF Research Database (Denmark)

    Hansen, Susan; Dahl, Ronald; Hoffmann, Hans Jürgen

    2008-01-01

    at a high rate has a median of 163 microl (IQR 156-170) with TruCount tubes. Leukocyte concentrations of 26 healthy volunteers were measured twice on up to four occasions with a Bürker-Türk chamber, by single platform technology (SPT) with TruCount tubes and on the same data set using CVA. Total leukocyte...... concentrations determined by CVA correlated better with measurements in a Bürker-Türk (BT) chamber than with SPT. Concentrations determined with CVA were 1.86% higher than with BT whereas SPT data were 5.35% higher than BT (p...LeukoGating can be established using microscopy as a reference, and is comparable to BT chamber and SPT determination. Leukocyte concentrations can be measured with CVA on flow cytometers in research and clinical settings....

  14. Portable dual field gradient force multichannel flow cytometer device with a dual wavelength low noise detection scheme

    Science.gov (United States)

    James, Conrad D; Galambos, Paul C; Derzon, Mark S; Graf, Darin C; Pohl, Kenneth R; Bourdon, Chris J

    2012-10-23

    Systems and methods for combining dielectrophoresis, magnetic forces, and hydrodynamic forces to manipulate particles in channels formed on top of an electrode substrate are discussed. A magnet placed in contact under the electrode substrate while particles are flowing within the channel above the electrode substrate allows these three forces to be balanced when the system is in operation. An optical detection scheme using near-confocal microscopy for simultaneously detecting two wavelengths of light emitted from the flowing particles is also discussed.

  15. Automated urinalysis: first experiences and a comparison between the Iris iQ200 urine microscopy system, the Sysmex UF-100 flow cytometer and manual microscopic particle counting.

    Science.gov (United States)

    Shayanfar, Noushin; Tobler, Ulrich; von Eckardstein, Arnold; Bestmann, Lukas

    2007-01-01

    Automated analysis of insoluble urine components can reduce the workload of conventional microscopic examination of urine sediment and is possibly helpful for standardization. We compared the diagnostic performance of two automated urine sediment analyzers and combined dipstick/automated urine analysis with that of the traditional dipstick/microscopy algorithm. A total of 332 specimens were collected and analyzed for insoluble urine components by microscopy and automated analyzers, namely the Iris iQ200 (Iris Diagnostics) and the UF-100 flow cytometer (Sysmex). The coefficients of variation for day-to-day quality control of the iQ200 and UF-100 analyzers were 6.5% and 5.5%, respectively, for red blood cells. We reached accuracy ranging from 68% (bacteria) to 97% (yeast) for the iQ200 and from 42% (bacteria) to 93% (yeast) for the UF-100. The combination of dipstick and automated urine sediment analysis increased the sensitivity of screening to approximately 98%. We conclude that automated urine sediment analysis is sufficiently precise and improves the workflow in a routine laboratory. In addition, it allows sediment analysis of all urine samples and thereby helps to detect pathological samples that would have been missed in the conventional two-step procedure according to the European guidelines. Although it is not a substitute for microscopic sediment examination, it can, when combined with dipstick testing, reduce the number of specimens submitted to microscopy. Visual microscopy is still required for some samples, namely, dysmorphic erythrocytes, yeasts, Trichomonas, oval fat bodies, differentiation of casts and certain crystals.

  16. Quantitative evaluation of radiation dose by γ-H2AX on a microfluidic chip in a miniature fluorescence cytometer

    International Nuclear Information System (INIS)

    Wang, Junsheng; Song, Wendong; Song, Yongxin; Xu, Dan; Zhang, Min; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2014-01-01

    Evaluation of radiation dose is very important for the detection of radiation damage. γ-H2AX is a popular biological dosimeter to evaluate the radiation effect. Typically, bulky and expensive commercial flow cytometers are used to detect γ-H2AX. This paper presents a miniaturized and high sensitive cytometer using a microfluidic chip for evaluating the radiation dose by detecting the mean immunofluorescence intensity of γ-H2AX. A compact optical focusing system and a shift-phase differential amplifier are designed to improve the detection sensitivity. Sample lymphocyte cells are stained by FITC fluorescent dye after being irradiated by UVC. Comparison experiments between the developed miniature cytometer and a commercial flow cytometer were conducted under different radiation doses. The developed microfluidic cytometer also demonstrates a good linear correlation between the measured fluorescence intensity and the irradiation dose with a detection limit similar to that of the commercial flow cytometer. The developed cytometer can evaluate quantitatively the radiation dose by the mean fluorescence intensity of γ-H2AX with a significantly smaller amount of blood samples than a commercial flow cytometer. - Highlights: • A new microfluidic cytometer for evaluating irradiation dose was developed. • The utility of this biosensor is verified by comparison experiments using FCM. • The developed cytometer is small size, high sensitivity, low cost, and simple. • The cytometer can dramatically reduce sample consumption and analysis time

  17. Assessment of Cumulative Trauma Disorder (CTD) Risk for 3 Different Tasks Constructing and Repairing Multi-Layer Insulation (MLI) Blankets, Preparing the Dough for a Pizza, and Operating the Becton-Dickinson FACSAria Flow Cytometer

    Science.gov (United States)

    Gentzler, Marc; Kline, Martin; Palmer, Andrew; Terrone, Mark

    2007-01-01

    The Cumulative Trauma Disorder (CTD) risks for three different tasks using McCauley-Bell and Badiru's (1993) formula based on task, personal, and organizational factors were examined. For the Multi-Layer Insulation (MLI) blanket task, the results showed that the task, personal, and organizational risks were at about the same level. The personal risk factors for this task were evaluated using a hypothetical female employee age 52. For the pizza dough task, it was shown that the organizational risk was particularly high, with task related factors also at quite dangerous levels. On the other hand, there was a very low level of personal risk factors, based on a female age 17. The flow cytometer task was assessed with three different participants, a11 of whom had quite disparate levels of personal risk, which slightly affected the overall CTD risk. This reveals how individual difference variables certainly need to be considered. The task and organizational risks for this task were rated at about the same moderate level. The overall CTD risk averaged across the three participants was .335, indicating some risk. Compruing across the tasks revealed that the pizza dough task created the greatest overall CTD risk by far (.568), with the MLI (.325) and flow cytometer task (.335) having some risk associated with them. Future research should look into different tasks for more of a comparison

  18. Small Submersible Robust Microflow Cytometer for Quantitative Detection of Phytoplankton, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Translume will develop an extremely robust, inexpensive micro flow cytometer (mFCM) for quantitative detection of phytoplankton. This device will be designed to be...

  19. Microflow Cytometers with Integrated Hydrodynamic Focusing

    Directory of Open Access Journals (Sweden)

    Martin Schmidt

    2013-04-01

    Full Text Available This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex principle. The sample—A suspension of micro particles or blood cells—is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.

  20. High throughput imaging cytometer with acoustic focussing.

    Science.gov (United States)

    Zmijan, Robert; Jonnalagadda, Umesh S; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn; Glynne-Jones, Peter

    2015-10-31

    We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.

  1. A new concept and a comprehensive evaluation of SYSMEX UF-1000i  flow cytometer to identify culture-negative urine specimens in patients with UTI.

    Science.gov (United States)

    Monsen, T; Ryden, P

    2017-09-01

    Urinary tract infections (UTIs) are among the most common bacterial infections in men and urine culture is gold standard for diagnosis. Considering the high prevalence of culture-negative specimens, any method that identifies such specimens is of interest. The aim was to evaluate a new screening concept for flow cytometry analysis (FCA). The outcomes were evaluated against urine culture, uropathogen species and three conventional screening methods. A prospective, consecutive study examined 1,312 urine specimens, collected during January and February 2012. The specimens were analyzed using the Sysmex UF1000i FCA. Based on the FCA data culture negative specimens were identified in a new model by use of linear discriminant analysis (FCA-LDA). In total 1,312 patients were included. In- and outpatients represented 19.6% and 79.4%, respectively; 68.3% of the specimens originated from women. Of the 610 culture-positive specimens, Escherichia coli represented 64%, enterococci 8% and Klebsiella spp. 7%. Screening with FCA-LDA at 95% sensitivity identified 42% (552/1312) as culture negative specimens when UTI was defined according to European guidelines. The proposed screening method was either superior or similar in comparison to the three conventional screening methods. In conclusion, the proposed/suggested and new FCA-LDA screening method was superior or similar to three conventional screening methods. We recommend the proposed screening method to be used in clinic to exclude culture negative specimens, to reduce workload, costs and the turnaround time. In addition, the FCA data may add information that enhance handling and support diagnosis of patients with suspected UTI pending urine culture [corrected].

  2. Battery operated preconcentration-assisted lateral flow assay.

    Science.gov (United States)

    Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

    2017-07-11

    Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

  3. Shellfish Hemocyte Data from Flow Cytometers

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Various immune function tests using shellfish hemolymph have been developed to determine shellfish health. These tests including viability, phagocytosis, adhesion,...

  4. Microfluidic Multichannel Flow Cytometer, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Techshot, Inc. proposes continued research and development of an on-orbit cell counter culminating in a deliverable hand-held blood cell counter in the form of a...

  5. Use of laminar flow patterning for miniaturised biochemical assays

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Krühne, Ulrich; Beyer, M.

    2004-01-01

    Laminar flow in microfluidic chambers was used to construct low (one dimensional) density arrays suitable for miniaturized biochemical assays. By varying the ratio of flows of two guiding streams flanking a sample stream, precise focusing and positioning of the latter was achieved, and reactive s...... species carried in the sample stream were deposited on functionalized chip surfaces as discrete 50 mm wide lanes. Using different model systems we have confirmed the method's suitability for qualitative screening and quantification tasks in receptor-ligand assays, recording biotin...

  6. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

  7. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Lago-Cachón, D., E-mail: dlagocachon@gmail.com [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Rivas, M., E-mail: rivas@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Martínez-García, J.C., E-mail: jcmg@uniovi.es [Dpto. de Física, Universidad de Oviedo, Edificio Departamental Este, Campus de Viesques, 33204 Gijón (Spain); Oliveira-Rodríguez, M., E-mail: oliveiramyriam@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); Blanco-López, M.C., E-mail: cblanco@uniovi.es [Dpto. de Química Física y Analítica, Universidad de Oviedo, C/Julián Clavería 8, 33006 Oviedo (Spain); García, J.A., E-mail: joseagd@uniovi.es [Dpto. de Física, Universidad de Oviedo, Escuela de Marina, Campus de Viesques, 33204 Gijón (Spain)

    2017-02-01

    Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies. - Highlights: • A method for quantification of Lateral Flow Assays is proposed. • MNP induce an increase of the impedance on a RF-current carrying copper sensor. • Magnetic nanoparticles (MNP) can be detected flowing over the sensing element.

  8. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    International Nuclear Information System (INIS)

    Lago-Cachón, D.; Rivas, M.; Martínez-García, J.C.; Oliveira-Rodríguez, M.; Blanco-López, M.C.; García, J.A.

    2017-01-01

    Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies. - Highlights: • A method for quantification of Lateral Flow Assays is proposed. • MNP induce an increase of the impedance on a RF-current carrying copper sensor. • Magnetic nanoparticles (MNP) can be detected flowing over the sensing element.

  9. Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays.

    Science.gov (United States)

    Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagström, Anna E V; Kourentzi, Katerina; Conrad, Jacinta C; Willson, Richard C

    2015-12-01

    We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ∼100 times lower than those of previously reported IgE assays.

  10. The effect of an optimized imaging flow cytometry analysis template on sample throughput in the reduced culture cytokinesis-block micronucleus assay

    International Nuclear Information System (INIS)

    Rodrigues, M.A.; Beaton-Green, L.A.; Wilkins, R.C.; Probst, C.E.

    2016-01-01

    In cases of overexposure to ionizing radiation, the cytokinesis-block micronucleus (CBMN) assay can be performed in order to estimate the dose of radiation to an exposed individual. However, in the event of a large-scale radiation accident with many potentially exposed casualties, the assay must be able to generate accurate dose estimates to within ±0.5 Gy as quickly as possible. The assay has been adapted to, validated and optimized on the ImageStream"X imaging flow cyto-meter. The ease of running this automated version of the CBMN assay allowed investigation into the accuracy of dose estimates after reducing the volume of whole blood cultured to 200 μl and reducing the culture time to 48 h. The data analysis template used to identify binucleated lymphocyte cells (BNCs) and micronuclei (MN) has since been optimized to improve the sensitivity and specificity of BNC and MN detection. This paper presents a re-analysis of existing data using this optimized analysis template to demonstrate that dose estimations from blinded samples can be obtained to the same level of accuracy in a shorter data collection time. Here, we show that dose estimates from blinded samples were obtained to within ±0.5 Gy of the delivered dose when data collection time was reduced by 30 min at standard culture conditions and by 15 min at reduced culture conditions. Reducing data collection time while retaining the same level of accuracy in our imaging flow cytometry-based version of the CBMN assay results in higher throughput and further increases the relevancy of the CBMN assay as a radiation bio-dosimeter. (authors)

  11. A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

    Science.gov (United States)

    Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

    2015-12-15

    Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays

    Directory of Open Access Journals (Sweden)

    Helen V. Hsieh

    2017-05-01

    Full Text Available Immunochromatographic or lateral flow assays (LFAs are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor’s office in Manhattan to a rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads, biological reagents (e.g., antibodies, blocking reagents and buffers and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness.

  13. Detection of Shiga Toxins by Lateral Flow Assay

    Directory of Open Access Journals (Sweden)

    Kathryn H. Ching

    2015-04-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC produce shiga toxins (Stxs that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

  14. Multiplex immunoassay for persistent organic pollutants in tilapia: comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

    NARCIS (Netherlands)

    Meimaridou, A.; Haasnoot, W.; Shelver, W.L.; Franek, M.; Nielen, M.W.F.

    2013-01-01

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with

  15. Numerical and experimental characterization of solid-state micropore-based cytometer for detection and enumeration of biological cells.

    Science.gov (United States)

    Guo, Jinhong; Chen, Liang; Ai, Ye; Cheng, Yuanbing; Li, Chang Ming; Kang, Yuejun; Wang, Zhiming

    2015-03-01

    Portable diagnostic devices have emerged as important tools in various biomedical applications since they can provide an effective solution for low-cost and rapid clinical diagnosis. In this paper, we present a micropore-based resistive cytometer for the detection and enumeration of biological cells. The proposed device was fabricated on a silicon wafer by a standard microelectromechanical system processing technology, which enables a mass production of the proposed chip. The working principle of this cytometer is based upon a bias potential modulated pulse, originating from the biological particle's physical blockage of the micropore. Polystyrene particles of different sizes (7, 10, and 16 μm) were used to test and calibrate the proposed device. A finite element simulation was developed to predict the bias potential modulated pulse (peak amplitude vs. pulse bandwidth), which can provide critical insight into the design of this microfluidic flow cytometer. Furthermore, HeLa cells (a type of tumor cell lines) spiked in a suspension of blood cells, including red blood cells and white blood cells, were used to assess the performance for detecting and counting tumor cells. The proposed microfluidic flow cytometer is able to provide a promising platform to address the current unmet need for point-of-care clinical diagnosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Microflow Cytometer with a Rectangular Quasi-Flat-Top Laser Spot

    Directory of Open Access Journals (Sweden)

    Jingjing Zhao

    2016-09-01

    Full Text Available This work develops a microflow cytometer, based on a microfluidic chip for three-dimensional (3D hydrodynamic focusing and a binary optical element (BOE for shaping and homogenizing a laser beam. The microfluidic chip utilizes sheath flows to confine the sample flow along the channel centerline with a narrow cross section. In addition to hydrodynamic focusing, secondary flows are generated to strengthen the focusing in the vertical direction. In experiments, the chip was able to focus the sample flow with cross sections of 15 μm high and 8–30 μm wide at 5 m/s, under the condition of the sample flow rates between 10 and 120 μL/min. Instead of using the conventional elliptical Gaussian spot for optical detection, we used a specially designed BOE and obtained a 50 μm × 10 μm rectangular quasi-flat-top spot. The microflow cytometer combining the chip and the BOE was tested to count 3, 5, and 7 μm fluorescence microbeads, and the experimental results were comparable to or better than those derived from two commercial instruments.

  17. Spaceflight Flow Cytometry: Design Challenges and Applications

    Science.gov (United States)

    Pappas, Dimitri; Kao, Shih-Hsin; Jeevarajan, Antony S.

    2004-01-01

    Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health and a wide array of biochemistry and immunology assays. While flow cytometry has been widely used for cellular analysis in both clinical and research settings, the requirements for proper operation in spaceflight impose constraints on any instrument designs. The challenges of designing a spaceflight-ready flow cytometer are discussed, as well as some preliminary results using a prototype system.

  18. Automated processing integrated with a microflow cytometer for pathogen detection in clinical matrices.

    Science.gov (United States)

    Golden, J P; Verbarg, J; Howell, P B; Shriver-Lake, L C; Ligler, F S

    2013-02-15

    A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose-response curves, demonstrating the potential for quantification of pathogen load in buffer and serum. Published by Elsevier B.V.

  19. Papel da imunofenotipagem por citometria de fluxo no diagnóstico diferencial das pancitopenias e das linfocitoses The role of flow cytometer-based immunophenotyping in the differential diagnosis of pancytopenia and of lymphocytosis

    Directory of Open Access Journals (Sweden)

    Eduardo M. Rego

    2009-01-01

    Full Text Available A imunofenotipagem por citometria de fluxo (CMF é atualmente uma ferramenta indispensável para o diagnóstico hematopatológico. Nos últimos anos muitos progressos foram alcançados em instrumentação, novos anticorpos e fluorocromos e programas de análise. Consequentemente, houve um grande avanço no conhecimento da patogênese das neoplasias hematológicas e novos marcadores diagnósticos e prognósticos foram descritos. Revisamos aqui a contribuição destas novas técnicas no diagnóstico diferencial de pacientes com bi- ou pancitopenia e linfocitose. São apresentados os achados mais frequentes e as dificuldades na interpretação dos resultados. Além disto, a importância do uso concomitante de um conjunto de outras técnicas diagnósticas é demonstrada.The use of flow cytometry for immunophenotyping is currently an essential tool in the diagnosis of hematological abnormalities. In recent years, new equipment, antibodies, fluorochromes and computer programs have become available. As a result, a better understanding of the pathogeneses of hematological malignancies has emerged and new markers with diagnostic and prognostic relevance have been described. Here we review how this new technology may contribute to the differential diagnosis of patients with bi- or pancytopenia and of lymphocytosis. The common findings as well as the difficulties in interpreting the results obtained by flow cytometry will be discussed. The importance of the concomitant analyses by different methods is also demonstrated.

  20. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... daughter cells, leading to a characteristic flow cytometric profile where a ... cell recognition without any impact on bone marrow hemato- ... cells of various cancer cells that load CFSE concentration ... (B) Target cells (R1) were further analyzed in an FL1/FL3 dot plot, ..... hematopoietic cell transplantation.

  1. Nanoparticle-based assays in automated flow systems: A review

    Energy Technology Data Exchange (ETDEWEB)

    Passos, Marieta L.C. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Pinto, Paula C.A.G., E-mail: ppinto@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Santos, João L.M., E-mail: joaolms@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Saraiva, M. Lúcia M.F.S., E-mail: lsaraiva@ff.up.pt [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Araujo, André R.T.S. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda, Av. Dr. Francisco de Sá Carneiro, n° 50, 6300-559 Guarda (Portugal)

    2015-08-19

    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section.

  2. Nanoparticle-based assays in automated flow systems: A review

    International Nuclear Information System (INIS)

    Passos, Marieta L.C.; Pinto, Paula C.A.G.; Santos, João L.M.; Saraiva, M. Lúcia M.F.S.; Araujo, André R.T.S.

    2015-01-01

    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section

  3. Performance evaluation of FlowCytomix assays to quantify cytokines in patients with rheumatoid arthritis

    Science.gov (United States)

    Wang, Xuefeng; Dong, Liyang; Liang, Yong; Ni, Hongchang; Tang, Jun; Xu, Chengcheng; Zhou, Yuepeng; Su, Yuting; Wang, Jun; Chen, Deyu; Mao, Chaoming

    2015-01-01

    Objectives: To compare the cytokine profile in RA patients and healthy control by using two methods-FlowCytomix assay and traditional ELISA. Methods: Cytokine levels were evaluated by FlowCytomix assay and ELISA in serum and supernatants of peripheral blood mononuclear cells (PBMC) cultures with and without stimulation by phytohaemagglutinin (PHA). Results: The levels of IL-6, IL-1β, and TNF-α were significantly higher in sera of RA patients than those of healthy controls. The levels of IL-22, IL-6, IL-1β, TNF-α, and IL-10 were higher in unstimulated PBMC culture supernatant of RA patients than those of healthy controls. PHA stimulation significantly increased the production of proinflammatory cytokines from PBMC with RA patients. Compared with detectable cytokine levels in sera, cytokine concentration in the supernatant of PBMCs was remarkably higher. FlowCytomix and ELISA showed significant correlation in detecting cytokines. However, the FlowCytomix assay detected more cytokines than ELISA. Conclusion: The supernatant of PBMCs provide a fine condition for the study of cytokine production because of the lack of interference factors in sera. The FlowCytomix assay is more sensitive than ELISA in detecting cytokines from RA patients. Multiple cytokine signatures using FlowCytomix assay may represent a more realistic approach in the future of personalized medicine in RA. PMID:26629129

  4. Designs, formats and applications of lateral flow assay: A literature review

    Directory of Open Access Journals (Sweden)

    Muhammad Sajid

    2015-11-01

    Full Text Available This manuscript provides a brief overview of latest research involving the use of lateral flow assay for qualitative and quantitative analysis in different areas. The excellent features and versatility of detection formats make these strips an ideal choice for point of care applications. We outline and critically discuss detection formats, molecular recognition probes, labels, and detection systems used in lateral flow assay. Applications in different fields along with selected examples from the literature have been included to show analytical performance of these devices. At the end, we summarize accomplishments, weaknesses and future challenges in the area of lateral flow strips.

  5. Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections.

    Science.gov (United States)

    Galiana, Antonio; Coy, Javier; Gimeno, Adelina; Guzman, Noemi Marco; Rosales, Francisco; Merino, Esperanza; Royo, Gloria; Rodríguez, Juan Carlos

    2017-01-01

    Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients. In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays. Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines). SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively. This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship), in order for the results to be applied appropriately to the management of patients`infectious processes.

  6. Flow Cytometry Technician | Center for Cancer Research

    Science.gov (United States)

    PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) of the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of cancer and cancer cells. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Technician will be responsible for: Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Monitoring lab supply levels and order lab supplies, perform various record keeping responsibilities Assist in the training of scientific end users on the use of flow cytometry in their research, as well as how to operate and troubleshoot the bench-top analyzer instruments Experience with sterile technique and tissue culture

  7. Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

    Science.gov (United States)

    Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

    2017-10-23

    Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

  8. Correlation between ELISA and ML Flow assays applied to 60 Brazilian patients affected by leprosy

    NARCIS (Netherlands)

    Da Silva, Rozana C.; Lyon, Sandra; Lyon, Ana C.; Grossi, Maria A. F.; Lyon, Silvia H.; Bührer-Sékula, Samira; Antunes, Carlos M. F.

    2010-01-01

    Serological tests can be helpful in classifying leprosy patients as having either the paucibacillary or the multibacillary form. The aim of this study was to evaluate the concordance between two serological assays, i.e. ML Flow and ELISA, in a population of leprosy patients in Brazil. The

  9. Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections.

    Directory of Open Access Journals (Sweden)

    Antonio Galiana

    Full Text Available Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients.In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays.Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines. SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively.This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship, in order for the results to be applied appropriately to the management of patients`infectious processes.

  10. A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

    Science.gov (United States)

    Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

    2018-06-04

    A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

  11. A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

    Directory of Open Access Journals (Sweden)

    Gentry-Nielsen Martha J

    2006-07-01

    Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

  12. Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer.

    Science.gov (United States)

    Zhang, Haohai; Chan, Leo Li-Ying; Rice, William; Kassam, Nasim; Longhi, Maria Serena; Zhao, Haitao; Robson, Simon C; Gao, Wenda; Wu, Yan

    2017-08-01

    Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5ng/mL, with concurrent K d binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

    Directory of Open Access Journals (Sweden)

    Helene Andersson-Svahn

    2011-11-01

    Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  14. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  15. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

    Science.gov (United States)

    Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

  16. Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

    Science.gov (United States)

    Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

    2008-09-15

    Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

  17. A flow cytometric assay technology based on quantum dots-encoded beads

    International Nuclear Information System (INIS)

    Wang Haiqiao; Liu Tiancai; Cao Yuancheng; Huang Zhenli; Wang Jianhao; Li Xiuqing; Zhao Yuandi

    2006-01-01

    A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs' unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement

  18. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    Directory of Open Access Journals (Sweden)

    Iole Macchia

    2013-01-01

    Full Text Available The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs and tumor-associated antigens (TAAs and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM- based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

  19. A portable battery powered microfluidic impedance cytometer with smartphone readout: towards personal health monitoring.

    Science.gov (United States)

    Talukder, Niloy; Furniturewalla, Abbas; Le, Tuan; Chan, Matthew; Hirday, Shreyas; Cao, Xinnan; Xie, Pengfei; Lin, Zhongtian; Gholizadeh, Azam; Orbine, Steve; Javanmard, Mehdi

    2017-06-01

    We present a portable system for personalized blood cell counting consisting of a microfluidic impedance cytometer and portable analog readout electronics, feeding into an analog-to-digital converter (ADC), and being transmitted via Bluetooth to a user-accessible mobile application. We fabricated a microfluidic impedance cytometer with a novel portable analog readout. The novel design of the analog readout, which consists of a lock-in-amplifier followed by a high-pass filter stage for subtraction of drift and DC offset, and a post-subtraction high gain stage, enables detection of particles and cells as small as 1 μm in diameter, despite using a low-end 8-bit ADC. The lock-in-amplifier and the ADC were set up to receive and transmit data from a Bluetooth module. In order to initiate the system, as well as to transmit all of the data, a user friendly mobile application was developed, and a proof-of-concept trial was run on a blood sample. Applications such as personalized health monitoring require robust device operation and resilience to clogging. It is desirable to avoid using channels comparable in size to the particles being detected thus requiring high levels of sensitivity. Despite using low-end off-the-shelf hardware, our sensing platform was capable of detecting changes in impedance as small as 0.032%, allowing detection of 3 μm diameter particles in a 300 μm wide channel. The sensitivity of our system is comparable to that of a high-end bench-top impedance spectrometer when tested using the same sensors. The novel analog design allowed for an instrument with a footprint of less than 80 cm 2 . The aim of this work is to demonstrate the potential of using microfluidic impedance spectroscopy for low cost health monitoring. We demonstrated the utility of the platform technology towards cell counting, however, our platform is broadly applicable to assaying wide panels of biomarkers including proteins, nucleic acids, and various cell types.

  20. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Science.gov (United States)

    Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  1. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

    Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  2. Flow cytometric measurement of DNA level and steroid hormone receptor assay in breast cancer

    International Nuclear Information System (INIS)

    Zubrikhina, G.N.; Kuz'mina, Eh.V.; Bassalyk, L.S.; Murav'eva, N.I.

    1989-01-01

    DNA level measured by flow cytometry and estrogen and progesteron receptors assayed in tissue samples obtained from 85 malignant and 16 benign lesions of the breast. All the benign tumors revealed 2c DNA content and most of them were receptor-negative, while 74.1% of breast carcinomas displayed aneuploidy. Three patients (3.5%) had two lines of aneuploid cells. Many aneuploid tumors were receptor-negative. Preoperative radiation treatmet (14-20 Gy) did not significantly influence the level of steroid hormone receptors in tumors. Estrogen receptor level was higher in menopausal patients than in premenopausal ones

  3. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis

    NARCIS (Netherlands)

    Smits, Henk L.; Abdoel, Theresia H.; Solera, Javier; Clavijo, Encarnacion; Diaz, Ramon

    2003-01-01

    To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG

  4. Use of the Brucella IgM and IgG flow assays in the serodiagnosis of human brucellosis in an area endemic for brucellosis

    NARCIS (Netherlands)

    Irmak, Hasan; Buzgan, Turan; Evirgen, Omer; Akdeniz, Hayrettin; Demiroz, A. Pekcan; Abdoel, Theresia H.; Smits, Henk L.

    2004-01-01

    The clinical utility of two complementary tests for brucellosis, the Brucella IgM and IgG flow assays, was evaluated in a hospital in eastern Turkey. The results show that the flow assays are convenient diagnostic tests for use in endemic areas. A positive result in the flow assays was obtained in

  5. Isothermal Amplification and Lateral-Flow Assay for Detecting Crown-Gall-Causing Agrobacterium spp.

    Science.gov (United States)

    Fuller, Skylar L; Savory, Elizabeth A; Weisberg, Alexandra J; Buser, Jessica Z; Gordon, Michael I; Putnam, Melodie L; Chang, Jeff H

    2017-09-01

    Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

  6. Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

    Directory of Open Access Journals (Sweden)

    Lyerly Herbert K

    2008-03-01

    Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

  7. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    Science.gov (United States)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  8. Instrument for real-time pulse-shape analysis of slit-scan flow cytometry signals

    NARCIS (Netherlands)

    van Oven, C.; Aten, J. A.

    1990-01-01

    An instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse-shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system.

  9. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

    OpenAIRE

    Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

    2007-01-01

    Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. ...

  10. Comparison of flow cytometry and immunohistochemistry in non-radioisotopic murine lymph node assay using bromodeoxyuridine.

    Science.gov (United States)

    Jung, Kyoung-Mi; Bae, Il-Hong; Kim, Bae-Hwan; Kim, Wang-Ki; Chung, Jin-Ho; Park, Young-Ho; Lim, Kyung-Min

    2010-02-01

    Non-radioisotopic local lymph node assay (LLNA) employing 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FACS) or immunohistochemistry (IHC) is gaining attention due to a regulatory issue of using radioisotope, (3)H-thymidine, in vivo in traditional LLNA. In this study, to compare the performance of these non-radioisotopic endpoints, 7 chemicals with known sensitizing potencies were examined in LLNA. Mice were topically treated with chemicals or vehicle on both ears for 3 days. After intraperitoneal injection of BrdU, bilateral lymph nodes were isolated separately and undergone respectively, FACS or IHC to determine BrdU incorporated lymph node cells (LNCs). Weight and histology of treated ears were also examined to evaluate chemical-induced edema and irritation. Both FACS and IHC could successively identify the skin sensitizers from non-sensitizers. Comparison of FACS and IHC with traditional LLNA revealed that FACS has a higher sensitivity although both assays produced comparable sensitivity and performance to traditional LLNA. In conclusion, non-radioisotopic LLNA using FACS and IHC can successfully detect sensitizers with a good correlation to traditional LLNA. Notably, FACS showed almost equivalent sensitivity and accuracy to traditional LLNA. 2009 Elsevier Ireland Ltd. All rights reserved.

  11. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    Directory of Open Access Journals (Sweden)

    Sónia Troeira Henriques

    Full Text Available The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  12. Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

    Science.gov (United States)

    Dolz, María; O'Connor, José-Enrique; Lequerica, Juan L

    2004-10-01

    The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.

