PubMed Health⌕ Search

Biomedical subjects

P Wilding

Publications and source records attributed to P Wilding.

At least 19 recordsLinked to original sources
PubMed2003

Microchip PCR.

Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties

Equipment Design↗
PubMed2001

Microchip module for blood sample preparation and nucleic acid amplification reactions.

A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 microm x 254 microm microchannels for transporting human whole blood and reagents in and out of an 8--9 microL dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 microL) in an integrated cell isolation--PCR microchip containing a series of 3.5-microm feature-sized "weir-type" filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays.

Cell Separation↗
PubMed2001

Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene.

The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing Campylobacter jejuni. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed.

Autoanalysis↗
PubMed1999

Effects of a 99mTc-labeled murine immunoglobulin M antibody to CD15 antigens on human granulocyte membranes in healthy volunteers.

UNLABELLED: An injectible, 99mTc-labeled, murine immunoglobulin M antibody to stage-specific embryonic antigen-1 has been developed that can localize infections by binding to CD15 glycoproteins expressed on the cell membranes of human granulocytes in vivo after systemic administration. The purpose of this study was to measure its clinical effects on healthy people. METHODS: Multiple blood samples were aspirated before and after the intravenous administration of about 125 microg antibody labeled with approximately 370 MBq (10.0 mCi) 99mTc in 10 healthy human volunteers. Complete blood cell counts were performed at each time point. Whole-body scans were acquired contemporaneously with a dual-head gamma camera. The fraction of the administered dose at each time point was quantified in 18 regions of interest. Statistical analyses included paired t tests. RESULTS: Administration was associated with a transient decrease in the concentration of red and white blood cells in the whole blood. The effect always began within 3 min of administration. Its nadir was always reached 15-20 min after administration. There was full recovery with mild overcompensation in about an hour. The hematocrit dropped by a mean of 3.8% (P<0.002), whereas the total white blood cell count fell 44.0%+/-3.1% (P<0.001). The effect was most pronounced on the number of circulating granulocytes, which fell from 5.7+/-2.1 to 3.2+/-1.3x10(3)/microL blood. The drop paralleled a decrease in the percentage of whole blood radioactivity bound to the white blood cell membranes, which peaked at 50.4%+/-7.6% at 3 min after injection and then fell to 26.1%+/-9.3% over the next 30+/-13.4 min before recovering to 40.7%+/-8.2% at 2 h. Image analysis showed that the effect was temporally associated with an increase in the amount of radioactivity within the liver and the spleen. Recovery was associated with a decrease in hepatosplenic radioactivity. No evidence of cell destruction or agglutination could be detected. CONCLUSION: This study confirmed that administration of this radiolabeled antibody is associated with a transient decrease in the number of circulating granulocytes. However, there also seems to be a secondary hemodilutionlike effect on all blood components that has not been reported previously. The effect appears to be clinically silent and very short-lived.

Adult↗
PubMed1998

Integrated cell isolation and polymerase chain reaction analysis using silicon microfilter chambers.

White blood cells are isolated from whole blood in silicon-glass 4.5-microliter microchips containing a series of 3.5-micron feature-sized 'weir-type' filters, formed by an etched silicon dam spanning the flow chamber. Genomic DNA targets, e.g., dystrophin gene, can be directly amplified using the polymerase chain reaction (PCR) from the white cells isolated on the filters. This dual function microchip provides a means to simplify nucleic acid analyses by integrating in a single device two key steps in the analytical procedure, namely, cell isolation and PCR.

Cell Separation↗
PubMed1998

Degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human DNA on microchip-based devices.

Random amplification of the human genome using the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) was performed in a silicon-glass chip. An aliquot of the DOP-PCR amplified genomic DNA was then introduced into another silicon-glass chip for a locus-specific, multiplex PCR of the dystrophin gene exons in order to detect deletions causing Duchenne/Becker muscular dystrophy. Amplicons were analyzed by both conventional capillary electrophoresis and microchip electrophoresis and results were compared to those obtained using standard non-chip-based PCR assays. Results from microchip electrophoresis were consistent with those from conventional capillary electrophoresis. Whole genome amplification products obtained by DOP-PCR proved to be a suitable template for multiplex PCR as long as amplicon size was < 250 bp. Successful detection and resolution of all PCR products from the multiplex PCR clearly shows the feasibility of performing complex PCR assays using microfabricated devices.

DNA↗
PubMed1997

Effect of long-term treatment with salmeterol on asthma control: a double blind, randomised crossover study.

