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Results for “Detection technology”

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PubMed1996

The activated coagulation time: suitability for monitoring heparin effect and neutralization during pediatric cardiac surgery.

OBJECTIVES: To determine how the stage of surgery affects the relationship between activated clotting time (ACT) and heparin effect in children undergoing cardiac surgery using cardiopulmonary bypass (CPB) and to compare the results of ACT determinations made with two different coagulation timers using different clot detection technologies and activator compositions. DESIGN: Prospective, paired observation. SETTING: Tertiary care children's hospital affiliated with an academic medical center. PARTICIPANTS: Fifty-eight children scheduled for nonprimary cardiac surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: ACTs were measured by two different commercially available automated coagulation timers (Hepcon Hemostasis Management System [HP] and the Hemochron model 801 [HM]) at four different time points over the course of cardiac surgery requiring CPB in patients ranging in age from 0.16 to 19 years. Simultaneous determinations of whole blood heparin concentration using heparin-protamine titrations were made as well. When the two methods of ACT determination were compared, baseline ACTs were not significantly different. HP ACT prolongation after heparin administration but before bypass was significantly less than HM ACT prolongation (median ACT range HM > or = 999 seconds; HP, 560 to 679; p = 0.006). Twenty-one percent of the HP ACTs and none of the HM ACTs fell below the 480 seconds required at this institution for the initiation of CPB (p = 0.008). Both instruments showed a significant further prolongation of ACT after initiation of bypass (median ACT range HP > or = 999 seconds; HM > or = 999; p < 0.001 for both), whereas the heparin concentration decreased significantly (before, 3.5 +/- 0.2 U/mL; after, 2.7 +/- 0.1; p < 0.001). After termination of CPB and heparin neutralization, no significant difference between the ACTs was found. However, four HP ACTs were > or = 999 seconds despite simultaneous HM baseline values and whole blood heparin concentrations of zero. Heparin concentration correlated with ACT prolongation using both the HM (Spearman p = 0.36; p = 0.02) and the HP (Spearman p = 0.57; p = 0.0025) instruments before, but not 10 minutes after, initiation of bypass. CONCLUSIONS: In pediatric cardiac surgery, the relationship between ACT and heparin concentration changes depending on when during the surgery the ACT is measured. ACT prolongation in children anticoagulated for CPB correlates poorly with heparin concentrations during CPB. HP and HM ACT tests are not interchangeable. The HM ACT is a better indicator of heparin neutralization than the HP ACT. On the other hand, continued prolongation of the HP ACT after heparin neutralization may be related to risk of postoperative hemorrhagic complications. If devices from different manufactures are freely substituted for each other, clinical practice may be altered in an uncontrolled manner.

Adolescent↗

PubMed1995

Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis.

A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative T06 as they passed through the approximately 2 micrometer diameter focused laser beam. Amplified photoelectron pulses from the photomultiplier are grouped into bins of 360-450 micros in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were used to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. Statistical analyses showed that (i) the number of M13 molecules counted versus concentration was linear with slope = 1, (ii) the average burst duration was consistent with the expected transit time of a single molecule through the laser beam, and (iii) the number of detected molecules was consistent with single molecule detection. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The autocorrelation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of approximately 100 DNA molecules/band or better.

DNA↗

PubMed2004

Electrochemical coding of single-nucleotide polymorphisms by monobase-modified gold nanoparticles.