  13. Rapid assay for cell age response to radiation by electronic volume flow cell sorting

    International Nuclear Information System (INIS)

    Freyer, J.P.; Wilder, M.E.; Raju, M.R.

    1987-01-01

    A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. Sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [ 3 H]thymidine autoradiography of the sorted cell populations, it is demonstrated that resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. Variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. Advantages of this method include: (1) no treatment of the cells is required, thus, this method is noncytotoxic; (2) no cell cycle progression is needed to obtain different cell age compartments; (3) the cell population can be held in complete growth medium at any desired temperature during sorting; (4) a complete radiation age - response assay can be plated in 2 h. Applications of this method are discussed, along with some technical limitations. (author)

  14. Clinical utility of the cryptococcal antigen lateral flow assay in a diagnostic mycology laboratory.

    Directory of Open Access Journals (Sweden)

    Brendan J McMullan

    Full Text Available BACKGROUND: Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA to detect cryptococcal antigen (CRAG is reportedly more rapid and convenient than standard latex agglutination (LA, but has not yet been evaluated in a diagnostic laboratory setting. METHODS: One hundred and six serum, 42 cerebrospinal fluid (CSF, and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients. RESULTS: Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56 from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI 93.6-100% compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7-96.1%. Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56 this did not reach statistical significance (p = 0.063. Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients. Agreement between titres obtained by both methods (n = 38 samples was imperfect; correlation between log-transformed titres (r was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively. CONCLUSIONS: Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.

  15. Flow cytometry protocols

    National Research Council Canada - National Science Library

    Jaroszeski, Mark J; Heller, Richard

    1998-01-01

    ... are individually analyzed, and it is typical for flow cytometers to quantitatively process thousands of individual particles in a matter of seconds. This a powerful analytic feat particularly if one relates it to the time required to examine several thousand individual cells using a microscope. This leaves little doubt regarding why the field of flow cytometry has...

  16. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

    Science.gov (United States)

    Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

    2007-08-31

    Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

  17. Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer.

    Science.gov (United States)

    Kessel, Sarah; Cribbes, Scott; Bonasu, Surekha; Rice, William; Qiu, Jean; Chan, Leo Li-Ying

    2017-09-01

    The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better

  18. The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan

    NARCIS (Netherlands)

    Mizanbayeva, Sulushash; Smits, Henk L.; Zhalilova, Katya; Abdoel, Theresia H.; Kozakov, Stanislaw; Ospanov, Kenes S.; Elzer, Philip H.; Douglas, James T.

    2009-01-01

    Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers > or = 1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high

  19. Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

    Science.gov (United States)

    Ott, Laura E.; Carson, Susan

    2014-01-01

    Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

  20. A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay.

    Science.gov (United States)

    Du, Xin-Jun; Zhou, Tian-Jiao; Li, Ping; Wang, Shuo

    2017-08-01

    Salmonella is a major foodborne pathogen that is widespread in the environment and can cause serious human and animal disease. Since conventional culture methods to detect Salmonella are time-consuming and laborious, rapid and accurate techniques to detect this pathogen are critically important for food safety and diagnosing foodborne illness. In this study, we developed a rapid, simple and portable Salmonella detection strategy that combines thermophilic helicase-dependent amplification (tHDA) with a lateral flow assay to provide a detection result based on visual signals within 90 min. Performance analyses indicated that the method had detection limits for DNA and pure cultured bacteria of 73.4-80.7 fg and 35-40 CFU, respectively. Specificity analyses showed no cross reactions with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Enterobacter aerogenes, Shigella and Campylobacter jejuni. The results for detection in real food samples showed that 1.3-1.9 CFU/g or 1.3-1.9 CFU/mL of Salmonella in contaminated chicken products and infant nutritional cereal could be detected after 2 h of enrichment. The same amount of Salmonella in contaminated milk could be detected after 4 h of enrichment. This tHDA-strip can be used for the rapid detection of Salmonella in food samples and is particularly suitable for use in areas with limited equipment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan.

    Science.gov (United States)

    Mizanbayeva, Sulushash; Smits, Henk L; Zhalilova, Katya; Abdoel, Theresia H; Kozakov, Stanislaw; Ospanov, Kenes S; Elzer, Philip H; Douglas, James T

    2009-09-01

    Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers > or = 1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high sensitivity of these assays. The strong reactivity in the LFA observed for the majority (92.1%) of the samples from the patients with culture-confirmed brucellosis together with the user-friendliness of the assay procedure makes the LFA ideal for the confirmation of brucellosis in endemic areas in Kazakhstan. The Rose Bengal test lacked sensitivity in particular for patients with chronic brucellosis therefore limiting its value as a quick screening assay. The study emphasizes the importance of the LFA as a useful, rapid, and easy-to-perform tool in the diagnostic testing of brucellosis.

  2. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  3. Flow cytometric chemosensitivity assay using JC‑1, a sensor of mitochondrial transmembrane potential, in acute leukemia.

    Science.gov (United States)

    Yokosuka, Tomoko; Goto, Hiroaki; Fujii, Hisaki; Naruto, Takuya; Takeuchi, Masanobu; Tanoshima, Reo; Kato, Hiromi; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Yokota, Shumpei

    2013-12-01

    The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples. We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA. In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003). This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.

  4. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

    Science.gov (United States)

    Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A; Ferry, Berne

    2011-04-01

    Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  5. Installation of a flow cytometry facility and some applications in radiobiology

    International Nuclear Information System (INIS)

    Walsh, M.; Kellington, J.P.

    1988-01-01

    Flow cytometry has enormous potential in many areas of experimental pathology. Details of the installation and commissioning of a flow cytometer at the Harwell Laboratory are described. Following an explanation of the principles of flow cytometry, several applications to specific problems in radiobiology are discussed. Also included are results of some preliminary studies with the Harwell flow cytometer on samples such as blood, bone marrow, macrophages and cell cultures, and a discussion of future applications. (author)

  6. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    Czech Academy of Sciences Publication Activity Database

    Pospíchalová, V.; Svoboda, Jan; Dave, Z.; Kotrbová, A.; Kaiser, K.; Klemová, D.; Ilkovics, L.; Hampl, A.; Crha, I.; Jandáková, E.; Minář, L.; Weinberger, V.; Bryja, Vítězslav

    2015-01-01

    Roč. 4, March 31 (2015), s. 25530 ISSN 2001-3078 Institutional support: RVO:61388971 ; RVO:68081707 Keywords : exosomes * microvesicles * extracellular vesicles Subject RIV: EE - Microbiology, Virology

  7. Method of detaching adherent cells for flow cytometry

    KAUST Repository

    Kaur, Mandeep

    2015-12-24

    In one aspect, a method for detaching adherent cells can include adding a cell lifting solution to the media including a sample of adherent cells and incubating the sample of adherent cells with the cell lifting solution. No scraping or pipetting is needed to facilitate cell detachment. The method do not require inactivation of cell lifting solution and no washing of detaching cells is required to remove cell lifting solution. Detached cells can be stained with dye in the presence of cell lifting solution and are further analyzed using flow cytometer. The method has been tested using 6 different cell lines, 4 different assays, two different plate formats (96 and 384 well plates) and two different flow cytometry instruments. The method is simple to perform, less time consuming, with no cell loss and makes high throughput flow cytometry on adherent cells a reality.

  8. Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

    Science.gov (United States)

    Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

    2017-09-01

    Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  9. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

    Science.gov (United States)

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen's' reports and fish community monitorings.

  10. Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

    Science.gov (United States)

    2013-01-01

    Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

  11. A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies

    International Nuclear Information System (INIS)

    Marcusson-Staahl, Maritha; Cederbrant, Karin

    2003-01-01

    A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies. The classical 51 Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems. Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development. The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The final format of the assay includes 0.5 ml peripheral blood, 1.25x10 5 effector cells per sample, and collection of 500 target events by flow-cytometry. When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08%. The corresponding cytotoxic activity decreased from 68 to 8%. Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension. Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells. The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies. Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled

  12. Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

    Science.gov (United States)

    Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

    2014-12-01

    The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

    Science.gov (United States)

    Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

    2013-03-01

    Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

  14. Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.

    Science.gov (United States)

    Bobosha, Kidist; Tjon Kon Fat, Elisa M; van den Eeden, Susan J F; Bekele, Yonas; van der Ploeg-van Schip, Jolien J; de Dood, Claudia J; Dijkman, Karin; Franken, Kees L M C; Wilson, Louis; Aseffa, Abraham; Spencer, John S; Ottenhoff, Tom H M; Corstjens, Paul L A M; Geluk, Annemieke

    2014-05-01

    Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to

  15. A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

    Science.gov (United States)

    Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

    2017-10-01

    In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

  16. A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.

    Directory of Open Access Journals (Sweden)

    Erika Hammarlund

    Full Text Available Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar could not be measured by plaque assay (PA, focus-forming assay (FFA, or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6 and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine. Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

  17. Development of a paper-based vertical flow SERS assay for citrulline detection using aptamer-conjugated gold nanoparticles

    Science.gov (United States)

    Locke, Andrea; Deutz, Nicolaas; Coté, Gerard

    2018-02-01

    Research toward development of point-of-care (POC) technologies is emerging as a means for diagnosis and monitoring of patients outside the hospital. These POC devices typically utilize assays capable of detecting low level biomarkers indicative of specific diseases. L-citrulline, an α-amino acid produced in the intestinal mucosa cells, is one such biomarker typically found circulating within the plasma at physiological concentrations of 40 μM. Researchers have found that intestinal enterocyte malfunction causes its level to be significantly lowered, establishing it as a potential diagnostic biomarker for gut function. Our research group has proposed the development of a surface enhanced Raman spectroscopy (SERS) based assay, using vertical flow paper fluidics, for citrulline detection. The assay consists of a fluorescently active, Raman reporter labeled aptamer conjugated on gold nanoparticles. The aptamer changes its confirmation on binding to its target, which in turn changes the distance between the Raman active molecule and the nanoparticle surface. These particles were embedded within a portable chip consisting of cellulose-based paper. After the chips were loaded with different concentrations of free L-citrulline in phosphate buffer, time was given for the assay to interact with the sample. A handheld Raman spectrometer (638 nm; Ocean Optics) was used to measure the SERS intensity. Results showed decrease in intensity with increasing concentration of L-citrulline (0-50μM).

  18. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  19. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

    NARCIS (Netherlands)

    Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

    2016-01-01

    In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

  20. α-amylase assay and action pattern determination using radioactive substrate, HPLC, and a radioactive flow detector

    International Nuclear Information System (INIS)

    Marsili, R.T.; Ostapenko, H.

    1987-01-01

    A new assay system is presented for the analysis of α-amylase. The disappearance of 14 C-labeled starch substrate and the appearance of its radioactive degradation products were monitored by HPLC and a radioactive flow detector/integrator. The hydrolysis of radioactive substrate was proportional to enzyme concentration when two commercially available α-amylase preparations of Bacillus subtilis origin were studied. The method demonstrated an average recovery of 101.7 +/- 6.5% when modified food starch was spiked with amylase and analyzed. In addition, the method was shown to be useful for predicting detailed action patterns of various types of amylases

  1. Technical discussions II - Flow cytometric analysis

    NARCIS (Netherlands)

    Cunningham, A; Cid, A; Buma, AGJ

    In this paper the potencial of flow cytometry as applied to the aquatic life sciences is discussed. The use of flow cytometry for studying the ecotoxicology of phytoplankton was introduced. On the other hand, the new flow cytometer EUROPA was presented. This is a multilaser machine which has been

  2. Nuclear assay of coal. Volume 6. Mass flow devices for coal handling

    International Nuclear Information System (INIS)

    Anon.

    1979-01-01

    The mass of coal entering the boiler per unit time is an essential parameter for determinig the total rate of heat input. The mass flow rate of coal on a conveyor belt is generally determined as a product of the instantaneous mass of material on a short section of the belt and the belt velocity. Belt loading could be measured by conventional transducers incorporating mechanical or electromechanical weighers or by gamma-ray attenuation gauge. This report reviews the state of the art in mass flow devices for coal handling. The various methods are compared and commented upon. Special design issues are discussed relative to incorporating a mass flow measuring device in a Continuous On-Line Nuclear Analysis of Coal (CONAC) system

  3. Nuclear assay of coal. Volume 6. Mass flow devices for coal handling. Final report

    International Nuclear Information System (INIS)

    Gozani, T.; Elias, E.; Bevan, R.

    1980-04-01

    The mass of coal entering the boiler per unit time is an essential parameter for determining the total rate of heat input. The mass flow rate of coal on a conveyor belt is generally determined as a product of the instantaneous mass of material on a short section of the belt and the belt velocity. Belt loading could be measured by conventional transducers incorporating mechanical or electromechanical weighers or by gamma-ray attenuation gauge. This report reviews the state of the art in mass flow devices for coal handling. The various methods are compared and commented upon. Special design issues are discussed relative to incorporating a mass flow measuring device in a Continuous On-Line Nuclear Analysis of Coal (CONAC) system

  4. Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Paul F. Rühle

    2016-08-01

    Full Text Available The monitoring of immune cells gained great significance in prognosis and prediction of therapy responses. For analyzing blood samples, the multicolor flow cytometry has become the method of choice as it combines high specificity on single cell level with multiple parameters and high throughput. Here, we present a modular assay for the detailed immunophenotyping of blood (DIoB that was optimized for an easy and direct application in whole blood samples. The DIoB assay characterizes 34 immune cell subsets that circulate the peripheral blood including all major immune cells such as T cells, B cells, natural killer (NK cells, monocytes, dendritic cells (DCs, neutrophils, eosinophils, and basophils. In addition, it evaluates their functional state and a few non-leukocytes that also have been associated with the outcome of cancer therapy. This DIoB assay allows a longitudinal and close-meshed monitoring of a detailed immune status in patients requiring only 2.0 mL of peripheral blood and it is not restricted to peripheral blood mononuclear cells. It is currently applied for the immune monitoring of patients with glioblastoma multiforme (IMMO-GLIO-01 trial, NCT02022384, pancreatic cancer (CONKO-007 trial, NCT01827553, and head and neck cancer (DIREKHT trial, NCT02528955 and might pave the way for immune biomarker identification for prediction and prognosis of therapy outcome.

  5. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Vanparys, Caroline, E-mail: caroline.vanparys@ua.ac.be [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Depiereux, Sophie; Nadzialek, Stephanie [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); Robbens, Johan; Blust, Ronny [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Kestemont, Patrick [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); De Coen, Wim [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); European Chemicals Agency (ECHA), Helsinki (Finland)

    2010-09-15

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC{sub 50} value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R{sup 2} = 0.98), the estrogen receptor (ER) binding (R{sup 2} = 0.84) and the ER transcription activation assay (R{sup 2} = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies

  6. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    International Nuclear Information System (INIS)

    Vanparys, Caroline; Depiereux, Sophie; Nadzialek, Stephanie; Robbens, Johan; Blust, Ronny; Kestemont, Patrick; De Coen, Wim

    2010-01-01

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC 50 value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R 2 = 0.98), the estrogen receptor (ER) binding (R 2 = 0.84) and the ER transcription activation assay (R 2 = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of

  7. Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.

    Directory of Open Access Journals (Sweden)

    Kidist Bobosha

    2014-05-01

    Full Text Available BACKGROUND: Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. METHODS: The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC. For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP reporter technology was applied in all LFAs. RESULTS: Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. CONCLUSIONS: Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required

  8. Quality control in the application of flow cytometric assays of genetic damage due to environmental contaminants

    International Nuclear Information System (INIS)

    McCreedy, C.D.; Jagoe, C.H.; Brisbin, I.L. Jr.; Wentworth, R.W.; Dallas, C.E.

    1995-01-01

    Clinical technologies, such as flow cytometry, are increasingly adopted by environmental toxicologists to identify resource damage associated with exposure to xenobiotics. One application of flow cytometry allows the rapid determination of the DNA content of large numbers of individual cells, and can be used to detect aneuploidy or other genetic abnormalities. The laboratory has used this methodology in studies of genetic toxicology of fish, birds, arid mammals exposed to organic pollutants, metals and radionuclides, However, without appropriate quality controls, false positive results and other artifacts can arise from sample handling and preparations, inter and intra-individual variations, instrument noise and other sources. The authors describe the routine measures this laboratory employs to maintain quality control of genomic DNA analysis, including the control of staining conditions, machine standardization, pulse-width doublet discrimination, and, in particular, the use of internal controls and the use of time as a cytometric parameter. Neglect of these controls can produce erroneous results, leading to conclusions of genetic abnormalities when none are present. Conversely, attention to these controls, routinely used in clinical settings, facilitates the interpretation of flow cytometric data and allows the application of this sensitive indicator of genotoxic effects to a variety of environmental problems

  9. A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays

    Directory of Open Access Journals (Sweden)

    Linda G. Lee

    2013-10-01

    Full Text Available We demonstrate a fluorescence lateral flow system that has excellent sensitivity and wide dynamic range. The illumination system utilizes an LED, plastic lenses and plastic and colored glass filters for the excitation and emission light. Images are collected on an iPhone 4. Several fluorescent dyes with long Stokes shifts were evaluated for their signal and nonspecific binding in lateral flow. A wide range of values for the ratio of signal to nonspecific binding was found, from 50 for R-phycoerythrin (R-PE to 0.15 for Brilliant Violet 605. The long Stokes shift of R-PE allowed the use of inexpensive plastic filters rather than costly interference filters to block the LED light. Fluorescence detection with R-PE and absorbance detection with colloidal gold were directly compared in lateral flow using biotinylated bovine serum albumen (BSA as the analyte. Fluorescence provided linear data over a range of 0.4–4,000 ng/mL with a 1,000-fold signal change while colloidal gold provided non-linear data over a range of 16–4,000 ng/mL with a 10-fold signal change. A comparison using human chorionic gonadotropin (hCG as the analyte showed a similar advantage in the fluorescent system. We believe our inexpensive yet high-performance platform will be useful for providing quantitative and sensitive detection in a point-of-care setting.

  10. Flow cytometry-based assay to evaluate human serum MUC1-Tn antibodies

    DEFF Research Database (Denmark)

    Van Elssen, Catharina H M J; Clausen, Henrik; Germeraad, Wilfred T V

    2011-01-01

    Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due to the underglycosylat......Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due...... to detect antibodies binding to the underglycosylated MUC1 protein. This cellular system is complementary to the previously published methods to detect MUC1 serum antibodies, since the antibodies to the native protein are evaluated and therefore it can be effectively used for MUC1 antibody monitoring...... in vaccination studies as well as for functional assays....

  11. Cathepsin D immobilized capillary reactors for on-flow screening assays.

    Science.gov (United States)

    Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

    2018-03-20

    The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood.

    Science.gov (United States)

    Hewitt, Rachel E; Vis, Bradley; Pele, Laetitia C; Faria, Nuno; Powell, Jonathan J

    2017-10-01

    Pigment grade titanium dioxide is composed of sub-micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell-particle associations could be determined in immune cells of human whole blood at "real life" concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle-bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  13. HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

    Science.gov (United States)

    Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

    2016-03-01

    Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

  14. Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

    Science.gov (United States)

    Ahn, Ilyoung; Kim, Tae-Sung; Jung, Eun-Sun; Yi, Jung-Sun; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Jung, Mi-Sook; Jeon, Eun-Young; Yeo, Kyeong-Uk; Jo, Ji-Hoon; Park, Jung-Eun; Kim, Chang-Yul; Park, Yeong-Chul; Seong, Won-Keun; Lee, Ai-Young; Chun, Young Jin; Jeong, Tae Cheon; Jeung, Eui Bae; Lim, Kyung-Min; Bae, SeungJin; Sohn, Soojung; Heo, Yong

    2016-10-01

    Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Four-dimensional characterization of thrombosis in a live-cell, shear-flow assay: development and application to xenotransplantation.

    Directory of Open Access Journals (Sweden)

    Donald G Harris

    Full Text Available Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.Confluent wild-type (WT, n = 48 and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11 porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm2 and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells and total fluorescence (a metric of clot volume. Aggregation was calculated by the fluorescence/SA ratio (FR. WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2% and aggregation (FR 120 ± 1 a.u., indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each; importantly, aggregation was only marginally affected (11% reduction, P < 0.0001.This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to

  16. Screening for urinary tract infection with the Sysmex UF-1000i urine flow cytometer

    NARCIS (Netherlands)

    Broeren, M.A.C.; Bahceci, C.; Vader, H.; Arents, N.L.A.

    2011-01-01

    The diagnosis of urinary tract infection (UTI) by urine culture is time-consuming and can produce up to 60 to 80% negative results. Fast screening methods that can reduce the necessity for urine cultures will have a large impact on overall turnaround time and laboratory economics. We have evaluated

  17. Fiber free plug and play on-chip scattering cytometer module – for implementation in microfluidic point of care devices

    DEFF Research Database (Denmark)

    Jensen, Thomas Glasdam; Kutter, Jörg Peter

    2010-01-01

    In this paper, we report on recent progress toward the development of a plug and play on-chip cytometer based on light scattering. By developing a device that does not depend on the critical alignment and cumbersome handling of fragile optical fibers, we approach a device that is suitable for non...

  18. The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

    Science.gov (United States)

    Nantakomol, Duangdao; Imwong, Malika; Soontarawirat, Ingfar; Kotjanya, Duangporn; Khakhai, Chulalak; Ohashi, Jun; Nuchnoi, Pornlada

    2009-05-01

    Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. (c) 2008 Clinical Cytometry Society.

  19. Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis

    NARCIS (Netherlands)

    Zeytinoğlu, Ayşin; Turhan, Ajda; Altuğlu, Imre; Bilgiç, Altinay; Abdoel, Theresia H.; Smits, Henk L.

    2006-01-01

    The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that

  20. Development of SCAR markers and PCR assays for single or simultaneous species-specific detection of Phytophthora nicotianae and Pythium helicoides in ebb-and-flow irrigated kalanchoe.

    Science.gov (United States)

    Ahonsi, Monday O; Ling, Yin; Kageyama, Koji

    2010-11-01

    Phytophthora nicotianae and Pythium helicoides are important water-borne oomycete pathogens of irrigated ornamentals particularly ebb-and-flow irrigated kalanchoe in Japan. We developed novel PCR-based sequence characterized amplified region markers and assays for rapid identification and species-specific detection of both pathogens in separate PCR reactions or simultaneously in a duplex PCR.

  1. Early diagnosis of influenza virus a using surface-enhanced Raman scattering-based lateral flow assay

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyun Ji; Choo, Jae Bum [Dept. of Bionano Technology, Hanyang University, Ansan (Korea, Republic of); Yang, Sung Chul [School of Architectural Engineering, Hongik University, Sejong (Korea, Republic of)

    2016-12-15

    We report a surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) kit for the rapid diagnosis of influenza virus A. Influenza virus A is highly infectious and causes acute respiratory diseases. Therefore, it is important to diagnose the virus early to prevent a pandemic and to provide appropriate treatment to the patient and vaccination of high-risk individuals. Conventional diagnostic tests, including virus cell culture and real-time polymerase chain reaction, take longer than 1 day to confirm the disease. In contrast, a commercially available rapid influenza diagnostic test can detect the infection within 30 min, but it is hard to confirm viral infection using only this test because of its low sensitivity. Therefore, the development of a rapid and simple test for the early diagnosis of influenza infection is urgently needed. To resolve these problems, we developed a SERS-based LFA kit in which the gold nanoparticles in the commercial rapid kit were replaced with SERS-active nano tags. It is possible to quantitatively detect the influenza virus A with high sensitivity by measuring the enhanced Raman signal of these SERS nano tags on the LFA strip. The limit of detection (LOD) using our proposed SERS-based LFA kit was estimated to be 1.9 × 10{sup 4} PFU/mL, which is approximately one order of magnitude more sensitive than the LOD determined from the colorimetric LFA kit.

  2. Early diagnosis of influenza virus a using surface-enhanced Raman scattering-based lateral flow assay

    International Nuclear Information System (INIS)

    Park, Hyun Ji; Choo, Jae Bum; Yang, Sung Chul

    2016-01-01

    We report a surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) kit for the rapid diagnosis of influenza virus A. Influenza virus A is highly infectious and causes acute respiratory diseases. Therefore, it is important to diagnose the virus early to prevent a pandemic and to provide appropriate treatment to the patient and vaccination of high-risk individuals. Conventional diagnostic tests, including virus cell culture and real-time polymerase chain reaction, take longer than 1 day to confirm the disease. In contrast, a commercially available rapid influenza diagnostic test can detect the infection within 30 min, but it is hard to confirm viral infection using only this test because of its low sensitivity. Therefore, the development of a rapid and simple test for the early diagnosis of influenza infection is urgently needed. To resolve these problems, we developed a SERS-based LFA kit in which the gold nanoparticles in the commercial rapid kit were replaced with SERS-active nano tags. It is possible to quantitatively detect the influenza virus A with high sensitivity by measuring the enhanced Raman signal of these SERS nano tags on the LFA strip. The limit of detection (LOD) using our proposed SERS-based LFA kit was estimated to be 1.9 × 10"4 PFU/mL, which is approximately one order of magnitude more sensitive than the LOD determined from the colorimetric LFA kit

  3. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    Energy Technology Data Exchange (ETDEWEB)

    Cary,; Bruce, R [Santa Fe, NM; Stubben, Christopher J [Los Alamos, NM

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  4. Teaching Phagocytosis Using Flow Cytometry

    Directory of Open Access Journals (Sweden)

    John Boothby

    2009-12-01

    Full Text Available Investigative microbiology on protists in a basic teaching laboratory environment is limited by student skill level, ease of microbial culture and manipulation, instrumentation, and time. The flow cytometer is gaining use as a mainstream instrument in research and clinical laboratories, but has had minimal application in teaching laboratories. Although the cost of a flow cytometer is currently prohibitive for many microbiology teaching environments and the number of trained instructors and teaching materials is limited, in many ways the flow cytometer is an ideal instrument for teaching basic microbiology. We report here on a laboratory module to study phagocytosis in Tetrahymena sp. using flow cytometry in a basic microbiology teaching laboratory. Students and instructors found the flow cytometry data analysis program, Paint-A-GatePRO-TM, to be very intuitive and easy to learn within a short period of time. Assessment of student learning about Tetrahymena sp., phagocytosis, flow cytometry, and investigative microbiology using an inquiry-based format demonstrated an overall positive response from students.

  5. Sorting catalytically active polymersome nanoreactors by flow cytometry

    NARCIS (Netherlands)

    Nallani, M.; Woestenenk, R.; de Hoog, H.P.M.; van Dongen, S.F.M.; Boezeman, J.; Cornelissen, J.J.L.M.; Nolte, R.J.M.; van Hest, J.C.M.

    2009-01-01

    A strategy that involves a versatile one-step preparation procedure of enzyme filled porous and stable polymeric catalytically active nanoreactors (polymersomes) by flow cytometry was reported. A 1:1 mixture of the polymerase dispersions was analyzed in a Coulter Epics Elite Flow Cytometer, while

  6. Evaluation of a multiple-cycle, recombinant virus, growth competition assay that uses flow cytometry to measure replication efficiency of human immunodeficiency virus type 1 in cell culture.

    Science.gov (United States)

    Dykes, Carrie; Wang, Jiong; Jin, Xia; Planelles, Vicente; An, Dong Sung; Tallo, Amanda; Huang, Yangxin; Wu, Hulin; Demeter, Lisa M

    2006-06-01

    Human immunodeficiency virus type 1 (HIV-1) replication efficiency or fitness, as measured in cell culture, has been postulated to correlate with clinical outcome of HIV infection, although this is still controversial. One limitation is the lack of high-throughput assays that can measure replication efficiency over multiple rounds of replication. We have developed a multiple-cycle growth competition assay to measure HIV-1 replication efficiency that uses flow cytometry to determine the relative proportions of test and reference viruses, each of which expresses a different reporter gene in place of nef. The reporter genes are expressed on the surface of infected cells and are detected by commercially available fluorescence-labeled antibodies. This method is less labor-intensive than those that require isolation and amplification of nucleic acids. The two reporter gene products are detected with similar specificity and sensitivity, and the proportion of infected cells in culture correlates with the amount of viral p24 antigen produced in the culture supernatant. HIV replication efficiencies of six different drug-resistant site-directed mutants were reproducibly quantified and were similar to those obtained with a growth competition assay in which the relative proportion of each variant was measured by sequence analysis, indicating that recombination between the pol and reporter genes was negligible. This assay also reproducibly quantified the relative fitness conferred by protease and reverse transcriptase sequences containing multiple drug resistance mutations, amplified from patient plasma. This flow cytometry-based growth competition assay offers advantages over current assays for HIV replication efficiency and should prove useful for the evaluation of patient samples in clinical trials.

  7. Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

    Science.gov (United States)

    Borrebaeck, C; Börjeson, J; Mattiasson, B

    1978-06-15

    Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

  8. Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay

    Science.gov (United States)

    Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

    2016-01-01

    The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 104 CFU mL−1 or 105 CFU mL−1 for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R2) of 0.916–0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water. PMID:26884128

  9. Air sampling to assess potential generation of aerosolized viable bacteria during flow cytometric analysis of unfixed bacterial suspensions

    Science.gov (United States)

    Carson, Christine F; Inglis, Timothy JJ

    2018-01-01

    This study investigated aerosolized viable bacteria in a university research laboratory during operation of an acoustic-assisted flow cytometer for antimicrobial susceptibility testing by sampling room air before, during and after flow cytometer use. The aim was to assess the risk associated with use of an acoustic-assisted flow cytometer analyzing unfixed bacterial suspensions. Air sampling in a nearby clinical laboratory was conducted during the same period to provide context for the existing background of microorganisms that would be detected in the air. The three species of bacteria undergoing analysis by flow cytometer in the research laboratory were Klebsiella pneumoniae, Burkholderia thailandensis and Streptococcus pneumoniae. None of these was detected from multiple 1000 L air samples acquired in the research laboratory environment. The main cultured bacteria in both locations were skin commensal and environmental bacteria, presumed to have been disturbed or dispersed in laboratory air by personnel movements during routine laboratory activities. The concentrations of bacteria detected in research laboratory air samples were reduced after interventional cleaning measures were introduced and were lower than those in the diagnostic clinical microbiology laboratory. We conclude that our flow cytometric analyses of unfixed suspensions of K. pneumoniae, B. thailandensis and S. pneumoniae do not pose a risk to cytometer operators or other personnel in the laboratory but caution against extrapolation of our results to other bacteria and/or different flow cytometric experimental procedures. PMID:29608197

  10. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

    Science.gov (United States)

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

    2015-01-01

    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

  11. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Directory of Open Access Journals (Sweden)

    Anna Grazia Recchia

    Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  12. Comparison of Lateral Flow Assay, Kidney Inhibition Swab, and Liquid Chromatography-Tandem Mass Spectrometry for the Detection of Penicillin G Residues in Sow Urine.

    Science.gov (United States)

    Shelver, Weilin L; Chakrabarty, Shubhashis; Smith, David J

    2017-03-01

    Sows (n = 126) were administered penicillin G; urine, collected at slaughter, was screened by kidney inhibition swab (KIS; 4 h testing time) and then stored at -80 °C (∼1200 days) until analysis by lateral flow assay (LF, ∼5 min testing time) and tandem quadrupole LC-MS/MS (TQ) analysis. The stability of penicillin in urine during storage was verified using TQ analyses. Quantitative results were well-correlated (R 2 = 0.98) with only a ∼10% decrease in penicillin concentration during the 3-year storage period. KIS retesting of stored samples returned results consistent with the original analyses. Lateral flow assay results were highly correlated with the KIS and TQ results. A KIS positive sample, which was not confirmed by TQ or LF, was assayed by Triple-TOF LC-MS to determine the cause of the apparent false positive. This study suggests LF can be used to quickly and efficiently screen for penicillin G residues before slaughter.

