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At least 595 records · Page 33Linked to original sources
PubMed2004

[New method for quantification of cell swelling using infrared differential interference microscopy].

We have developed a novel device for the quantification of swelling of cells in acute brain slices. We can also carry out detailed real-time monitoring of hippocampal cells. The device we developed is based on an infrared differential interference contrast microscopy (IR-DIC) and a custom-made real-time computerized image analysis system for the quantification of the morphological dynamics of cells in slice preparations. We applied the coefficient of variation (CV) of light intensity in IR-DIC images to quantify the change in morphological dynamics. There were notable close relationships among the edema formations, the light transmittance, extent of changes in CV, and features of fEPSP during ischemic insult. We also applied this method for the evaluation of neuroprotective effects of mannitol. The dose-dependent improvement on the deteriorated hippocampal slices could be obtained by administration of mannitol (10, 50, and 100 mM) after 10-min ischemia. The present results indicate that CV is a reliable quantification index for edema formation of brain tissue and confirm that applying CV for the analysis in addition to the light transmittance analysis presents additional important information on brain tissue sweling.

Animals↗
PubMed2007

A sensitive gait parameter for quantification of arthritis in rats.

Quantification of arthritis is helpful for investigating pain mechanisms of arthritis and for developing new drugs. We assessed and identified a feasible parameter for quantification of rat arthritis using a novel gait analyzing system. Knee-joint injection with small doses of lambda-carrageenan decreased swing time ratio (STR, swing time of the non-treated hindlimb/swing time of the lambda-carrageenan-injected hindlimb) in a dose-dependent manner. Intraperitoneal treatment with indomethacin restored the decreased STR dose-dependently. The arthritis could not be accurately quantified by swing time and swelling, common indices of arthritis. These results show that STR is a sensitive, reliable parameter for quantification of arthritis.

Animals↗
PubMed2006

Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase A gene.

The presence of cyanobacterial bloom in water supply reservoirs can cause potential health hazards. In this study, we aimed at the quantification of microcystin-producing cyanobacteria based on the microcystin synthetase A (mcyA) gene using real-time PCR. To perform a highly sensitive real-time PCR assay, the novel primer MSR-2R was designed and a coprecipitation DNA extraction method was used in this study. Cyanobacterial cells could be collected efficiently by coprecipitation with other bacteria suspended in solution even in the case of low concentrations of cyanobacteria. The detection limit of the method was found to be 8.8 cells per reaction. When cyanobacterial growth was monitored in pure culture, the cell concentration determined by real-time PCR positively correlated with the cell concentration determined from direct microscopic count. Furthermore, we could detect and quantify the mcyA gene in lake water samples using real-time PCR. It was concluded that the quantification of the mcyA gene based on real-time PCR is a powerful tool for the rapid quantification of microcystin-producing cyanobacteria in environmental samples.

Bacterial Proteins↗
PubMed2006

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard.

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.

Food, Genetically Modified↗
PubMed2005

Identification and quantification of 8 sulfonylureas with clinical toxicology interest by liquid chromatography-ion-trap tandem mass spectrometry and library searching.

BACKGROUND: Identification of sulfonylureas in blood may be useful in the evaluation of hypoglycemic crises of unknown origin. The aim of the present study was to develop a highly selective liquid chromatography-electrospray tandem mass spectrometry (MS-MS) method using an ion-trap detector for rapid screening, identification, and quantification of sulfonylureas in human plasma. METHODS: After standard liquid-liquid extraction with glisoxepide as an internal standard, 8 sulfonylureas (glibenclamide, glipizide, gliclazide, glibornuride, glimepiride, carbutamide, chlorpropamide, and tolbutamide) were eluted from a C18 column within 10 min with an isocratic mobile phase. Drugs were identified and quantified in full-scan MS-MS mode by use of a homemade MS-MS library. We used the assay in 134 cases of hypoglycemic crises of unknown origin. RESULTS: No ion suppression effect was noted for the analytes at their specific retention-time windows. For all drugs, assay validation showed good linearity (r2>0.990) and acceptable imprecision and recovery based on commonly used criteria of acceptance. The mean extraction recoveries were 63%-87% for 5 sulfonylureas but <45% for 3 (carbutamide, chlorpropamide, and tolbutamide). Nevertheless, the high sensitivity of the MS instrument made possible detection and quantification of all 8 drugs at subtherapeutic to toxic concentrations with good precision. Sulfonylureas were found in 9 hypoglycemic patients. CONCLUSION: The described assay method allows accurate, rapid identification and quantification of 8 sulfonylureas in human plasma and can be used for specific diagnosis of factitious hypoglycemia caused by ingestion of these drugs.