OBJECTIVES: To determine the effect of adding salmeterol 50 micrograms twice daily for six months to current treatment in subjects with asthma who control their inhaled corticosteroid dose according to a management plan. DESIGN: A double blind, randomised crossover study. SETTING: Nottingham. SUBJECTS: 101 subjects with mild or moderate asthma taking at least 200 micrograms twice daily of beclomethasone dipropionate or budesonide. INTERVENTIONS: Salmeterol 50 micrograms twice daily and placebo for six months each, with a one month washout. Subjects adjusted inhaled steroid dose according to guidelines. MAIN OUTCOME MEASURE: Reduction in inhaled steroid use, exacerbations of asthma, and use of oral steroids. RESULTS: Data were available for 87 subjects. When compared with placebo salmeterol treatment was associated with a 17% reduction in inhaled steroid use (95% confidence interval 12% to 22%) with no significant difference in the number of subjects who had an exacerbation (placebo 25%, salmeterol 16%) or use of oral steroids. For secondary end points salmeterol treatment was associated with higher morning and evening peak expiratory flow and forced expiratory volume in one second; a reduction in symptoms, bronchodilator use and airway responsiveness to methacholine; and no effect on serum potassium concentration, 24 hour heart rate, or the final forced expiratory volume in one second achieved during a salbutamol dose-response study. CONCLUSIONS: In subjects who adjusted their inhaled steroid treatment according to guidelines the addition of salmeterol 50 micrograms twice daily was associated with a reduction in inhaled steroid use and improved lung function and symptom control.

Administration, Inhalation↗
PubMed1997

Micromachined analytical devices: microchips for semen testing.

Micromachined devices (microchips) have been designed and tested for a range of clinically important assays. In this study we compare sperm motility determined using disposable glass microchips and a conventional Makler chamber. The 17 x 14 mm glass microchips contained three etched test structures each comprising either duplicate or quadruplicate analytical microchannels. Semen samples with sperm counts ranging from 21 to 78 million sperm per ml and forward progression scores of from 1+ to 3+ were evaluated and swimming times ranging from 360 s (3.3+ progression) to 770 s (1+,2 forward progression) observed in the microchips. Motility determined by the time taken for sperm to swim to the end of a microchannel (100 microns wide x 40 microns deep x 10 mm long) in the microchip correlated with forward progression of the sperm determined by the conventional Makler chamber method. This study demonstrates the feasibility of microchips for sperm motility testing and suggests that this technique would be applicable to the study of other types of motile cells.

Biotechnology↗
PubMed1997

Adenosine monophosphate and histamine induced bronchoconstriction: repeatability and protection by terbutaline.

BACKGROUND: Inhaled adenosine monophosphate (AMP) is thought to cause bronchoconstriction in asthmatic patients indirectly through mast cell mediator release. It may therefore be a more sensitive marker of airway inflammation in asthma and hence more specific for epidemiological surveys of asthma than challenges that act directly on airway smooth muscle such as histamine. There is some uncertainty as to how repeatable the measurement is and this is important if it is to be used for epidemiological studies. METHODS: The response to histamine and AMP challenges and the protection afforded by terbutaline (500 micrograms) against these two challenges was measured on two occasions two weeks apart in 20 subjects with asthma (19 completed the study). The response to histamine and AMP was measured as the provocative dose causing a 20% fall in forced expiratory volume in one second (PD20) and the protection afforded by terbutaline in doubling doses (DD). Repeatability was assessed as the limits of agreement. RESULTS: Although terbutaline had a slightly greater protective effect against AMP than histamine on both the first (delta PD20 = 2.66 versus 2.11 DD) and second occasion (2.56 and 2.15 DD), the differences were not statistically significant. The limits of agreement for the two histamine and two AMP challenges after placebo were from 3.06 to -3.5 and from 3.78 to -4.54 DD respectively, and these values did not differ significantly. The agreement limits between the first PD20 histamine and PD20 AMP values after placebo were similar, being from 3.73 to -3.72 DD after allowing for the 17.8-fold higher PD20 values for AMP compared with histamine. CONCLUSIONS: Terbutaline caused a slightly greater inhibition of the bronchoconstrictor response to AMP than histamine but the differences were small and non-significant. Any differences in repeatability between AMP and histamine challenges are small and in this study were not significant. The fact that the agreement between histamine and AMP PD20 values was similar to the agreement between repeat histamine or repeat AMP PD20 values suggests that, within an asthmatic population, PD20 AMP may not be providing different information from that provided by PD20 histamine.

Adenosine Monophosphate↗
PubMed1997

Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identification of carrier status by use of entangled solution capillary electrophoresis.

Use of capillary electrophoresis, a new and useful analytical tool, offers a variety of advantages for nucleic acid analyses, including rapid analysis, automation, high resolution, qualitative and quantitative results, and low consumption of both sample and reagents. We report the first example of the use of entangled solution capillary electrophoresis (ESCE) and laser-induced fluorescence detection (LIF) for separation-based diagnostics in the quantitative analysis of multiplex PCR products for determination of carrier status of Duchenne/ Becker muscular dystrophy (DMD/BMD). This approach greatly improved the speed, resolution, and sensitivity of information needed for the diagnosis of DMD/BMD compared with that from conventional diagnostic methods, and is of general utility for diagnosis of genetic diseases.