  13. Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

    Science.gov (United States)

    Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

    2018-01-01

    The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  14. A paper-polymer centrifugal device for low-cost sample pre-concentration and colorimetric lateral flow assay enhancement

    CSIR Research Space (South Africa)

    Wiederoder, MS

    2016-10-01

    Full Text Available This study describes a novel hybrid paper-polymer centrifugal microfluidic device for pre-concentration of E.coli and lateral flow immunoassay enhancement for water quality verification. The device balances rotational centrifugal force...

  15. Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

    Science.gov (United States)

    Hommatsu, Manami; Okahashi, Hisamitsu; Ohta, Keisuke; Tamai, Yusuke; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2013-01-01

    A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.

  16. Multi-color CD34⁺ progenitor-focused flow cytometric assay in evaluation of myelodysplastic syndromes in patients with post cancer therapy cytopenia.

    Science.gov (United States)

    Tang, Guilin; Jorgensen, L Jeffrey; Zhou, Yi; Hu, Ying; Kersh, Marian; Garcia-Manero, Guillermo; Medeiros, L Jeffrey; Wang, Sa A

    2012-08-01

    Bone marrow assessment for myelodysplastic syndrome (MDS) in a patient who develops cytopenia(s) following cancer therapy is challenging. With recent advances in multi-color flow cytometry immunophenotypic analysis, a CD34(+) progenitor-focused 7-color assay was developed and tested in this clinical setting. This assay was first performed in 73 MDS patients and 53 non-MDS patients (developmental set). A number of immunophenotypic changes were differentially observed in these two groups. Based on the sensitivity, specificity and reproducibility, a core panel of markers was selected for final assessment that included increased total CD34(+) myeloblasts; decreased stage I hematogones; altered CD45/side scatter; altered expression of CD13, CD33, CD34, CD38, CD117, and CD123; aberrant expression of lymphoid or mature myelomonocytic antigens on CD34(+) myeloblasts; and several marked alterations in maturing myelomonocytic cells. The data were translated into a simplified scoring system which was then used in 120 patients with cytopenia(s) secondary to cancer therapy over a 2-year period (validation set). With a median follow-up of 11 months, this assay demonstrated 89% sensitivity, 94% specificity, and 92% accuracy in establishing or excluding a diagnosis of MDS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. A micro flow cytometry system for study of marine phytoplankton from costal waters of Hong Kong

    KAUST Repository

    Yunyang Ling,

    2010-01-01

    Although conventional flow cytometers (CFCs) have been widely used for study of marine biology, most CFCs are too bulky to be used for field study in ocean and have corrosion problem due to salty samples. A new computer-controlled micro flow cytometer (MFC) system has been successfully developed using MEMS technology. We demonstrate that this new MFC can analyze mixture of two species of marine phytoplankton: Chlorella autotrophica and Rhodomonas. The results from our MFC are consistent with those from digital fluorescence microscopy. ©2010 IEEE.

  18. Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

    Science.gov (United States)

    Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

    2016-12-27

    The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

  19. Water excretion mechanisms of the kidney studied in the rabbit using tritiated water during the stop-flow assay

    International Nuclear Information System (INIS)

    Morel, F.; Amiel, CI.; Falbriard, A.

    1960-01-01

    The pattern of water turnover in the kidney and the mechanisms of water transfer into the urine have been studied in the rabbit using tritiated water as a tracer and the stop-flow technique. The experiments have given the following results: a) During the interruption of the diuresis, the injected tritiated water is completely exchanged with the water of the renal cortex, but the tracer does not reach the deep regions of the kidney, despite the fact that the blood circulation is maintained in these regions; this suggests that the vascular loops of the vasa recta function as a mechanism of water exchange by countercurrent. b) During the osmotic polyuria following the stop-flow period, the concentration gradient of tritiated water inside the kidney diminishes progressively. The concentration of the tracer in the urine is at all time similar to that existing in the deep medulla and the renal papilla and markedly different from that of the cortex or arterial blood. This fact shows that the molecules of water in the urine excreted do not come from either the glomerular filtrate or the convoluted tubules but from the water contained in the deep regions of the kidney. Also these results indicate that the walls of the collecting ducts have a very high permeability to water diffusion. Reprint of a paper published in Revue francaise d'etudes cliniques et biologiques, Vol. IV, no. 8, p. 773-779, 1959 [fr

  20. Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus

    Science.gov (United States)

    Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

    2001-05-01

    Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

  1. Copper (II) and zinc (II) complexes with flavanone derivatives: Identification of potential cholinesterase inhibitors by on-flow assays.

    Science.gov (United States)

    Sarria, André Lucio Franceschini; Vilela, Adriana Ferreira Lopes; Frugeri, Bárbara Mammana; Fernandes, João Batista; Carlos, Rose Maria; da Silva, Maria Fátima das Graças Fernandes; Cass, Quezia Bezerra; Cardoso, Carmen Lúcia

    2016-11-01

    Metal chelates strongly influence the nature and magnitude of pharmacological activities in flavonoids. In recent years, studies have shown that a promising class of flavanone-metal ion complexes can act as selective cholinesterase inhibitors (ChEIs), which has led our group to synthesize a new series of flavanone derivatives (hesperidin, hesperetin, naringin, and naringenin) complexed to either copper (II) or zinc (II) and to evaluate their potential use as selective ChEIs. Most of the synthesized complexes exhibited greater inhibitory activity against acetylcholinesterase (AChE) than against butyrylcholinesterase (BChE). Nine of these complexes constituted potent, reversible, and selective ChEIs with inhibitory potency (IC 50 ) and inhibitory constant (K i ) ranging from 0.02 to 4.5μM. Copper complexes with flavanone-bipyridine derivatives afforded the best inhibitory activity against AChE and BChE. The complex Cu(naringin)(2,2'-bipyridine) (11) gave IC 50 and K i values of 0.012±0.002 and 0.07±0.01μM for huAChE, respectively, which were lower than the inhibitory values obtained for standard galanthamine (IC 50 =206±30.0 and K i =126±18.0μM). Evaluation of the inhibitory activity of this complex against butyrylcholinesterase from human serum (huBChE) gave IC 50 and K i values of 8.0±1.4 and 2.0±0.1μM, respectively. A Liquid Chromatography-Immobilized Capillary Enzyme Reactor by UV detection (LC-ICER-UV) assay allowed us to determine the IC 50 and K i values and the type of mechanism for the best inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Determination of CD4+ and CD8+ lymphocytes with the cytosphere assay: a comparative study with flow cytometry and the immunoalkaline phosphatase method

    DEFF Research Database (Denmark)

    Gernow, A; Lisse, I M; Böttiger, B

    1995-01-01

    number of CD4+ lymphocytes determined by CA showed a good correlation with results obtained by FC (correlation coefficients were 0.92 and 0.74 in Denmark and The Ivory Coast, respectively) and IA (correlation coefficients were 0.94 and 0.66 in Denmark and The Ivory Coast, respectively). However, for HIV......The proportion and absolute numbers of CD4+ and CD8+ lymphocytes in peripheral blood were determined using a new manual method, the cytosphere assay (CA). This method uses small latex beads coated with monoclonal antibodies directed against the CD4 and CD8 receptors, respectively. The CA...... was compared with two other methods for determination of T lymphocyte subsets, flow cytometry (FC) and the immunoalkaline phosphatase (IA) method, by testing HIV-seropositive and HIV-seronegative samples from Denmark (44) and Ivory Coast (79). For HIV-seropositive samples, both the proportion and the absolute...

  3. Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

    Science.gov (United States)

    Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

    2011-07-18

    Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

  4. Spectrophotometric flow-injection analysis assay of tetracycline antibiotics using a dual light-emitting diode based detector

    Directory of Open Access Journals (Sweden)

    Prinya Masawat

    2008-02-01

    Full Text Available In this paper, a small dual light-emitting diode (LED-based detector for FIA process analyser has been designed. The detector’s optical parts comprise a flow-through cell, a dual-blue LED and a photodiode. Neither mirrors nor lenses are used. The optical path for the first LED detects the blank, while the other LED detects the sample. The detector’s electronic components including a signal amplifier and an A/D converter are integrated on one small board connected to a PC for measuring the results. The designed spectrophotometric detector was used for the determination of tetracycline antibiotics. Uranyl acetate was used as a reagent forming orange-red complexes with the drugs in N,N– dimethylformamide. The complexes show absorption maxima at 410, 416 and 408 nm for tetracycline hydrochloride (TCH, chlortetracycline hydrochloride (CTCH, and doxycycline hydrochloride (DCH, respectively. The detection limit was found to be 0.38, 0.75, 1.44 µg mL-1 and the linear range was obtained at 1.0-3.0, 3.0-5.0, and 3.0-10.0 µg mL-1 for TCH, CTCH and DCH, respectively. The proposed method has been successfully applied to the determination of tetracycline antibiotic residues in milk samples. Moreover, this method is an environmentally friendly approach and suitable for routine analysis.

  5. Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

    Science.gov (United States)

    Bradford, Jolene A; Buller, Gayle; Suter, Michael; Ignatius, Michael; Beechem, Joseph M

    2004-10-01

    Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon

  6. Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2

    International Nuclear Information System (INIS)

    Barbezan, Angélica B.; Santos, Carla J.B.; Carvalho, Luma R.; Vieira, Daniel P.; Villavicêncio, Anna L.C.H.; Santelli, Glaucia M.M.

    2017-01-01

    Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line. (author)

  7. Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2

    Energy Technology Data Exchange (ETDEWEB)

    Barbezan, Angélica B.; Santos, Carla J.B.; Carvalho, Luma R.; Vieira, Daniel P.; Villavicêncio, Anna L.C.H., E-mail: abarbezan@ipen.br [Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil); Santelli, Glaucia M.M. [Universidade de São Paulo (USP), SP (Brazil). Departamento de Biologia Celular e do Desenvolvimento

    2017-07-01

    Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line. (author)

  8. Comparison of a flow assay for brucellosis antibodies with the reference cELISA test in West African Bos indicus.

    Directory of Open Access Journals (Sweden)

    Barend M deC Bronsvoort

    Full Text Available Brucellosis is considered by the Food and Agricultural Organisation and the World Health Organisation as one of the most widespread zoonoses in the world. It is a major veterinary public health challenge as animals are almost exclusively the source of infection for people. It is often undiagnosed in both human patients and the animal sources and it is widely acknowledged that the epidemiology of brucellosis in humans and animals is poorly understood, particularly in sub-Saharan Africa. It is therefore important to develop better diagnostic tools in order to improve our understanding of the epidemiology and also for use in the field for disease control and eradication. As with any new diagnostic test, it is essential that it is validated in as many populations as possible in order to characterise its performance and improve the interpretation of its results. This paper describes a comparison between a new lateral flow assasy (LFA for bovine brucellosis and the widely used cELISA in a no gold standard analysis to estimate test performance in this West African cattle population. A Bayesian formulation of the Hui-Walter latent class model incorporated previous studies' data on sensitivity and specificity of the cELISA. The results indicate that the new LFA is very sensitive (approximately 87% and highly specific (approximately 97%. The analysis also suggests that the current cut-off of the cELSIA may not be optimal for this cattle population but alternative cut-offs did not significantly change the estimates of the LFA. This study demonstrates the potential usefulness of this simple to use test in field based surveillance and control which could be easily adopted for use in developing countries with only basic laboratory facilities.

  9. Comparison of a Flow Assay for Brucellosis Antibodies with the Reference cELISA Test in West African Bos indicus

    Science.gov (United States)

    Bronsvoort, Barend M. deC.; Koterwas, Bronwyn; Land, Fiona; Handel, Ian G.; Tucker, James; Morgan, Kenton L.; Tanya, Vincent N.; Abdoel, Theresia H.; Smits, Henk L.

    2009-01-01

    Brucellosis is considered by the Food and Agricultural Organisation and the World Health Organisation as one of the most widespread zoonoses in the world. It is a major veterinary public health challenge as animals are almost exclusively the source of infection for people. It is often undiagnosed in both human patients and the animal sources and it is widely acknowledged that the epidemiology of brucellosis in humans and animals is poorly understood, particularly in sub-Saharan Africa. It is therefore important to develop better diagnostic tools in order to improve our understanding of the epidemiology and also for use in the field for disease control and eradication. As with any new diagnostic test, it is essential that it is validated in as many populations as possible in order to characterise its performance and improve the interpretation of its results. This paper describes a comparison between a new lateral flow assasy (LFA) for bovine brucellosis and the widely used cELISA in a no gold standard analysis to estimate test performance in this West African cattle population. A Bayesian formulation of the Hui-Walter latent class model incorporated previous studies' data on sensitivity and specificity of the cELISA. The results indicate that the new LFA is very sensitive (∼87%) and highly specific (∼97%). The analysis also suggests that the current cut-off of the cELSIA may not be optimal for this cattle population but alternative cut-offs did not significantly change the estimates of the LFA. This study demonstrates the potential usefulness of this simple to use test in field based surveillance and control which could be easily adopted for use in developing countries with only basic laboratory facilities. PMID:19381332

  10. A Raman Flow Cytometer: An Innovative Microfluidic Approach for Continuous Label-Free Analysis of Cells via Raman Spectroscopy

    KAUST Repository

    De Grazia, Antonio

    2015-01-01

    of calcium fluoride (which gives a negligible signal in Raman analyses), a photosensitive resist containing a pattern with channels and another slide of calcium fluoride in order for the channels to be sealed on both sides. The chip is, in turn, connected

  11. Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM).

    Science.gov (United States)

    Lee, Yong Sun; Yi, Jung-Sun; Seo, Souk Jin; Kim, Joo Hwan; Jung, Mi-Sook; Seo, Im-Kwon; Ahn, Ilyoung; Ko, Kyungyuk; Kim, Tae Sung; Lim, Kyung Min; Sohn, Soojung

    2017-02-01

    The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

    DEFF Research Database (Denmark)

    Nielsen, Morten Hjuler; Beck-Nielsen, Henning; Andersen, Morten Nørgaard

    2014-01-01

    BACKGROUND AND AIM: Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold...

  13. Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

    Science.gov (United States)

    Page, Brent T; Shields, Christine E; Merz, William G; Kurtzman, Cletus P

    2006-09-01

    This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

  14. Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

    Science.gov (United States)

    Lee, Jong-Hwan; Seo, Hyuk Seong; Kwon, Jung-Hyuk; Kim, Hee-Tae; Kwon, Koo Chul; Sim, Sang Jun; Cha, Young Joo; Lee, Jeewon

    2015-07-15

    Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

    Directory of Open Access Journals (Sweden)

    Jose E. VIDAL

    2015-09-01

    Full Text Available SUMMARYAIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex or enzyme-linked immunoassay (EIA has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered. CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcusspecies. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die.

  16. Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

    Science.gov (United States)

    Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

    2017-06-28

    Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization

  17. Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

    Directory of Open Access Journals (Sweden)

    Michael Degtyarev

    Full Text Available Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication, takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.

  18. Effective delivery of hydrophobic drugs to breast (MCF-7) and Liver (HepG2) cancer cells: A detailed investigation using Cytotoxicity assays, fluorescence imaging and flow cytometry.

    Science.gov (United States)

    Manatunga, Danushika C; de Silva, Rohini M; Nalin de Silva, K M; Neelika Malavige, Gathsaurie; Wijeratne, Dulharie T; Williams, Gareth R; Jayasinghe, Chanika D; Udagama, Preethi V

    2018-04-03

    This study aimed to develop a drug carrier system consisting of a polymer containing hydroxyapatite (HAp) shell and a magnetic core of iron oxide nanoparticles. Doxorubicin and/or curcumin were loaded into the carrier via a simple diffusion deposition approach, with encapsulation efficiencies (EE) for curcumin and doxorubicin of 93.03 ± 0.3% and 97.37 ± 0.12% respectively. The co-loading of curcumin and doxorubicin led to a total EE of 76.02 ± 0.48%. Release studies were carried out at pH 7.4 and 5.3, and revealed higher release was at pH 5.3 expressing the potential application in tumor microenvironments. Cytotoxicity assays, fluorescence imaging and flow cytometry showed the formulations could effectively inhibit the growth of MCF-7 and HEpG2 cancer cells, being more potent than the free drug molecules both in dose and time dependent manner. Additionally, hemolysis tests and cytotoxicity evaluations determined the drug-loaded carriers to be non-toxic towards non-cancerous cells. These formulations thus have great potential in the development of new cancer therapeutics. Copyright © 2018. Published by Elsevier B.V.

  19. A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing.

    Science.gov (United States)

    Yurinskaya, Valentina; Aksenov, Nikolay; Moshkov, Alexey; Model, Michael; Goryachaya, Tatyana; Vereninov, Alexey

    2017-10-01

    A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.

  20. Asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised HIV-infected patients in São Paulo, Brazil.

    Science.gov (United States)

    Vidal, José E; Toniolo, Carolina; Paulino, Adriana; Colombo, Arnaldo; Dos Anjos Martins, Marilena; da Silva Meira, Cristina; Pereira-Chioccola, Vera Lucia; Figueiredo-Mello, Claudia; Barros, Tiago; Duarte, Jequelie; Fonseca, Fernanda; Alves Cunha, Mirella; Mendes, Clara; Ribero, Taiana; Dos Santos Lazera, Marcia; Rajasingham, Radha; Boulware, David R

    2016-12-01

    To determine the prevalence of asymptomatic cryptococcal antigen (CRAG) using lateral flow assay (LFA) in hospitalised HIV-infected patients with CD4 counts 18 years old without prior cryptococcal meningitis, without clinical suspicion of cryptococcal meningitis, regardless of antiretroviral (ART) status, and with CD4 counts <200 cells/μl. Serum CRAG was tested by LFA in all patients, and whole blood CRAG was tested by LFA in positive cases. We enrolled 163 participants of whom 61% were men. The duration of HIV diagnosis was a median of 8 (range, 1-29) years. 26% were antiretroviral (ART)-naïve, and 74% were ART-experienced. The median CD4 cell count was 25 (range, 1-192) cells/μl. Five patients (3.1%; 95%CI, 1.0-7.0%) were asymptomatic CRAG-positive. Positive results cases were cross-verified by performing LFA in whole blood. 3.1% of HIV-infected inpatients with CD4 <200 cells/μl without symptomatic meningitis had cryptococcal antigenemia in São Paulo, suggesting that routine CRAG screening may be beneficial in similar settings in South America. Our study reveals another targeted population for CRAG screening: hospitalised HIV-infected patients with CD4 <200 cells/μl, regardless of ART status. Whole blood CRAG LFA screening seems to be a simple strategy to prevention of symptomatic meningitis. © 2016 John Wiley & Sons Ltd.

  1. Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

    Science.gov (United States)

    SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

    2013-01-01

    ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

  2. Utilizing the ultrasensitive Schistosoma up-converting phosphor lateral flow circulating anodic antigen (UCP-LF CAA) assay for sample pooling-strategies.

    Science.gov (United States)

    Corstjens, Paul L A M; Hoekstra, Pytsje T; de Dood, Claudia J; van Dam, Govert J

    2017-11-01

    Methodological applications of the high sensitivity genus-specific Schistosoma CAA strip test, allowing detection of single worm active infections (ultimate sensitivity), are discussed for efficient utilization in sample pooling strategies. Besides relevant cost reduction, pooling of samples rather than individual testing can provide valuable data for large scale mapping, surveillance, and monitoring. The laboratory-based CAA strip test utilizes luminescent quantitative up-converting phosphor (UCP) reporter particles and a rapid user-friendly lateral flow (LF) assay format. The test includes a sample preparation step that permits virtually unlimited sample concentration with urine, reaching ultimate sensitivity (single worm detection) at 100% specificity. This facilitates testing large urine pools from many individuals with minimal loss of sensitivity and specificity. The test determines the average CAA level of the individuals in the pool thus indicating overall worm burden and prevalence. When requiring test results at the individual level, smaller pools need to be analysed with the pool-size based on expected prevalence or when unknown, on the average CAA level of a larger group; CAA negative pools do not require individual test results and thus reduce the number of tests. Straightforward pooling strategies indicate that at sub-population level the CAA strip test is an efficient assay for general mapping, identification of hotspots, determination of stratified infection levels, and accurate monitoring of mass drug administrations (MDA). At the individual level, the number of tests can be reduced i.e. in low endemic settings as the pool size can be increased as opposed to prevalence decrease. At the sub-population level, average CAA concentrations determined in urine pools can be an appropriate measure indicating worm burden. Pooling strategies allowing this type of large scale testing are feasible with the various CAA strip test formats and do not affect

  3. Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

    Science.gov (United States)

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sahul Hameed, A S; Paknikar, Kishore M

    2017-06-01

    White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (K d ,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

  4. B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry.

    Science.gov (United States)

    Jung, Kyoung-Mi; Jang, Won-Hee; Lee, Yong-Kyoung; Yum, Young Na; Sohn, Soojung; Kim, Bae-Hwan; Chung, Jin-Ho; Park, Young-Ho; Lim, Kyung-Min

    2012-03-25

    Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, (3)H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220+ and CD3e+. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  5. Predictive capacity of a non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: Comparison of a cutoff approach and inferential statistics.

    Science.gov (United States)

    Kim, Da-Eun; Yang, Hyeri; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Choi, Jin Kyu; Jung, Mi-Sook; Jeon, Eun-Young; Heo, Yong; Yeo, Kyung-Wook; Jo, Ji-Hoon; Park, Jung Eun; Sohn, Soo Jung; Kim, Tae Sung; Ahn, Il Young; Jeong, Tae-Cheon; Lim, Kyung-Min; Bae, SeungJin

    2016-01-01

    In order for a novel test method to be applied for regulatory purposes, its reliability and relevance, i.e., reproducibility and predictive capacity, must be demonstrated. Here, we examine the predictive capacity of a novel non-radioisotopic local lymph node assay, LLNA:BrdU-FCM (5-bromo-2'-deoxyuridine-flow cytometry), with a cutoff approach and inferential statistics as a prediction model. 22 reference substances in OECD TG429 were tested with a concurrent positive control, hexylcinnamaldehyde 25%(PC), and the stimulation index (SI) representing the fold increase in lymph node cells over the vehicle control was obtained. The optimal cutoff SI (2.7≤cutoff <3.5), with respect to predictive capacity, was obtained by a receiver operating characteristic curve, which produced 90.9% accuracy for the 22 substances. To address the inter-test variability in responsiveness, SI values standardized with PC were employed to obtain the optimal percentage cutoff (42.6≤cutoff <57.3% of PC), which produced 86.4% accuracy. A test substance may be diagnosed as a sensitizer if a statistically significant increase in SI is elicited. The parametric one-sided t-test and non-parametric Wilcoxon rank-sum test produced 77.3% accuracy. Similarly, a test substance could be defined as a sensitizer if the SI means of the vehicle control, and of the low, middle, and high concentrations were statistically significantly different, which was tested using ANOVA or Kruskal-Wallis, with post hoc analysis, Dunnett, or DSCF (Dwass-Steel-Critchlow-Fligner), respectively, depending on the equal variance test, producing 81.8% accuracy. The absolute SI-based cutoff approach produced the best predictive capacity, however the discordant decisions between prediction models need to be examined further. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    International Nuclear Information System (INIS)

    Tanner, Scott D.; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I.

    2007-01-01

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration

  7. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    Energy Technology Data Exchange (ETDEWEB)

    Tanner, Scott D. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)], E-mail: sd.tanner@utoronto.ca; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)

    2007-03-15

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

  8. High throughput imaging cytometer with acoustic focussing† †Electronic supplementary information (ESI) available: High throughput imaging cytometer with acoustic focussing. See DOI: 10.1039/c5ra19497k Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

    Science.gov (United States)

    Zmijan, Robert; Jonnalagadda, Umesh S.; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn

    2015-01-01

    We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint. PMID:29456838

  9. Predictive value of the flow cytometric PCNA - assay (proliferating cell nuclear antigen) in head and neck tumors after accelerated-hyperfractionated radiochemotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Wenz, F; Lohr, F; Rudat, V; Dietz, A; Flentje, M; Wannenmacher, M

    1995-07-01

    Purpose/Objective: Proliferation of surviving tumor cells during fractionated radiotherapy may limit tumor control, especially in rapidly proliferating tumors. It has been widely accepted, that this may play a major role in head and neck tumors. Several methods for the assessment of tumor proliferation have been developed, however, most of them are either laborious, invasive or potentially toxic. Today, the gold standard is the flow cytometric BrdUrd assay. We present a flow cytometric method for detection of PCNA, which is an intranuclear proliferation associated protein, in solid human head and neck tumors and how these data correlate with outcome. Materials and Methods: Pretherapeutic biopsies of 20 inoperable patients with squamous cell carcinoma of the head and neck (T3-4N2M0) were examined. The tissue was disaggregated with pepsin/HCl, antibody staining was performed using the clone PC10. Biparametric flow cytometry was performed after a FITC conjugated secondary antibody and propidiumjodine staining was applied. The PCNA-index (i.e. percentage PCNA-positive cells), the DNA-index and the S-phase fraction (SPF, euploid tumors only) were determined. The therapy consisted of combined accelerated-hyperfractionated radiochemotherapy (66 Gy in 5 wks, concomittant boost of 1.6 Gy/d in wks 4+5, Carboplatin in wks 1+5). The median follow-up time was 14 mths (5 - 28), the clinical partners (V.R., A.D.) were 'blinded' towards the PCNA-values. Results: 13 patients suffered from disease progession and 11 died. The actuarial median survival and disease free survival (DFS) were 14.4 and 10.7 mths, respectively. The PCNA-values ranged from 3.2 to 70% (median 9%), there were 7 aneuploid and 13 euploid tumors. SFP in the euploid tumors ranged from 4 to 14.5% (median 10.5%). Neither SFP nor ploidy had a significant influence on the outcome. The patients were divided according to their PCNA-value in higher (n=10) and lower (n=10) than the median. The survival and DFS were 13

  10. The use of flow cytometry in radiation biology

    International Nuclear Information System (INIS)

    Szekely, J.G.; Raaphorst, G.P.; Lobreau, A.U.; Einspenner, M.; Sargent, M.; Azzam, E.I.

    1989-09-01

    The flow cytometer has been used in a number of projects at the Whiteshell Nuclear Research Establishment, Pinawa, Manitoba, Canada. In this report we have summarized the methods and results obtained in the cellular radiobiology program. The techniques used in the program included live/dead analysis in lymphocytes, identification of bone-marrow subsets, chromosome analysis, cell-cycle analysis, cell sorting and the quantification of surface antigens

  11. [Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

    Science.gov (United States)

    Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

    2015-01-01

    To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance

  12. Amphiphilic mediated sample preparation for micro-flow cytometry

    Science.gov (United States)

    Clague, David S [Livermore, CA; Wheeler, Elizabeth K [Livermore, CA; Lee, Abraham P [Irvine, CA

    2009-03-17

    A flow cytometer includes a flow cell for detecting the sample, an oil phase in the flow cell, a water phase in the flow cell, an oil-water interface between the oil phase and the water phase, a detector for detecting the sample at the oil-water interface, and a hydrophobic unit operatively connected to the sample. The hydrophobic unit is attached to the sample. The sample and the hydrophobic unit are placed in an oil and water combination. The sample is detected at the interface between the oil phase and the water phase.

  13. Mechanism and kinetics of the loss of poorly soluble drugs from liposomal carriers studied by a novel flow field-flow fractionation-based drug release-/transfer-assay.

    Science.gov (United States)

    Hinna, Askell Hvid; Hupfeld, Stefan; Kuntsche, Judith; Bauer-Brandl, Annette; Brandl, Martin

    2016-06-28

    Liposomes represent a versatile drug formulation approach e.g. for improving the water-solubility of poorly soluble drugs but also to achieve drug targeting and controlled release. For the latter applications it is essential that the drug remains associated with the liposomal carrier during transit in the vascular bed. A range of in vitro test methods has been suggested over the years for prediction of the release of drug from liposomal carriers. The majority of these fail to give a realistic prediction for poorly water-soluble drugs due to the intrinsic tendency of such compounds to remain associated with liposome bilayers even upon extensive dilution. Upon i.v. injection, in contrast, rapid drug loss often occurs due to drug transfer from the liposomal carriers to endogenous lipophilic sinks such as lipoproteins, plasma proteins or membranes of red blood cells and endothelial cells. Here we report on the application of a recently introduced in vitro predictive drug transfer assay based on incubation of the liposomal drug carrier with large multilamellar liposomes, the latter serving as a biomimetic model sink, using flow field-flow fractionation as a tool to separate the two types of liposomes. By quantifying the amount of drug remaining associated with the liposomal drug carrier as well as that transferred to the acceptor liposomes at distinct times of incubation, both the kinetics of drug transfer and release to the water phase could be established for the model drug p-THPP (5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine). p-THPP is structurally similar to temoporfin, a photosensitizer which is under clinical evaluation in a liposomal formulation. Mechanistic insights were gained by varying the donor-to-acceptor lipid mass ratio, size and lamellarity of the liposomes. Drug transfer kinetics from one liposome to another was found rate determining as compared to redistribution from the outermost to the inner concentric bilayers, such that the overall

  14. An assessment of software for flow cytometry analysis in banana plants

    Directory of Open Access Journals (Sweden)

    Renata Alves Lara Silva

    2014-02-01

    Full Text Available Flow cytometry is a technique that yields rapid results in analyses of cell properties such as volume, morphological complexity and quantitative DNA content, and it is considered more convenient than other techniques. However, the analysis usually generates histograms marked by variations that can be produced by many factors, including differences between the software packages that capture the data generated by the flow cytometer. The objective of the present work was to evaluate the performance of four software products commonly used in flow cytometry based on quantifications of DNA content and analyses of the coefficients of variation associated with the software outputs. Readings were obtained from 25 ‘NBA’ (AA banana leaf samples using the FACSCalibur (BD flow cytometer, and 25 histograms from each software product (CellQuest™, WinMDI™, FlowJo™ and FCS Express™ were analyzed to obtain the estimated DNA content and the coefficient of variation (CV of the estimates. The values of DNA content obtained from the software did not differ significantly. However, the CV analysis showed that the precision of the WinMDI™ software was low and that the CV values were underestimated, whereas the remaining software showed CV values that were in relatively close agreement with those found in the literature. The CellQuest™ software is recommended because it was developed by the same company that produces the flow cytometer used in the present study.

  15. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  16. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  17. A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer.

    Science.gov (United States)

    Cribbes, Scott; Kessel, Sarah; McMenemy, Scott; Qiu, Jean; Chan, Leo Li-Ying

    2017-06-01

    Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.

  18. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  19. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert

    2016-01-01

    ) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

  20. Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes.

    Science.gov (United States)

    Telford, William G

    2004-09-01

    Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370-375-nm range have been evaluated as laser sources for cuvette-based flow cytometers. Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1. NUVLDs at the 8-10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8-10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources. These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments. Copyright 2004 Wiley-Liss, Inc.

