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At least 469 records · Page 26Linked to original sources

PubMed2000

Distribution of Tsc1 protein detected by immunohistochemistry in various normal rat tissues and the renal carcinomas of Eker rat: detection of limited colocalization with Tsc1 and Tsc2 gene products in vivo.

We and others previously demonstrated that hereditary mutation and a subsequent second hit in the rat homolog of tuberous sclerosis gene (Tsc2) are responsible for Eker renal carcinomas (RC). In humans, alteration in the TSC2 gene is known to cause the tuberous sclerosis complex (TSC) that results in hamartomatous lesions in multiple organs, but the function of TSC2 is not fully understood. In recent years, a second gene (TSC1) responsible for human TSC has been cloned, and binding between TSC1 and TSC2 proteins was reported. In this study, to clarify associations between Tsc proteins in vivo, the expression of Tsc1 protein was detected by immunohistochemistry, and compared with Tsc2 expression. Tsc1 protein was expressed in the nervous system and in many endocrine tissues, including pancreatic islets, the parathyroids, testis, and ovary. Tsc1 was also detected in the many epithelial tissues of organs, such as kidney, uterus, small and large intestine, and liver. Our results indicate overlapping, but not identical, organ distributions of Tsc1 and Tsc2 proteins. At the intracellular distribution, double fluorescent immunolabeling allowed the determination that only a partial portion of Tsc1 signals overlapped with Tsc2 in some organs. These results suggest the existence of co-localizing and independent forms of Tsc proteins in endogenous expressions. Additionally, relatively high expression of Tsc1 protein was detected in RC in the Tsc2 mutant (Eker) rat.

Animals↗

PubMed2006

Multiplexed analyte and oligonucleotide detection on microarrays using several redox enzymes in conjunction with electrochemical detection.

We show that multiple enzyme tags may be used in an immunoassay format or for the detection of sequence-specific DNA on microarrays. The assays may be multiplexed and monitored under separate solution and voltage differences. Thus, the detection method relies on an electrochemical detection format, whereby multiple enzymes can be sensed. In our case we utilize horseradish peroxidase, laccase, and glucose dehydrogenase as enzymes attached to specific antibodies or to streptavidin.

Antibodies↗

PubMed1999

Confirmation of the detection of HLA-A0104N in a family: a PCR-SSP reaction for the allelic detection of HLA-A0104N.

The allele A*0104N has been detected in a family with a patient requiring a bone marrow transplant. The allele was found as a consequence of a discrepant result when family members were typed using serology and polymerase chain reaction using sequence-specific primers (PCR-SSP). Serological typing gave an apparent HLA-A 'blank' while PCR-SSP revealed the presence of an A*01 allele in three family members who were serologically negative for A1. Sequencing-based typing (SBT) was then used to establish that the allele was A*0104N. A PCR-SSP reaction was subsequently designed and used for the allelic detection of A*0104N. The study highlights the potential risks involved if molecular technology is used for typing, unless all non-expressed alleles are specifically detected.

Alleles↗

PubMed2002

Pre-screening for antigen detectability in cells: a TEM-based solid phase digital immunogold detection method utilizing ultra low volumes of reagents.

Rapid and sensitive pre-screening for the presence of antigens in cell samples and confirmation of reactivity of antibodies, before proceeding with electron microscopy, is highly desirable. Most of the methods developed for this purpose are generally not very efficient and suitable for dealing with very small volumes of sample and reagents. In this work we present a simple, sensitive and rapid solid phase transmission electron microscope (TEM) based method for the detection of picogram (pg) levels of soluble antigens using as little as 10 micro L of reagents. Protein was adsorbed onto grids coated with polystyrene films to form the solid phase. The presence of antigen was detected using immunogold labelling. Gold particles adhering to the film were visualized and counted in a TEM providing a digital signal. This method was 100-fold more sensitive than dot blot in detection of rabbit IgG. We have demonstrated the utility of this technique by screening for Vitreoscilla haemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold labelling transmission electron microscopy of cell sections.

Animals↗

PubMed2002

Failure of atrial flutter detection by a pacemaker with a dedicated atrial flutter detection algorithm.

