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PubMed1995

Automated discrimination and quantification of idiopathic pulmonary fibrosis from normal lung parenchyma using generalized fractal dimensions in high-resolution computed tomography images.

RATIONALE AND OBJECTIVES: We computed generalized fractal dimensions for high-resolution computed tomography (HRCT) images to investigate their value in the discrimination and quantification of idiopathic pulmonary fibrosis (IPF) from normal lung parenchyma. METHODS: A probability distribution that was based on the pixel value in each image was used to compute capacity, information, and higher fractal dimensions for a series of 52 HRCT slices obtained from four patients. Qualitative classification of normal, mild, moderate, and severe IPF cases was achieved by computing the following parameter: DD = D0 - 2D1 + D2, where D0, D1, and D2 represents the capacity, information, and pair correlation dimensions, respectively. A multiple linear regression analysis using morphometric quantification for the set of 52 slices was tested for all possible combinations of the parameters D0, D1, D2, and D3. The generalizability of the model was tested by predicting the extent of IPF for each patient from a regression model computed with the remaining slices in the database. RESULTS: The best regression results were obtained using the independent parameters D1 and D2 to quantify the extent of diseased lung parenchyma. The technique was tested with 48 slices from 12 new patients. The results indicated that the extent of IPF could be predicted within the confidence limits given by the regression analysis. CONCLUSION: The extent of IPF can be predicted well within the 90% confidence interval given by the model. The width of the confidence interval decreases as the number of slices used in the linear regression model increases. This operator-independent quantitative technique may be useful in the follow-up of patients with IPF.

Confidence Intervals↗
PubMed2003

Flow encoded NMR spectroscopy for quantification of metabolite flow in intact plants.

An NMR flow quantification technique applicable to metabolite flow in plants is presented. It combines flow sensitive magnetization preparation with slice selective spectroscopy. Flow encoded NMR spectroscopy is described to quantify, for the first time, flow velocities of metabolites in plants non-invasively. Flow sensitivity is introduced by magnetization preparation based on a stimulated echo experiment, prior to slice selective spectroscopy. For flow quantification eight different flow-weighted spectra are collected. With this flow preparation very slow flow velocities down to 0.1mm/s can be detected and small amounts of flowing metabolites can be observed despite the large background signal of stationary and flowing water. Important sequence optimization steps include appropriate choice of experimental parameters used for flow encoding as well as complete balancing of eddy currents from the flow encoding gradients. The method was validated in phantom experiments and applied in vivo. Examples of quantitative flow measurements of water and metabolites in phantoms and plants are provided to demonstrate the reliability and the performance of flow encoded spectroscopy.

Magnetic Resonance Spectroscopy↗
PubMed2000

PCR-based quantification of Pneumocystis carinii in in vitro systems.

In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant.

Animals↗
PubMed2002

Diffusion-enhanced visualization and quantification of vascular anomalies in three-dimensional rotational angiography: results of an in-vitro evaluation.

Three-dimensional rotational angiography (3DRA) is a new and promising technique for obtaining high-resolution isotropic 3D images of vascular structures. However, due to the relatively high noise level and the presence of other background structures in clinical 3DRA images, noise reduction is inevitable. In this paper, we evaluate a number of linear and nonlinear diffusion techniques for this purpose. Specifically, we analyze the effects of these techniques on the threshold-based visualization and quantification of vascular anomalies in 3DRA images. The results of in-vitro experiments indicate that edge-enhancing anisotropic diffusion filtering is most suitable: the increase in the user-dependency of visualizations and quantifications is considerably less with this technique compared to linear filtering techniques, and it is better at reducing noise near edges than isotropic nonlinear diffusion. However, in view of the memory and computation-time requirements of this technique, the latter scheme may be considered a useful alternative.

Algorithms↗
PubMed1999

Correcting for toxic inhibition in quantification of genotoxic response in the umuC test.