Chromatography, Liquid↗
PubMed2006

Real-time RT-PCR quantification of human telomerase reverse transcriptase splice variants in tumor cell lines and non-small cell lung cancer.

BACKGROUND: We developed and validated a real-time reverse transcription (RT)-PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (alpha+beta+, alpha-beta+, alpha+beta-, alpha-beta-) in tumor cell lines and non-small cell lung cancer (NSCLC). METHODS: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript-specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. RESULTS: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The alpha+beta- splice variant showed the highest expression and alpha-beta+ and alpha-beta- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the alpha+beta- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by alpha+beta+, with the alpha-beta+ and alpha-beta- splice variants having the lowest expression. In the NSCLC tumors, the alpha+beta+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. CONCLUSIONS: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT alpha+beta+ splice variant may be an independent negative prognostic factor for NSCLC patients.

Carcinoma, Non-Small-Cell Lung↗
PubMed2005

Quantification of proviral DNA load of human immunodeficiency virus type 2 subtypes A and B using real-time PCR.

HIV-2 infection is confined mostly to West Africa. Seven HIV-2 subtypes have so far been described; only HIV-2 subtypes A and B are prevalent, the others being considered self-limiting infections at the epidemiological level. The main limitation for the HIV-2 DNA proviral quantification is the lack of HIV-2 DNA standard. We designed and tested a new HIV-2 primer couple that amplifies both the HIV-2 ROD strain and HIV-1 LAV/BRU strain. These HIV-2 primers were used to quantified an HIV-2 standard comparatively to a standard widely used in proviral DNA HIV-1 quantification, i.e., the 8E5 cell line transfected by a single defective integrated provirus of HIV-1 BRU/LAV by cell. The primers and probe used to quantify HIV-2 DNA are located in a long terminal repeat (LTR) region with low variability. These primers amplify both HIV-2 subtypes A and B. The relevance of the follow-up of the infected patients by the quantification of the proviral DNA HIV-2 is currently studied.

Cell Line↗
PubMed2006

Plasma cell quantification in bone marrow by computer-assisted image analysis.

BACKGROUND: Minor and major criteria for the diagnosis of multiple meloma according to the definition of the WHO classification include different categories of the bone marrow plasma cell count: a shift from the 10-30% group to the > 30% group equals a shift from a minor to a major criterium, while the < 10% group does not contribute to the diagnosis. Plasma cell fraction in the bone marrow is therefore critical for the classification and optimal clinical management of patients with plasma cell dyscrasias. The aim of this study was (i) to establish a digital image analysis system able to quantify bone marrow plasma cells and (ii) to evaluate two quantification techniques in bone marrow trephines i.e. computer-assisted digital image analysis and conventional light-microscopic evaluation. The results were compared regarding inter-observer variation of the obtained results. MATERIAL AND METHODS: Eighty-seven patients, 28 with multiple myeloma, 29 with monoclonal gammopathy of undetermined significance, and 30 with reactive plasmocytosis were included in the study. Plasma cells in H&E- and CD138-stained slides were quantified by two investigators using light-microscopic estimation and computer-assisted digital analysis. The sets of results were correlated with rank correlation coefficients. Patients were categorized according to WHO criteria addressing the plasma cell content of the bone marrow (group 1: 0-10%, group 2: 11-30%, group 3: > 30%), and the results compared by kappa statistics. RESULTS: The degree of agreement in CD138-stained slides was higher for results obtained using the computer-assisted image analysis system compared to light microscopic evaluation (corr.coeff. = 0.782), as was seen in the intra- (corr.coeff. = 0.960) and inter-individual results correlations (corr.coeff. = 0.899). Inter-observer agreement for categorized results (SM/PW: kappa 0.833) was in a high range. CONCLUSIONS: Computer-assisted image analysis demonstrated a higher reproducibility of bone marrow plasma cell quantification. This might be of critical importance for diagnosis, clinical management and prognostics when plasma cell numbers are low, which makes exact quantifications difficult.

Algorithms↗
PubMed1982

Accurate quantification of nonesterified and total cholesterol in plasma and urine from crude lipid extracts by stable isotope dilution and field desorption mass spectrometry.