Detection of atrial flutter may be difficult for pacemakers with automatic mode switching algorithms. The Medtronic Kappa 700 device pacemakerfamily offers a dedicated Blanked Flutter Search algorithm specifically designed for the detection of atrial flutter. This report describes how spontaneous atrioventricular conduction, resulting in a sensed QRS complex at a critical time, inhibited the function of the Blanked Flutter Search algorithm to sense "concealed" or "blanked" atrialflutter. This observation underscores the need for further refinement of pacemaker algorithms for the detection of atrial tachyarrhythmias.

Aged↗

PubMed2001

Radiologist detection of microcalcifications with and without computer-aided detection: a comparative study.

AIM: To compare the sensitivity and specificity of microcalcification detection by radiologists alone and assisted by a computer-aided detection (CAD) system. MATERIALS AND METHODS: Films of 106 patients were masked, randomized, digitized and analysed by the CAD-system. Five readers interpreted the original mammograms and were blinded to demographics, medical history and earlier films. Forty-two mammograms with malignant microcalcifications, 40 with benign microcalcifications and 24 normal mammograms were included. Results were recorded on a standardized image interpretation form. The mammograms with suspicious areas flagged by the CAD-system were displayed on mini-monitors and immediately re-reviewed. The interpretation was again recorded on a new copy of the standard form and classified according to six groups. RESULTS: Forty-one out of 42 (98%) malignant microcalcifications and 32 of 40 (80%) benign microcalcifications were flagged by the CAD-system. There was an average of 1.2 markers per image. The sensitivity for malignant microcalcifications detection by mammographers without and with the CAD-system ranged from 81% to 98% and from 88% to 98%, respectively. The mean difference without and with CAD-system was 2.2% (range 0-7%). CONCLUSION: No statistically significant changes in sensitivity were found when experienced mammographers were assisted by the CAD-system, with no significant compromise in specificity.

Breast Diseases↗

PubMed1997

Temporal and Quantitative Analysis of Chimerism in Liver and Kidney Transplant Patients: An Enhanced Fluorescence Detection System Allows for More Sensitive Quantitative Detection.

Background: Because spontaneous microchimerism has been reported in stable renal and hepatic allografts, the presence of donor-derived cells in recipient tissues was investigated in kidney and liver tranplant recipients. Methods and Results: Human lymphocyte antigen class II markers and Y-chromosome sequences in male donor-to-female recipient transplants were used for chimeric analysis. Human lymphocyte antigen typing was performed by group-specific polymerase chain reaction amplification and restriction fragment length polymorphism analysis, X-chromosome- and Y-chromosome-specific primers were used in a multiplex polymerase chain reaction analysis. Quantitative Y-chromosome analysis was performed using energy-transfer fluorescence from a nested primer system. Patients who had rejected their grafts were also analyzed, as were a group who were analyzed for chimerism at the time of transplant (day 1) and sequentially at various intervals for up to 3 months. Of 23 long-term kidney patients analyzed, 16 were chimeric by human lymphocyte antigen or sex-determination analysis. In 2 patients whose graft had failed no chimerism was observed. Chimerism in liver patients was detectable on the day of transplant and was maintained for 30 to 120 days as measured at 5-day intervals (these patients continue to be monitored). Quantititative analysis suggested that the ratio of donor to recipient cells was variable in a patient and ranged from greater than 1 in 10(4) to less than 1 in 10(5). An enhanced fluorescence energy-transfer detection system was adopted to increase sensitivity of the polymerase chain reaction detection of chimerism and to quantitate the results. Conclusions: The results indicate that cells from the donor organ migrate into recipient tissues early after transplantation. These cells persist in a majority of patients for at least 3 to 4 years. It has been proposed that tolerance is related to the presence of these "passenger" leukocytes and that dendritic cells play the most important role. The data suggest that the establishment of chimerism plays an important role in graft acceptance in a majoritiy of the kidney and liver patients in this study. These findings also suggest that the levels of chimeric cells, "a quantitative chimerism," may be important in establishing tolerance but further studies are required to support this contention.

Journal Article↗

PubMed2005

Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies.