An improved procedure for quantification of results from the umuC tests for genotoxicity is presented. The calculation method better separates toxic growth inhibition (cytotoxicity) from genotoxic effects than currently used methods and therefore, greatly extends the applicability of genotoxicity tests on environmental samples. The basic principle is to normalize the genotoxic response compensating for both decreasing biomass and growth rate reduction that results from cytotoxicity. The improved method and the currently used method was compared for umuC tests on the pure compounds: methylmethanesulfonate (MMS), N-methyl-N'-nitro-N-nitroguanidine (MNNG), sodium azide (NaN3), and 4-nitroquinoline-1-oxide (4-NQO). For compounds with no or low cytotoxicity, the two calculation methods gave practically identical results, while for highly cytotoxic compounds, the traditional method overestimated genotoxicity. umuC tests were also carried out on leachate polluted groundwater sampled downgradient of a landfill (Grindsted, Denmark). All polluted samples showed high cytotoxicity concomitant with high genotoxicity when the results were quantified in the traditional way. The new method showed that these results were in fact false positive, as the apparent genotoxicity was a result of cytotoxicity. Based on the mathematical analysis leading to the improved procedure for correction for cytotoxicity, it is suggested to alter the present test design of the umuC test in order to obtain well-defined exposure concentrations as well as mathematical consistency in the quantification of genotoxicity.

4-Nitroquinoline-1-oxide↗
PubMed2002

Application of real-time quantitative polymerase chain reaction to quantification of glutamate receptor gene expression in the vestibular brainstem and cerebellum.

Reverse transcription-polymerase chain reaction (RT-PCR) is a powerful tool to detect specific gene expression from a small amount of tissue, which is superior to the traditional RNA assays such as Northern blotting and in situ hybridization (ISH) in terms of sensitivity. However, conventional RT-PCR is not suitable for quantification due to its exponential nature. Recently, a real-time quantitative PCR method has been developed to overcome the weak points of RT-PCR, e.g. quantification. Here we describe the use of real-time quantitative PCR using a fluorescent TaqMan probe, to study the regional differences in expression of glutamate receptor subunit/subtype genes (NR1, NR2A, GluR2, KA2, mGluR1, mGluR7) in the central vestibular system including the vestibular nucleus complex, inferior olive and cerebellar flocculus. We found that real-time quantitative PCR yielded similar results to other techniques such as ISH but offered several advantages in terms of relative speed and ability to detect low levels of gene expression. We suggest that real-time quantitative PCR is a useful method to study gene expression for other neurotransmitter receptors in the vestibular brainstem and cerebellum, and is also expected to be more accurate to assess the changes in gene expression following any treatment.

Animals↗
PubMed1999

Quantification of neurotrophin-3 mRNA in the rat hippocampal subregions using the RT-PCR-based coamplification method.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) is a suitable method for determining the expression levels of rare mRNAs in small amounts of tissue. To compare the mRNA expression levels across specific brain regions, we adopted an RT-PCR method in which a target gene was coamplified with an endogenous internal standard gene in single reaction tubes. Use of the endogenous internal standard can control fluctuations in target quantification resulting from various factors, including tube-to-tube variation in amplification efficiency and variation in mRNA content among the total RNAs prepared from different tissues. In this study, we quantitatively determined the mRNA expression levels for NT-3, a member of the neurotrophin family of growth factors, in the hippocampal subregions: the entorhinal cortex, dentate gyrus and CA1. NT-3 gene was simultaneously coamplified with an endogenous internal standard gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT), in the same reaction tube. Using this RT-PCR coamplification method, we detected a regional difference in the NT-3 mRNA expression levels across the hippocampal subregions. Our method can serve as a useful quantification method to investigate molecular signaling cascades in a specific cortical region.

Animals↗
PubMed2001

Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

BACKGROUND: The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE: To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY: Viral load quantification of each patient's first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS: Retrospective evaluation of first quantitative values suggested that 82.4% (n=168/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33/54). CONCLUSION: The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

DNA, Viral↗
PubMed2002

Rapid detection and quantification of CMV DNA in urine using LightCycler-based real-time PCR.

