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At least 631 records · Page 35Linked to original sources
PubMed2002

High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.

BACKGROUND: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification. METHODS: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros. RESULTS: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was approximately 12% for the major and 25% for the minor species. The detection limit was <1 micromol/L. CONCLUSION: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker.

Calibration↗
PubMed2002

CT quantification of pulmonary emphysema: assessment of lung structure and function.

Accurate diagnosis and quantification of pulmonary emphysema in vivo is important to understand the natural history of the disease, to assess the extent of the disease, and to evaluate and follow-up therapeutic interventions. Because pulmonary emphysema is defined by pathology, new diagnostic methods for quantification should be validated by reference to pathological and histological standards. Recent studies have addressed the capability of computed tomography (CT) to accurately quantify pulmonary emphysema. These studies that have been overviewed in this article have been based on CT scans obtained after deep inspiration or expiration, on subjective visual grading, and on objective measurements of attenuation values by using dedicated software providing numerical data on two-dimensional and on three-dimensional approaches, and compared CT data with pulmonary function tests. More recently, fractal and textural analyses were applied to CT scans to assess the presence, extent, and types of emphysema. Quantitative CT has already been used in patient selection for surgical treatment of pulmonary emphysema and in pharmacotherapeutical trials. However, despite numerous and extensive studies already available, this technique has not yet been standardized, and important questions about how to best use CT for the quantification of pulmonary emphysema remain to be addressed.

Age Factors↗
PubMed1992

[Quantification of nucleolar organizers (NORs) in renal carcinomas. Comparison with the nuclear grade].

Twenty three renal tumours: 6 clear cell carcinomas, 3 granular cell carcinomas, 8 mixed cell carcinomas, 3 tubulopapillary tumours, 2 spindle cell carcinomas and 1 oncocytoma, were retrospectively submitted to a staining technique which reveals argyrophilic proteins related to Nucleolar Organizer Regions (NORs). The semiautomated quantification of the number and mean surface area of the argyrophilic granules was performed by means of a quantimeter. The results were compared to the nuclear grade, which constitutes the reference histological prognostic factor. A very good correlation was observed between NORs and nuclear grade. The mean number of argyrophilic granules per nucleus (n) and more particularly their surface area (s) were very significantly increased in high-grade carcinomas (n = 1.96), s = 3.72) in comparison with low-grade carcinomas (n = 1.50, s = 1.35). Quantification of NORs therefore allowed objective differentiation between a low nuclear grade and a high nuclear grade. The quantification method used emphasised the value of not only measuring the number but also the surface area of the argyrophilic granules.

Cell Nucleus↗
PubMed2003

Microbial quantification in activated sludge: the hits and misses.

Since the implementation of the activated sludge process for treating wastewater, there has been a reliance on chemical and physical parameters to monitor the system. However, in biological nutrient removal (BNR) processes, the microorganisms responsible for some of the transformations should be used to monitor the processes with the overall goal to achieve better treatment performance. The development of in situ identification and rapid quantification techniques for key microorganisms involved in BNR are required to achieve this goal. This study explored the quantification of Nitrospira, a key organism in the oxidation of nitrite to nitrate in BNR. Two molecular genetic microbial quantification techniques were evaluated: real-time polymerase chain reaction (PCR) and fluorescence in situ hybridisation (FISH) followed by digital image analysis. A correlation between the Nitrospira quantitative data and the nitrate production rate, determined in batch tests, was attempted. The disadvantages and advantages of both methods will be discussed.

Bioreactors↗
PubMedDate not supplied

Quantification of PCR products using microparticles and flow cytometry.

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.

Animals↗
PubMed2004

Quantification of allantoin in various Zea mays L. hybrids by RP-HPLC with UV detection.

A RP-HPLC method for quantification of allantoin in silk of fifteen maize hybrids (Zea mays L., Poaceae) was described. Following extraction of the plant material with an acetone-water (7:3, VN) mixture, filtration and dilution, the extracts were analyzed without previous chemical derivatization. Separation and quantification were achieved using an Alltech Econosil C18 column under isocratic conditions at 40 degrees C. The mobile phase flow (20% methanol--80% water with 5 mM sodium laurylsulfate added at pH 2.5, adjusted with 85% orthophosphoric acid; pH of water phase was finally adjusted at 6.0 by addition of triethylamine) was maintained at 1.0 mL/min. Column effluent was monitored at 235 nm. This simple procedure afforded efficient separation and quantification of allantoin in plant material, without interference of polyphenols or other plant constituents of medium to high polarity, or similar UV absorption. Our study revealed that the silk of all investigated maize hybrids could be considered relatively rich in allantoin, covering the concentration range between 215 and 289 mg per 100 g of dry plant material.