AIMS: To develop and evaluate multiplex PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae, using real-time and conventional PCR detection methodologies. METHODS: Two real-time multiplex PCR assays, using nuclease (TaqMan-ABI7500) and hybridisation (LightCycler) probe formats, and a third assay using conventional PCR with solid-phase hybridisation and colour detection, were developed. The porA pseudogene was targeted for N. gonorrhoeae, and the major outer membrane protein gene for C. trachomatis. A total of 145 urogenital specimens were tested in all assays, and the results were compared with the Roche Cobas Amplicor assay. RESULTS: There was little difference in clinical sensitivity and specificity, result discrimination and test cost for the three in-house assays. Our results showed that competitive inhibition of the PCR occurred in some samples that were positive for both organisms. CONCLUSIONS: These results highlight the suitability and versatility of three multiplex PCR methods for the detection of C. trachomatis and N. gonorrhoeae.

Adolescent↗

PubMed2004

Detection of brain oedema using magnetic induction tomography: a feasibility study of the likely sensitivity and detectability.

The detection and continuous monitoring of brain oedema is of particular interest in clinical applications because existing methods (invasive measurement of the intracranial pressure) may cause considerable distress for the patients. A new non-invasive method for continuous monitoring of an oedema promises the use of multi-frequency magnetic induction tomography (MIT). MIT is an imaging method for reconstructing the changes of the conductivity deltakappa in a target object. The sensitivity of a single MIT-channel to a spherical oedematous region was analysed with a realistic model of the human brain. The model considers the cerebrospinal fluid around the brain, the grey matter, the white matter, the ventricle system and an oedema (spherical perturbation). Sensitivity maps were generated for different sizes and positions of the oedema when using a coaxial coil system. The maps show minimum sensitivity along the coil axis, and increasing values when moving the perturbation towards the brain surface. Parallel to the coil axis, however, the sensitivity does not vary significantly. When assuming a standard deviation of 10(-7) for the relative voltage change due to the system's noise, a centrally placed oedema with a conductivity contrast of 2 with respect to the background and a radius of 20 mm can be detected at 100 kHz. At higher frequencies the sensitivity increases considerably, thus suggesting the capability of multi-frequency MIT to detect cerebral oedema.

Brain Edema↗

PubMed1989

Detection of DNA adducts by high-performance liquid chromatography with electrochemical detection.

A method for the detection of rare adducts in DNA has been developed by combining the resolution of high-performance liquid chromatography with the specificity and sensitivity of electrochemical detection. Many adducts are electrochemically active, while the normal bases, except for guanine, are not. Enzymatic hydrolysis of DNA is used to obtain the deoxynucleosides for analysis, or where appropriate, acid hydrolysis or thermal depurination of DNA is used to free the adduct base for analysis. Various types of DNA damage have been induced by in vitro exposure of DNA to acrolein, dimethyl sulfate, sodium nitrite, ascorbate/Cu2+ and gamma-irradiation. Several adducts are detected at a level of one adduct in 10(5)-10(6) normal bases in micrograms of DNA. The method is also useful for measuring O6-methylguanine (O6MeGua) in DNA from rats treated with N-nitrosodimethylamine and 8-hydroxydeoxyguanosine (oh8dG), and O6-MeGua in DNA from bacteria treated with hydrogen peroxide and dimethyl sulfate. oh8dG, which appears to be the most suitable marker for measuring the steady-state level of oxidative DNA damage, can be measured at fmol levels in DNA from normal rat tissues. The method is applicable to the analysis of DNA base damage caused by major endogenous processes relevant to aging, such as deamination, methylation and oxidation. The analysis of DNA adducts with this simple assay also may be potentially useful for studies on carcinogenesis and as a tool in studies on the epidemiology of cancer.

Animals↗

PubMed1997

Early detection of perinatal human immunodeficiency virus (HIV) type 1 infection using HIV RNA amplification and detection. New York City Perinatal HIV Transmission Collaborative Study.