  1. Automated Blood Sample Preparation Unit (ABSPU) for Portable Microfluidic Flow Cytometry.

    Science.gov (United States)

    Chaturvedi, Akhil; Gorthi, Sai Siva

    2017-02-01

    Portable microfluidic diagnostic devices, including flow cytometers, are being developed for point-of-care settings, especially in conjunction with inexpensive imaging devices such as mobile phone cameras. However, two pervasive drawbacks of these have been the lack of automated sample preparation processes and cells settling out of sample suspensions, leading to inaccurate results. We report an automated blood sample preparation unit (ABSPU) to prevent blood samples from settling in a reservoir during loading of samples in flow cytometers. This apparatus automates the preanalytical steps of dilution and staining of blood cells prior to microfluidic loading. It employs an assembly with a miniature vibration motor to drive turbulence in a sample reservoir. To validate performance of this system, we present experimental evidence demonstrating prevention of blood cell settling, cell integrity, and staining of cells prior to flow cytometric analysis. This setup is further integrated with a microfluidic imaging flow cytometer to investigate cell count variability. With no need for prior sample preparation, a drop of whole blood can be directly introduced to the setup without premixing with buffers manually. Our results show that integration of this assembly with microfluidic analysis provides a competent automation tool for low-cost point-of-care blood-based diagnostics.

  2. Sample handling for kinetics and molecular assembly in flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Sklar, L.A. [Los Alamos National Lab., NM (United States). National Flow Cytometry Resource]|[Univ. of New Mexico, Albuquerque, NM (United States). School of Medicine; Seamer, L.C.; Kuckuck, F.; Prossnitz, E.; Edwards, B. [Univ. of New Mexico, Albuquerque, NM (United States). School of Medicine; Posner, G. [Northern Arizona Univ., Flagstaff, AZ (United States). Dept. of Chemistry

    1998-07-01

    Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. The authors have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, the authors used fast valves to overcome the disparity between mixing 100 {micro}ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 {micro}l/sec. Particles were detected within 100 msec after mixing, but turbulence was created which lasted for 1 sec after injection of the sample into the flow cytometer. They used optical criteria to discriminate particles which were out of alignment due to the turbulent flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system they were able to automate sample handling and delivery with intervals of a few seconds. The authors used a fluidic approach to defeat turbulence caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of turbulence was reduced to {approximately} 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.

  3. Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

    International Nuclear Information System (INIS)

    Ma Yongjun; Zhou Min; Jin Xiaoyong; Zhang Ziyu; Teng Xiulan; Chen Hui

    2004-01-01

    A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0x10 -8 to 0.1 μg ml -1 for herring sperm DNA and 2.0x10 -6 to 0.2 μg ml -1 for calf thymus DNA with 3σ detection limits of 8.3x10 -9 μg ml -1 for herring sperm DNA and 3.5x10 -7 μg ml -1 for calf thymus DNA, respectively. The relative standard deviation for 1.0x10 -4 μg ml -1 herring sperm DNA was 0.99% and 2.0x10 -3 μg ml -1 for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper

  4. Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

    Energy Technology Data Exchange (ETDEWEB)

    Ma Yongjun; Zhou Min; Jin Xiaoyong; Zhang Ziyu; Teng Xiulan; Chen Hui

    2004-01-09

    A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0x10{sup -8} to 0.1 {mu}g ml{sup -1} for herring sperm DNA and 2.0x10{sup -6} to 0.2 {mu}g ml{sup -1} for calf thymus DNA with 3{sigma} detection limits of 8.3x10{sup -9} {mu}g ml{sup -1} for herring sperm DNA and 3.5x10{sup -7} {mu}g ml{sup -1} for calf thymus DNA, respectively. The relative standard deviation for 1.0x10{sup -4} {mu}g ml{sup -1} herring sperm DNA was 0.99% and 2.0x10{sup -3} {mu}g ml{sup -1} for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper.

  5. Flow cytometry and integrated imaging

    Directory of Open Access Journals (Sweden)

    V. Kachel

    2000-06-01

    Full Text Available It is a serious problem to relate the results of a flow cytometric analysis of a marine sample to different species. Images of particles selectively triggered by the flow cytometric analysis and picked out from the flowing stream give a valuable additional information on the analyzed organisms. The technical principles and problems of triggered imaging in flow are discussed, as well as the positioning of the particles in the plane of focus, freezing the motion of the quickly moving objects and what kinds of light sources are suitable for pulsed illumination. The images have to be stored either by film or electronically. The features of camera targets and the memory requirements for storing the image data and the conditions for the triggering device are shown. A brief explanation of the features of three realized flow cytometric imaging (FCI systems is given: the Macro Flow Planktometer built within the EUROMAR MAROPT project, the Imaging Module of the European Plankton Analysis System, supported by the MAST II EurOPA project and the most recently developed FLUVO VI universal flow cytometer including HBO 100- and laser excitation for fluorescence and scatter, Coulter sizing as well as bright field and and phase contrast FCI.

  6. A Microfluidic Cytometer for Complete Blood Count With a 3.2-Megapixel, 1.1- μm-Pitch Super-Resolution Image Sensor in 65-nm BSI CMOS.

    Science.gov (United States)

    Liu, Xu; Huang, Xiwei; Jiang, Yu; Xu, Hang; Guo, Jing; Hou, Han Wei; Yan, Mei; Yu, Hao

    2017-08-01

    Based on a 3.2-Megapixel 1.1- μm-pitch super-resolution (SR) CMOS image sensor in a 65-nm backside-illumination process, a lens-free microfluidic cytometer for complete blood count (CBC) is demonstrated in this paper. Backside-illumination improves resolution and contrast at the device level with elimination of surface treatment when integrated with microfluidic channels. A single-frame machine-learning-based SR processing is further realized at system level for resolution correction with minimum hardware resources. The demonstrated microfluidic cytometer can detect the platelet cells (< 2 μm) required in CBC, hence is promising for point-of-care diagnostics.

  7. Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

    Science.gov (United States)

    Carulli, Giovanni; Marini, Alessandra; Sammuri, Paola; Domenichini, Cristiana; Ottaviano, Virginia; Pacini, Simone; Petrini, Mario

    2015-01-01

    The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

  8. Lateral Flow Urine Lipoarabinomannan Assay (LF-LAM) for Diagnosis of Active Tuberculosis in HIV-Infected Adults: a Prospective Cohort Study

    Science.gov (United States)

    Na Songkhla, Munjit; Tantipong, Hutsaya; Tongsai, Sasima; Angkasekwinai, Nasikarn

    2017-01-01

    result. Urine LAM strip test can remain positive for up to 4 weeks even after anti-TB treatment. Conclusion Urine LAM assay gave the best performance for diagnosis of active TB in advanced HIV-infected patients and provide an additional benefit of a greater simplicity, speed, with a more easily obtainable sample. Disclosures All authors: No reported disclosures.

  9. Simultaneous ultrasound and photoacoustics based flow cytometry

    Science.gov (United States)

    Gnyawali, Vaskar; Strohm, Eric M.; Tsai, Scott S. H.; Kolios, Michael C.

    2018-04-01

    We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.

  10. Automated urinalysis: first experiences and a comparison between the Iris iQ200 urine microscopy system, the Sysmex UF-100 flow cytometer and manual microscopic particle counting

    OpenAIRE

    Shayanfar, Noushin; Tobler, Ulrich; von Eckardstein, Arnold; Bestmann, Lukas

    2007-01-01

    Background: Automated analysis of insoluble urine components can reduce the workload of conventional microscopic examination of urine sediment and is possibly helpful for standardization. We compared the diagnostic performance of two automated urine sediment analyzers and combined dipstick/automated urine analysis with that of the traditional dipstick/microscopy algorithm. Methods: A total of 332 specimens were collected and analyzed for insoluble urine components by microscopy and automated ...

  11. Automated analysis of flow cytometric data for measuring neutrophil CD64 expression using a multi-instrument compatible probability state model.

    Science.gov (United States)

    Wong, Linda; Hill, Beth L; Hunsberger, Benjamin C; Bagwell, C Bruce; Curtis, Adam D; Davis, Bruce H

    2015-01-01

    Leuko64™ (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of state modeling (PSM). Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis. PSM with GemStone correlates well with the expert manual analysis, r(2) = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00-7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r(2) = 1.000. Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher

  12. P-gp Expression and Rh 123 Efflux Assay Have no Impact on Survival in Egyptian Pediatric Acute Lymphoblastic Leukemia Patients

    International Nuclear Information System (INIS)

    Kamel, A.M.; El-Sharkawy, N.; Yassin, D.; Shaaban, Kh.; Hussein, H.; Sidhom, I; Abo El-Naga, S.; Ameen, M.; El-Hattab, O.; Aly El-Din, N.H.

    2005-01-01

    In a previous work we have studied MDR status in terms of P-glycoprotein (P-gp) expression and Rhodamine 123 efflux assay in Egyptian acute leukemia patients. We have reported results comparable to the literature as regards ANLL both in pediatric and adult cases. However, higher figures were encountered for the functional assay in ALL. As our ALL cases especially in pediatric age group show worse prognosis compared to literature, we hypothesized that the higher percentage of cases with positive Rh 123 efflux assay might be a contributing factor. Material and Methods: A total of 108 cases were studied including 80 ALL and 28 ANLL. ALL cases included 48 male and 32 female with an age range of 6m to 18 yrs and a median of 7 yrs. ANLL cases included 18 male and 10 female with an age range of 6m to 18 yrs and a median of 8 yrs. P-gp expression was evaluated using 4E3 and DIC2 mAb, analyzed by Coulter XL flow cytometer and expressed as a ratio at a cut off of ≤1.1 and/or ≤5% positive cells. For the evaluation of MDR function Rh 123 efflux assay using cyclosporine as a blocker and expressed as a ratio at a cutoff of ≤1. 1 and/or ≤ 10% positive cells was performed. MDR expression and function were correlated to age, Hb, TLC, CD34 expression, immuno phenotype and DNA index in ALL, FAB subtypes in ANLL as well as to CR, DFS and EFS in ALL. In ALL, P-gp expression was encountered in 26.4% of cases. Positive Rh efflux was reported in 61.5%. No correlation was encountered between neither expression nor functional assay with age, Hb, TLC, CD34 expression or immuno phenotype. CR was achieved in 89.8%; neither P-gp expression nor Rh123 efflux had an impact on CR except for Rh 123 efflux in T-ALL where a cutoff of 1.25 could predict CR at a total accuracy of 70.6%. DFS was 92.3% while EFS was 72.2% for the whole group. No significant difference was encountered neither between cases expressing or lacking P-gp nor between cases with negative or positive Rh 123 efflux assay

  13. Evaluation of the Diagnostic Accuracy of a Typhoid IgM Flow Assay for the Diagnosis of Typhoid Fever in Cambodian Children Using a Bayesian Latent Class Model Assuming an Imperfect Gold Standard

    Science.gov (United States)

    Moore, Catrin E.; Pan-Ngum, Wirichada; Wijedoru, Lalith P. M.; Sona, Soeng; Nga, Tran Vu Thieu; Duy, Pham Thanh; Vinh, Phat Voong; Chheng, Kheng; Kumar, Varun; Emary, Kate; Carter, Michael; White, Lisa; Baker, Stephen; Day, Nicholas P. J.; Parry, Christopher M.

    2014-01-01

    Rapid diagnostic tests are needed for typhoid fever (TF) diagnosis in febrile children in endemic areas. Five hundred children admitted to the hospital in Cambodia between 2009 and 2010 with documented fever (≥ 38°C) were investigated using blood cultures (BCs), Salmonella Typhi/Paratyphi A real-time polymerase chain reactions (PCRs), and a Typhoid immunoglobulin M flow assay (IgMFA). Test performance was determined by conventional methods and Bayesian latent class modeling. There were 32 cases of TF (10 BC- and PCR-positive cases, 14 BC-positive and PCR-negative cases, and 8 BC-negative and PCR-positive cases). IgMFA sensitivity was 59.4% (95% confidence interval = 41–76), and specificity was 97.8% (95% confidence interval = 96–99). The model estimate sensitivity for BC was 81.0% (95% credible interval = 54–99). The model estimate sensitivity for PCR was 37.8% (95% credible interval = 26–55), with a specificity of 98.2% (95% credible interval = 97–99). The model estimate sensitivity for IgMFA (≥ 2+) was 77.9% (95% credible interval = 58–90), with a specificity of 97.5% (95% credible interval = 95–100). The model estimates of IgMFA sensitivity and specificity were comparable with BCs and better than estimates using conventional analysis. PMID:24218407

  14. The cytometric future: it ain't necessarily flow!

    Science.gov (United States)

    Shapiro, Howard M

    2011-01-01

    Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology.

  15. Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

    DEFF Research Database (Denmark)

    Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne

    2007-01-01

    Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...... describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture...

  16. An HTS-compatible 3D colony formation assay to identify tumor-specific chemotherapeutics.

    Science.gov (United States)

    Horman, Shane R; To, Jeremy; Orth, Anthony P

    2013-12-01

    There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

  17. Appraisal of within- and between-laboratory reproducibility of non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: comparison of OECD TG429 performance standard and statistical evaluation.

    Science.gov (United States)

    Yang, Hyeri; Na, Jihye; Jang, Won-Hee; Jung, Mi-Sook; Jeon, Jun-Young; Heo, Yong; Yeo, Kyung-Wook; Jo, Ji-Hoon; Lim, Kyung-Min; Bae, SeungJin

    2015-05-05

    Mouse local lymph node assay (LLNA, OECD TG429) is an alternative test replacing conventional guinea pig tests (OECD TG406) for the skin sensitization test but the use of a radioisotopic agent, (3)H-thymidine, deters its active dissemination. New non-radioisotopic LLNA, LLNA:BrdU-FCM employs a non-radioisotopic analog, 5-bromo-2'-deoxyuridine (BrdU) and flow cytometry. For an analogous method, OECD TG429 performance standard (PS) advises that two reference compounds be tested repeatedly and ECt(threshold) values obtained must fall within acceptable ranges to prove within- and between-laboratory reproducibility. However, this criteria is somewhat arbitrary and sample size of ECt is less than 5, raising concerns about insufficient reliability. Here, we explored various statistical methods to evaluate the reproducibility of LLNA:BrdU-FCM with stimulation index (SI), the raw data for ECt calculation, produced from 3 laboratories. Descriptive statistics along with graphical representation of SI was presented. For inferential statistics, parametric and non-parametric methods were applied to test the reproducibility of SI of a concurrent positive control and the robustness of results were investigated. Descriptive statistics and graphical representation of SI alone could illustrate the within- and between-laboratory reproducibility. Inferential statistics employing parametric and nonparametric methods drew similar conclusion. While all labs passed within- and between-laboratory reproducibility criteria given by OECD TG429 PS based on ECt values, statistical evaluation based on SI values showed that only two labs succeeded in achieving within-laboratory reproducibility. For those two labs that satisfied the within-lab reproducibility, between-laboratory reproducibility could be also attained based on inferential as well as descriptive statistics. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Harmonization of radiobiological assays: why and how?

    International Nuclear Information System (INIS)

    Prasanna, Pataje G.

    2014-01-01

    The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

  19. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  20. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  1. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  2. A quantitative comet infection assay for influenza virus

    Science.gov (United States)

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

  3. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  4. Flow Injection Analysis

    DEFF Research Database (Denmark)

    Hansen, Elo Harald

    1998-01-01

    Learning objectives:* To provide an introduction to automated assays* To describe the basic principles of FIA * To demonstrate the capabilities of FIA in relation to batch assays and conventional continuous flow systems* To show that FIA allows one to augment existing analytical techniques* To sh...... how FIA offers novel analytical procedures which are not feasible by conventional means* To hightlight the potentials of FIA in selected practical assays...

  5. Solid phase assays

    International Nuclear Information System (INIS)

    Reese, M.G.; Johnson, L.R.; Ransom, D.K.

    1980-01-01

    In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

  6. Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status.

    Science.gov (United States)

    D'Hondt, Liesbet; Höfte, Monica; Van Bockstaele, Erik; Leus, Leen

    2011-10-01

    Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  7. Studying circulation times of liver cancer cells by in vivo flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Liu, G; Li, Y; Fan, Z; Guo, J; Tan, X; Wei, X, E-mail: xwei@fudan.edu.cn [Institutes of Biomedical Sciences, Fudan University, 138 Yi Xue Yuan Road, Shanghai, 200032 (China)

    2011-02-01

    Hepatocellular carcinoma (HCC) may metastasize to lung kidney and many other organs. The survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed 'in vivo flow cytometer' combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines high-metastatic HCCLM3 cells and low-metastatic HepG2 cells which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

  8. Factor IX assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  9. Factor VIII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  10. Factor II assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003674.htm Factor II assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  11. Factor VII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003676.htm Factor VII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  12. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

    2013-01-01

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

  13. Flow karyotyping and flow instrumentation development. Final report

    Energy Technology Data Exchange (ETDEWEB)

    GEngh, G.J. van den

    1997-11-01

    The project had three major aims: improvement of technology for high-speed cell and chromosome sorting; the use of such instrumentation in genome analysis; applying the principles developed and the lessons learned to automated processes for the genome program. The work was a continuation of studies that were started at the Lawrence Livermore National Laboratory before the PI moved to the University of Washington. At Livermore, a high-speed sorter for the selection of human chromosomes was built. The instrument incorporated Livermore`s advanced sorter technology. The engineering focused on improving robustness and reliability so that the full potential of high-speed sorting would become available to the biological research laboratory. The new instrument, dubbed MoFlo for modular flow cytometer, proved to be a very practical and efficient tool during the chromosome isolation phase of the gene-library project. Its reliability and ease of operation exceeded that of the commercial instruments. The technology was licensed to two companies.

  14. Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

    Science.gov (United States)

    Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

    2005-01-01

    This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer

  15. Assay method and compositions

    International Nuclear Information System (INIS)

    1977-01-01

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  16. A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

    International Nuclear Information System (INIS)

    Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

    2015-01-01

    Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

  17. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  18. Rover waste assay system

    International Nuclear Information System (INIS)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-01-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  19. Radioreceptor assay for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

    1975-04-01

    Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

  20. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  1. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  2. Assays for calcitonin receptors

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

  3. Microchemiluminescent assay system

    Energy Technology Data Exchange (ETDEWEB)

    Kiel, J.L.

    1986-04-09

    The patent concerns a microchemiluminescent assay system, which can be used to detect ionizing radiation, heat or specific substances. The method involves the use of a complex formed from serum albumin and a luminescer which, in the presence of ionizing radiation (heat, or a specific analyte), will emit light in an amount proportional to the amount of radiation, etc. (U.K.).

  4. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  5. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  6. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a...

  7. Monitoring environmental exposures with semen assays

    International Nuclear Information System (INIS)

    Anon.

    1979-01-01

    Semen studies in humans and animals have yielded extensive and compelling evidence that sperm can be used to assess reproductive potential and diagnose pathology. More recent studies on mutagens and carcinogens both at this and other laboratories suggest that a combination of mouse and human assays can be an efficient, effective approach to monitoring for reproductive hazards in the environment. We are investigating the potential of using variability in sperm morphology and DNA content to quantify and monitor the effects of environmental agents on the human testes. Here we review the status of human and mouse assays for environmental surveillance, discuss the genetic and fertility implications of chemically induced semen changes, and describe the high-speed flow methods being developed to automate sperm assays

  8. Development of a biochip-based assay integrated in a global strategy for identification of fusion transcripts in acute myeloid leukemia: a work flow for acute myeloid leukemia diagnosis.

    Science.gov (United States)

    Giusiano, S; Formisano-Tréziny, C; Benziane, A; Maroc, N; Picard, C; Hermitte, F; Taranger-Charpin, C; Gabert, J

    2010-08-01

    Three major types of rearrangements are involved in acute myeloid leukemias (AML): t(8;21)(q22;q22), inv(16)(p13q22), and 11q23/MLL abnormalities. Their precise identification becomes essential for diagnosis, prognosis, and therapeutic choices. Resulting fusion transcripts (FT) are also powerful markers for monitoring the efficacy of treatment, the minimal residual disease (MRD) and could become therapeutic targets. Today, the challenge is to propose an individual follow-up for each patient even for those with a rare fusion event. In this study, we propose a biochip-based assay integrated in a global strategy for identification of rare FT in AML, after fluorescence in situ hybridization detection, as described by the World Health Organization classification. Using cell lines, we developed and validated a biochip-based assay called the AMLFusionChip that identifies every FT of AML1-ETO, CBFbeta-MYH11 as well as MLL-AF9, MLL-ENL, MLL-AF6, and MLL-AF10. The original design of our AMLFusionChip.v01 enables the identification of these FT wherever the breakpoint on the partner gene may be. In case of biochip negative result, our 3'RACE amplification strategy enables to clone and then sequence the new translocation partner. This AMLFusionChip strategy fits into the concept of personalized medicine for the largest number of patients.

  9. Radioreceptor assay for oxyphenonium

    International Nuclear Information System (INIS)

    Ensing, K.; Zeeuw, R.A. de

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

  10. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  11. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    Levin, G.V.; Straat, P.A.

    1981-01-01

    A radiorespirometic assay device is described in which the presence of microorganisms in a sample is determined by placing the sample in contact with a metabolisable radioactive labelled substrate, collecting any gas evolved, exposing a photosensitive material to the gas and determining if a spot is produced on the material. A spot indicates the presence of radioactivity showing that the substrate has been metabolized by a microorganism. Bacteria may be detected in body fluids, hospital operating rooms, water, food, cosmetics and drugs. (U.K.)

  12. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  13. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  14. Improving shuffler assay accuracy

    International Nuclear Information System (INIS)

    Rinard, P.M.

    1995-01-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

  15. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  16. Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

    KAUST Repository

    Cerezo, Maria I.

    2017-02-17

    Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

  17. An acoustic prion assay

    Directory of Open Access Journals (Sweden)

    Gordon Hayward

    2016-12-01

    Full Text Available An acoustic prion assay has been demonstrated for sheep brain samples. Only five false positives and no false negatives were observed in a test of 45 positive and 45 negative samples. The acoustic prion sensor was constructed using a thickness shear mode quartz resonator coated with a covalently bound recombinant prion protein. The characteristic indicator of a scrapie infected sheep brain sample was an observed shoulder in the frequency decrease in response to a sample.The response of the sensor aligns with a conformational shift in the surface protein and with the propagation mechanism of the disease. This alignment is evident in the response timing and shape, dependence on concentration, cross species behaviour and impact of blood plasma. This alignment is far from sufficient to prove the mechanism of the sensor but it does offer the possibility of a rapid and inexpensive additional tool to explore prion disease. Keywords: Prions, Thickness shear mode quartz sensor

  18. Assay of oestrogen

    International Nuclear Information System (INIS)

    Edwards, J.C.

    1981-01-01

    A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125 I-labelled oestriol during the assay procedure and the steroid-protein bound 125 I-labelled oestriol is precipitated along with the antibody-bound 125 I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

  19. Smart fast blood counting of trace volumes of body fluids from various mammalian species using a compact custom-built microscope cytometer (Conference Presentation)

    Science.gov (United States)

    Smith, Zachary J.; Gao, Tingjuan; Lin, Tzu-Yin; Carrade-Holt, Danielle; Lane, Stephen M.; Matthews, Dennis L.; Dwyre, Denis M.; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Cell counting in human body fluids such as blood, urine, and CSF is a critical step in the diagnostic process for many diseases. Current automated methods for cell counting are based on flow cytometry systems. However, these automated methods are bulky, costly, require significant user expertise, and are not well suited to counting cells in fluids other than blood. Therefore, their use is limited to large central laboratories that process enough volume of blood to recoup the significant capital investment these instruments require. We present in this talk a combination of a (1) low-cost microscope system, (2) simple sample preparation method, and (3) fully automated analysis designed for providing cell counts in blood and body fluids. We show results on both humans and companion and farm animals, showing that accurate red cell, white cell, and platelet counts, as well as hemoglobin concentration, can be accurately obtained in blood, as well as a 3-part white cell differential in human samples. We can also accurately count red and white cells in body fluids with a limit of detection ~3 orders of magnitude smaller than current automated instruments. This method uses less than 1 microliter of blood, and less than 5 microliters of body fluids to make its measurements, making it highly compatible with finger-stick style collections, as well as appropriate for small animals such as laboratory mice where larger volume blood collections are dangerous to the animal's health.

  20. Flow cytometry: design, development and experimental validation

    International Nuclear Information System (INIS)

    Seigneur, Alain

    1987-01-01

    The flow cytometry techniques allow the analysis and sorting of living biologic cells at rates above five to ten thousand events per second. After a short review, we present in this report the design and development of a 'high-tech' apparatus intended for research laboratories and the experimental results. The first part deals with the physical principles allowing morphologic and functional analysis of cells or cellular components. The measured parameters are as follows: electrical resistance pulse sizing, light scattering and fluorescence. Hydrodynamic centering is used, and in the same way, the division of a water-stream into droplets leading to electrostatic sorting of particles. The second part deals with the apparatus designed by the 'Commissariat a l'Energie Atomique' (C.E.A.) and industrialised by 'ODAM' (ATC 3000). The last part of this thesis work is the performance evaluations of this cyto-meter. The difference between the two size measurement methods are analyzed: electrical resistance pulse sizing versus small-angle light scattering. By an original optics design, high sensitivity has been reached in the fluorescence measurement: the equivalent noise corresponds to six hundred fluorescein isothiocyanate (FITC) molecules. The sorting performances have also been analyzed and the cell viability proven. (author) [fr

  1. A Calorimetric Assay For Enzymatic Saccharification Of Biomass

    DEFF Research Database (Denmark)

    Murphy, Leigh; Borch, Kim; McFarland, K.C.

    2010-01-01

    A limited selection of assay and screening methodologies for cellulolytic enzymes has been stated as a restriction in biomass research. In this report we test the potential of isothermal calorimetry for this purpose. The primary observable in this technique (the heat flow in Watts), scales with t...... of the regulation and functional mechanism of cellulases....

  2. Assaying Cellular Viability Using the Neutral Red Uptake Assay.

    Science.gov (United States)

    Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

    2017-01-01

    The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

  3. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  4. Solution assay instrument operations manual

    International Nuclear Information System (INIS)

    Li, T.K.; Marks, T.; Parker, J.L.

    1983-09-01

    An at-line solution assay instrument (SAI) has been developed and installed in a plutonium purification and americium recovery process area in the Los Alamos Plutonium Processing Facility. The instrument was designed for accurate, timely, and simultaneous nondestructive analysis of plutonium and americium in process solutions that have a wide range of concentrations and americium/plutonium ratios and for routine operation by process technicians who lack instrumentation background. The SAI, based on transmission-corrected, high-resolution gamma-ray spectroscopy, has two measurement stations attached to a single multichannel analyzer/computer system. To ensure the quality of assay results, the SAI has an internal measurement control program, which requires daily and weekly check runs and monitors key aspects of all assay runs. For a 25-ml sample, the assay precision is 5 g/l within a 2000-s count time

  5. Radioligand assay in reproductive biology

    International Nuclear Information System (INIS)

    Korenman, S.G.; Sherman, B.M.

    1975-01-01

    Radioligand assays have been developed for the principal reproductive steroids and peptide hormones. Specific binding reagents have included antibodies, plasma binders, and intracellular receptors. In each assay, problems of specificity, sensitivity, and nonspecific inhibitors were encountered. Many features of the endocrine physiology in childhood, during puberty, and in adulthood have been characterized. Hormonal evaluations of endocrine disorders of reproduction are characterized on the basis of their characteristic pathophysiologic alterations. (U.S.)

  6. Assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

    International Nuclear Information System (INIS)

    Mehdi, S.; Wiseman, J.S.

    1989-01-01

    An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection

  7. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  8. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.

    2013-01-01

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  9. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  10. Human semen assays for workplace monitoring

    International Nuclear Information System (INIS)

    Wyrobek, A.J.; Gledhill, B.L.

    1978-01-01

    Decades of human semen studies have yielded compelling evidence that sperm can be used to access reproductive potential and diagnose pathology. With these studies as background, the small number of detailed semen studies of men exposed to physical and chemical agents point with optimism to the application of human semen assays as efficient, effective means to monitor for reproductive hazards in the workplace. Sperm are the most accessible of human gonadal tissue and provide a means of monitoring exposure induced changes in the human testes, changes which may result in infertility and increased frequencies of genetically abnormal gametes. The focus on semen has precipitated the development of new sperm bioassays which use older conventional andrological methods (i.e., sperm counts, motility, and morphology) as well as recently developed high speed flow and scanning methods for automated cytological analyses. The status of these sperm assays for workplace surveillance is reviewed, procedures are suggested with examples of use, and their effectiveness is evaluated. The available mouse models of induced semen changes are briefly described and the importance of these models for evaluating the genetic implications of findings in human semen is discussed

  11. Barcoded microchips for biomolecular assays.

    Science.gov (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  12. Measurement of Soluble Biomarkers by Flow Cytometry

    OpenAIRE

    Antal-Szalm?s, P?ter; Nagy, B?la; Debreceni, Ildik? Beke; Kappelmayer, J?nos

    2013-01-01

    Microparticle based flow cytometric assays for determination of the level of soluble biomarkers are widely used in several research applications and in some diagnostic setups. The major advantages of these multiplex systems are that they can measure a large number of analytes (up to 500) at the same time reducing assay time, costs and sample volume. Most of these assays are based on antigen-antibody interactions and work as traditional immunoassays, but nucleic acid alterations ? by using spe...

  13. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

    Directory of Open Access Journals (Sweden)

    Josep M. Gasol

    2000-06-01

    Full Text Available Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

  14. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  15. Automation of the dicentric chromosome assay and related assays

    International Nuclear Information System (INIS)

    Balajee, Adayabalam S.; Dainiak, Nicholas

    2016-01-01

    Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

  16. Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays.

    Science.gov (United States)

    Staats, Janet S; Enzor, Jennifer H; Sanchez, Ana M; Rountree, Wes; Chan, Cliburn; Jaimes, Maria; Chan, Ray Chun-Fai; Gaur, Amitabh; Denny, Thomas N; Weinhold, Kent J

    2014-07-01

    The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Assay strategies and methods for phospholipases

    International Nuclear Information System (INIS)

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

  18. Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

    Science.gov (United States)

    Eustaquio, Trisha; Leary, James F

    2012-01-01

    Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells.