Rapidly increasing information about the human genome requires a fast and simple method for the detection of single-nucleotide polymorphisms (SNPs). To date, the conventional SNP detection technologies have been unable to identify all possible SNPs and needed further development in cost, speed, and sensitivity. Here we describe a novel method to discriminate and code all possible combinations. SNPs were coded by monitoring the changes in the electrochemical signal of the monobase-modified colloidal gold (Au) nanoparticles. First, a chitosan layer was formed on the alkanethiol self-assembled monolayer-modified Au nanoparticle. The monobases were then attached onto the chitosan-coated Au nanoparticles through their 5' phosphate group via the formation of a phosphoramidate bond with the free amino groups of chitosan. The size of the surface-modified Au nanoparticle was found to be 8.46 +/- 1.53 nm by using atomic force microscopy. If there is a SNP in DNA and the mismatched bases are complementary to the monobase, Au nanoparticles accumulate on the electrode surface in the presence of DNA polymerase I (Klenow fragment), thus resulting in a significant change in the Au oxide wave. In this report, monobase-modified Au nanoparticles show not only the presence of a SNP, but also identify which bases are involved within the pair. Especially, the identification of a transversion SNP, which contains a couple of the same pyrimidine or purine bases, is greatly simplified. A model study was performed by using a synthetic 21-base DNA probe related to tumor necrosis factor (TNF-alpha) along with its all possible mutant combinations. This versatile nanoparticle-based electrochemical protocol is a promising candidate for coding all mutational changes.

Base Sequence↗

PubMed2005

Toward single-calibrant quantification in HPLC. A comparison of three detection strategies: evaporative light scattering, chemiluminescent nitrogen, and proton NMR.

There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance (1H NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10mM in microtiter plate (MTP) format.

Journal Article↗

PubMed2001

Stochastic sensors inspired by biology.

Sensory systems use a variety of membrane-bound receptors, including responsive ion channels, to discriminate between a multitude of stimuli. Here we describe how engineered membrane pores can be used to make rapid and sensitive biosensors with potential applications that range from the detection of biological warfare agents to pharmaceutical screening. Notably, use of the engineered pores in stochastic sensing, a single-molecule detection technology, reveals the identity of an analyte as well as its concentration.

Biosensing Techniques↗

PubMed2006

The US Food and Drug Administration perspective on cancer biomarker development.

Despite the intense interest in biomarker development for cancer management, few biomarker assays for diagnostic uses have been submitted to the US Food and Drug Administration (FDA). What challenges must researchers overcome to bring cancer-detection technologies to the market and, therefore, into clinical use?

Biological Assay↗

PubMed2006

Cellular imaging in drug discovery.

Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell or organism, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. This review describes how cellular imaging technologies contribute to the drug discovery process.

Animals↗

PubMed2003

Intermolecular interactions with/within cell membranes and the trinity of membrane potentials: kinetics and imaging.

The interactions of (macro-)molecules with biological membranes underlies much of cell biology. This paper outlines many of the factors that must be taken into account in order to understand fully the nature of these interactions. These include some roles of the membrane potentials including features of the surface and dipole potentials. Several fluorescence detection technologies directed towards these are outlined that offer high-resolution experimental determination of the intermolecular interactions by measuring small changes of these potentials resulting from specific interactions of many kinds of molecular species. The possibilities for making single-cell spatial imaging measurements of such interactions is also described. Examples are used to indicate the feasibility of identifying and tracking localized interactions on the membrane surface in real-time. Some of this work points to the possibility that the membrane dipole potential spatially varies about the cell surface, particularly within membrane microdomains such as 'rafts'. Such variation is suggested to underlie the altered behaviour of signalling systems within rafts and offer the means of an additional level of biological control.

Animals↗

PubMed2003

A study of candidate genes for psoriasis near HLA-C in Chinese patients with psoriasis.