Allantoin↗
PubMed2004

Quantification of plasma DNA as a screening tool for lung cancer.

BACKGROUND: Recent studies suggest that circulating DNA may be a potential tumor marker for lung cancer, but most of these studies are conducted between healthy controls and lung cancer patients, with few or no benign lung disease patients included. The objective of this study was to evaluate the performance of plasma DNA quantification in discriminating lung cancer from the healthy and benign lung disease. METHODS: Plasma DNA was extracted with a QIAamp DNA Blood Midi kit and quantified by a PicoGreen dsDNA quantitation kit in 44 healthy individuals, 36 benign lung disease patients and 67 lung cancer patients. Discrimination power was evaluated by the receiver operating characteristic curve. RESULTS: Plasma DNA values were significantly increased in lung cancer patients, especially in those with metastases, and in benign lung disease patients compared with that in the healthy individuals (P < 0.001, respectively). The values in lung cancer patients were significantly increased compared with that in the benign lung disease patients (P < 0.001). The area under the curve was 0.96 [95% confidence interval (CI) 0.92 - 0.99] for the healthy versus lung cancer, 0.73 (95% CI 0.64 - 0.83) for lung cancer versus benign lung disease, and 0.86 (95% CI 0.80 - 0.91) for lung cancer versus the healthy and benign lung disease. CONCLUSIONS: Plasma DNA quantification has a strong power to discriminate lung cancer from the healthy and from the healthy and benign lung disease, less power to discriminate lung cancer from benign lung disease. Plasma DNA quantification may be useful as a screening tool for lung cancer.

DNA↗
PubMed2004

Assessment of myocardial reperfusion after myocardial infarction using automatic 3-dimensional quantification and template matching.

UNLABELLED: Assessment of perfusion defect extent is essential for determining prognosis after a myocardial infarction (MI), but quantification methods usually rely on segmental analysis, which may lack accuracy. We present an automated voxel-based and template-based approach for precise quantification of perfusion defect extent and reperfusion evolution. METHODS: Coronary angiography and stress/reinjection (201)Tl tomography were performed prospectively on 49 patients with recent MI (45 men; mean age +/- SD, 54 +/- 10 y), before and 3 mo after revascularization (40 angioplasties and 9 bypasses). Perfusion defect extent was quantified using expert 16-segment visual scoring of the slices and a 3-dimensional (3D) method with spatial normalization between times 1 and 2. Briefly, the latter automatically extracted myocardial edges, matched them to a reference template, and compared the perfusion intensity in each voxel with the intensity of the corresponding voxel in a control population of 100 healthy subjects. RESULTS: Reocclusion occurred in 12 patients within 3 mo of surgery (all had undergone angioplasty). The perfusion gain between times 1 and 2, assessed by visual analysis, was significantly higher in permeable patients than in reoccluded patients: 12.4% +/- 13.3% and 2.3% +/- 8.2% of the initial stress defect, respectively (P = 0.02). Proportional gains, measured with the quantitative 3D method, were 4.5% +/- 3.6% and 1.9% +/- 2.7%, respectively (P = 0.02). Furthermore, the 3D method allowed measurement within the initial ischemic defect (reversible part of the stress defect at time 1), the extent of myocardium whose perfusion improved at time 2 (reperfusion), and the extent of myocardium whose perfusion remained unchanged (residual ischemia). A voxel-by-voxel analysis of these regions revealed that the proportion of reperfusion was significantly higher in permeable patients than in reoccluded patients: 60.0% +/- 21.3% versus 40.0% +/- 22.5%, respectively (P = 0.008). This was cumbersome to quantify using visual analysis and did not reach statistical significance, likely because of segmental division (partial-volume effect) and absence of spatial normalization. CONCLUSION: The 3D voxel-based quantification allows satisfying assessment of reperfusion 3 mo after MI. Moreover, the automated analysis using spatial normalization should facilitate a reproducible assessment of large populations over time.

Aged↗
PubMed1992

Indium-111 whole-body retention: a method for quantification of disease activity in inflammatory bowel disease.