  19. One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

    Science.gov (United States)

    Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A; Naivar, Mark A; Lόpez, Gabriel P; Graves, Steven W

    2012-07-01

    Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Simplified flow cytometric assay to detect minimal residual disease in childhood with acute lymphoblastic leukemia Detecção de doença residual mínima em crianças com leucemia linfoblástica aguda por citometria de fluxo

    Directory of Open Access Journals (Sweden)

    Elizabete Delbuono

    2008-08-01

    Full Text Available The detection of minimal residual disease (MRD is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL providing crucial information on the response to treatment and risk of relapse. However, the high cost of these techniques restricts their use in countries with limited resources. Thus, we prospectively studied the use of flow cytometry (FC with a simplified 3-color assay and a limited antibody panel to detect MRD in the bone marrow (BM and peripheral blood (PB of children with ALL. BM and PB samples from 40 children with ALL were analyzed on days (d 14 and 28 during induction and in weeks 24-30 of maintenance therapy. Detectable MRD was defined as > 0.01% cells expressing the aberrant immunophenotype as characterized at diagnosis among total events in the sample. A total of 87% of the patients had an aberrant immunophenotype at diagnosis. On d14, 56% of the BM and 43% of the PB samples had detectable MRD. On d28, this decreased to 45% and 31%, respectively. The percentage of cells with the aberrant phenotype was similar in both BM and PB in T-ALL but about 10 times higher in the BM of patients with B-cell-precursor ALL. Moreover, MRD was detected in the BM of patients in complete morphological remission (44% on d14 and 39% on d28. MRD was not significantly associated to gender, age, initial white blood cell count or cell lineage. This FC assay is feasible, affordable and readily applicable to detect MRD in centers with limited resources.A detecção de doença residual mínima (DRM é um importante fator prognóstico na leucemia linfóide aguda (LLA infantil e fornece informações sobre a resposta ao tratamento e o risco de recaída. Entretanto, os altos custos das técnicas utilizadas limitam seu uso nos países em desenvolvimento. Desta forma, realizamos um estudo prospectivo para avaliar a citometria de fluxo (CF, utilizando três fluorescências e um painel limitado de anticorpos monoclonais, como método de detec

  1. Nondestructive assay of sale materials

    International Nuclear Information System (INIS)

    Rodenburg, W.W.; Fleissner, J.G.

    1981-01-01

    This paper covers three primary areas: (1) reasons for performing nondestructive assay on SALE materials; (2) techniques used; and (3) discussion of investigators' revised results. The study shows that nondestructive calorimetric assay of plutonium offers a viable alternative to traditional wet chemical techniques. For these samples, the precision ranged from 0.4 to 0.6% with biases less than 0.2%. Thus, for those materials where sampling errors are the predominant source of uncertainty, this technique can provide improved accuracy and precision while saving time and money as well as reducing the amount of liquid wastes to be handled. In addition, high resolution gamma-ray spectroscopy measurements of solids can provide isotopic analysis data in a cost effective and timely manner. The timeliness of the method can be especially useful to the plant operator for production control and quality control measurements

  2. Comet Assay in Cancer Chemoprevention.

    Science.gov (United States)

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  3. Radioreceptor assay for somatomedin A

    Energy Technology Data Exchange (ETDEWEB)

    Takano, K [Tokyo Women' s Medical Coll. (Japan)

    1975-04-01

    Measurement method of somatomedian A by radioreceptor assay using the human placenta membrane was described and discussed. Binding rate of /sup 125/I-somatomedin A to its receptors was studied under various conditions of time and temperature of the incubation, and pH of the system. The influence of somatomedin A, porcine insulin, and porcine calcitonin, on /sup 125/I-somatomedin A bound receptors was studied, and these hormones showed the competitive binding to somatomedin A receptors in some level. The specificity, recovery rate, and clinical applications of somatomedin A were also discussed. Radioreceptor assay for somatomedine A provided easier, faster, and more accurate measurements than conventional bioassay. This technique would be very useful to study somatomedin A receptor and functions of insulin.

  4. Assay of vitamin B12

    International Nuclear Information System (INIS)

    Tovey, K.C.; Carrick, D.T.

    1982-01-01

    A radioassay is described for vitamin B12 which involves denaturing serum protein binding proteins with alkali. In the denaturation step a dithiopolyol and cyanide are used and in the intrinsic factor assay step a vitamin B12 analogue such as cobinamide is used to bind with any remaining serum proteins. The invention also includes a kit in which the dithiopolyol is provided in admixture with the alkali. The dithiopolyol may be dithiothreitol or dithioerythritol. (author)

  5. Flow cytometric analysis of microbial contamination in food industry technological lines--initial study.

    Science.gov (United States)

    Józwa, Wojciech; Czaczyk, Katarzyna

    2012-04-02

    Flow cytometry constitutes an alternative for traditional methods of microorganisms identification and analysis, including methods requiring cultivation step. It enables the detection of pathogens and other microorganisms contaminants without the need to culture microbial cells meaning that the sample (water, waste or food e.g. milk, wine, beer) may be analysed directly. This leads to a significant reduction of time required for analysis allowing monitoring of production processes and immediate reaction in case of contamination or any disruption occurs. Apart from the analysis of raw materials or products on different stages of manufacturing process, the flow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. In the present work samples comprising smears from 3 different surfaces of technological lines from fruit and vegetable processing company from Greater Poland were analysed directly with flow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Flow cytometric analysis of the surface of food industry production lines enable the preliminary evaluation of microbial contamination within few minutes from the moment of sample arrival without the need of sample pretreatment. The presented method of fl ow cytometric initial evaluation of microbial state of food industry technological lines demonstrated its potential for developing a robust, routine method for the rapid and labor-saving detection of microbial contamination in food industry.

  6. Assay of ribulose bisphosphate carboxylase

    International Nuclear Information System (INIS)

    Pike, C.; Berry, J.

    1987-01-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, [ 14 C]-NaHCO 3 , and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30 0 . The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number

  7. Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.

    Science.gov (United States)

    Wyant, Tim; Estevam, Jose; Yang, Lili; Rosario, Maria

    2016-03-01

    Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society.

  8. Rotating flow

    CERN Document Server

    Childs, Peter R N

    2010-01-01

    Rotating flow is critically important across a wide range of scientific, engineering and product applications, providing design and modeling capability for diverse products such as jet engines, pumps and vacuum cleaners, as well as geophysical flows. Developed over the course of 20 years' research into rotating fluids and associated heat transfer at the University of Sussex Thermo-Fluid Mechanics Research Centre (TFMRC), Rotating Flow is an indispensable reference and resource for all those working within the gas turbine and rotating machinery industries. Traditional fluid and flow dynamics titles offer the essential background but generally include very sparse coverage of rotating flows-which is where this book comes in. Beginning with an accessible introduction to rotating flow, recognized expert Peter Childs takes you through fundamental equations, vorticity and vortices, rotating disc flow, flow around rotating cylinders and flow in rotating cavities, with an introduction to atmospheric and oceanic circul...

  9. Flow visualization

    CERN Document Server

    Merzkirch, Wolfgang

    1974-01-01

    Flow Visualization describes the most widely used methods for visualizing flows. Flow visualization evaluates certain properties of a flow field directly accessible to visual perception. Organized into five chapters, this book first presents the methods that create a visible flow pattern that could be investigated by visual inspection, such as simple dye and density-sensitive visualization methods. It then deals with the application of electron beams and streaming birefringence. Optical methods for compressible flows, hydraulic analogy, and high-speed photography are discussed in other cha

  10. Radiosotopic assay and binder therefor

    International Nuclear Information System (INIS)

    Caston, J.D.; Kamen, B.A.

    1976-01-01

    A rapid and less costly radioisotopic assay for measuring the concentration of folate in blood serum is described. This procedure utilizes 3 H-pteroylmonoglutamate, unlabeled 5-methyltetrahydrofolic acid, and a partially purified folate binder, such as for example a folate binder extracted from hog kidney. The procedure involves radioisotopically relating the bound amounts of a labeled folate and a known folate at various concentrations of the known folate in a system containing a predetermined amount of the labeled folate, a predetermined amount of the binder factor for the folates, and a predetermined amount of defolated test serum. 16 claims, 8 drawing figures

  11. Flow regimes

    International Nuclear Information System (INIS)

    Kh'yuitt, G.

    1980-01-01

    An introduction into the problem of two-phase flows is presented. Flow regimes arizing in two-phase flows are described, and classification of these regimes is given. Structures of vertical and horizontal two-phase flows and a method of their identification using regime maps are considered. The limits of this method application are discussed. The flooding phenomena and phenomena of direction change (flow reversal) of the flow and interrelation of these phenomena as well as transitions from slug regime to churn one and from churn one to annular one in vertical flows are described. Problems of phase transitions and equilibrium are discussed. Flow regimes in tubes where evaporating liquid is running, are described [ru

  12. Fluorescent multiplex cell flow systems and methods

    KAUST Repository

    Merzaban, Jasmeen

    2017-06-01

    Systems and methods are provided for simultaneously assaying cell adhesion or cell rolling for multiple cell specimens. One embodiment provides a system for assaying adhesion or cell rolling of multiple cell specimens that includes a confocal imaging system containing a parallel plate flow chamber, a pump in fluid communication with the parallel plate flow chamber via a flow chamber inlet line and a cell suspension in fluid communication with the parallel plate flow chamber via a flow chamber outlet line. The system also includes a laser scanning system in electronic communication with the confocal imaging system, and a computer in communication with the confocal imaging system and laser scanning system. In certain embodiments, the laser scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least seven cell specimens in the parallel plate flow chamber.

  13. Antioxidants and the Comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

    2009-01-01

    It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

  14. Rotor assembly and assay method

    Science.gov (United States)

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  15. Flow Cytometry Method as a Diagnostic Tool for Pleural Fluid Involvement in a Patient with Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    MUZAFFER KEKLIK

    2012-01-01

    Full Text Available

    Multiple myeloma is a malignant proliferation of plasma cells that mainly affects bone marrow. Pleural effusions secondary to pleural myelomatous involvement have rarely been reported in the literature. As it is rarely detected, we aimed to report a case in which pleural effusion of a multiple myeloma was confirmed as true myelomatous involvement by flow cytometry method. A 52-years old man presented to our clinic with chest and back pain lasting for 3 months. On the chest radiography, pleural fluid was detected in left hemithorax. Pleural fluid flow cytometry was performed. In the flow cytometry, CD56, CD38 and CD138 found to be positive, while CD19 was negative. True myelomatous pleural effusions are very uncommon, with fewer than 100 cases reported worldwide. Flow cytometry is a potentially useful diagnostic tool for clinical practice. We presented our case; as it has been rarely reported, although flow cytometer is a simple method for detection of pleural fluid involvement in multiple myeloma.

  16. Flow Cytometry Method as a Diagnostic Tool for Pleural Fluid Involvement in a Patient with Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Muzaffer Keklik

    2012-10-01

    Full Text Available Multiple myeloma is a malignant proliferation of plasma cells that mainly affects bone marrow. Pleural effusions secondary to pleural myelomatous involvement have rarely been reported in the literature. As it is rarely detected, we aimed to report a case in which pleural effusion of a multiple myeloma was confirmed as true myelomatous involvement by flow cytometry method. A 52-years old man presented to our clinic with chest and back pain lasting for 3 months. On the chest radiography, pleural fluid was detected in left hemithorax. Pleural fluid flow cytometry was performed. In the flow cytometry, CD56, CD38 and CD138 found to be positive, while CD19 was negative. True myelomatous pleural effusions are very uncommon, with fewer than 100 cases reported worldwide. Flow cytometry is a potentially useful diagnostic tool for clinical practice. We presented our case; as it has been rarely reported, although flow cytometer is a simple method for detection of pleural fluid involvement in multiple myeloma.

  17. Data transformation methods for multiplexed assays

    Science.gov (United States)

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  18. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

  19. A perspective for biomedical data integration: Design of databases for flow cytometry

    Directory of Open Access Journals (Sweden)

    Lakoumentas John

    2008-02-01

    Full Text Available Abstract Background The integration of biomedical information is essential for tackling medical problems. We describe a data model in the domain of flow cytometry (FC allowing for massive management, analysis and integration with other laboratory and clinical information. The paper is concerned with the proper translation of the Flow Cytometry Standard (FCS into a relational database schema, in a way that facilitates end users at either doing research on FC or studying specific cases of patients undergone FC analysis Results The proposed database schema provides integration of data originating from diverse acquisition settings, organized in a way that allows syntactically simple queries that provide results significantly faster than the conventional implementations of the FCS standard. The proposed schema can potentially achieve up to 8 orders of magnitude reduction in query complexity and up to 2 orders of magnitude reduction in response time for data originating from flow cytometers that record 256 colours. This is mainly achieved by managing to maintain an almost constant number of data-mining procedures regardless of the size and complexity of the stored information. Conclusion It is evident that using single-file data storage standards for the design of databases without any structural transformations significantly limits the flexibility of databases. Analysis of the requirements of a specific domain for integration and massive data processing can provide the necessary schema modifications that will unlock the additional functionality of a relational database.

  20. A general method for bead-enhanced quantitation by flow cytometry

    Science.gov (United States)

    Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

    2009-01-01

    Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

  1. Microfluidic passive permeability assay using nanoliter droplet interface lipid bilayers.

    Science.gov (United States)

    Nisisako, Takasi; Portonovo, Shiva A; Schmidt, Jacob J

    2013-11-21

    Membrane permeability assays play an important role in assessing drug transport activities across biological membranes. However, in conventional parallel artificial membrane permeability assays (PAMPA), the membrane model used is dissimilar to biological membranes physically and chemically. Here, we describe a microfluidic passive permeability assay using droplet interface bilayers (DIBs). In a microfluidic network, nanoliter-sized donor and acceptor aqueous droplets are alternately formed in cross-flowing oil containing phospholipids. Subsequently, selective removal of oil through hydrophobic pseudo-porous sidewalls induces the contact of the lipid monolayers, creating arrayed planar DIBs between the donor and acceptor droplets. Permeation of fluorescein from the donor to the acceptor droplets was fluorometrically measured. From the measured data and a simple diffusion model we calculated the effective permeabilities of 5.1 × 10(-6) cm s(-1), 60.0 × 10(-6) cm s(-1), and 87.6 × 10(-6) cm s(-1) with donor droplets at pH values of 7.5, 6.4 and 5.4, respectively. The intrinsic permeabilities of specific monoanionic and neutral fluorescein species were obtained similarly. We also measured the permeation of caffeine in 10 min using UV microspectroscopy, obtaining a permeability of 20.8 × 10(-6) cm s(-1). With the small solution volumes, short measurement time, and ability to measure a wide range of compounds, this device has considerable potential as a platform for high-throughput drug permeability assays.

  2. A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

    Science.gov (United States)

    Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

    2017-08-01

    Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  3. Making transuranic assay measurements using modern controllers

    International Nuclear Information System (INIS)

    Kuckertz, T.H.; Caldwell, J.T.; Medvick, P.A.; Kunz, W.E.; Hastings, R.D.

    1987-01-01

    This paper describes methodology and computer-controlled instrumentation developed at the Los Alamos National Laboratory that accurately performs nondestructive assays of large containers bearing transuranic wastes and nonradioactive matrix materials. These assay systems can measure fissile isotopes with 1-mg sensitivity and spontaneous neutron-emitting isotopes at a 10-mg sensitivity. The assays are performed by neutron interrogation, detection, and counting in a custom assay chamber. An International Business Machines Personal Computer (IBM-PC) is used to control the CAMAC-based instrumentation system that acquires the assay data. 6 refs., 7 figs

  4. Non destructive assay (NDA) techniques

    International Nuclear Information System (INIS)

    Mafra Guidicini, Olga; Llacer, Carlos D.; Rojo, Marcelo

    2001-01-01

    In the IAEA Safeguards System the basic verification method used is nuclear material accountancy, with containment and surveillance as important complementary measures. If nuclear material accountancy is to be effective, IAEA inspectors have to make independent measurements to verify declared material quantities. Most of the equipment available to the inspectors is designed to measure gamma rays and/or neutrons emitted by various nuclear materials. Equipment is also available to measure the gross weight of an item containing nuclear material. These types of measurement techniques are generally grouped under the title of nondestructive assay (NDA). The paper describes the NDA techniques and instruments used to verify the total amount of nuclear material held at a nuclear facility. (author)

  5. Assay of cysteine dioxygenase activity

    International Nuclear Information System (INIS)

    Bagley, P.J.; Stipanuk, M.H.

    1990-01-01

    It has been proposed that rat liver contains two cysteine dioxygenase enzymes which convert cysteine to cysteinesulfinic acid, one which is stimulated by NAD + and has a pH optimum of 6.8 and one which is not stimulated by NAD + and has a pH optimum of 9.0. This led the authors to reinvestigate assay conditions for measuring cysteine dioxygenase activity in rat liver homogenate. An HPLC method, using an anion exchange column (Dionex Amino-Pac trademark PA1 (4x250 mm)) was used to separate the [ 35 S]cysteinesulfinic acid produced from [ 35 S]cysteine in the incubation mixture. They demonstrated that inclusion of hydroxylamine prevented further metabolism of cysteinesulfinic acid. which occurred rapidly in the absence of hydroxylamine

  6. Flow visualization

    International Nuclear Information System (INIS)

    Weinstein, L.M.

    1991-01-01

    Flow visualization techniques are reviewed, with particular attention given to those applicable to liquid helium flows. Three techniques capable of obtaining qualitative and quantitative measurements of complex 3D flow fields are discussed including focusing schlieren, particle image volocimetry, and holocinematography (HCV). It is concluded that the HCV appears to be uniquely capable of obtaining full time-varying, 3D velocity field data, but is limited to the low speeds typical of liquid helium facilities. 8 refs

  7. A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum

    DEFF Research Database (Denmark)

    Lloyd, Yukie M.; Ngati, Elise P.; Salanti, Ali

    2017-01-01

    Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation....... A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads...

  8. Flow regimes

    International Nuclear Information System (INIS)

    Liles, D.R.

    1982-01-01

    Internal boundaries in multiphase flow greatly complicate fluid-dynamic and heat-transfer descriptions. Different flow regimes or topological configurations can have radically dissimilar interfacial and wall mass, momentum, and energy exchanges. To model the flow dynamics properly requires estimates of these rates. In this paper the common flow regimes for gas-liquid systems are defined and the techniques used to estimate the extent of a particular regime are described. Also, the current computer-code procedures are delineated and introduce a potentially better method is introduced

  9. Expert system for transuranic waste assay

    Energy Technology Data Exchange (ETDEWEB)

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  10. Expert system for transuranic waste assay

    International Nuclear Information System (INIS)

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs

  11. Radiometric assays for glycerol, glucose, and glycogen

    International Nuclear Information System (INIS)

    Bradley, D.C.; Kaslow, H.R.

    1989-01-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

  12. Automated amperometric plutonium assay system

    International Nuclear Information System (INIS)

    Burt, M.C.

    1985-01-01

    The amperometric titration for plutonium assay has been used in the nuclear industry for over twenty years and has been in routine use at the Hanford Engineering Development Laboratory since 1976 for the analysis of plutonium oxide and mixed oxide fuel material for the Fast Flux Test Facility. It has proven itself to be an accurate and reliable method. The method may be used as a direct end point titration or an excess of titrant may be added and a back titration performed to aid in determination of the end point. Due to the slowness of the PuVI-FeII reaction it is difficult to recognize when the end point is being approached and is very time consuming if the current is allowed to decay to the residual value after each titrant addition. For this reason the back titration in which the rapid FeII-CrVI reaction occurs is used by most laboratories. The back titration is performed by the addition of excess ferrous solution followed by two measured aliquots of standard dichromate with measurement of cell current after each addition

  13. TRU assay system and measurements

    International Nuclear Information System (INIS)

    Brodzinski, R.L.

    1984-02-01

    The measurement of the transuranic content of nuclear products or process residues has become increasingly important for the recovery of fissionable material from spent fuel elements, the identification of commercial fuel elements which have not yet reached full burnup, the measurement and recovery of transuranics from discarded or stored waste materials, the determination of the transuranic content in high gamma activity waste material scheduled for disposal, compliance with 10CFR61 by land burial operators/shippers, and the satisfaction of accountability requirements. Active neutron interrogation techniques measure either the prompt neutrons or the beta delayed neutrons from fission products following induced fission. These techniques normally only measure fissile transuranics ( 235 U, 239 Pu, and 241 Pu) and are commonly applied only to contact handleable waste. Passive neutron interrogation techniques, on the other hand, are capable of measuring all transuranics except 235 U with adequate sensitivity and will work on both contact handleable and high gamma activity wastes. Since the passive techniques are senstitive to a wider spectrum of transuranic isotopes than the active techniques, substantially less complex and less expensive than the active systems, and they have proven techniques for measuring small quantities of TRU in high gamma activity packages, the passive neutron TRU assay technology was chosen for development into the instruments discussed in this paper

  14. Micronucleus assay for radiation workers

    International Nuclear Information System (INIS)

    Balasem, A.N.; Ali, A.S.K.

    1997-01-01

    Micronucleus assay was performed on 49 radiation workers and 22 healthy volunteers. Radiation workers were subdivided into two groups according to their employments durations in the radiation field. Group a consisted of 18 radiation workers who have been in this work between 5 and 22 years. Group b included 31 employees who have been classified as radiation workers for 1 to 4.5 years. Statistical analysis showed significant variations between the yields of micronuclei in groups A and B as well as between group A and a group of healthy controls. Meanwhile no significant difference was noticed between the yields of micronuclei in group B and the corresponding values in the healthy controls. The possible effect of age in the induction of micronuclei was discussed and a comparison with the yield of chromosomal aberrations was described. It seems that cytokinesis- blocking method may be used to detect the radiation-induced micronuclei in workers exposed occupationally to ionizing radiation in levels below the maximum permissible limit of 0.05 Sv per year

  15. PAME: plasmonic assay modeling environment

    Directory of Open Access Journals (Sweden)

    Adam Hughes

    2015-08-01

    Full Text Available Plasmonic assays are an important class of optical sensors that measure biomolecular interactions in real-time without the need for labeling agents, making them especially well-suited for clinical applications. Through the incorporation of nanoparticles and fiberoptics, these sensing systems have been successfully miniaturized and show great promise for in-situ probing and implantable devices, yet it remains challenging to derive meaningful, quantitative information from plasmonic responses. This is in part due to a lack of dedicated modeling tools, and therefore we introduce PAME, an open-source Python application for modeling plasmonic systems of bulk and nanoparticle-embedded metallic films. PAME combines aspects of thin-film solvers, nanomaterials and fiber-optics into an intuitive graphical interface. Some of PAME’s features include a simulation mode, a database of hundreds of materials, and an object-oriented framework for designing complex nanomaterials, such as a gold nanoparticles encased in a protein shell. An overview of PAME’s theory and design is presented, followed by example simulations of a fiberoptic refractometer, as well as protein binding to a multiplexed sensor composed of a mixed layer of gold and silver colloids. These results provide new insights into observed responses in reflectance biosensors.

  16. An ultrafiltration assay for lysyl oxidase

    International Nuclear Information System (INIS)

    Shackleton, D.R.; Hulmes, D.J.

    1990-01-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

  17. Assay development status report for total cyanide

    International Nuclear Information System (INIS)

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN - ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation

  18. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  19. New automated pellet/powder assay system

    International Nuclear Information System (INIS)

    Olsen, R.N.

    1975-01-01

    This paper discusses an automated, high precision, pellet/ powder assay system. The system is an active assay system using a small isotopic neutron source and a coincidence detection system. The handling of the pellet powder samples has been automated and a programmable calculator has been integrated into the system to provide control and data analysis. The versatile system can assay uranium or plutonium in either active or passive modes

  20. Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

    International Nuclear Information System (INIS)

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

  1. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    Science.gov (United States)

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  2. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    Energy Technology Data Exchange (ETDEWEB)

    Cary, Robert B.

    2018-04-17

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  3. 233U Assay A Neutron NDA System

    Energy Technology Data Exchange (ETDEWEB)

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-11-17

    The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

  4. 233U Assay A Neutron NDA System

    International Nuclear Information System (INIS)

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-01-01

    The assay of highly enriched 233 U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched 235 U do not convert easily over to the assay of 233 U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with γ ray isotopics information should give a good overall determination of 233 U material now stored in bldg. 3019 at the Oak Ridge National Laboratory

  5. Safeguards and Non-destructive Assay

    International Nuclear Information System (INIS)

    Carchon, R.; Bruggeman, M.

    2001-01-01

    SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

  6. Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays.

    Science.gov (United States)

    Souberbielle, Jean-Claude; Fayol, Véronique; Sault, Corinne; Lawson-Body, Ethel; Kahan, André; Cormier, Catherine

    2005-02-01

    The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P 50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

  7. Femtosecond laser fabrication of fiber based optofluidic platform for flow cytometry applications

    Science.gov (United States)

    Serhatlioglu, Murat; Elbuken, Caglar; Ortac, Bulend; Solmaz, Mehmet E.

    2017-02-01

    Miniaturized optofluidic platforms play an important role in bio-analysis, detection and diagnostic applications. The advantages of such miniaturized devices are extremely low sample requirement, low cost development and rapid analysis capabilities. Fused silica is advantageous for optofluidic systems due to properties such as being chemically inert, mechanically stable, and optically transparent to a wide spectrum of light. As a three dimensional manufacturing method, femtosecond laser scanning followed by chemical etching shows great potential to fabricate glass based optofluidic chips. In this study, we demonstrate fabrication of all-fiber based, optofluidic flow cytometer in fused silica glass by femtosecond laser machining. 3D particle focusing was achieved through a straightforward planar chip design with two separately fabricated fused silica glass slides thermally bonded together. Bioparticles in a fluid stream encounter with optical interrogation region specifically designed to allocate 405nm single mode fiber laser source and two multi-mode collection fibers for forward scattering (FSC) and side scattering (SSC) signals detection. Detected signal data collected with oscilloscope and post processed with MATLAB script file. We were able to count number of events over 4000events/sec, and achieve size distribution for 5.95μm monodisperse polystyrene beads using FSC and SSC signals. Our platform shows promise for optical and fluidic miniaturization of flow cytometry systems.

  8. Relationship between conventional culture and flow cytometry for the diagnosis of urinary tract infection.

    Science.gov (United States)

    García-Coca, Marta; Gadea, Ignacio; Esteban, Jaime

    2017-06-01

    Urine culture is the gold standard for the diagnosis of urinary tract infections (UTI). The use of flow cytometry analyzers (FCA) prior to culture allows for the quantification and recognition of cell components in urine to be automated and makes it possible to relate these data to the urine pathogens subsequently identified in cultures. Urine samples were assessed with the Sysmex UF-1000i analyzer. Those that met the criteria for culture (> 25 leukocytes/μL or > 385 bacteria/μL) were subjected to quantitative urine culture on chromogenic agar. Counts of red blood cells (RBC), white blood cells (WBC), epithelial cells (EC), and the kind of microorganisms identified in cultures were evaluated. A total of 17,483 samples were processed by FCA. Of these, 9057 met the criteria for culture. Urine cultures were reduced by 48.2%. The most common urine pathogen was Escherichia coli (60.3%). Negative urine cultures were significantly (p flow cytometer for screening urine samples allows for a reduction in the number of urine cultures. WBC values correlate well with the main urine pathogens related to UTI. The results observed for Enterococcus spp. suggest a low impact of these pathogens as a cause of UTI.

  9. Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry.

    Science.gov (United States)

    Headland, Sarah E; Jones, Hefin R; D'Sa, Adelina S V; Perretti, Mauro; Norling, Lucy V

    2014-06-10

    Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStream(X) Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStream(X) could be used effectively to advance this scientific field.

  10. A micro flow cytometry system for study of marine phytoplankton from costal waters of Hong Kong

    KAUST Repository

    Yunyang Ling,; Hongbin Liu,; Yi-Kuen Lee,

    2010-01-01

    cytometer (MFC) system has been successfully developed using MEMS technology. We demonstrate that this new MFC can analyze mixture of two species of marine phytoplankton: Chlorella autotrophica and Rhodomonas. The results from our MFC are consistent

  11. Radiobiological and PK assays at advance Centre for Training Research and Education in Cancer (ACTREC)

    International Nuclear Information System (INIS)

    Sastri, Goda Jayant; Gota, Vikram

    2014-01-01

    Radiobiological, pharmacokinetic and biodistribution studies are of paramount importance for drug development and more so in the development of newer radiation modulators. Radiobiological studies have now graduated from simple cell survival and viability assays to more complex molecular and imaging studies to study radiation modulation both in in-vitro and in-vivo models. Tata Memorial Centre and its research centre (ACTREC) is a premiere cancer centre in India dedicated to cancer research. The Department of Radiation Oncology treats approximately 7000 new patients in a year and is uniquely placed to do both translational radiation and clinical research in the field of drug development. The Clinical Biology Lab of the Department of Radiation Oncology at ACTREC in collaboration with other labs at ACTREC has standardized cell survival assays, DNA damage assays such as Gamma H2AX assay (by flow as well as confocal microscopy), Micronuclei assay and COMET assays using CASP software for quantification. We have also done apoptotic assays. These assays have been conducted for development newer drug formulations (for e.g liposomal radiosensitizers). We also have a strong imaging division having sophisticated microscopes (confocal and single molecule super resolution microscopes) for in-vitro optical imaging and a dedicated preclinical PET/CT/SPECT for in-vivo imaging. The clinical 3T MRI and PET/CT is being used to study the effect of hypoxia in various cancers

  12. A radiochemical assay for biotin in biological materials

    International Nuclear Information System (INIS)

    Hood, R.L.

    1975-01-01

    A radiochemical assay for biotin is described. The assay was sensitive to one nanogram and simple enough for routine biotin analyses. The assay yielded results which were comparable to those obtained from a microbiological assay using Lactobacillus plantarum. (author)

  13. Radioreceptor assay: theory and applications to pharmacology

    International Nuclear Information System (INIS)

    Perret, G.; Simon, P.

    1984-01-01

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising [fr

  14. Assessing sediment contamination using six toxicity assays

    Directory of Open Access Journals (Sweden)

    Allen G. BURTON Jr.

    2001-08-01

    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  15. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    DEFF Research Database (Denmark)

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred

    2016-01-01

    and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2...

  16. 21 CFR 864.7525 - Heparin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  17. Assay in engine of agricultural tractor with biofuel

    Energy Technology Data Exchange (ETDEWEB)

    Lopes, Reny Adilmar Prestes; Meyer, Wagner [Universidade Estadual de Maringa (DEA/CCA/UEM), Cidade Gaucha, PR (Brazil). Centro de Ciencias Agrarias. Dept. de Engenharia Agricola], E-mail: raplopes@uem.br; Pinheiro Neto, Raimundo; Pinheiro, Andreia Cristina [Universidade Estadual de Maringa (DAG/CCA/UEM), PR (Brazil). Centro de Ciencias Agrarias. Dept. de Agronomia; Laurindo, Jose Carlos [Instituto de Tecnologia do Parana (CERBIO/TECPAR), Curitiba, PR (Brazil). Centro Brasileiro de Referencia em Biocombustiveis; Biazzono, Sergio Luis [Instituto de Tecnologia do Parana (TECPAR), Maringa, PR (Brazil). Inspecao Veicular

    2008-07-01

    The use of biofuel in tractors of diesel engines and agricultural harvester, in the operations of soil preparation and harvest, is a good option of fuel economy for the agriculturist. For a good performance of the machine a good regulation is necessary. The experiment was carried through in the Experimental Farm Iguatemi of the State University of Maringa, Maringa - PR. A tractor Massey Ferguson MF275 was used for the assay connected to be even grating. It carried through if the assays of consumption of diesel (100%) and biofuel (diesel 80% + vegetable oil 20%). To carry through the assay tractor + grating with three openings and without load was used to be even set. The rotation without load and of work was of 1900 rpm and mean speed of 6 km h{sup -1}. The hourly consumption was verified by a test tube and a fluxgate OVAL Flow mate M III - LSF 45L0-M2 connected to data logger CR23X. The hourly consumption was express in L h{sup -1}. The engine of the tractor presented similar behavior of fuel consumption for diesel and biofuel. The mean values of consumption had been inside of the specified one for the manufacturer. Mixture 80% diesel + 20% vegetable oil can be used as biofuel in the engine in study. (author)

  18. Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul [Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul (Korea, Republic of); Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo [Seoul National University college of Medicine, Seoul (Korea, Republic of)

    2009-08-15

    Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2+-1.7%, 3.9+-2.1%, 7.1+-6.2%, 11.2+-7.2%. The CVs by random assay were 2.1+-1.7%, 4.8+-3.1%, 3.6+-4.8%, and 7.4+-6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

  19. Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

    International Nuclear Information System (INIS)

    Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul; Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo

    2009-01-01

    Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2±1.7%, 3.9±2.1%, 7.1±6.2%, 11.2±7.2%. The CVs by random assay were 2.1±1.7%, 4.8±3.1%, 3.6±4.8%, and 7.4±6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

  20. Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

    International Nuclear Information System (INIS)

    Garaj-Vrhovac, V.; Kopjar, N.