Early diagnosis of perinatally transmitted human immunodeficiency virus type 1 (HIV) infection can guide early interventions. HIV coculture and DNA polymerase chain reaction (DNA-PCR) detect few HIV-infected infants at birth and 90%-100% by age 3 months. Because extracellular HIV RNA may appear soon after infection, a plasma HIV RNA assay was compared with DNA-PCR for early detection of perinatally infected infants. Blood-draw specimens (108) obtained at the same time from 49 HIV-infected infants and 10 specimens from 8 uninfected infants were tested. HIV RNA and DNA-PCR positivity rates were 56% and 33%, respectively, in 36 specimens from 36 infants <28 days of age (binomial test, P = .001). Among 81 specimens obtained after age 14 days, 79 (98%) were positive by HIV RNA testing. No HIV-infected infant specimens were DNA-PCR-positive and HIV RNA-negative. All specimens from 8 uninfected infants were HIV RNA-negative. These results suggest that plasma HIV RNA was detectable earlier and more reliably than HIV DNA in perinatal infection.

Female↗

PubMed2003

Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit.

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.

Bacterial Proteins↗

PubMedDate not supplied

Immunoassay detection of drugs in racing horses: detection of ethacrynic acid and bumetanide in equine urine by ELISA.

We have raised antibodies and developed one-step enzyme-linked immunosorbent assays (ELISA) for the diuretics ethacrynic acid and bumetanide as part of a panel of pre- and post-race tests for high potency drugs in racing horses. These ELISA tests are rapid (completed within one hour), sensitive, and can be read by eye. The ELISA detects ethacrynic acid at a drug concentration for half-maximal inhibition (I-50) of about 2.5 ng/mL for the parent drug. After dosing horses intravenously with 5 mg ethacrynic acid per horse, the parent drug or its metabolites are detectable in urine for at least 8 hours. The bumetanide ELISA has an I-50 for the parent drug of about 2.0 ng/mL and will detect bumetanide or its metabolites for about 8 hours in urine after intravenous administration of a 1.7-mg dose per horse. Both antibodies are relatively specific for each drug and do not cross-react with other commonly used diuretics or other acidic compounds often found in post-race equine urine samples. Ethacrynic acid and bumetanide are potent diuretics suspected of being illegally substituted for furosemide in certain racing jurisdictions. Development of these rapid, sensitive, and simple tests for these agents will allow more effective pre- and post-race control of the use of these agents in racing horses. Both tests have recently uncovered several "positives" for these medications in a midwestern racing jurisdiction.

Animals↗

PubMedDate not supplied

Detection of 2-amino-5-chloropyridine in urine as a parameter of zopiclone (Imovane) intake using HPLC with diode array detection.

A qualitative screening technique was developed for the detection of 2-amino-5-chloropyridine, a newly identified decomposition product of zopiclone and metabolites after alkaline hydrolysis of urine samples. The method was elaborated using a standard operation procedure (Merck Tox Screening System), combining a solid-phase extraction with reversed-phase high-performance liquid chromatography and diode array detection. The limit of detection, expressed as zopiclone concentrations in spiked urine, was found to be 0.5 microg/mL. Field urine samples from a volunteer were positive for 2-amino-5-chloropyridine until 16 h after ingestion of one therapeutic dose of Imovane (7.5 mg zopiclone).

Administration, Oral↗

PubMed2000

Breast density as a predictor of mammographic detection: comparison of interval- and screen-detected cancers.

BACKGROUND: Screening mammography is the best method to reduce mortality from breast cancer, yet some breast cancers cannot be detected by mammography. Cancers diagnosed after a negative mammogram are known as interval cancers. This study investigated whether mammographic breast density is related to the risk of interval cancer. METHODS: Subjects were selected from women participating in mammographic screening from 1988 through 1993 in a large health maintenance organization based in Seattle, WA. Women were eligible for the study if they had been diagnosed with a first primary invasive breast cancer within 24 months of a screening mammogram and before a subsequent one. Interval cancer case subjects (n = 149) were women whose breast cancer occurred after a negative or benign mammographic assessment. Screen-detected control subjects (n = 388) were diagnosed after a positive screening mammogram. One radiologist, who was blinded to cancer status, assessed breast density by use of the American College of Radiology Breast Imaging Reporting and Data System. RESULTS: Mammographic sensitivity (i.e., the ability of mammography to detect a cancer) was 80% among women with predominantly fatty breasts but just 30% in women with extremely dense breasts. The odds ratio (OR) for interval cancer among women with extremely dense breasts was 6.14 (95% confidence interval [CI] = 1.95-19.4), compared with women with extremely fatty breasts, after adjustment for age at index mammogram, menopausal status, use of hormone replacement therapy, and body mass index. When only those interval cancer cases confirmed by retrospective review of index mammograms were considered, the OR increased to 9.47 (95% CI = 2.78-32.3). CONCLUSION: Mammographic breast density appears to be a major risk factor for interval cancer.