A real-time quantitative PCR-hybridisation assay was developed for the detection of human cytomegalovirus DNA in clinical material. The assay is based on a LightCycler (LC) and provides both rapid results (<1 h) and quantification over a broad dynamic range (2 x 10(3)-5 x 10(8) CMV DNA copies/ml). Given that the assay showed a 3-fold increase in sensitivity compared to detection of early antigen fluorescent foci (DEAFF) testing of urine samples, we investigated the practicality of testing surveillance such specimens from immunocompromised patients at risk of CMV disease. Over a 12-month period, CMV DNA was detected in 81 (7%) of 1154 urine samples examined. A total of 28 patients tested positive; urine viral loads were higher in 13 infants being investigated for suspected congenital infection (median 1.6 x 10(5) copies/ml) compared with 15 transplant recipients (median 9 x 10(3) copies/ml). Urine samples could be tested directly without processing such that results were available in <1h. Real-time PCR provided information on the quantification of CMV DNA in urine and proved a reliable method for the surveillance of immunocompromised patients at risk of CMV disease. This approach should facilitate a better understanding of the epidemiology and natural history of CMV disease. Moreover, LC-based quantitative PCR is a potentially valuable tool for the management of CMV disease; assisting with the prompt initiation of treatment and assessing therapeutic response.

Cytomegalovirus↗
PubMed2005

Single monochrome real-time RT-PCR assay for identification, quantification, and breakpoint cluster region determination of t(9;22) transcripts.

t(9;22) generates the BCR-ABL fusion gene, the hallmark of chronic myeloid leukemia (CML) but also found in acute lymphoblastic leukemia (ALL). Multiple chimeric transcripts translate to proteins of 190 or 210 kd and, rarely, 230 kd. CML typically carries p210 BCR-ABL while ALL is most often associated with p190. Detection and quantification of these fusion transcripts is useful in clinical management. We have exploited the unique melting profiles of these transcripts to design a new, simple, and cost-effective assay based on monochrome multiplex real-time RT-PCR for identification and quantification of each of these transcripts (b3-a2, b2-a2, and e1-a2) without further manipulation. The sensitivity of this assay was 10(-4) for e1-a2 and 10(-5) for b3-a2/b2-a2, which is appropriate for detection of minimal residual disease (MRD). Inter- and intra-assay variation was minimal. We applied this assay to assess the distribution of p190 and p210 in 260 childhood ALL samples from India. BCR-ABL was detected in 19 (7.3%), including one T-ALL. Eight patients (3.1%) demonstrated mBCR-ABL (p190) and 11 (4.2%) had MBCR-ABL (p210). Transcript levels varied markedly (up to 3000-fold) but e1-a2 were generally expressed at higher levels than b3/b2-a2 (P = 0.05). This simple real-time multiplex assay can thus be easily applied to monitor patients with ALL as well as CML.

Adolescent↗
PubMed2005

Reliable transcript quantification by real-time reverse transcriptase-polymerase chain reaction in primary neuroblastoma using normalization to averaged expression levels of the control genes HPRT1 and SDHA.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) represents a sensitive and efficient technique to determine expression levels of target genes in multiple samples and is increasingly used in clinical oncology to evaluate the patient's outcome or to detect minimal residual disease. Normalization of raw data are required to obtain comparable results between different specimens and is usually achieved by correlating transcript abundances of target genes with those of a single control gene with putatively stable expression levels. In this study, expression stability of six supposed control genes was evaluated in 64 samples of primary neuroblastoma and HPRT1 and SDHA mRNA levels were shown to exhibit the least expression variability among the samples. Because application of more than one control gene may enhance reliability of real-time RT-PCR results, various normalization factors consisting of the geometrical mean of multiple control gene expression values were calculated and evaluated by mRNA quantification of 14 target genes. Comparison with transcript levels determined by oligonucleotide-array expression analysis revealed that target gene mRNA quantification became most consistent after normalization to averaged expression levels of HPRT1 and SDHA. This normalization factor was in addition demonstrated to be not associated with stage of disease or MYCN amplification status of the tumor. Thus, these data indicate that the geometrical mean of HPRT1 and SDHA transcript levels represents a suitable internal control for biological and clinical studies investigating differential gene expression in primary neuroblastoma by real-time RT-PCR.