    2003-01-01

    Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

  1. Comparison of non-magnetic and magnetic beads in bead-based assays

    NARCIS (Netherlands)

    Hansenová Maňásková, S.; van Belkum, A.; Endtz, H.P.; Bikker, F.J.; Veerman, E.C.I.; van Wamel, W.J.B.

    2016-01-01

    Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens

  2. Flow chemistry vs. flow analysis.

    Science.gov (United States)

    Trojanowicz, Marek

    2016-01-01

    The flow mode of conducting chemical syntheses facilitates chemical processes through the use of on-line analytical monitoring of occurring reactions, the application of solid-supported reagents to minimize downstream processing and computerized control systems to perform multi-step sequences. They are exactly the same attributes as those of flow analysis, which has solid place in modern analytical chemistry in several last decades. The following review paper, based on 131 references to original papers as well as pre-selected reviews, presents basic aspects, selected instrumental achievements and developmental directions of a rapidly growing field of continuous flow chemical synthesis. Interestingly, many of them might be potentially employed in the development of new methods in flow analysis too. In this paper, examples of application of flow analytical measurements for on-line monitoring of flow syntheses have been indicated and perspectives for a wider application of real-time analytical measurements have been discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Nano-immunosafety: issues in assay validation

    International Nuclear Information System (INIS)

    Boraschi, Diana; Italiani, Paola; Oostingh, Gertie J; Duschl, Albert; Casals, Eudald; Puntes, Victor F; Nelissen, Inge

    2011-01-01

    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  4. Radioactive wastes assay technique and equipment

    International Nuclear Information System (INIS)

    Lee, K. M.; Hong, D. S; Kim, T. K.; Bae, S. M.; Shon, J. S.; Hong, K. P.

    2004-12-01

    The waste inventory records such as the activities and radio- nuclides contained in the waste packages are to be submitted with the radioactive wastes packages for the final disposal. The nearly around 10,000 drums of waste stocked in KAERI now should be assayed for the preparation of the waste inventory records too. For the successive execution of the waste assay, the investigation into the present waste assay techniques and equipment are to be taken first. Also the installation of the waste assay equipment through the comprehensive design, manufacturing and procurement should be proceeded timely. As the characteristics of the KAERI-stocked wastes are very different from that of the nuclear power plant and those have no regular waste streams, the application of the in-direct waste assay method using the scaling factors are not effective for the KAERI-generated wastes. Considering for the versal conveniency including the accuracy over the wide range of waste forms and the combination of assay time and sensitivity, the TGS(Tomographic Gamma Scanner) is appropriate as for the KAERI -generated radioactive waste assay equipment

  5. A multiwell format assay for heparanase.

    Science.gov (United States)

    Behzad, Farhad; Brenchley, Paul E C

    2003-09-15

    This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

  6. Vortical flows

    International Nuclear Information System (INIS)

    Wu, Jie-Zhi; Ma, Hui-Yang; Zhou, Ming-De

    2015-01-01

    This book is a comprehensive and intensive book for graduate students in fluid dynamics as well as scientists, engineers and applied mathematicians. Offering a systematic introduction to the physical theory of vortical flows at graduate level, it considers the theory of vortical flows as a branch of fluid dynamics focusing on shearing process in fluid motion, measured by vorticity. It studies vortical flows according to their natural evolution stages,from being generated to dissipated. As preparation, the first three chapters of the book provide background knowledge for entering vortical flows. The rest of the book deals with vortices and vortical flows, following their natural evolution stages. Of various vortices the primary form is layer-like vortices or shear layers, and secondary but stronger form is axial vortices mainly formed by the rolling up of shear layers. Problems are given at the end of each chapter and Appendix, some for helping understanding the basic theories, and some involving specific applications; but the emphasis of both is always on physical thinking.

  7. Vortical flows

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jie-Zhi [Peking Univ., Beijing (China). College of Engineering; Ma, Hui-Yang [Univ. of Chinese Academy of Sciences, Beijing (China). Dept. of Physics; Zhou, Ming-De [Arizona Univ., Tucson, AZ (United States). Dept. of Aerospace and Mechanical Engineering

    2015-11-01

    This book is a comprehensive and intensive book for graduate students in fluid dynamics as well as scientists, engineers and applied mathematicians. Offering a systematic introduction to the physical theory of vortical flows at graduate level, it considers the theory of vortical flows as a branch of fluid dynamics focusing on shearing process in fluid motion, measured by vorticity. It studies vortical flows according to their natural evolution stages,from being generated to dissipated. As preparation, the first three chapters of the book provide background knowledge for entering vortical flows. The rest of the book deals with vortices and vortical flows, following their natural evolution stages. Of various vortices the primary form is layer-like vortices or shear layers, and secondary but stronger form is axial vortices mainly formed by the rolling up of shear layers. Problems are given at the end of each chapter and Appendix, some for helping understanding the basic theories, and some involving specific applications; but the emphasis of both is always on physical thinking.

  8. Evaluation of different in vitro assays of inherent sensitivity as predictors of radiotherapy response

    International Nuclear Information System (INIS)

    Schwartz, J.L.; Chicago Univ., IL; Beckett, M.A.; Mustafi, R.; Weichselbaum, R.R.; Vaughan, A.T.M.

    1991-01-01

    The inherent sensitivity of cells within a tumor plays an important role in the response of the tumor to radiotherapy. Clonogenic assays show that cells established from in-field radiotherapy failures are significantly more resistant to radiation than cell lines established from pre-treatment samples. Clonogenic assays fail to predict tumor response to radiotherapy, however. The failure might be due to the small sample size in this study, or the complicating factors of staging, surgery, and chemotherapy, and/or in vivo selection by radiotherapy for resistant tumor cells. In vitro selection for resistant cell lines does not appear to be a complicating factor. Nonclonogenic assays such as those that measure DNA strand break rejoining rates (filter elution, pulse-field gel electrophoresis) or chromosome structure (flow cytometric analysis) show promise as alternative rapid assays of radiation sensitivity and possibly tumor response. 16 refs., 2 figs

  9. Examination of a high resolution laser optical plankton counter and FlowCAM for measuring plankton concentration and size

    Science.gov (United States)

    Kydd, Jocelyn; Rajakaruna, Harshana; Briski, Elizabeta; Bailey, Sarah

    2018-03-01

    Many commercial ships will soon begin to use treatment systems to manage their ballast water and reduce the global transfer of harmful aquatic organisms and pathogens in accordance with upcoming International Maritime Organization regulations. As a result, rapid and accurate automated methods will be needed to monitoring compliance of ships' ballast water. We examined two automated particle counters for monitoring organisms ≥ 50 μm in minimum dimension: a High Resolution Laser Optical Plankton Counter (HR-LOPC), and a Flow Cytometer with digital imaging Microscope (FlowCAM), in comparison to traditional (manual) microscopy considering plankton concentration, size frequency distributions and particle size measurements. The automated tools tended to underestimate particle concentration compared to standard microscopy, but gave similar results in terms of relative abundance of individual taxa. For most taxa, particle size measurements generated by FlowCAM ABD (Area Based Diameter) were more similar to microscope measurements than were those by FlowCAM ESD (Equivalent Spherical Diameter), though there was a mismatch in size estimates for some organisms between the FlowCAM ABD and microscope due to orientation and complex morphology. When a single problematic taxon is very abundant, the resulting size frequency distribution curves can become skewed, as was observed with Asterionella in this study. In particular, special consideration is needed when utilizing automated tools to analyse samples containing colonial species. Re-analysis of the size frequency distributions with the removal of Asterionella from FlowCAM and microscope data resulted in more similar curves across methods with FlowCAM ABD having the best fit compared to the microscope, although microscope concentration estimates were still significantly higher than estimates from the other methods. The results of our study indicate that both automated tools can generate frequency distributions of particles

  10. Nondestructive assay measurements applied to reprocessing plants

    International Nuclear Information System (INIS)

    Ruhter, Wayne D.; Lee, R. Stephen; Ottmar, Herbert; Guardini, Sergio

    1999-01-01

    Nondestructive assay for reprocessing plants relies on passive gamma-ray spectrometry for plutonium isotopic and plutonium mass values of medium-to-low-density samples and holdup deposits; on active x-ray fluorescence and densitometry techniques for uranium and plutonium concentrations in solutions; on calorimetry for plutonium mass in product; and passive neutron techniques for plutonium mass in spent fuel, product, and waste. This paper will describe the radiation-based nondestructive assay techniques used to perform materials accounting measurements. The paper will also discuss nondestructive assay measurements used in inspections of reprocessing plants [ru

  11. Thermometric enzyme linked immunosorbent assay: TELISA.

    Science.gov (United States)

    Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

    1977-08-11

    A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

  12. Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays

    Directory of Open Access Journals (Sweden)

    Crabb Brendan S

    2010-06-01

    Full Text Available Abstract Background Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. Methods The D10 P. falciparum line was transfected to express green fluorescent protein (GFP. In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Results Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Conclusions Flow cytometry based assays using GFP

  13. Split Beta-Lactamase Complementation Assay

    Indian Academy of Sciences (India)

    IAS Admin

    A Search for the Molecular Better Half! Vaishali Verma ... These assays comprise of a protein molecule, ... ciferase, beta-galactosidase, GFP, g3p of M13 filamentous ph- .... sensors of protein–protein interactions, Nature Biotechnology, Vol.20,.

  14. Linearization of the bradford protein assay.

    Science.gov (United States)

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  15. 21 CFR 866.3210 - Endotoxin assay.

    Science.gov (United States)

    2010-04-01

    ... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a... intended for use in conjunction with other laboratory findings and clinical assessment of the patient to...

  16. Passive nondestructive assay of nuclear materials

    International Nuclear Information System (INIS)

    Reilly, D.; Ensslin, N.; Smith, H. Jr.; Kreiner, S.

    1991-03-01

    The term nondestructive assay (NDA) is applied to a series of measurement techniques for nuclear fuel materials. The techniques measure radiation induced or emitted spontaneously from the nuclear material; the measurements are nondestructive in that they do not alter the physical or chemical state of the nuclear material. NDA techniques are characterized as passive or active depending on whether they measure radiation from the spontaneous decay of the nuclear material or radiation induced by an external source. This book emphasizes passive NDA techniques, although certain active techniques like gamma-ray absorption densitometry and x-ray fluorescence are discussed here because of their intimate relation to passive assay techniques. The principal NDA techniques are classified as gamma-ray assay, neutron assay, and calorimetry. Gamma-ray assay techniques are treated in Chapters 1--10. Neutron assay techniques are the subject of Chapters 11--17. Chapters 11--13 cover the origin of neutrons, neutron interactions, and neutron detectors. Chapters 14--17 cover the theory and applications of total and coincidence neutron counting. Chapter 18 deals with the assay of irradiated nuclear fuel, which uses both gamma-ray and neutron assay techniques. Chapter 19 covers perimeter monitoring, which uses gamma-ray and neutron detectors of high sensitivity to check that no unauthorized nuclear material crosses a facility boundary. The subject of Chapter 20 is attribute and semiquantitative measurements. The goal of these measurements is a rapid verification of the contents of nuclear material containers to assist physical inventory verifications. Waste and holdup measurements are also treated in this chapter. Chapters 21 and 22 cover calorimetry theory and application, and Chapter 23 is a brief application guide to illustrate which techniques can be used to solve certain measurement problems

  17. Optical assay for biotechnology and clinical diagnosis.

    Science.gov (United States)

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  18. Calibration method for a radwaste assay system

    International Nuclear Information System (INIS)

    Dulama, C.; Dobrin, R.; Toma, Al.; Paunoiu, C.

    2004-01-01

    A waste assay system entirely designed and manufactured in the Institute for Nuclear Research is used in radwaste treatment and conditioning stream to ensure compliance with national repository radiological requirements. Usually, waste assay systems are calibrated by using various experimental arrangements including calibration phantoms. The paper presents a comparative study concerning the efficiency calibration performed by shell source method and a semiempirical, computational method based on a Monte Carlo algorithm. (authors)

  19. Evaluation of cell proliferative activity after irradiation using immunohistochemical approach and flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Tamada, Takashi (Okayama Univ. (Japan). School of Medicine)

    1992-06-01

    To evaluate a proliferative activity of post-irradiated malignant cells, we studied the kinetics of HeLa cells using immunohistochemical approach and flow cytometry. HeLa cells were stained with two proliferation-associated monoclonal antibodies, Ki-67 and anti-DNA polymerase {alpha} antibody. Nucleoli of non-irradiated cells were granularly stained with Ki-67. After irradiation, only the center of nuclei was diffusely stained with Ki-67. One hundred forty-four hours after low-dose irradiation, the staining patterns became the same as the control. On the other hand, after high-dose irradiation, the center of nuclei was weakly stained. DNA polymerase {alpha} was diffusely labelled with nuclei of the control. It was located around the border of nuclei of low-dose irradiated cells like a ring. But after high-dose irradiation, it was granularly distributed in the periphery of nuclei. FITC conjugated Ki-67/PI two parameter analysis was done by a single laser flow cytometer. Twenty-four hours after irradiation, DNA-histograms showed the accumulation to G{sub 2}/M phase and the increase of DNA content of G{sub 2}/M cells, as exposure dose was increased. Two parameter analysis showed the increase of FITC uptake of G{sub 2}/M phase as dose increased. These changes of flow cytometry were remarkably observed after 24 hours' incubation. It was shown that the difference of Ki-67 antigen and DNA polymerase {alpha} appearance depended on the irradiation dose. These findings suggest that immunohistochemical staining with Ki-67 or anti-DNA polymerase {alpha} antibody and flow cytometry using Ki-67 are available to evaluate cell damages after irradiation. (author).

  20. Rapid detection of microbial contamination in grape juice by flow cytometry

    Directory of Open Access Journals (Sweden)

    Marielle Bouix

    1999-03-01

    Full Text Available This study presents an application of flow cytometry to evaluate rapidly the viable micro-organisms in grape juice. In this method, viable cells are firstly specitically labelled with a fluorescent reagent. The sample is then injected into the flow cytometer where the labelled micro-organisms are individually illuminated by a laser beam. The emission of fluorescence is measured. The system counts the number of fluorescent events and prints out a histogram of the fluorescence intensity which is characteristic of the micro-organism being analysed. In laboratory conditions, preliminary trials have been undertaken with an artificially inoculated grape juice with pure yeast and bacteria cultures. This method succeeded in counting simultaneously yeasts and bacteria within 15 minutes, with a high degree of sensitivity, 5.103 yeasts perml and 5.104 bacteria per ml. This technique can also be applied to the detection of mould contamination and the test has been done with Botrytis spores. The method makes direct cell counts possible and is capable of analysing 30 samples per hour. It can be automatised and easily used in industrial laboratory. During the last harvest, more than a thousand of must samples were controled using this technique. The results let to determine the yeast contamination level of a grape juice tank even before unloading. The results obtained by flow cytometry were compared to the plate count reference method. The correlation between cytometry and count by plate culture was 99 p. cent for the threshold of 5.1 04 yeasts/ml which seemed to point out a high contamination. By using this flow cytometry method during the harvest period, the results were supplied in real time. This allowed a rapid selection of the musts, depending upon the scale of their contamination and improved the quality of the wine by corrective actions.

  1. Granular flow

    DEFF Research Database (Denmark)

    Mitarai, Namiko; Nakanishi, Hiizu

    2012-01-01

    Granular material is a collection of macroscopic particles that are visible with naked eyes. The non-equilibrium nature of the granular materials makes their rheology quite different from that of molecular systems. In this minireview, we present the unique features of granular materials focusing...... on the shear flow of dry granular materials and granule-liquid mixture....

  2. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Roth, G.J.; Machuga, E.T.

    1982-01-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

  3. Nondestructive assay methods for irradiated nuclear fuels

    International Nuclear Information System (INIS)

    Hsue, S.T.; Crane, T.W.; Talbert, W.L. Jr.; Lee, J.C.

    1978-01-01

    This report is a review of the status of nondestructive assay (NDA) methods used to determine burnup and fissile content of irradiated nuclear fuels. The gamma-spectroscopy method measures gamma activities of certain fission products that are proportional to the burnup. Problems associated with this method are migration of the fission products and gamma-ray attenuation through the relatively dense fuel material. The attenuation correction is complicated by generally unknown activity distributions within the assemblies. The neutron methods, which usually involve active interrogation and prompt or delayed signal counting, are designed to assay the fissile content of the spent-fuel elements. Systems to assay highly enriched spent-fuel assemblies have been tested extensively. Feasibility studies have been reported of systems to assay light-water reactor spent-fuel assemblies. The slowing-down spectrometer and neutron resonance absorption methods can distinguish between the uranium and plutonium fissile contents, but they are limited to the assay of individual rods. We have summarized the status of NDA techniques for spent-fuel assay and present some subjects in need of further investigation. Accuracy of the burnup calculations for power reactors is also reviewed

  4. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Jennifer Pasquier

    2015-06-01

    Full Text Available The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp. The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading, we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  5. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

    Directory of Open Access Journals (Sweden)

    Vinci Maria

    2012-03-01

    Full Text Available Abstract Background There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K-mammalian target of rapamycin (mTOR inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG (heat shock protein 90 (HSP90 inhibitor and CCT130234 (in-house phospholipase C (PLCγ inhibitor. Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration

  6. Selection of non-destructive assay methods: Neutron counting or calorimetric assay?

    International Nuclear Information System (INIS)

    Cremers, T.L.; Wachter, J.R.

    1994-01-01

    The transition of DOE facilities from production to D ampersand D has lead to more measurements of product, waste, scrap, and other less attractive materials. Some of these materials are difficult to analyze by either neutron counting or calorimetric assay. To determine the most efficacious analysis method, variety of materials, impure salts and hydrofluorination residues have been assayed by both calorimetric assay and neutron counting. New data will be presented together with a review of published data. The precision and accuracy of these measurements are compared to chemistry values and are reported. The contribution of the gamma ray isotopic determination measurement to the overall error of the calorimetric assay or neutron assay is examined and discussed. Other factors affecting selection of the most appropriate non-destructive assay method are listed and considered

  7. High-throughput assay of 9 lysosomal enzymes for newborn screening.

    Science.gov (United States)

    Spacil, Zdenek; Tatipaka, Haribabu; Barcenas, Mariana; Scott, C Ronald; Turecek, Frantisek; Gelb, Michael H

    2013-03-01

    There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS). The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories. HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.

  8. Controlling variation in the comet assay

    Directory of Open Access Journals (Sweden)

    Andrew Richard Collins

    2014-10-01

    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  9. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  10. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Directory of Open Access Journals (Sweden)

    Bas-jan Van Der Leede

    2015-08-01

    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

  11. Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

    Directory of Open Access Journals (Sweden)

    François Vromman

    Full Text Available Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

  12. In vivo flow cytometry for blood cell analysis using differential epi-detection of forward scattered light

    Science.gov (United States)

    Paudel, Hari P.; Jung, Yookyung; Raphael, Anthony; Alt, Clemens; Wu, Juwell; Runnels, Judith; Lin, Charles P.

    2018-02-01

    The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient's blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

  13. Flow Game

    DEFF Research Database (Denmark)

    Olsen, Jesper Lind

    2003-01-01

    Flow Game er et dialogspil, der kan bruges som ledelsesværktøj, ledertræning, samtaletræning, coachingtræning og ideudvikling m.m. Gennem dilemmakort provokeres en dialog og teori-U inspireret afklaring- og udviklingsproces, hvor der enten arbejdes på en gruppes eller et individs vision/innovatio......Flow Game er et dialogspil, der kan bruges som ledelsesværktøj, ledertræning, samtaletræning, coachingtræning og ideudvikling m.m. Gennem dilemmakort provokeres en dialog og teori-U inspireret afklaring- og udviklingsproces, hvor der enten arbejdes på en gruppes eller et individs vision...

  14. Random assay in radioimmunoassay: Feasibility and application compared with batch assay

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

    2016-12-15

    The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

  15. Media Flow

    DEFF Research Database (Denmark)

    Kabel, Lars

    2016-01-01

    News and other kinds of journalistic stories, 16-17 hours a day, all year round, on all platforms, also the moderated social media. The key research thesis behind this article is that the continuous and speedy stream of news stories and media content now is becoming the centre of the production...... processes and the value creation in converged multimedia newsrooms. The article identify new methods and discuss editorial challenges in handling media flow....

  16. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  17. Immune chromatography: a quantitative radioimmunological assay

    International Nuclear Information System (INIS)

    Davis, J.W.; Demetriades, M.; Bowen, J.M.

    1984-01-01

    Immune chromatography, a radioimmunological binding assay, employs paper chromatography to separate immune complexes from free antigen and antibodies. During chromatography free antigen and antibodies become distributed throughout the paper, while immune complexes remain near the bottoms of the strips. The chromatographic differences can be made quantitative by using either iodinated antigens or antibodies. Under these conditions nanogram quantities of antigen can be detected or antibodies in sera diluted several 1000-fold. The immune chromatography assay can also be performed as an indirect assay, since the paper strips are cut from nitrocellulose paper. In this case the immune components are absorbed by the paper during chromatography. Antigen is then detected with an iodinated second antibody. The indirect immune chromatography assay is particularly useful for identifying different sera that react with the same antigen. Reaction with the first serum before chromatography reduces the amount of antigen available to the second serum following chromatography. In addition to characterizing the immune chromatography procedure, we discuss the possible applications of chromatography assays for the quantitation of other types of molecular binding interactions. (Auth.)

  18. Evaluation of three gentamicin serum assay techniques

    International Nuclear Information System (INIS)

    Matzke, G.R.; Gwizdala, C.; Wery, J.; Ferry, D.; Starnes, R.

    1982-01-01

    This investigation was designed to compare the enzyme-modified immunoassay (Syva--EMIT) with a radioimmunoassay (New England Nuclear--RIA) and the radiometric assay (Johnston--BACTEC) to determine the optimal assay for use in our aminoglycoside dosing service. The serum concentration determinations obtained via the three assay methods were analyzed by linear regression analysis. Significant positive correlations were noted between the three assay techniques (p less than 0.005) during both sample collection phases. The coefficients of determination for EMIT vs BACTEC and RIA vs BACTEC were 0.73 and 0.83 during phase 1, respectively, and 0.65 and 0.68 during phase 2, respectively. The slope of the regression lines also varied markedly during the two phases; 0.49 and 0.42 for EMIT and for RIA vs BACTEC, respectively, during phase 1 compound with 1.12 and 0.77, respectively, during phase 2. The differences noted in these relationships during phase 1 and 2 may be related to the alteration of the pH of the control sera utilized in the BACTEC assay. In contrast, RIA vs EMIT regression analysis indicated that existence of a highly significant relationship (p less than 0.0005 and r2 . 0.90). The EMIT technique was the easiest and most accurate for determination of serum gentamicin concentrations, whereas the BACTEC method was judged unacceptable for clinical use

  19. Standard guide for making quality nondestructive assay measurements

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2009-01-01

    1.1 This guide is a compendium of Quality Measurement Practices for performing measurements of radioactive material using nondestructive assay (NDA) instruments. The primary purpose of the guide is to assist users in arriving at quality NDA results, that is, results that satisfy the end user’s needs. This is accomplished by providing an acceptable and uniform basis for the collection, analysis, comparison, and application of data. The recommendations are not compulsory or prerequisites to achieving quality NDA measurements, but are considered contributory in most areas. 1.2 This guide applies to the use of NDA instrumentation for the measurement of nuclear materials by the observation of spontaneous or stimulated nuclear radiations, including photons, neutrons, or the flow of heat. Recommended calibration, operating, and assurance methods represent guiding principles based on current NDA technology. The diversity of industry-wide nuclear materials measurement applications and instrumentation precludes disc...

  20. Design of an error-free nondestructive plutonium assay facility

    International Nuclear Information System (INIS)

    Moore, C.B.; Steward, W.E.

    1987-01-01

    An automated, at-line nondestructive assay (NDA) laboratory is installed in facilities recently constructed at the Savannah River Plant. The laboratory will enhance nuclear materials accounting in new plutonium scrap and waste recovery facilities. The advantages of at-line NDA operations will not be realized if results are clouded by errors in analytical procedures, sample identification, record keeping, or techniques for extracting samples from process streams. Minimization of such errors has been a primary design objective for the new facility. Concepts for achieving that objective include mechanizing the administrative tasks of scheduling activities in the laboratory, identifying samples, recording and storing assay data, and transmitting results information to process control and materials accounting functions. These concepts have been implemented in an analytical computer system that is programmed to avoid the obvious sources of error encountered in laboratory operations. The laboratory computer exchanges information with process control and materials accounting computers, transmitting results information and obtaining process data and accounting information as required to guide process operations and maintain current records of materials flow through the new facility

  1. A new semiquantitative radiometric opsonin assay

    International Nuclear Information System (INIS)

    Yamamura, M.; Valdimarsson, H.

    1978-01-01

    A new semiquantitative radiometric opsonin assay is described. It was found that the opsonin activity generated by incubating brewer's yeast, Saccharomyces cerevisiae, in medium containing less than 5% human serum was exclusively complement dependent. In contrast, C.albicans was effectively opsonized in the absence of complement. Antibodies and the early classical complement pathway did not contribute to the opsonization of S.cerevisiae and neither did C5-9. The brewer's yeast assay can therefore be used for measuring selectively the opsonizing capacity of the alternative pathway. Sera from approximately 7% of apparently healthy adult controls consistently failed to generate significant opsonin activity while 8 out of 26 patients with suspected immune deficiency of unknown cause were defective in this assay. All opsonin deficient sera so far tested had haemolytically normal alternative pathway and Factor B activity. (author)

  2. Evaluation of a molybdenum assay canister

    International Nuclear Information System (INIS)

    Yoshizumi, T.T.; Keener, S.J.

    1988-01-01

    The performance characteristics of a commercial molybdenum assay canister were evaluated. The geometrical variation of the technetium-99m (/sup 99m/Tc) activity reading was studied as a function of the elution volume for the standard vials. It was found that the /sup 99m/Tc canister activity reading was ∼ 5% lower than that of the standard method. This is due to attenuation by the canister wall. However, the effect of the geometric variation on the clinical dose preparation was found to be insignificant. The molybdenum-99 ( 99 Mo) contamination level was compared by two methods: (1) the commercial canister and (2) the standard assay kit. The 99 Mo contamination measurements with the canister indicated consistently lower readings than those with the standard 99 Mo assay kit. The authors conclude that the canister may be used in the clinical settings. However, the user must be aware of the problems and the limitations associated with this canister

  3. Elements of nondestructive assay (NDA) technology

    International Nuclear Information System (INIS)

    Anon.

    1981-01-01

    This session provides an introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards. The purpose of the sessions is to enable participants to: (1) discuss the general principles and major applications of NDA; (2) describe situations in which NDA is particularly useful for nuclear safeguards purposes; (3) distinguish between various passive and active gamma-ray and neutron NDA methods; (4) describe several NDA instruments that measure gamma rays, and identify assay situations particularly suited to gamma-ray techniques; (5) describe several NDA instruments that measure neutrons, and identify assay situations particularly suited to neutron techniques; (6) discuss the role of calorimetry in the NDA of plutonium-bearing materials; and (7) compare the advantages and disadvantages of various NDA methods for different types of nuclear materials

  4. Development of an integrated assay facility

    International Nuclear Information System (INIS)

    Molesworth, T.V.; Bailey, M.; Findlay, D.J.S.; Parsons, T.V.; Sene, M.R.; Swinhoe, M.T.

    1990-01-01

    The I.R.I.S. concept proposed the use of passive examination and active interrogation techniques in an integrated assay facility. A linac would generate the interrogating gamma and neutron beams. Insufficiently detailed knowledge about active neutron and gamma interrogation of 500 litre drums of cement immobilised intermediate level waste led to a research programme which is now in its main experimental stage. Measurements of interrogation responses are being made using simulated waste drums containing actinide samples and calibration sources, in an experimental assay assembly. Results show that responses are generally consistent with theory, but that improvements are needed in some areas. A preliminary appraisal of the engineering and economic aspects of integrated assay shows that correct operational sequencing is required to achieve the short cycle time needed for high throughput. The main engineering features of a facility have been identified

  5. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  6. Have you stress tested your assay?

    Directory of Open Access Journals (Sweden)

    Zheng Cao

    2016-08-01

    Full Text Available Objectives: When a clinical assay is stressed with extraordinarily high volume of specimens over a short period of time, extra caution may be needed to avoid systematic errors and biases. Here we report our experience with a HgbA1c assay used for high volume wellness screening purpose, to illustrate the importance of stress testing during assay validation. Design and Methods: Over 15,000 whole blood specimens were tested for HgbA1c in a period of 2 months. HgbA1c was tested by an immunoturbidimetric method on a high through-put automation line. The HgbA1c population distribution in our study was compared to that from the NHANES database. Daily distributions of HgbA1c values ≥6%, means and medians were plotted. Correlation studies were performed between the high through-put immunoturbidimetric assay and a medium through-put HPLC method. Results: We observed a shift of HgbA1c distribution to the higher values compared to the NHANES. A bias of 15–20% was noted from further stress testing where large number of samples were batched and tested using the immunoturbidimetric assay. A 5–7% higher bias remained after implementing a cuvette washing program after each HgbA1c sample. We hypothesized this bias was caused by build-up of blood cell fragments in the cuvettes when continuous whole blood samples are run through the system. Our experience suggests stress testing needs to be incorporated early in the test validation process for high volume batched screening applications. This seemingly extra validation step may save significant troubleshooting and retesting efforts down the road. Keywords: Hemoglobin A1c, Immunoturbidimetric assay, HPLC, Quality assurance, Systematic bias, High volume, Automation

  7. Rapid colorimetric assay for gentamicin injection.

    Science.gov (United States)

    Tarbutton, P

    1987-01-01

    A rapid colorimetric method for determining gentamicin concentration in commercial preparations of gentamicin sulfate injection was developed. Methods currently available for measuring gentamicin concentration via its colored complex with cupric ions in alkaline solution were modified to reduce the time required for a single analysis. The alkaline copper tartrate (ACT) reagent solution was prepared such that each milliliter contained 100 mumol cupric sulfate, 210 mumol potassium sodium tartrate, and 1.25 mmol sodium hydroxide. The assay involves mixing 0.3 mL gentamicin sulfate injection 40 mg/mL (of gentamicin), 1.0 mL ACT reagent, and 0.7 mL water; the absorbance of the resulting solution at 560 nm was used to calculate the gentamicin concentration in the sample. For injections containing 10 mg/mL of gentamicin, the amount of the injection was increased to 0.5 mL and water decreased to 0.5 mL. The concentration of gentamicin in samples representing 11 lots of gentamicin sulfate injection 40 mg/mL and 8 lots of gentamicin sulfate injection 10 mg/mL was determined. The specificity, reproducibility, and accuracy of the assay were assessed. The colored complex was stable for at least two hours. Gentamicin concentration ranged from 93.7 to 108% and from 95 to 109% of the stated label value of the 40 mg/mL and the 10 mg/mL injections, respectively. No components of the preservative system present in the injections interfered with the assay. Since other aminoglycosides produced a colored complex, the assay is not specific for gentamicin. The assay was accurate and reproducible over the range of 4-20 mg of gentamicin. This rapid and accurate assay can be easily applied in the hospital pharmacy setting.