BACKGROUND: Genetic analyses have identified the HLA-Cw6 allele as the major risk allele for psoriasis in many racial groups. However, by serological typing, HLA-Cw6 is not considered a risk factor in Chinese psoriatics. There are several susceptibility genes for psoriasis residing in chromosome 6p near the HLA-C locus, including the corneodesmosin (CDSN) gene, the octamer transcription factor-3 (POU5F1) gene, the major histocompatibility complex class I chain-related gene A (MICA), and the gene for tumour necrosis factor (TNF)-alpha. However, the information about their role in psoriasis in Chinese patients is limited. OBJECTIVES: We aimed to determine whether Cw6 and the genetic polymorphism of the CDSN gene, POU5F1 gene, MICA gene and the gene for TNF-alpha promoter region were associated with an increased risk of psoriasis in Chinese patients. METHODS: We conducted a case-control association study in 105 Chinese patients with psoriasis vulgaris and 160 control subjects of similar ages. Genotypes of Cw6, the CDSN gene, the POU5F1 gene, and the gene for the TNF-alpha promoter region were determined by polymerase chain reaction (PCR) followed by restriction enzyme digestion. Genotyping of MICA was determined by PCR combined with fluorescent-based automated fragment detection technology. Results The allele frequencies showed no differences between patients and controls for the POU5F1 gene, MICA gene and the gene for TNF-alpha promoter region. The frequency of the HLA-Cw6 allele in the psoriasis group was significantly higher than that in the control group (18.6% vs. 6.56%, P < 0.00005). For the CDSN gene, patients were more likely to have C allele at position +619 (P = 0.006) and C allele at position +1243 (P = 0.007), but the significance disappeared after correction for multiple testing (Pc > 0.05). CONCLUSIONS: HLA-Cw6 remains the most significant susceptibility gene in Chinese patients with psoriasis. However, the role of the CDSN gene in the pathogenesis of psoriasis deserves further scrutiny.

Adolescent↗

PubMed2002

Detection of dysplastic intestinal adenomas using enzyme-sensing molecular beacons in mice.

BACKGROUND & AIMS: Proteases play key roles in the pathogenesis of tumor growth and invasion. This study assesses the expression of cathepsin B in dysplastic adenomatous polyps. METHODS: Aged Apc(Min/+) mice served as an experimental model for familial adenomatous polyposis. The 4 experimental groups consisted of (a) animals injected with a novel activatable, cathepsin B sensing near infrared fluorescence (NIRF) imaging probe; (b) animals injected with a nonspecific NIRF; (c) uninjected control animals; and (d) non-APC(Min/+) mice injected with the cathepsin B probe. Lesions were analyzed by immunohistochemistry, Western blotting, reverse transcription-polymerase chain reaction, and optical imaging. RESULTS: Cathepsin B was consistently overexpressed in adenomatous polyps. When mice were injected intravenously with the cathepsin reporter probe, intestinal adenomas became highly fluorescent indicative of high cathepsin B enzyme activity. Even microscopic adenomas were readily detectable by fluorescence, but not light, imaging. The smallest lesion detectable measured 50 microm in diameter. Adenomas in the indocyanine green and/or noninjected group were only barely detectable above the background. CONCLUSIONS: The current experimental study shows that cathepsin B is up-regulated in a mouse model of adenomatous polyposis. Cathepsin B activity can be used as a biomarker to readily identify such lesions, particularly when contrasted against normal adjacent mucosa. This detection technology can be adapted to endoscopy or tomographic optical imaging methods for screening of suspicious lesions and potentially for molecular profiling in vivo.

Adenomatous Polyps↗

PubMed2004

Colorectal cancer chemoprevention--an overview of the science.

The development and dissemination of sophisticated detection technologies have recently exposed the high prevalence of preinvasive colorectal neoplasia in the adult U.S. population. Although cancer screening and surveillance provide opportunities for risk stratification, they achieve risk reduction only when coupled with effective interventions. This review surveys the lead compounds for colorectal cancer prevention and the measures by which they may be prioritized for clinical testing. Clinical trials remain the rate-limiting step in agent development, and novel trial designs are needed to hasten agent identification and testing for cancer prevention. Innovative research models include the nesting of prevention end points within cancer treatment trials and within trials testing promising preventive compounds intended for nononcologic indications.

Chemoprevention↗

PubMed2002

Epidermal growth factor receptor expression and measurement in solid tumors.

The efficacies of targeted therapies for cancer treatment are dependent on the expression of the targeted molecule in the tumors of the treated patient population. Immunohistochemistry is an attractive assay format for determining protein expression in biopsies of solid tumors, and is widely used in pathology laboratories. An optimized immunohistochemistry assay for the epidermal growth factor receptor (EGFR) has been developed for the detection of EGFR in colorectal, head and neck, and other tumors that express EGFR, and the assay is being used in colorectal cancer trials of IMC-C225 to select patients for treatment. The specificity of the assay and the detection technology used will be described, along with other assay methodologies and applications for EGFR testing.