Quantification of disease activity in inflammatory bowel disease (IBD) has been by measurement of fecal excretion of 111In-granulocytes. The difficulties of this method prompted us to evaluate quantification of whole-body 111In retention, expressed as a percentage of whole-body activity at 3 hr following injection, as an alternative method. The patient stood in front of the uncollimated gamma camera at a distance of 4 m and counts were collected over 2 min. The geometric mean was taken of posterior and anterior counts and compared with a 111In standard. The lower limit of the 95% confidence interval for whole-body retention in normals was 90%. Forty-five studies were performed on 33 patients with IBD. They were assessed in two groups, one to whom routine instructions for the collection of feces were given (Group A) but who did not always comply. The other group received oral and written instructions and were also monitored during the collection period (Group B) and reported full fecal collection. Although in Group A the correlation between fecal excretion and whole-body retention was good (r = 0.7, n = 32; p less than 0.001), in Group B the relationship between fecal excretion and whole-body retention was significantly better (r = 0.95, n = 18; p less than 0.001). On average, 111In whole-body retention was consistent with findings obtained during imaging: 111In excretion (100-whole-body retention) was 7.8% +/- 4.9% in 5 normal scans, 10% +/- 5.9% in 17 (+) scans, 22.3% +/- 8% in 20 (++) scans and 57% +/- 16% in 8 ( ) scans. We conclude that imaging is more sensitive than whole-body retention and fecal excretion in the detection of disease, but for quantification, whole-body retention is an accurate reliable alternative to fecal excretion.

Adult↗
PubMed2005

[Development and application of a method for rapid quantification of recombinant ICOS-mIg fusion protein].

AIM: To set up a double antibody sandwich ELISA for rapid quantification of recombinant ICOS-mIg fusion protein and evaluate its sensitivity, specificity and linear detection range. METHODS: Recombinant ICOS-mIg fusion protein was prepared by molecular biology technique. Sheep anti-mouse IgG and horse anti-mouse IgG-HRP conjugate were selected as coating antibody and detection antibody, respectively for sandwich ELISA by pair-matching experiments. A standard curve for determining the recombinant ICOS-mIg fusion protein was plotted and applied for quantification of ICOS-mIg fusion protein. RESULTS: A sandwich ELISA for quantification of the recombinant ICOS-mIg fusion protein was developed. Its linear detection was 7.8 to 500 microg/L(R(2)=0.9911, P<0.0001). CONCLUSION: The recombinant ICOS-mIg fusion protein can be determined by this sandwich ELISA rapidly and precisely.

Animals↗
PubMed2005

Comparison of manual and automated quantification methods of 123I-ADAM.

UNLABELLED: 123I-ADAM is a novel radioligand for imaging of the brain serotonin transporters (SERTs). Traditionally, the analysis of brain receptor studies has been based on observer-dependent manual region of interest definitions and visual interpretation. Our aim was to create a template for automated image registrations and volume of interest (VOI) quantifications and to show that an automated quantification method of 123I-ADAM is more repeatable than the manual method. PATIENTS, METHODS: A template and a predefined VOI map was created from 123I-ADAM scans done for healthy volunteers (n = 15). Scans of another group of healthy persons (HS, n = 12) and patients with bulimia nervosa (BN, n = 10) were automatically fitted to the template and specific binding ratios (SBRs) were calculated by using the VOI map. Manual VOI definitions were done for the HS and BN groups by both one and two observers. The repeatability of the automated method was evaluated by using the BN group. RESULTS: For the manual method, the interobserver coefficient of repeatability was 0.61 for the HS group and 1.00 for the BN group. The introobserver coefficient of repeatability for the BN group was 0.70. For the automated method, the coefficient of repeatability was 0.13 for SBRs in midbrain. CONCLUSION: An automated quantification gives valuable information in addition to visual interpretation decreasing also the total image handling time and giving clear advantages for research work. An automated method for analysing 123I-ADAM binding to the brain SERT gives repeatable results for fitting the studies to the template and for calculating SBRs, and could therefore replace manual methods.

Automation↗
PubMed2005

Automated template quantification for DNA sequencing facilities.

The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n=198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/microL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by >or=20% from the requested concentration (500 ng/microL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/microL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown.

Plasmids↗
PubMedDate not supplied

[Quantification of caffeine from energy drinks with mass spectrometry method (LC/MS)].