Levels of free and total cholesterol were quantified in control sera and human urine using [2H7]cholesterol or [13C2]cholesterol as internal standard and field desorption mass spectrometry for the quantification. These analyses are performed using a crude chloroform extract without chromatographic separation of the constituents. Due to the extremely soft ionization conditions in field desorption, the constituents of the crude lipid extract predominantly form molecular ion species and only a very low amount of fragment ions of low relative abundance thus minimizing the possibility of ion overlap in the mass spectrum. Mass spectrometric quantification of cholesterol in two control sera resulted in data within the range of assigned concentrations. The standard deviations of the mass spectrometric data were around +/- 2%. Generally, 50 microliters of serum or plasma or 500 microliters of urine are extracted and the mass spectrometric quantification performed using an aliquot of 2-10%. A characteristic pattern of intact cholesteryl esters is also obtained.

Body Fluids↗
PubMed1999

Quantification of low abundance natriuretic peptide receptor mRNA in rat tissues.

Natriuretic peptides are important regulators of vascular resistance and volume and electrolyte homeostasis. The quantification of natriuretic peptide receptor (NPR) mRNA is important for the understanding of the regulation of this humoral system, but is difficult due to low expression of the NPR mRNA. We report here on the evaluation of a polymerase chain reaction (PCR)-aided transcript titration assay for quantification of all three NPR subtypes (NPR-A, NPR-B, and NPR-C) mRNA. A multispecific internal standard RNA with parts of NPR-A, NPR-B, NPR-C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nucleotide sequences was constructed and reverse transcription of standard and sample RNA (400 ng) was performed in parallel for all three NPRs and GAPDH. The specific PCR yielded differently sized products, which were quantified by high performance liquid chromatography (HPLC). The determination of specific mRNA concentrations was not influenced by cDNA input and did not depend on the PCR cycle number. Linearity between sample RNA input and mRNA concentration was demonstrated. Application of the evaluated method showed that the NPR-A mRNA expression was the most abundant of the three natriuretic peptide receptor mRNAs in rat lungs, glomeruli and left ventricles, followed by the NPR-C mRNA and the NPR-B mRNA expression. Thus, the described method allows the reliable quantification of the specific mRNA expression of all three NPRs with small amounts of RNA. The presented method might foster future research on the regulation of this humoral system in cardiovascular and kidney diseases.

Animals↗
PubMed2001

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry.

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.

Animals↗
PubMed2004

Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay.

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.

Animals↗
PubMed2001

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.

Binding, Competitive↗
PubMed2002

Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis.

The rapid development of molecular genetic analysis tools has made it possible to analyze most biological materialfound at the scene of a crime. Evidence materials containing DNA quantities too low to be analyzed using nuclear markers can be analyzed using the highly abundant mtDNA. However, there is a shortage of sensitive nDNA and mtDNA quantification assays. In this study, an assay for the quantification of very small amounts of DNA, based on the real-time Taq-Man assay, has been developed. This analysis will provide an estimate of the total number of nDNA copies and the total number of mtDNA molecules in a particular evidence material. The quantification is easy to perform, fast, and requires a minimum of the valuable DNA extracted from the evidence materiaL The results will aid in the evaluation of whether the specific sample is suitable for nDNA or mtDNA analysis. Furthermore, the optimal amount of DNA to be used in further analysis can be estimated ensuring that the analysis is successful and that the DNA is retained for future independent analysis. This assay has significant advantages over existing techniques because of its high sensitivity, accuracy, and the combined analysis of nDNA and mtDNA. Moreover, it has the potential to provide additional information about the presence of inhibitors in forensic samples. Subsequent mitochondrial and nuclear analysis of quantified samples illustrated the potential to predict the number of DNA copies required for a successful analysis in a certain typing assay.

Cell Nucleus↗
PubMed2006

Quantification of ammonia-oxidizing bacteria populations in full-scale sewage activated sludge systems and assessment of system variables affecting their performance.

This study carried out quantification of ammonia-oxidizing bacteria (AOB) populations in 12 full-scale sewage activated sludge systems that were different in ammonia removals and treatment processes during three different seasons. Experiment was divided into 3 parts: 1) analysis of AOB communities by PCR-DGGE-cloning-sequencing of 16S rRNA genes; 2) development of four real-time PCR primer sets for quantification of the particular AOB of interest; and 3) quantification of AOB populations by using the newly developed real-time PCR primer sets. The results suggested that all the primer sets gave good reproducibility and specificity for PCR amplification with the detection limits of 10(2) copies/PCR reaction. Although the 12 systems were different in several aspects, one of the identified sequence types of Nitrosomonas oligotropha cluster was the dominant AOB in every system and every season studied. However, the other sequence type of this cluster was not significantly involved in ammonia removals in the systems. The occurrence of N. communis cluster in the systems seemed to depend on the remaining oxygen concentrations in the sludge floc and thus the activity of aerobic heterotrophs in the aeration tanks. N. europaea-Nitrosococcus. mobilis solely existed in one A20 system of which the influent contained twice the chloride concentrations than those of other systems.