Adult↗

PubMed2001

Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides.

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region ( approximately 1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T(g)) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either (32)P-end-labelling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T(g) and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.

Alkylation↗

PubMed2004

Can acceleromyography detect low levels of residual paralysis? A probability approach to detect a mechanomyographic train-of-four ratio of 0.9.

BACKGROUND: The incidence of residual paralysis, i.e., a mechanomyographic train-of-four (TOF) ratio (T4/T1) less than 0.9, remains frequent. Routine acceleromyography has been proposed to detect residual paralysis in clinical practice. Although acceleromyographic data are easy to obtain, they differ from mechanomyographic data, with which they are not interchangeable. The current study aimed to determine (1) the acceleromyographic TOF ratio that detects residual paralysis with a 95% probability, and (2) the impact of calibration and normalization on this predictive acceleromyographic value. METHODS: In 60 patients, recovery from neuromuscular block was assessed simultaneously with mechanomyography and acceleromyography. To obtain calibrated acceleromyographic TOF ratios in group A, the implemented calibration modus 2 was activated in the TOF-Watch S; to obtain uncalibrated acceleromyographic TOF ratios in group B, the current was manually set at 50 mA (n = 30 for each). In addition, data in group B were normalized (i.e., dividing the final TOF ratio by the baseline value). The agreement between mechanomyography and acceleromyography was assessed by calculating the intraclass correlation coefficient. Negative predictive values were calculated for detecting residual paralysis from acceleromyographic TOFs of 0.9, 0.95, and 1.0. RESULTS GROUP A: : For a mechanomyographic TOF of 0.9 or greater, the corresponding acceleromyographic TOF was 0.95 (range, 0.86-1.0), and the negative predictive values for acceleromyographic TOFs of 0.9, 0.95, and 1.0 were 37% (95% CI, 20-56%), 70% (95% CI, 51-85%), and 97% (95% CI, 83-100%), respectively. Group B: Without normalization, an acceleromyographic TOF of 0.97 (range, 0.68-1.18) corresponded to a mechanomyographic TOF of 0.9 or greater, with negative predictive values for acceleromyographic TOFs of 0.9, 0.95, and 1.0 being 40% (95% CI, 23-59%), 60% (95% CI, 41-77%), and 77% (95% CI, 58-90%), respectively. After normalization, an acceleromyographic TOF of 0.89 (range, 0.63-1.06) corresponded to a mechanomyographic TOF of 0.9 or greater, and the negative predictive values of acceleromyographic TOFs of 0.9, 0.95, and 1.0 were 89% (95% CI, 70-98%), 92% (95% CI, 75-99%), and 96% (95% CI, 80-100%), respectively. CONCLUSION: To exclude residual paralysis reliably when using acceleromyography, TOF recovery to 1.0 is mandatory.

Adult↗

PubMed1996

A rapid immunoassay method for the direct detection of PCR products: application to detection of TEM beta-lactamase genes.

A rapid immunoassay for the detection of specific PCR products is described in which a positive PCR amplification result is detected, usually in less than 5 min, by applying a few drops of the diluted PCR end-product to a small immunoassay sample device. The method was evaluated in comparison with conventional susceptibility tests and isoelectric focusing (IEF) for the detection of TEM-family beta-lactamase genes in 477 Escherichia coli isolates from urine samples. Of 187 isolates identified as presumptive TEM beta-lactamase producers by conventional methods, 185 generated a positive signal in the PCR immunoassay system. Two further signal-positive isolates were recognised when the PCR was repeated. In addition, one of the 276 ampicillin-susceptible isolates gave a positive signal in repeated PCR-immunoassay experiments despite being ampicillin susceptible and failing to give a TEM-type enzyme band in iso-electric focusing experiments.

Bacteriuria↗