A high performance liquid chromatography coupled with mass spectrometry method (LC/MS) for quantification of caffeine from energy drinks was elaborated. It was utilised a Zorbax SB-C18, 100 mm x 3.0 mm i.d., 3.5 microm column with a mobile phase containing methanol/ solution 0.2% formic acid in water. Detection and quantification of caffeine was based on monitoring the protonated molecular ion abundance (m/z 194.9). The quantification was made using the external standard method. The calibration curve were made on range 0.26-26 microg/ml for caffeine with a correlation coefficient greater than 0.995. There were analysed 12 energy drinks with a caffeine content between 13 and 34 mg/100 ml.

Beverages↗
PubMed2006

Quantification of cortisol, cortisol immunoreactive metabolites, and immunoglobulin A in serum, saliva, urine, and feces for noninvasive assessment of stress in reindeer.

This study was designed to develop reliable methods for quantification of cortisol and cortisol immunoreactive metabolites (C-CIM) and immunoglobulin A (IgA) in reindeer serum, saliva, urine, and feces as tools for the objective noninvasive assessment of well-being and immunocompetence in reindeer. Although C-CIM was readily quantifiable by radioimmunoassay in serum, urine, and feces, the levels in saliva samples were low, rendering quantification unreliable. Whereas IgA concentrations were high in feces samples, they were much lower, albeit quantifiable, in serum and urine; the levels in saliva samples were too low for quantification with the use of an enzyme-linked immunosorbent assay that we developed. Further studies are in progress to validate the usefulness of fecal levels of C-CIM and IgA in the assessment of welfare in reindeer.

Acute Disease↗
PubMed2006

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification.

AIM: The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA. METHODS: Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 10(2) to 10(5) copies of target pCMV-435 plasmid with 10(4) copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 10(4) copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve. RESULTS: Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia. CONCLUSIONS: The QC-PCR assay could detect from 10(2) to over 10(7) copies of HCMV DNA with a range of linearity between 10(2) and 10(5) genomes.

Adult↗
PubMed2006

[Relative quantification of cytokeratin 19 transcription in oral squamous cell carcinoma tissues by fluorescent quantitive real-time RT-PCR].

OBJECTIVE: To investigate the relative quantification of cytokeratin 19 transcription in oral squamous cell carcinoma tissues by fluorescent quantitive real-time reverse transcription-polymerase chain reaction ((RT-PCR). METHODS: CK19mRNA level was detected by fluorescent quantitive real-time RT-PCR in cancerous and para-cancerous tissues from 31 oral squamous cell carcinoma patients. According to the 2(-DeltaDeltaCt) equation, the relative quantification fold of CK19mRNA level was calculated in cancerous tissues compared with para-cancerous tissues. RESULTS: CK19mRNA levels in cancerous tissues were 2.21 folds higher than those in para-cancerous tissues, and the amplicon was specific. CK19mRNA level in cancerous tissue correlated significantly with pathological differentiation degree, the poorer the differentiation was, the higher the CK19mRNA level became. CONCLUSIONS: Fluorescent quantitive real-time RT-PCR is accurate and reliable in the detection of relative quantification of CK19 transcription in oral squamous cell carcinoma tissues.

Adult↗
PubMed1991

Quantification of valvular regurgitation by Doppler echocardiography.

One of the widest uses of color Doppler echocardiography is for the quantification of valvular regurgitation. In this article we review the physics and instrumentation factors related to the most commonly applied method, that of planimetering areas of regurgitant jet spray within the receiving chamber, and relate features such as instrument gain, pulse repetition frequency, and important physical parameters such as receiving chamber compliance and driving pressure to the application of this method for precordial as well as transesophageal echocardiography. New approaches to the quantification of valvular insufficiency, such as measurement of the momentum of the jet and study of the acceleration flow in the proximal flow convergence region within the chamber of origin of an insufficiency jet and proximal to the orifice, are described and preliminary results given for a new method of quantification of volume flow across regurgitant orifices.

Compliance↗
PubMed1991

Direct quantification of HIV-1 infectivity for monocyte--macrophages using an infectious focus assay.

Monocyte--macrophages are important target cells and reservoirs for HIV. The existing methods for the quantification of infectious virus in HIV stocks are not totally satisfactory for use with macrophage cultures. We have developed an infectious focus assay for the direct quantification of virions infectious for human peripheral blood monocyte-derived macrophages adhering to plastic microtitre plates. The combination of an HIV-1 p24-antigen-specific monoclonal antibody and a beta-galactosidase-linked second antibody resulted in a sensitive and very specific assay. With 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as substrate, the assay proved to be as sensitive as p24 antigen quantification in culture supernatants.

Gene Products, gag↗