Antibodies, Monoclonal↗

PubMed2003

Acute volume reduction with aortic valve replacement immediately improves ventricular mechanics in patients with aortic regurgitation.

OBJECTIVES: Few data have been available regarding the immediate response in ventricular mechanics to acute volume reduction caused by aortic valve replacement for aortic regurgitation. METHODS: We studied 9 patients in the operating room immediately before and after the institution of cardiopulmonary bypass. Left ventricular pressure and cross-sectional area (a surrogate of left ventricular volume) were measured with a catheter-tip manometer and a transesophageal echocardiographic system equipped with automated border-detection technology. Left ventricular pressure-area loops were constructed, and the caval occlusion method was used to obtain the slope of the end-systolic pressure-area relationship and the end-systolic area associated with 100 mm Hg. From the steady-state beats, stroke area was obtained by subtracting the minimum area from the maximum area. Effective arterial elastance, a measure of ventricular afterload, was calculated from end-systolic pressure, and stroke area as follows: effective arterial elastance equals end-systolic pressure divided by stroke area. RESULTS: Reductions in maximum area (21.0 +/- 8.5 to 16.0 +/- 6.8 cm(2) [SD])and minimum area (15.3 +/- 8.4 to 12.0 +/- 6.1 m(2)) shifted the baseline pressure-area loops to the left. The slope of the end-systolic pressure-area relationship (11.6 +/- 4.8 to 16.0 +/- 7.5 mm Hg/cm(2)) and afterload (effective arterial elastance, 17.9 +/- 11.6 to 26.3 +/- 16.4 mm Hg/cm(2)) were increased, and the end-systolic area associated with 100 mm Hg was reduced (18.3 +/- 10.0 to 13.7 +/- 5.8 cm(2)). CONCLUSION: Correction of volume overload reduced preload (minimum area), shifted the end-systolic pressure-area relationship to the left (decreased end-systolic area), and improved ventricular contractility (increased slope of the end-systolic pressure-area relationship). The result indicated that acute volume reduction favorably influenced ventricular mechanical parameters immediately after the operation.

Adult↗

PubMed1995

Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.

Base Sequence↗

PubMed2000

Continuous emissions monitoring using spark-induced breakdown spectroscopy.

A new technology for monitoring airborne heavy metals on aerosols and particulates based on spark-induced breakdown spectroscopy (SIBS) was evaluated at a joint U.S. Environmental Protection Agency (EPA)/U.S. Department of Energy test at the rotary kiln incinerator simulator (RKIS) facility at EPA/Research Triangle Park, NC, in September 1997. The instrument was configured to measure lead and chromium in a simulated combustion flue gas in real time and in situ at target levels of 15 and 75 micrograms/dry standard cubic meters. Actual metal concentrations were measured during the tests using EPA Reference Method (RM) 29. The SIBS technology detected both lead and chromium at the low- and high-level concentrations. Additionally, the hardware performed without failure for more than 100 hr of operation and acquired data for 100% of the RM tests. The chromium data were well correlated with concentration increases resulting from duct operations and pressure fluctuations that are known to entrain dust.

Aerosols↗

PubMed2004

Methods to reduce traffic crashes involving deer: what works and what does not.

More than 1.5 million traffic crashes involving deer, producing at least $1.1 billion in vehicle damage and about 150 fatalities, are estimated to occur annually in the United States. Deer-related crashes are increasing as both deer populations and vehicular travel increase. Many methods have been used in attempts to reduce deer crashes, often with little scientific foundation and limited evaluation. This article summarizes the methods and reviews the evidence of their effectiveness and the situations in which each may be useful. The only widely accepted method with solid evidence of effectiveness is well-designed and maintained fencing, combined with underpasses or overpasses as appropriate. Herd reduction is controversial but can be effective. Deer whistles appear useless. Roadside reflectors appear to have little long-term effect, although additional well-designed evaluations are needed before firm conclusions can be drawn. Both temporary passive signs and active signs appear promising in specific situations, but considerable research is required to evaluate long-term driver response and to improve and test deer detection technology for active signs. Other methods using advanced technology require substantial additional research and evaluation.