  8. Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)

    Science.gov (United States)

    Ou, Fang; McGoverin, Cushla; Swift, Simon; Vanholsbeeck, Frédérique

    2017-03-01

    Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios. Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

  9. An open-source solution for advanced imaging flow cytometry data analysis using machine learning.

    Science.gov (United States)

    Hennig, Holger; Rees, Paul; Blasi, Thomas; Kamentsky, Lee; Hung, Jane; Dao, David; Carpenter, Anne E; Filby, Andrew

    2017-01-01

    Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using "user-friendly" platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery

  10. Elements of nondestructive assay (NDA) technology

    International Nuclear Information System (INIS)

    Hatcher, C.R.; Smith, H.

    1984-01-01

    A thorough introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards is presented. The general principles and major applications of NDA are discussed and situations in which NDA is particularly useful for nuclear safeguards purposes are described. Various passive and active γ-ray and neutron methods are examined and assay situations particularly suited to γ-ray techniques, or to neutron techniques are identified. The role of calorimetry in the NDA of plutonium-bearing materials is also discussed. The advantages and disadvantages of various NDA methods for different types of nuclear materials are mentioned

  11. Assay of low-level plutonium effluents

    International Nuclear Information System (INIS)

    Hsue, S.T.; Hsue, F.; Bowersox, D.F.

    1981-01-01

    In the plutonium recovery section at the Los Alamos National Laboratory, an effluent solution is generated that contains low plutonium concentration and relatively high americium concentration. Nondestructive assay of this solution is demonstrated by measuring the passive L x-rays following alpha decay. Preliminary results indicate that an average deviation of 30% between L x-ray and alpha counting can be achieved for plutonium concentrations above 10 mg/L and Am/Pu ratios of up to 3; for plutonium concentrations less than 10 mg/L, the average deviation is 40%. The sensitivity of the L x-ray assay is approx. 1 mg Pu/L

  12. A sensitive assay for Staphylococcus aureus nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Kohli, J K; Vakil, B V; Patil, M S; Pandey, V N; Pradhan, D S [Bhabha Atomic Reserach Centre, Bombay (India). Biochemistry Div.

    1989-10-01

    A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured ({sup 3}H) thymidine labelled DNA from E.coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate. (author). 26 refs., 3 figs ., 3 tabs.

  13. Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats

    International Nuclear Information System (INIS)

    Mizushima, Yutaka; Takeichi, Noritoshi; Minami, Akio; Kasai, Masaharu; Itaya, Toshiyuki

    1981-01-01

    KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12 - 15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay ( 51 Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing hosts. (author)

  14. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

    Directory of Open Access Journals (Sweden)

    Angela Filomena

    2017-10-01

    Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  15. Use of the microculture kinetic assay of apoptosis to determine chemosensitivities of leukemias.

    Science.gov (United States)

    Kravtsov, V D; Greer, J P; Whitlock, J A; Koury, M J

    1998-08-01

    Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate-labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell

  16. Flow Control

    Science.gov (United States)

    2013-04-08

    can be written as â fj (t) =WO tanh( WIx (t)+bI)+bO, (38) where WI , WO are the input and output matrices, respectively, and bI and bO are the input...applications, present on envisioned airborne optical platforms . One of the problems is that all adaptive optical systems rely on mechanically moving some...of successfully controlling the optical aberration due to the flow over the aperture of airborne optical platforms . As outlined above, systems

  17. Liquor oligoclonal bands assay: interpretation, correlation with other laboratory assays and importance for diagnostics of neurological disorders

    OpenAIRE

    Bagdonas, Dovydas

    2017-01-01

    Aim: to analyse the possible relationship between liquor IgG oligoclonal bands assay and other laboratory assays in neurological patients. Objectives: to determine the frequency of oligoclonal bands in neurological patients; to compare the results between serum and liquor laboratory assays in dependence of oligoclonal bands assay results; to evaluate the relationships between oligoclonal bands assay and serological-immunological assays for infectious diseases, gender, age and neurological ...

  18. Astrophysical Flows

    Science.gov (United States)

    Pringle, James E.; King, Andrew

    2003-07-01

    Almost all conventional matter in the Universe is fluid, and fluid dynamics plays a crucial role in astrophysics. This new graduate textbook provides a basic understanding of the fluid dynamical processes relevant to astrophysics. The mathematics used to describe these processes is simplified to bring out the underlying physics. The authors cover many topics, including wave propagation, shocks, spherical flows, stellar oscillations, the instabilities caused by effects such as magnetic fields, thermal driving, gravity, shear flows, and the basic concepts of compressible fluid dynamics and magnetohydrodynamics. The authors are Directors of the UK Astrophysical Fluids Facility (UKAFF) at the University of Leicester, and editors of the Cambridge Astrophysics Series. This book has been developed from a course in astrophysical fluid dynamics taught at the University of Cambridge. It is suitable for graduate students in astrophysics, physics and applied mathematics, and requires only a basic familiarity with fluid dynamics.• Provides coverage of the fundamental fluid dynamical processes an astrophysical theorist needs to know • Introduces new mathematical theory and techniques in a straightforward manner • Includes end-of-chapter problems to illustrate the course and introduce additional ideas

  19. Water excretion mechanisms of the kidney studied in the rabbit using tritiated water during the stop-flow assay; Les mecanismes d'excretion de l'eau par le rein etudies a l'aide d'eau tritiee chez le lapin au cours de l'epreuve de diurese interrompue

    Energy Technology Data Exchange (ETDEWEB)

    Morel, F.; Amiel, CI.; Falbriard, A. [Commissariat a l' energie atomique et aux energies alternatives - CEA, Service de Biologie (France)

    1960-07-01

    The pattern of water turnover in the kidney and the mechanisms of water transfer into the urine have been studied in the rabbit using tritiated water as a tracer and the stop-flow technique. The experiments have given the following results: a) During the interruption of the diuresis, the injected tritiated water is completely exchanged with the water of the renal cortex, but the tracer does not reach the deep regions of the kidney, despite the fact that the blood circulation is maintained in these regions; this suggests that the vascular loops of the vasa recta function as a mechanism of water exchange by countercurrent. b) During the osmotic polyuria following the stop-flow period, the concentration gradient of tritiated water inside the kidney diminishes progressively. The concentration of the tracer in the urine is at all time similar to that existing in the deep medulla and the renal papilla and markedly different from that of the cortex or arterial blood. This fact shows that the molecules of water in the urine excreted do not come from either the glomerular filtrate or the convoluted tubules but from the water contained in the deep regions of the kidney. Also these results indicate that the walls of the collecting ducts have a very high permeability to water diffusion. Reprint of a paper published in Revue francaise d'etudes cliniques et biologiques, Vol. IV, no. 8, p. 773-779, 1959 [French] Les modalites du renouvellement de l'eau dans le rein et les mecanismes de son passage dans l'urine ont ete etudies chez le lapin a l'aide d'eau tritiee, employee comme indicateur au cours de l'epreuve de 'diurese interrompue' (stop flow). Les experiences effectuees montrent que: a) pendant l'interruption de la diurese, l'eau tritiee injectee s'echange completement avec l'eau contenue dans le cortex renal, mais elle n'atteint pratiquement pas les regions profondes du rein, bien que la circulation sanguine y soit maintenue; cette observation suggere que les anses

  20. Enzymatic assay for methotrexate in erythrocytes

    DEFF Research Database (Denmark)

    Schrøder, H; Heinsvig, E M

    1985-01-01

    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...

  1. Comet assay. Pt.1. Theory and practice

    International Nuclear Information System (INIS)

    Kruszewski, M.; Wojewodzka, M.; Iwanenko, T.

    1996-01-01

    Comet assay is a new method for measuring DNA breakage in a single cell. The main applications of the method are estimation of DNA single and double strand breaks, oxidative damage, pyrimidine dimers and (6-4)photoproducts, DNA-DNA and DNA-protein crosslinks. The method is used for studying DNA damage and its repair. (author).19 refs, 9 figs

  2. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Science.gov (United States)

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  3. Nondestructive assay of HTGR fuel rods

    International Nuclear Information System (INIS)

    Menlove, H.O.

    1974-01-01

    Performance characteristics of three different radioactive source NDA systems are compared for the assay of HTGR fuel rods and stacks of rods. These systems include the fast neutron Sb-Be assay system, the 252 Cf ''Shuffler,'' and the thermal neutron PAPAS assay system. Studies have been made to determinethe perturbation on the measurements from particle size, kernel Th/U ratio, thorium content, and hydrogen content. In addition to the total 235 U determination, the pellet-to-pellet or rod-to-rod uniformity of HTGR fuel rod stacks has been measured by counting the delayed gamma rays with a NaI through-hole in the PAPAS system. These measurements showed that rod substitutions can be detected easily in a fuel stack, and that detailed information is available on the loading variations in a uniform stack. Using a 1.0 mg 252 Cf source, assay rates of 2 to 4 rods/s are possible, thus facilitating measurement of 100 percent of a plant's throughput. (U.S.)

  4. The use of calorimetry for plutonium assay

    International Nuclear Information System (INIS)

    Mason, J.A.

    1982-12-01

    Calorimetry is a technique for measuring the thermal power of heat-producing substances. The technique may be applied to the measurement of plutonium-bearing materials which evolve heat as a result of alpha and beta decay. A calorimetric measurement of the thermal power of a plutonium sample, combined with a knowledge or measurement of the plutonium isotopic mass ratios of the sample provides a convenient and accurate, non-destructive measure of the total plutonium mass of the sample. The present report provides a description, and an assessment of the calorimetry technique applied to the assay of plutonium-bearing materials. Types and characteristics of plutonium calorimeters are considered, as well as calibration and operating procedures. The instrumentation used with plutonium calorimeters is described and the use of computer control for calorimeter automation is discussed. A critical review and assessment of plutonium calorimetry literature since 1970 is presented. Both fuel element and plutonium-bearing material calorimeters are considered. The different types of plutonium calorimeters are evaluated and their relative merits are discussed. A combined calorimeter and gamma-ray measurement assay system is considered. The design principles of plutonium assay calorimeters are considered. An automatic, computer-based calorimeter control system is proposed in conjunction with a general plutonium assay calorimeter design. (author)

  5. Endoproteolytic activity assay in malting barley

    Directory of Open Access Journals (Sweden)

    Blanca Gómez Guerrero

    2013-12-01

    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  6. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

  7. 21 CFR 864.7250 - Erythropoietin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250 Erythropoietin...

  8. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

  9. Radioligand assay for biotin in liver tissues

    International Nuclear Information System (INIS)

    Rettenmaier, R.

    1979-01-01

    A radioligand assay for biotin in liver tissue is described. 3 H-biotin is used as tracer and avidin as binder. The biotin-loaded avidin is separated from free biotin on dextran-coated charcoal, which leaves the avidin-biotin complex in the supernatant liquid. Thus, the avidin-biotin complex can easily be utilized for determination of the radioactivity. Calibration with known additions of biotin in the range 0.25-8.0 ng per assay sample yields a linear logit-log plot. The biotin is extracted from liver tissues by enzymatic proteolysis with papain. This treatment is optimized to liberate the bound forms of the vitamin. Microbiological parallel assays with Lactobacillus plantarum were in good agreement with the radioligand assay giving a regression coefficient of 0.974(n=44). The coefficient of variation was found to be 4.2% in the range 500-1200 ng of biotin per g of liver tissue (n=46). The method is simple and reliable and allows the simultaneous analysis of a considerable number of samples. (Auth.)

  10. Relationships between ytterbium precipitation assay, colorimetric ...

    African Journals Online (AJOL)

    digestion and metabolism of protein (Komolong et al., 2001). ... room temperature (25 °C) pending chemical analyses and in vitro ... assayed without sodium sulphite but with a heat-stable α-amylase due to the high ... of starch in the tree fruits.

  11. Nondestructive assay of boxed radioactive waste

    International Nuclear Information System (INIS)

    Gilles, W.P.; Roberts, R.J.; Jasen, W.G.

    1992-12-01

    This paper describes the problems related to the nondestructive assay (NDA) of boxed radioactive waste at the Hanford Site and how Westinghouse Hanford company (WHC) is solving the problems. The waste form and radionuclide content are described. The characteristics of the combined neutron and gamma-based measurement system are described

  12. ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

    Science.gov (United States)

    Oldaker, Teri; Whitby, Liam; Saber, Maryam; Holden, Jeannine; Wallace, Paul K; Litwin, Virginia

    2018-01-01

    Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  13. Production and assay of forskolin antibodies

    International Nuclear Information System (INIS)

    Ho, L.T.; Ho, R.J.

    1986-01-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using 3 H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added 3 H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC 50 was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound

  14. Assays for mammalian tyrosinase: a comparative study

    International Nuclear Information System (INIS)

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed

  15. Direct 125I-radioligand assays for serum progesterone compared with assays involving extraction of serum

    International Nuclear Information System (INIS)

    Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.; Macpherson, J.S.

    1982-01-01

    Two direct radioimmunoassays for progesterone in 50 μL of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11α-hemisuccinyl conjugate and the radioligand 125 I-labeled progesterone 11α-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum

  16. Evaluation of total PSA assay on vitros ECi and correlation with Kryptor-PSA assay.

    Science.gov (United States)

    Cassinat, B; Wacquet, M; Toubert, M E; Rain, J D; Schlageter, M H

    2001-01-01

    An increasing number of multiparametric immuno-analysers for PSA assays are available. As different immuno-assays may vary in their analytical quality and their accuracy for the follow-up of patients, expertise is necessary for each new assay. The PSA assay on the Vitros-ECi analyser has been evaluated and compared with the PSA assay from the Kryptor analyser. Variation coefficients were 0.91 to 1.98% for within-run assays, and 4.2% to 5.4% for interassay (PSA levels = 0.8 microgram/L to 33.6 micrograms/L). Dilution tests showed 93 to 136% recovery until 70 micrograms/L PSA. Functional sensitivity was estimated at 0.03 microgram/L. Equimolarity of the test was confirmed. Correlation of PSA levels measured with Vitros-ECi and Kryptor analysers displayed a correlation coefficient r2 of 0.9716. The half-lives and doubling times of PSA were similar using both methods. Vitros-ECi PSA assay meets the major criteria for the management of prostate cancer patients.

  17. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

  18. Short communication. Microculture syncytia assay for bovine leukemia virus

    Energy Technology Data Exchange (ETDEWEB)

    Paul, P.S.; Castro, A.E.; Pomeroy, K.A.; Johnson, D.W.; Muscoplat, C.C.

    1978-01-01

    A microculture syncytia assay for the detection of bovine leukemia virus (BLV) has been described and compared with the conventional macroculture assay. The microculture assay required fewer indicator cells, was as sensitive as the macroculture assay and provided a reproducible test for the detection and titration of BLV.

  19. A simple clot based assay for detection of procoagulant cell-derived microparticles.

    Science.gov (United States)

    Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

    2016-05-01

    Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

  20. Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium.

    OpenAIRE

    Felder Marcel; Sallin Pauline; Barbe Laurent; Haenni Beat; Gazdhar Amiq; Geiser Thomas; Guenat Olivier

    2012-01-01

    We present a microfluidic epithelial wound healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system a technique that is reported here ...

  1. Evaluation of furocoumarins as photosynthetic inhibitor by chlorophyll a fluorescence assay

    OpenAIRE

    Sampaio, Olívia Moreira; Silva, Maria Fátima das Graças Fernandes da; Veiga, Thiago Andre Moura [UNIFESP; King-Díaz, Beatriz; Lotina-Hennsen, Blas

    2012-01-01

    The evaluations of Chorophyll a fluorescence emitted by superior plants carry structural information and photosynthetic apparatus function. Quantitative analysis apparatus of fluorescence kinetic were measured by energy flows (ABS), (TR), (ET) and (DI), known as phenomenological phenomena of OJIP test. Four furocoumarins were isolated from Ruta graveolens (Rutaceae), and chorophyll a (Chl a) fluorescence assays were performed with these compounds to evaluate the photosynthesis inhibition pote...

  2. Radio Column Chromatographic Assay of H3-Labelled Substances

    International Nuclear Information System (INIS)

    Scharpenseel, H.W.; Menke, K.H.

    1962-01-01

    Combined radio-chromatographic investigations of H 3 -labelled substances are an integral part of the majority of biochemical experiments with H 3 -labelled compounds. H 3 -radio paper chromatography yields, in a scanner with a windowless flow counter, a counting efficiency of 0,5 -1,5%, depending largely on the thickness of the paper and the self-absorption of the labelled compound. The radio gas chromatography of tritiated compounds presents no major problem. Successful use is being made of a combination of a gas chromatograph with a flow ionization chamber and vibrating reed electrometer, a system originated by K. E. Wilzbach and P. Riessz, and improved by H. Dutton, L. Mason and L. Blair. Through the use of ''Teflon'' and silicone-rubber for the insulating parts of the flow ion chamber, it can be operated at close to 300 o C. Radio column chromatography with tritium holds little promise, when the column effluent is spread out as a shallow layer and slowly passes under a windowless flow counter or a scintillation counter, as was successfully tried with C 14 . Liquid scintillation spectrometry is likely to be the chosen method. Essentially, there are two different approaches feasible. These have been compared: 1. The column effluent is passed through a coil of plastic scintillator tubing, which is wound around a ''Plexiglas'' cylinder and placed in a bath of silicone oil in a light pipe with TiO 2 -reflector. Similarly, the HP-containing effluent can be directed through a test vial, filled - very much as in Steinberg's method - with plastic scintillator beads. These two approaches, that operate highly satisfactorily in the case of C 14 , offer low counting efficiencies of less than 1% for H 3 due to the unfavourable surface to volume ratio. 2. The column effluent is combined 1:30 with a mixture of 3:2 toluene/ethanol by the action of a magnet-vibrator before being assayed while passing through a K 40 -free glass - coiled between the analyser- and monitor

  3. Storage and Assay of Tritium in STAR

    International Nuclear Information System (INIS)

    Longhurst, Glen R.; Anderl, Robert A.; Pawelko, Robert J.; Stoots, Carl J.

    2005-01-01

    The Safety and Tritium Applied Research (STAR) facility at the Idaho National Engineering and Environmental Laboratory (INEEL) is currently being commissioned to investigate tritium-related safety questions for fusion and other technologies. The tritium inventory for the STAR facility will be maintained below 1.5 g to avoid the need for STAR to be classified as a Category 3 nuclear facility. A key capability in successful operation of the STAR facility is the ability to receive, inventory, and dispense tritium to the various experiments underway there. The system central to that function is the Tritium Storage and Assay System (SAS).The SAS has four major functions: (1) receiving and holding tritium, (2) assaying, (3) dispensing, and (4) purifying hydrogen isotopes from non-hydrogen species.This paper describes the design and operation of the STAR SAS and the procedures used for tritium accountancy in the STAR facility

  4. Identification of irradiated pepper with comet assay

    International Nuclear Information System (INIS)

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra; Iglesia Enriquez, Isora

    2007-01-01

    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  5. Radioligand purification prior to routine receptor assays

    International Nuclear Information System (INIS)

    Le Goff, J.-M.; Berthois, Y.; Martin, P.-M.

    1988-01-01

    The need to repurify the commercially available radioligands [ 3 H]estradiol and [ 3 H]testosterone before use in routine assays was investigated. Storage of these products for 2 months after delivery led to appreciable degradation of [ 3 H]estradiol compared to [ 3 H]testosterone. Unexpectedly, TLC and even HPLC procedures were ineffective in completely restoring the purity of [ 3 H]-estradiol and the unremoved polar products induced important variations in our estrogen receptor assays. An increase in non-specific binding and a concomitant decrease in total binding were observed resulting in an underestimation of specific binding sites and of the affinity constant. In some cases Scatchard analysis was not possible. The authors therefore strongly recommend the repurification of low-stability radioligands and propose an economic time-saving procedure for the purification of [ 3 H]estradiol by solvent differential partition which requires no high-cost investment in apparatus. (author)

  6. Assay of 25-OH vitamin D3

    International Nuclear Information System (INIS)

    Nayer, P. de; Thalasso, M.; Beckers, C.

    1977-01-01

    A simplified version of the competitive protein binding assay for 25-OH vit D3 derived from the method of Belsey et al. is presented. The procedure does not include a chromatography step, and is performed on an alcoolic extract of 0.1 ml plasma or serum. Normal rat serum (1:20,000) was used as binding protein. No β-lipoproteins were added to the assay buffer. A 10% displacement of the tracer was observed at 0.04 ng/tube, and 50% at 0.15 ng/tube, allowing for the measurement of 25-OH vit D3 concentrations between 2 ng/ml and 200 ng/ml. Mean values in a normal group was 23.1 +- 6.5 ng/ml (range 16-37 ng/ml, n = 11). (orig.) [de

  7. Developments in plutonium waste assay at AWE

    International Nuclear Information System (INIS)

    Miller, T J

    2009-01-01

    In 2002 a paper was presented at the 43rd Annual Meeting of the Institute of Nuclear Materials Management (INMM) on the assay of low level plutonium (Pu) in soft drummed waste (Miller 2002 INMM Ann. Meeting (Orlando, FL, 23-27 July 2002)). The technique described enabled the Atomic Weapons Establishment (AWE), at Aldermaston in the UK, to meet the stringent Low Level Waste Repository at Drigg (LLWRD) conditions for acceptance for the first time. However, it was initially applied to only low density waste streams because it relied on measuring the relatively low energy (60 keV) photon yield from Am-241 during growth. This paper reviews the results achieved when using the technique to assay over 10 000 waste packages and presents the case for extending the range of application to denser waste streams.

  8. Neutron Assay System for Confinement Vessel Disposition

    International Nuclear Information System (INIS)

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Mayo, Douglas R.; Miko, David K.; Salazar, William R.; Stange, Sy; Valdez, Jose I.; Vigil, Georgiana M.

    2012-01-01

    Los Alamos National Laboratory has a number of spherical confinement vessels (CVs) remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1-inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the CVs. The Confinement Vessel Assay System (CVAS) was developed to measure the amount of special nuclear material (SNM) in CVs before and after cleanout. Prior to cleanout, the system will be used to perform a verification measurement of each vessel. After cleanout, the system will be used to perform safeguards-quality assays of (le)100-g 239 Pu equivalent in a vessel for safeguards termination. The CVAS has been tested and calibrated in preparation for verification and safeguards measurements.

  9. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

    Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

  10. Expert system technology for nondestructive waste assay

    International Nuclear Information System (INIS)

    Becker, G.K.; Determan, J.C.

    1998-01-01

    Nondestructive assay waste characterization data generated for use in the National TRU Program must be of known and demonstrable quality. Each measurement is required to receive an independent technical review by a qualified expert. An expert system prototype has been developed to automate waste NDA data review of a passive/active neutron drum counter system. The expert system is designed to yield a confidence rating regarding measurement validity. Expert system rules are derived from data in a process involving data clustering, fuzzy logic, and genetic algorithms. Expert system performance is assessed against confidence assignments elicited from waste NDA domain experts. Performance levels varied for the active, passive shielded, and passive system assay modes of the drum counter system, ranging from 78% to 94% correct classifications

  11. LDLCHOLESTEROLEXAMINATION (LDL-C USINGHOMOGENEOUS ASSAY

    Directory of Open Access Journals (Sweden)

    Made DwiAmbara Putra

    2013-07-01

    Full Text Available Homogeneous method describe as a method that does not require separation of free and bound label. This method has the ability tofully automate the determination of LDL-C directly small sample volume sand short examination time. In addition this method use automated pipette and control of time and temperature more accurate. There are 5 methods i.e. Solubilization homogeneous LDL-C assay (SOL from KyowaMedex, Surfactant LDL-C assay (SUR from Daiichi Pure Chemicals, Protecting LDL-assay reagent (PRO from Wako Chemicals, LDL-C assaycatalase (CAT Denka Seiken and Calixarene of LDL-C assay (CAL from International Reagents Corporation. All method is to use a variety of detergents and other chemicals that cause blocking or dissolution of specific lipoprotein classes to achieve specificity for LDL. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  12. Assaying the reporter gene chloramphenicol acetyltransferase

    International Nuclear Information System (INIS)

    Crabb, D.W.; Minth, C.D.; Dixon, J.E.

    1989-01-01

    These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product

  13. The electrophoretic mobility shift assay (EMSA)

    OpenAIRE

    sprotocols

    2015-01-01

    The electrophoretic mobility shift assay (EMSA), also known as “gel shift assay”, is used to examine the binding parameters and relative affinities of protein and DNA interactions. We produced recombinant CCA1 protein and tested its binding affinity for the promoter fragments that contain CBS (AAAAATCT) or evening element (EE, AAAATATCT) (1) using a modified procedure adopted from published protocols (2,3).

  14. Nondestructive assay methodologies in nuclear forensics analysis

    International Nuclear Information System (INIS)

    Tomar, B.S.

    2016-01-01

    In the present chapter, the nondestructive assay (NDA) methodologies used for analysis of nuclear materials as a part of nuclear forensic investigation have been described. These NDA methodologies are based on (i) measurement of passive gamma and neutrons emitted by the radioisotopes present in the nuclear materials, (ii) measurement of gamma rays and neutrons emitted after the active interrogation of the nuclear materials with a source of X-rays, gamma rays or neutrons

  15. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  16. Quality control of estrogen receptor assays.

    Science.gov (United States)

    Godolphin, W; Jacobson, B

    1980-01-01

    Four types of material have been used for the quality control of routine assays of estrogen receptors in human breast tumors. Pieces of hormone-dependent Nb rat mammary tumors gave a precision about 40%. Rat uteri and rat tumors pulverized at liquid nitrogen temperature and stored as powder yielded precision about 30%. Powdered and lyophilised human tumors appear the best with precision as good as 17%.

  17. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

    Directory of Open Access Journals (Sweden)

    Snehal Shabrish

    2016-01-01

    Full Text Available Natural killer (NK cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA and calcium ionophore (Ca2+-ionophore instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p<0.0001. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

  18. Immunocapture loop-mediated isothermal amplification assays for the detection of canine parvovirus.

    Science.gov (United States)

    Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

    2017-11-01

    A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10 -1 , 10 -1 , and 10 -1 TCID 50 /mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Design of radiation dose tumor response assays

    International Nuclear Information System (INIS)

    Suit, H.D.; Hwang, T.; Hsieh, C.; Thames, H.

    1985-01-01

    The efficient utilization of animals in a radiation dose response assay for tumor control requires a definition of the goal, e.g., TCD50 or slope. A series of computer modelled ''experiments'' have been performed for each of a number of allocations of dose levels (DL) and number of animals/DL. The authors stipulated that the assumed TCD50 was .85 of true value; assumed slope was correct. They stipulated a binominal distribution of observed tumor control results at each dose level. A pilot assay used 6 tumors at 7 DL (from TCD1-TCD97). The second assay used 30 tumors assigned to 2,3,5 or 9 DL and to selected tumor control probabilities (TCP derived from the pilot run. Results from 100 test runs were combined with the pilot run for each of the combination of DL and TCP values. Logit regression lines were fitted through these ''data'' and the 95% CL around the TCD50 and the TCD37 values and the variances of the slopes were computed. These experiments were repeated using the method suggested by Porter (1980). Results show that a different strategy is needed depending upon the goal, viz. TCD50 or TCD37 vs slope. The differences between the two approaches are discussed

  20. New Application of the Comet Assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

  1. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier

    2014-01-01

    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  2. Chromosome aberration assays in barley (Hordeum vulgare)

    Energy Technology Data Exchange (ETDEWEB)

    Constantin, M J [Univ. of Tennessee, Knoxville; Nilan, R A

    1982-01-01

    Barley is an exceellent organism for studies of induced chromosome aberrations because of its few (2n = 2x = 14) relatively large chromosomes. Root-tip and shoot-tip cells have been used extensively for the study of ionizing radiation-induced chromosome aberrations. The general procedures are well known, the technology is simple and easy to learn, and the assays are relatively quick and inexpensive. Both root tips and shoot tips can be used for the study of chemical mutagens as well as ionizing radiations. Pollen mother cells are well suited for studying the effects of mutagens on meiotic chromosomes. The literature review for the Gene-Tox Program reported on 61 chemicals tested for their effects on barley chromosomes. Of these, 90% were reported to be either positive or positive dose-related, while 7% were negative and 3% were questionable. Barley assays based on chromosomal aberrations are useful to detect the clastogenic potency of chemicals under laboratory conditions. Indications are that the data from barley can be used to corroborate data obtained from other organisms. Among the classes of chemicals assayed were: alcohols and phenols; alkaloids; epoxides; alkyl sulfates; amides and sulfonamides; aromatic amines; aryl halides; aziridines; alkenes; carbamates; hydroazides; nitroaromatics; nitrosamides; nitrosources; phenothiazines; and polycyclic aromatic hydrocarbons.

  3. Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

    International Nuclear Information System (INIS)

    Schulster, D.

    1977-01-01

    Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

  4. A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

    International Nuclear Information System (INIS)

    Patibandla, Phani K; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

    2014-01-01

    Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (∼45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

  5. Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

    Science.gov (United States)

    Steudler, Susanne; Böhmer, Ulrike; Weber, Jost; Bley, Thomas

    2015-02-01

    Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  6. Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry

    Science.gov (United States)

    He, Shengbin; Hong, Xinyi; Huang, Tianxun; Zhang, Wenqiang; Zhou, Yingxing; Wu, Lina; Yan, Xiaomei

    2017-06-01

    A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

  7. Image cytometer method for automated assessment of human spermatozoa concentration

    DEFF Research Database (Denmark)

    Egeberg, D L; Kjaerulff, S; Hansen, C

    2013-01-01

    In the basic clinical work-up of infertile couples, a semen analysis is mandatory and the sperm concentration is one of the most essential variables to be determined. Sperm concentration is usually assessed by manual counting using a haemocytometer and is hence labour intensive and may be subject...

  8. Microfluidic Cytometer for Complete Blood Count Analysis, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — RMD proposes to develop a MEMS based complete blood count (CBC) instrument that can be used aboard a spacecraft. We will produce a microfluidic scale combination...

  9. Microfluidic Cytometer for Complete Blood Count Analysis, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — We will fabricate and test microfluidic designs for a micro-electromechanical system based complete blood count (CBC) analysis in separate modules and integrate them...