Ammonia↗
PubMed2003

Fully automated quantification of regional cerebral blood flow with three-dimensional stereotaxic region of interest template: validation using magnetic resonance imaging--technical note.

The previously reported three-dimensional stereotaxic region of interest (ROI) template (3DSRT-t) for the analysis of anatomically standardized technetium-99m-L,L-ethyl cysteinate dimer (99mTc-ECD) single photon emission computed tomography (SPECT) images was modified for use in a fully automated regional cerebral blood flow (rCBF) quantification software, 3DSRT, incorporating an anatomical standardization engine transplanted from statistical parametric mapping 99 and ROIs for quantification based on 3DSRT-t. Three-dimensional T2-weighted magnetic resonance images of 10 patients with localized infarcted areas were compared with the ROI contour of 3DSRT, and the positions of the central sulcus in the primary sensorimotor area were also estimated. All positions of the 20 lesions were in strict accordance with the ROI delineation of 3DSRT. The central sulcus was identified on at least one side of 210 paired ROIs and in the middle of 192 (91.4%) of these 210 paired ROIs among the 273 paired ROIs of the primary sensorimotor area. The central sulcus was recognized in the middle of more than 71.4% of the ROIs in which the central sulcus was identifiable in the respective 28 slices of the primary sensorimotor area. Fully automated accurate ROI delineation on anatomically standardized images is possible with 3DSRT, which enables objective quantification of rCBF and vascular reserve in only a few minutes using 99mTc-ECD SPECT images obtained by the RVR method.

Aged↗
PubMed1989

Blood flow in the carotid arteries: quantification by using phase-sensitive MR imaging.

The feasibility of MR phase-sensitive imaging for the quantification of blood flow in the carotid arteries was studied in two normal volunteers and six patients with carotid artery and/or cerebrovascular disease. The technique consists of sensitizing the phase of the MR signal to blood flow velocity gated to different times in the cardiac cycle. Flow velocities and volumes were measured by using transverse planes in the common, internal, and external carotid arteries, and flow curves were generated. Measurements made by using flow phantoms correlated well with calculated results. The MR measurements yielded values between 250 and 580 ml/min for the total flow through each of the common carotid arteries in the two normal volunteers. Markedly reduced flow (about 50% below normal) was detected in a patient whose arteriogram showed severe occlusion of the internal carotid artery. In a second patient, who had a large frontal intracranial arteriovenous malformation noted by arteriography, the MR-quantified flow was abnormally high (about 1 liter/min). In the remaining four patients, the findings on phase-sensitive quantification were consistent with those expected from clinical and other laboratory studies (including arteriography and sonography). These preliminary findings suggest that MR phase mapping may be a feasible tool for the noninvasive quantification of carotid blood flow.

Adult↗
PubMed2005

MR quantification of the washout changes in breast tumors under preoperative chemotherapy: feasibility and preliminary results.

OBJECTIVE: The objective of our study was to describe and determine the feasibility of an MR washout quantification method in patients with breast cancer under preoperative chemotherapy. MATERIALS AND METHODS: Nineteen patients with breast T2 or T3 tumors were enrolled in a previous study for tumor response evaluation during chemotherapy based on dynamic contrast-enhanced MRI. We retrospectively used the dynamic acquisition data to produce parametric images representing the washout pattern. Two radiologists unaware of the final pathologic results measured the volume of pixels exhibiting washout within the tumor before chemotherapy (volume 1), after two courses of chemotherapy (volume 2), and before surgery after four courses of chemotherapy (volume 3). The interobserver variability and intraobserver variability were calculated to evaluate the reproducibility of our method with the Pearson's correlation coefficient and the concordance correlation coefficient. We correlated the washout changes by means of a Student's t test and noted the histopathologic final outcome. RESULTS: A washout pattern was present in all patients on the initial MR study. The quantification method of the washout changes was reproducible with good interobserver agreement (r = 0.85, p < 10(-5)) and an excellent intraobserver agreement (r = 0.94, p < 10(-5)). A significant decrease of the washout volume was observed after two courses of chemotherapy (p = 0.004), whereas no significant modification was observed between two and four courses of chemotherapy (p = 0.52). CONCLUSION: Quantification of the washout variation in breast tumor based on the use of parametric images is feasible and reproducible. It may add information to the evaluation of tumor response to preoperative therapy.

Adult↗