Accidents, Traffic↗

PubMed2001

An analysis of spirochete load, strain, and pathology in a model of tick-transmitted Lyme borreliosis.

Four laboratory-grown, low-passage isolates of Borrelia burgdorferi sensu stricto, B31, JD-1, 910255, and N40, were incorporated into Ixodes scapularis ticks to examine the pathogenesis of these isolates in mice after tick transmission. All isolates induced multifocal, lymphoid nodular cystitis, subacute, multifocal, necrotizing myocarditis, and a localized periostitis and arthritis of the femorotibial joint 6-18 weeks after tick infestation. In terms of the number of mice that demonstrated pathology in bladder, heart, and joint, the highest incidence of lesions occurred 12 weeks after tick bite. Utilizing the Taqman quantitative polymerase chain reaction (q-PCR) fluorogenic detection technology to amplify a conserved region of the flagellin gene, a trend was demonstrated between the number of spirochetes in tissue with duration of pathology. The q-PCR assay developed for this study was sensitive and could reliably measure as few as 1 to 10 spirochetes in the target tissues tested. A higher percentage of B31- and N40-infected mice (92 and 100%, respectively) developed myocarditis than JD-1- or 910255-infected mice (67 and 46%, respectively) 12 weeks after tick bite. The amount of spirochetal DNA that could be amplified for heart at this time point was not statistically different between isolates, indicating a difference in virulence between B31 and N40 relative to JD-1 and 910225. N40-infected mice demonstrated a significantly higher spirochete load (an average of 1.23 spirochetes/mg of tissue, p = 0.045) in femorotibial joints 18 weeks after infection, with 60% of these mice maintaining lesions compared with those infected with B31 (13%), JD-1 (25%), or 910255 (50%), which averaged <0.5 spirochetes/mg of tissue. This mouse model of Lyme borreliosis, including the ability to monitor lesion development and spirochete load, can facilitate the testing of therapeutic regimens for the later stages of tick-transmitted Lyme disease and help investigate aspects of the immunopathogenesis of lesion development.

Animals↗

PubMed1998

Accurate, high-throughput "snapshot" detection of hMLH1 mutations by two-dimensional DNA electrophoresis.

The human genome and related projects have resulted in the isolation of a rapidly growing number of genes that cause susceptibility to human cancer. With rare exception, these genes are large and have disease-associated mutations scattered along the length of the genes. Therefore, the development of accurate and cost-efficient mutation detection tests that can scan entire genes singly or in combination is warranted. hMLH1, encoding a mismatch repair enzyme, is a susceptibility gene for hereditary nonpolyposis colon cancer syndrome. This gene comprises 19 exons; mutations are scattered, typical of many susceptibility genes. Here, we present a strategy that combines extensive PCR multiplexing and two-dimensional DNA electrophoresis (Two-Dimensional Gene Scanning, TDGS) to scan accurately for mutations that lie within the exons and splice junctions of hMLH1. All target fragments, designed to have optimal melting characteristics, were prepared in a two-stage PCR--a four-plex long-distance PCR followed by short PCR in two multiplex groups of 10 and 11 amplicons. The mixture of amplicons was subjected to two-dimensional electrophoresis: separation by size in the first dimension and by melting characteristics in the second. Using this design, 41 samples containing known hMLH1 sequence variants or no alterations were blindly subjected to TDGS. All mutations were detected; there were no genuine false-positive or false-negative results. These results confirm that TDGS is a generally applicable, rapid, accurate, and reproducible mutation detection technology that would serve large-scale molecular epidemiologic studies as well as clinical molecular diagnostic purposes.

Adaptor Proteins, Signal Transducing↗