  10. Absolute counting of neutrophils in whole blood using flow cytometry.

    Science.gov (United States)

    Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

    2014-12-01

    Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

  11. MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

    Science.gov (United States)

    Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

    2014-08-05

    Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

  12. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1984-01-01

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

  13. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  14. Ultrasonic flow meter

    NARCIS (Netherlands)

    Lötters, Joost Conrad; Snijders, G.J.; Volker, A.W.F.

    2014-01-01

    The invention relates to an ultrasonic flow meter comprising a flow tube for the fluid whose flow rate is to be determined. The flow meter comprises a transmitting element for emitting ultrasonic waves, which is provided on the outer jacket of the flow tube. A receiving element, which is provided on

  15. Biobarcode assay for the oral anticoagulant acenocoumarol.

    Science.gov (United States)

    Broto, Marta; Salvador, J Pablo; Galve, Roger; Marco, M Pilar

    2018-02-01

    A novel approach for therapeutic drug monitoring of oral anticoagulants (OA) in clinical samples is reported, based on a NP-based biobarcode assay. The proposed strategy uses specific antibodies for acenocumarol (ACL) covalently bound to magnetic particles (pAb236-MP) and a bioconjugate competitor (hACL-BSA) linked to encoded polystyrene probes (hACL-BSA-ePSP) on a classical competitive immunochemical format. By using this scheme ACL can be detected in low nM range (LOD, 0.96 ± 0.26, N = 3, in buffer) even in complex samples such as serum or plasma (LOD 4 ± 1). The assay shows a high reproducibility (%CV 1.1 day-to-day) and is robust, as it is demonstrated by the fact that ACL can be quantified in complex biological samples with a very good accuracy (slope = 0.97 and R 2 = 0.91, of the linear regression obtained when analyzing spiked vs measured values). Moreover, we have demonstrated that the biobarcode approach has the potential to overcome one of the main challenges of the multiplexed diagnostic, which is the possibility to measure in a single run biomarker targets present at different concentration ranges. Thus, it has been proven that the signal and the detectability can be modulated by just modifying the oligonucleotide load of the encoded probes. This fact opens the door for combining in the same assay encoded probes with the necessary oligonucleotide load to achieve the detectability required for each biomarker target. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Radiometric assays for the measurement of PSA

    International Nuclear Information System (INIS)

    Venkatesh, M.

    1997-01-01

    Prostate Specific Antigen, a serine protease enzyme, of M.W. ∼ 26-33 kDa, is widely considered to be a very useful marker for prostate cancer. It satisfies nearly all the requirements of an ideal 'Tumour Marker' and has hence attracted a lot of attention in the past decade. PSA is present in multiple forms in serum, with an appreciable fraction bound to the protease inhibitor α-1-antichymotrypsin (ACT) and to a small extent to other proteins such as α-2-macroglobulin (AMG) leaving the rest in the free form. The total PSA levels have been reported to have 80% sensitivity and 60% specificity towards the detection of prostate cancer. The lack of specificity occurs mainly due to the high levels of t-PSA in benign prostatic hypertrophy(BPH) apart from the cancer. The concept of free PSA has been introduced in the recent past and the ratio of free/total PSA levels have been shown to be advantageous in the differential diagnosis of BPH from prostate cancer. The f/t ratio is considered to be particularly useful in the grey zones of decision making (t-PSA levels 4-20 ng/mL). The need for the development of assays for total and free PSA is felt due to: a. the high incidence of prostate cancers being detected currently; b. the high cost of tests (higher for free PSA assay, and the cost becomes an important parameter when a patient has to be regularly monitored after therapy) that is not affordable for many patients; c. the potential for research in the area of prostate cancer management where the PSA (total and free) assays will be of great help

  17. Results of in vitro chemosensitivity assays

    International Nuclear Information System (INIS)

    Tanigawa, Nobuhiko; Morimoto, Hideki; Akita, Toshiaki; Inoue, Hiroshi; Tanaka, Takeo.

    1986-01-01

    The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60 % of specimens in HTCA and 58 % in SA produced significant growth in vitro. HTCA was 52 % (13/25) reliable for predicting in vivo sensitivity, and 95 % (36/38) reliable for in vivo resistance, whereas SA was 40 % (8/20) reliable for in vivo sensitivity and 88 % (21/24) for in vivo resistance. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48 % and 60 % in HTCA, and 46 % and 68 % in SA, respectively). (p < 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between : 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors. (J.P.N.)

  18. Assessing sediment contamination using six toxicity assays

    OpenAIRE

    Allen G. BURTON Jr.; Carolyn ROWLAND; Renato BAUDO; Monica BELTRAMI

    2001-01-01

    An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists ...

  19. ARIES nondestructive assay system operation and performance

    International Nuclear Information System (INIS)

    Cremers, Teresa L.; Hansen, Walter J.; Herrera, Gary D.; Nelson, David C.; Sampson, Thomas E.; Scheer, Nancy L.

    2000-01-01

    The ARIES (Advanced Recovery and Integrated Extraction System) Project is an integrated system at the Los Alamos Plutonium Facility for the dismantlement of nuclear weapons. The plutonium produced by the ARIES process was measured by an integrated nondestructive assay (NDA) system. The performance of the NDA systems was monitored by a measurement control program which is a part of a nuclear material control and accountability system. In this paper we will report the results of the measurements of the measurement control standards as well as an overview of the measurement of the ARIES process materials

  20. Test procedure for boxed waste assay system

    International Nuclear Information System (INIS)

    Wachter, J.

    1994-01-01

    This document, prepared by Los Alamos National Laboratory's NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system's embedded operating and data reduction software

  1. Assaying gene function by growth competition experiment.

    Science.gov (United States)

    Merritt, Joshua; Edwards, Jeremy S

    2004-07-01

    High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

  2. Automatic titrator for high precision plutonium assay

    International Nuclear Information System (INIS)

    Jackson, D.D.; Hollen, R.M.

    1986-01-01

    Highly precise assay of plutonium metal is required for accountability measurements. We have developed an automatic titrator for this determination which eliminates analyst bias and requires much less analyst time. The analyst is only required to enter sample data and start the titration. The automated instrument titrates the sample, locates the end point, and outputs the results as a paper tape printout. Precision of the titration is less than 0.03% relative standard deviation for a single determination at the 250-mg plutonium level. The titration time is less than 5 min

  3. First results with a radioreceptor-assay (TRAK-Assay) for TSH-receptor-autoantibodies

    International Nuclear Information System (INIS)

    Becker, W.; Reiners, C.; Boerner, W.

    1983-01-01

    A new radioreceptor-assay (TRAK-assay) for autoantibodies against TSH-receptors was tested in 48 untreated thyrotoxic patients (26 regional autonomies, 22 toxic diffuse goiters). None of the 26 patients with regional autonomy showed positive autoantibody-titers. 4 patients with toxic diffuse goiter and thyrotoxic exophthalmos were TRAK-positive. Positive titers of microsomal and thyreoglobulin autoantibodies could be seen in 8 of 9 patients with positive TRAK-titers. In accordance with the conventional methods for detecting thyroid-stimulating immunoglobulins the new TRAK-assay seems to be suited for differentiating between immunogenic toxic diffuse goiter (Graves' disease) and goiter with disseminated autonomy as well as for prediction of relapse. (orig.) [de

  4. Radioactive waste package assay facility. Volume 1. Application of assay technology

    International Nuclear Information System (INIS)

    Findlay, D.J.S.; Green, T.H.; Molesworth, T.V.; Staniforth, D.; Strachan, N.R.; Rogers, J.D.; Wise, M.O.; Forrest, K.R.

    1992-01-01

    This report, in three volumes, covers the work carried out by Taylor Woodrow Construction Ltd., and two major sub-contractors: Harwell Laboratory (AEA Technology) and Siemens Plessey Controls Ltd., on the development of a radioactive waste package assay facility, for cemented 500 litre intermediate level waste drums. In volume 1, the reasons for assay are considered together with the various techniques that can be used, and the information that can be obtained. The practical problems associated with the use of the various techniques in an integrated assay facility are identified, and the key parameters defined. Engineering and operational features are examined and provisional designs proposed for facilities at three throughput levels: 15,000, 750 and 30 drums per year respectively. The capital and operating costs for such facilities have been estimated. A number of recommendations are made for further work. 16 refs., 14 figs., 13 tabs

  5. Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values.

    Science.gov (United States)

    Galbraith, David W

    2009-08-01

    Flow cytometry provides a rapid, accurate, and simple means to determine nuclear DNA contents (C-value) within plant homogenates. This parameter is extremely useful in a number of applications in basic and applied plant biology; for example, it provides an important starting point for projects involving whole genome sequencing, it facilitates characterization of plant species within natural and agricultural settings, it allows facile identification of engineered plants that are euploid or that represent desired ploidy classes, it points toward studies concerning the role of C-value in plant growth and development and in response to the environment and in terms of evolutionary fitness, and, in uncovering new and unexpected phenomena (for example endoreduplication), it uncovers new avenues of scientific enquiry. Despite the ease of the method, C-values have been determined for only around 2% of the described angiosperm (flowering plant) species. Within this small subset, one of the most remarkable observations is the range of 2C values, which spans at least two orders of magnitude. In determining C-values for new species, technical issues are encountered which relate both to requirement for a method that can provide accurate measurements across this extended dynamic range, and that can accommodate the large amounts of debris which accompanies flow measurements of plant homogenates. In this study, the use of the Accuri C6 flow cytometer for the analysis of plant C-values is described. This work indicates that the unusually large dynamic range of the C6, a design feature, coupled to the linearity of fluorescence emission conferred by staining of nuclei using propidium iodide, allows simultaneous analysis of species whose C-values span that of almost the entire described angiosperms. Copyright 2009 International Society for Advancement of Cytometry.

  6. Flow of Aqueous Humor

    Science.gov (United States)

    ... Home Flow of Aqueous Humor Flow of Aqueous Humor Most, but not all, forms of glaucoma are ... remains normal when some of the fluid (aqueous humor) produced by the eye's ciliary body flows out ...

  7. Fluorescence lifetime assays: current advances and applications in drug discovery.

    Science.gov (United States)

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

  8. The Statistics of wood assays for preservative retention

    Science.gov (United States)

    Patricia K. Lebow; Scott W. Conklin

    2011-01-01

    This paper covers general statistical concepts that apply to interpreting wood assay retention values. In particular, since wood assays are typically obtained from a single composited sample, the statistical aspects, including advantages and disadvantages, of simple compositing are covered.

  9. Use of polymeric dyes in lignin biodegradation assays

    International Nuclear Information System (INIS)

    Gold, M.H.; Alic, M.; Glenn, J.K.

    1988-01-01

    This paper reviews the historical use of various 14 C-radiolabeled and unlabeled substrates to screen for ligninolytic activity. The disadvantages of these assays are presented. The authors describe the development of assays utilizing polymeric dyes

  10. MCNP efficiency calculations of INEEL passive active neutron assay system for simulated TRU waste assays

    International Nuclear Information System (INIS)

    Yoon, W.Y.; Meachum, T.R.; Blackwood, L.G.; Harker, Y.D.

    2000-01-01

    The Idaho National Engineering and Environmental Laboratory Stored Waste Examination Pilot Plant (SWEPP) passive active neutron (PAN) radioassay system is used to certify transuranic (TRU) waste drums in terms of quantifying plutonium and other TRU element activities. Depending on the waste form involved, significant systematic and random errors need quantification in addition to the counting statistics. To determine the total uncertainty of the radioassay results, a statistical sampling and verification approach has been developed. In this approach, the total performance of the PAN nondestructive assay system is simulated using the computer models of the assay system, and the resultant output is compared with the known input to assess the total uncertainty. The supporting steps in performing the uncertainty analysis for the passive assay measurements in particular are as follows: (1) Create simulated waste drums and associated conditions; (2) Simulate measurements to determine the basic counting data that would be produced by the PAN assay system under the conditions specified; and (3) Apply the PAN assay system analysis algorithm to the set of counting data produced by simulating measurements to determine the measured plutonium mass. The validity of this simulation approach was verified by comparing simulated output against results from actual measurements using known plutonium sources and surrogate waste drums. The computer simulation of the PAN system performance uses the Monte Carlo N-Particle (MCNP) Code System to produce a neutron transport calculation for a simulated waste drum. Specifically, the passive system uses the neutron coincidence counting technique, utilizing the spontaneous fission of 240 Pu. MCNP application to the SWEPP PAN assay system uncertainty analysis has been very useful for a variety of waste types contained in 208-ell drums measured by a passive radioassay system. The application of MCNP to the active radioassay system is also feasible

  11. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    Huggins, K.G.; Roitt, I.M.

    1979-01-01

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  12. Assaying environmental nickel toxicity using model nematodes.

    Directory of Open Access Journals (Sweden)

    David Rudel

    Full Text Available Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water, we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  13. Rapid multiple immunoenzyme assay of mycotoxins.

    Science.gov (United States)

    Urusov, Alexandr E; Zherdev, Anatoly V; Petrakova, Alina V; Sadykhov, Elchin G; Koroleva, Olga V; Dzantiev, Boris B

    2015-01-27

    Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin-polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

  14. Rapid Multiple Immunoenzyme Assay of Mycotoxins

    Directory of Open Access Journals (Sweden)

    Alexandr E. Urusov

    2015-01-01

    Full Text Available Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

  15. Rapid screening assay for calcium bioavailability studies

    International Nuclear Information System (INIS)

    Luhrsen, K.R.; Hudepohl, G.R.; Smith, K.T.

    1986-01-01

    Calcium bioavailability has been studied by numerous techniques. The authors report here the use of the gamma emitting isotope of calcium ( 47 Ca) in a whole body retention assay system. In this system, calcium sources are administered by oral gavage and subsequent counts are determined and corrected for isotopic decay. Unlike iron and zinc retention curves, which exhibit a 2-3 day equilibration period, calcium reaches equilibration after 24 hours. Autoradiographic analysis of the femurs indicate that the newly absorbed calcium is rapidly distributed to the skeletal system. Moreover, the isotope is distributed along the entire bone. Comparisons of calcium bioavailability were made using intrinsic/extrinsic labeled milk from two species i.e. rat and goat as well as CaCO 3 . In addition, extrinsic labeled cow milk was examined. In the rat, the extrinsic labeled calcium from milk was better absorbed than the intrinsic calcium. This was not the case in goat milk or the calcium carbonate which exhibited no significant differences. Chromatographic analysis of the labeled milk indicates a difference in distribution of the 47 Ca. From these data, the authors recommend the use of this assay system in calcium bioavailability studies. The labeling studies and comparisons indicate caution should be used, however, in labeling techniques and species milk comparison

  16. Assaying environmental nickel toxicity using model nematodes

    Science.gov (United States)

    Rudel, David; Douglas, Chandler; Huffnagle, Ian; Besser, John M.; Ingersoll, Christopher G.

    2013-01-01

    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegansand P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  17. Trofile HIV co-receptor usage assay.

    Science.gov (United States)

    Low, Andrew J; McGovern, Rachel A; Harrigan, P Richard

    2009-03-01

    The introduction of CCR5 antagonists increases the options available for constructing therapeutic drug regimens for HIV-positive patients. However, as these drugs do not inhibit HIV variants that use the CXCR4 co-receptor, a pretreatment test is required to determine accurately HIV co-receptor usage (tropism) before initiating CCR5 antagonist-based therapy. To discuss the Monogram Trofile assay as a diagnostic tool for determining HIV tropism by critically reviewing reported literature and available data. Monogram Trofile has become, largely by default, the de facto standard for HIV tropism assay. However, there is significant room for improvement in the speed, cost and availability of the test. Furthermore, the test is not quantitative, requires high-input HIV RNA viral loads, and produces results that are less biologically stable than expected. These technical considerations may limit the use of CCR5 antagonists in therapy. Nevertheless, this test is likely to remain the most widely used tropism diagnostic for the short term. We expect that a more practical and possibly more accurate method for measuring HIV tropism can be developed.

  18. The narrow therapeutic window of glycated hemoglobin and assay variability.

    Science.gov (United States)

    Hosseini, S S; Bibler, I; Charles, M A

    1999-12-01

    Glycated hemoglobin is measured by a variety of assays, each of which has a unique normal level. Our purpose is to show that among the different assays available in the United States, using the same patient's blood sample, assay results may vary widely and may more or less easily achieve a glycated hemoglobin value within the normal range. The following assays were compared using the same patient's blood sample for each pair of assays: glycohemoglobin affinity assay (GHB Reader; Isolab, Akron, OH) versus gel electrophoresis assay (n = 76); Isolab versus ion capture assay (IMX; Abbott Laboratories, Irving, TX) (n = 57); monoclonal antibody assay (DCA2000; Bayer Diagnostics, Pittsburgh, PA) versus IMX (n = 100); and high-performance liquid chromatography (HPLC) assay (Bio-Rad Variant A1c; Bio-Rad Laboratories, Richmond, CA) versus IMX assay (n = 55). Our analyses indicate that a relative ranking can be established for the ease of achieving a normal glycated hemoglobin level. The ranking indicates that the most stringent or difficult assays for achieving a normal level are the Isolab and DCA2000 assays. The intermediate assays are the IMX and Bio-Rad Variant, and the easiest method for achieving a normal value is the gel electrophoresis assay. Our results indicate that various glycated hemoglobin assays vary widely and are associated with more or less difficulty for an individual patient to achieve a glycated hemoglobin level within the normal range. These results are especially significant with respect to (1) the clinically narrow therapeutic window of glycated hemoglobin values in type 1 diabetes to avoid rapidly advancing severe hypoglycemia rates and chronic microvascular complication rates, and (2) the glycated hemoglobin threshold for rapidly advancing macrovascular disease in both type 1 and type 2 patients.

  19. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

    Science.gov (United States)

    Escadafal, Camille; Faye, Oumar; Sall, Amadou Alpha; Faye, Ousmane; Weidmann, Manfred; Strohmeier, Oliver; von Stetten, Felix; Drexler, Josef; Eberhard, Michael; Niedrig, Matthias; Patel, Pranav

    2014-03-01

    Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings.

  20. A protein chip membrane-capture assay for botulinum neurotoxin activity

    International Nuclear Information System (INIS)

    Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

    2008-01-01

    Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC 50 s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC 50 of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays

  1. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

    Directory of Open Access Journals (Sweden)

    Camille Escadafal

    2014-03-01

    Full Text Available BACKGROUND: Yellow fever (YF is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV, is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY: The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE: The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction and rapid processing time (<20 min. Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for

  2. Multiplexing a high-throughput liability assay to leverage efficiencies.

    Science.gov (United States)

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  3. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Science.gov (United States)

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  4. Clinical validation of the Tempus xO assay

    Science.gov (United States)

    Beaubier, Nike; Tell, Robert; Huether, Robert; Bontrager, Martin; Bush, Stephen; Parsons, Jerod; Shah, Kaanan; Baker, Tim; Selkov, Gene; Taxter, Tim; Thomas, Amber; Bettis, Sam; Khan, Aly; Lau, Denise; Lee, Christina; Barber, Matthew; Cieslik, Marcin; Frankenberger, Casey; Franzen, Amy; Weiner, Ali; Palmer, Gary; Lonigro, Robert; Robinson, Dan; Wu, Yi-Mi; Cao, Xuhong; Lefkofsky, Eric; Chinnaiyan, Arul; White, Kevin P.

    2018-01-01

    We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification. PMID:29899824

  5. 21 CFR 866.3305 - Herpes simplex virus serological assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3305 Herpes simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices...

  6. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Glial fibrillary acidic protein assay... Glial fibrillary acidic protein assay. (a) Purpose. Chemical-induced injury of the nervous system, i.e... paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate...

  7. GMR sensors and magnetic nanoparticles for immuno-chromatographic assays

    International Nuclear Information System (INIS)

    Marquina, C.; Teresa, J.M. de; Serrate, D.; Marzo, J.; Cardoso, F.A.; Saurel, D.; Cardoso, S.; Freitas, P.P.

    2012-01-01

    Conventional tests based on immunorecognition and on the use of coloured colloidal particles have still some drawbacks that limit their use: they do not provide a quantitative determination of the analyte, and their sensitivity is limited. Our strategy to overcome these disadvantages consists in the use of superparamagnetic core-shell nanoparticles to tag the analyte. The use of these magnetic labels allows us to quantify the amount of analyte present in our sample with a very high sensitivity, detecting their magnetic response by means of the suitable magnetic sensor. Our method is based on measuring the magnetoresistive response of a spin-valve giant magnetoresistive (GMR) sensor placed in proximity to the magnetic nanoparticles present in the lateral flow strip. Here, a brief description of our prototype and of the measurement procedure will be presented, as well as preliminary assays using our biosensor to detect the hCG pregnancy hormone in a solution. A crucial aspect to take into account in order to increase the sensitivity is the proper functionalisation of the nanoparticle shell, in order to achieve an oriented immobilisation of the antibodies to be used in the immunorecognition process. Several strategies to further increase the sensor sensitivity are suggested.

  8. GMR sensors and magnetic nanoparticles for immuno-chromatographic assays

    Energy Technology Data Exchange (ETDEWEB)

    Marquina, C., E-mail: clara@unizar.es [Instituto de Ciencia de Materiales de Aragon ICMA, CSIC-Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza (Spain); Departamento de Fisica de la Materia Condensada, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza (Spain); Teresa, J.M. de [Instituto de Ciencia de Materiales de Aragon ICMA, CSIC-Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza (Spain); Departamento de Fisica de la Materia Condensada, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza (Spain); Serrate, D. [Departamento de Fisica de la Materia Condensada, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza (Spain); Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, C/Mariano Esquillor s/n, 50018 Zaragoza (Spain); Marzo, J. [Instituto de Ciencia de Materiales de Aragon ICMA, CSIC-Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza (Spain); Cardoso, F.A. [INESC-MN-Instituto de Engenharia de Sistemas e Computadores-Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Rua Alves Redol 9, 1000-029 Lisbon (Portugal); Saurel, D. [Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, C/Mariano Esquillor s/n, 50018 Zaragoza (Spain); Cardoso, S.; Freitas, P.P. [INESC-MN-Instituto de Engenharia de Sistemas e Computadores-Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Rua Alves Redol 9, 1000-029 Lisbon (Portugal); and others

    2012-10-15

    Conventional tests based on immunorecognition and on the use of coloured colloidal particles have still some drawbacks that limit their use: they do not provide a quantitative determination of the analyte, and their sensitivity is limited. Our strategy to overcome these disadvantages consists in the use of superparamagnetic core-shell nanoparticles to tag the analyte. The use of these magnetic labels allows us to quantify the amount of analyte present in our sample with a very high sensitivity, detecting their magnetic response by means of the suitable magnetic sensor. Our method is based on measuring the magnetoresistive response of a spin-valve giant magnetoresistive (GMR) sensor placed in proximity to the magnetic nanoparticles present in the lateral flow strip. Here, a brief description of our prototype and of the measurement procedure will be presented, as well as preliminary assays using our biosensor to detect the hCG pregnancy hormone in a solution. A crucial aspect to take into account in order to increase the sensitivity is the proper functionalisation of the nanoparticle shell, in order to achieve an oriented immobilisation of the antibodies to be used in the immunorecognition process. Several strategies to further increase the sensor sensitivity are suggested.

  9. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  10. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    International Nuclear Information System (INIS)

    Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

    2006-01-01

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

  11. Dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics.

    Science.gov (United States)

    Lutz, Barry; Liang, Tinny; Fu, Elain; Ramachandran, Sujatha; Kauffman, Peter; Yager, Paul

    2013-07-21

    Lateral flow tests (LFTs) are an ingenious format for rapid and easy-to-use diagnostics, but they are fundamentally limited to assay chemistries that can be reduced to a single chemical step. In contrast, most laboratory diagnostic assays rely on multiple timed steps carried out by a human or a machine. Here, we use dissolvable sugar applied to paper to create programmable flow delays and present a paper network topology that uses these time delays to program automated multi-step fluidic protocols. Solutions of sucrose at different concentrations (10-70% of saturation) were added to paper strips and dried to create fluidic time delays spanning minutes to nearly an hour. A simple folding card format employing sugar delays was shown to automate a four-step fluidic process initiated by a single user activation step (folding the card); this device was used to perform a signal-amplified sandwich immunoassay for a diagnostic biomarker for malaria. The cards are capable of automating multi-step assay protocols normally used in laboratories, but in a rapid, low-cost, and easy-to-use format.

  12. LSDS Development for Isotopic Fissile Content Assay

    International Nuclear Information System (INIS)

    Lee, Yong Deok; Park, Chang Je; Park, Geun Il; Lee, Jung Won; Song, Kee Chan

    2010-01-01

    Concerning the sustainable energy supply and green house effect, nuclear energy became the most feasible option to meet the energy demand in Korea. However, the production of the spent nuclear fuel is the inevitable situation. Since the first nuclear power plant started to produce the electricity in Korea, the accumulated amount of spent fuels exceeded 10k tomes recently. The accumulation of the spent fuels is the big issue in the society. Therefore, as an option which strengthens the nuclear proliferation resistance and reduces the amount of spent fuels, sodium fast reactor (SFR) program linked with pyro-processing is under development to re-use the PWR spent fuel and produce the energy. In the process, the produced metallic material involves uranium and TRU (transuranic; neptunium, plutonium, and americium). The uranium-TRU is used to fabricate SFR fuel. The burning the recycled fuel in the reactor is to solve the current spent fuel storage problem and to minimize the actinides accumulation having long half-life. Generally, the spent fuel from PWR has unburned ∼1 % U235, produced ∼0.5 % plutonium from decay chain, ∼3 % fission products, ∼ 0.1 % minor actinides (MA) and uranium remainder. About 1.5 % fissile materials still exist in the spent fuel. Therefore, spent fuel is not only waste but energy resource. The direct and isotopic fissile content assay is the crucial technology for the spent fuel reuse. Additionally, the fissile content analysis will contribute to the optimum storage design and safe spent fuel management. Several nondestructive technologies have been developed for the spent fuel assay; gamma ray measurement, passive and active neutron measurements. Spent fuel emits intense gamma rays and neutrons by (a, n) and spontaneous fission. This intense background has the limitation on the direct analysis of fissile materials. Recently, to analyze the individual fissile content, leadslowing down spectrometer (LSDS) has been being developed in Korea

  13. Localized irradiations, Evaluation through ''comet assay''

    International Nuclear Information System (INIS)

    Giorgio, M.D.; Taja, M.R.; Nasazzi, N.B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

    2000-01-01

    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources Co 60 , Cs 137 or Ir 192 at workplaces for industrial gammagraphy. Severe skin reaction may develop at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models ''Contaminated Poisson'' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. This give an indication of the proportion of haemopoietic stem cell compartment involved in the overexposure. There are also different biophysical techniques that can give responses in biological dosimetry. The ''Comet Assay'' (single cell gel electrophoresis) is a sensitive and rapid method for DNA strand break detection in individual cells. The advantages of the technique include: collection of data at the level of individual cell; the need for small numbers of cells per sample; its sensitivity for detecting DNA damage and that virtually any eukaryote cell population is amenable to analysis. The objective of this work is to apply ''Comet Assay'' method to evaluate the effect of radiation on skin and subcutaneous tissues, differentiating irradiated from unirradiated body areas. It could provide a useful tool to estimate the extension and the dose in the irradiated region, contributing with the current techniques. In this first study, we evaluate the alkaline comet assay as a method for detection of DNA radiation induced damage in keratinocytes from primary culture obtained from full thickness skin biopsies of patients requiring grafts. Skin and, particularly, keratinocytes were selected as an appropriate cellular system due to: Skin, the first barrier

  14. Localized irradiations, evaluation through 'Comet Assay'

    International Nuclear Information System (INIS)

    Di Giorgio, Marina; Taja, Maria R.; Nasazzi, Nora B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.

    2000-01-01

    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources of Cobalt 60, Cesium 137 or Iridium 192 at work placed for industrial gammagraphy and other radiation sources. Severe skin reaction may developed at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models 'Contaminated Poisson' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. There are also different biophysical techniques that can give response in localized irradiations. Biological dosimetry is a necessary complement to physical and clinical dosimetries. Thus, there is increasing interest in the assessment of biological markers that permit the detection of radiation induced damage in the localized irradiations. The 'Comet Assay' (single cell gel electrophoresis) is a sensitive, rapid and relatively inexpensive method for measuring DNA damage in individual cells. Single cells are embedded in agarose on microscope slides, lysed to remove the majority of the proteins, electrophoresed, then stained with ethidium bromide in order to visualize the DNA. When visualized using a fluorescent microscope, DNA of undamaged cells appears as a spherical mass occupying the cavity formed by the lysed cell. Following radiation damage, the smaller the fragment size and the grater the number of fragments of DNA, the grater the percentage of DNA that it is able to migrate in an electric field, forming a comet image. The assay can be performed under alkaline conditions to examine DNA single strand breaks (SSBs), or in non denaturing (neutral) conditions to measure double strand breaks (DSBs) in individual

  15. Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.

    Science.gov (United States)

    2017-07-27

    The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

  16. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    Science.gov (United States)

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

  17. Stability-Indicating HPLC Assay for Determination of Idebenone in Pharmaceutical Forms

    Directory of Open Access Journals (Sweden)

    Sonoube Kombath

    2015-01-01

    Full Text Available A stability-indicating method was validated for the determination in pharmaceutical forms of idebenone a coenzyme Q10-like compound. The assay was achieved by liquid chromatography analysis using a reversed-phase C18 column and a detector set at 480 nm. The optimized mobile phase consisted of isocratic flow rate at 1.0 mL/min for 3 min with methanol. The linearity of the assay was demonstrated in the range of 3.0 to 8.0 mg/mL with a correlation coefficient r2>0.998. The limits of detection and quantification were 0.03 and 0.05 mg/mL, respectively. The intraday and interday precisions were less than 1.0%. Accuracy of the method ranged from 98.6 to 101.5% with RSD < 0.6%. Specificity of the assay showed no interference from tablets components and breakdown products formed by alkaline, acidic, oxidative, sunlight, and high temperature conditions. This method allows accurate and reliable determination of idebenone for drug stability assay in pharmaceutical studies.

  18. Some target assay uncertainties for passive neutron coincidence counting

    International Nuclear Information System (INIS)

    Ensslin, N.; Langner, D.G.; Menlove, H.O.; Miller, M.C.; Russo, P.A.

    1990-01-01

    This paper provides some target assay uncertainties for passive neutron coincidence counting of plutonium metal, oxide, mixed oxide, and scrap and waste. The target values are based in part on past user experience and in part on the estimated results from new coincidence counting techniques that are under development. The paper summarizes assay error sources and the new coincidence techniques, and recommends the technique that is likely to yield the lowest assay uncertainty for a given material type. These target assay uncertainties are intended to be useful for NDA instrument selection and assay variance propagation studies for both new and existing facilities. 14 refs., 3 tabs

  19. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  20. Time-resolved immunofluorometric assay of serum ferritin

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Yao [China Inst. of Atomic Energy, Beijing (China)

    2007-06-15

    This assay is a solid phase, two-site fluoroimmunometric assay based on the direct sandwish technique. Standards or samples containing ferritin are first reacted with immobilized anti-ferritin antibodies. Then the europium-lablled antibodies are reacted with the bound antigen. The range of this assay is 2-1000 ng/mL. The analytical sentivity is better than 0.05 ng/mL. The intra-assay variation and inter-assay variation are both below 5%; This kit was compared with Wallac DELFIA kit. The correlation is r=0.96. (authors)