Electrophoresis, Capillary↗
PubMed1996

Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis.

Ligase chain reaction (LCR) is a useful molecular technique for detecting known point mutations. We report the first example of the use of a disposable silicon-glass micro-chip for LCR and the first application of capillary electrophoresis (CE) to analyze samples amplified by LCR in a chip. Silicon-glass chips were manufactured using conventional photolithography and anodic bonding. The chips provide three distinct advantages for LCR: excellent thermal conductivity, a micro reaction volume ( < 10 microliters), and reproducible, low-cost manufacturing. Investigation and quantitation of amplification efficiency of LCR in a chip or in a tube requires an analytical technique that is faster and more convenient than the conventional slab gel methods. Slab gel electrophoresis uses relatively large amounts of sample and is labor-intensive and time-consuming, and thus is unsuitable for the separation and detection of LCR products. In contrast CE requires sample volume (original LCR products) of less than 1 microliter and is therefore well-suited to analysis of the micro-volume reaction mixture from chips. We combined CE with a sensitive laser induced fluorescence (LIF) detection system for the rapid separation and quantitative detection of LCR products amplified from the lacI gene in a silicon-glass chip. Comparative studies were made with LCR between tubes and silicon-glass chips. CE-LIF analysis is ideally suited to examination of micro-LCR amplification with high throughput. The technologies may find medical uses in disease diagnosis and research.

Base Sequence↗
PubMed1996

Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR.

The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube.

Base Sequence↗
PubMed1996

Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips.

We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip.

Antibodies↗
PubMed1996

Microchip-based Devices for Molecular Diagnosis of Genetic Diseases.

Microchips, constructed with a variety of microfabrication technologies (photolithography, micropatterning, microjet printing, light-directed chemical synthesis, laser stereochemical etching, and microcontact printing) are being applied to molecular biology. The new microchip-based analytical devices promise to solve the analytical problems faced by many molecular biologists (eg, contamination, low throughput, and high cost). They may revolutionize molecular biology and its application in clinical medicine, forensic science, and environmental monitoring. A typical biochemical analysis involves three main steps: (1) sample preparation, (2) biochemical reaction, and (3) detection (either separation or hybridization may be involved) accompanied by data acquisition and interpretation. The construction of a miniturized analyzer will therefore necessarily entail the miniaturization and integration of all three of these processes. The literature related to the miniaturization of these three processes indicates that the greatest emphasis so far is on the investigation and development of methods for the detection of nucleic acid, followed by the optimization of a biochemical reaction, such as the polymerase chain reaction. The first step involving sample preparation has received little attention. In this review the state of the art of, microchip-based, miniaturized analytical processes (eg, sample preparation, biochemical reaction, and detection of products) are outlined and the applications of microchip-based devices in the molecular diagnosis of genetic diseases are discussed.

Journal Article↗
PubMed1996

The effect of inhaled heparin on bronchial reactivity to sodium metabisulphite and methacholine in patients with asthma.

Inhaled heparin inhibits the early response to allergen and exercise-induced asthma, probably by inhibiting mast cell mediator release. Recent animal studies suggest that heparin might also inhibit cholinergic neurotransmission in asthma by restoring inhibitory M2 receptor function. We have tested the hypothesis that heparin inhibits neurally-mediated bronchoconstriction in asthma by examining the effect of inhaled heparin on bronchial reactivity to sodium metabisulphite. We also examined the effect of inhaled heparin on methacholine-induced bronchoconstriction to exclude a direct effect on airway smooth muscle. Eleven patients with mild asthma inhaled nebulized heparin (1,000 U.kg-1) or placebo (normal saline) in a randomized, double-blind fashion, 10 min before a challenge with sodium metabisulphite. Nine patients were also challenged with methacholine after the same dose of heparin or placebo. Inhaled heparin did not significantly change forced expiratory volume in one second (FEV1), nor did it alter the provocative dose of sodium metabisulphite or methacholine required to cause a 20% fall in FEV1 (PD20). Geometric mean sodium metabisulphite PD20 was 2.54 and 2.15 mumol after placebo and heparin, respectively (mean difference -0.24 doubling doses; 95% confidence interval (95% CI) -0.64-0.17). Geometric mean methacholine PD20 was 1.00 and 1.51 mumol after placebo and heparin, respectively (mean difference 0.6 doubling doses; 95% CI -0.25-1.5). Thus, heparin inhaled at doses sufficient to inhibit allergen and exercise-induced bronchoconstriction has no effect on the response to sodium metabisulphite and methacholine challenge in asthma. This argues against an inhibitory effect on neural pathways or airway smooth muscle.

Administration, Inhalation↗