Gene Expression↗
PubMed2002

Quantification of perphenazine in eurasian otter (Lutra lutra lutra) urine samples by gas chromatography-mass spectrometry.

Perphenazine enanthate has been used in wild animals as a tranquilizer during the period of adaptation to new environments to reduce stress, mortalities and injuries. A gas chromatographic procedure for the quantitative measurement of perphenazine in otter urine has been developed and validated. The method involved an enzymatic hydrolysis with beta-glucuronidase-arylsulfatase from Helix pomatia, followed by a solid-phase extraction with Bond Elut Certify cartridges. The resulting organic phase was evaporated, and the dry extract was derivatised with MSTFA to form the O-TMS derivative. The derivatised extracts were analysed by gas chromatography-mass spectrometry using SIM acquisition mode, measuring three diagnostic ions (m/z 246, 372 and 475). Another phenothiazine derivative, fluphenazine, was used as the internal standard (I.S.). Extraction recoveries for perphenazine and I.S. were 87.6 +/- 8.2% (n=4) and 106.7 +/- 13.4% (n=4), respectively. The calibration curves were linear in the range from 4 to 100 ng/ml (r2=0.99). The limits of detection and quantification were estimated as 1.2 and 3.5 ng/ml, respectively. Precision and accuracy obtained in intra-assay studies were in the ranges of 1.3-8.7 and 1.7-19.5%, respectively, using control samples containing 6, 16 and 60 ng/ml of perphenazine. In inter-assay experiments, precision ranged from 4.3 to 14.9% and accuracy from 3.1 to 11.8%. Examples of the application of the perphenazine quantification method in otter urines after administration of perphenazine enanthate are presented.

Animals↗
PubMed2002

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids.

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.

Amino Acids↗
PubMed2002

Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection.

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.

Chromatography, Liquid↗
PubMed2002

Simultaneous quantification of prostaglandins, isoprostane and thromboxane in cell-cultured medium using gas chromatography-mass spectrometry.

We have developed a simultaneous quantification method for prostaglandin (PG) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(4)]TXB(2) as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.

Calibration↗
PubMed2003

Measurement of paclitaxel in biological matrices: high-throughput liquid chromatographic-tandem mass spectrometric quantification of paclitaxel and metabolites in human and dog plasma.

A GLP-validated, sensitive and specific LC-MS-MS method for the quantification of paclitaxel and its 6-alpha- and 3'-p-hydroxy metabolites is presented. A 0.400 ml plasma aliquot is spiked with a (13)C(6)-labeled paclitaxel internal standard and extracted with 1 ml methyl-tert.-butyl ether. The ether is evaporated and the residue is reconstituted in 130 microl of 30% aqueous acetonitrile (ACN) containing 0.1% trifluoroacetic acid. Isocratic HPLC analysis is performed by injecting 50 microl of the reconstituted material onto a 50x2.1 mm C(18) column with an ACN-water-acetic acid (50:50:0.1) mobile phase at 200 microl/min flow. Detection is by positive ion electrospray followed by multiple reaction monitoring of the following transitions: paclitaxel (854>509 u), 6-alpha-hydroxy paclitaxel (870>525 u), 3'-p-hydroxy paclitaxel (870>509 u) and internal standard (860>509 u). Quantification is by peak area ratio against the 13C(6) internal standard. The method range is 0.117-117 nM (0.1-100 ng/ml) for paclitaxel and both metabolites using a 0.400 ml human or dog plasma sample. Analysis time per sample is less than 5 min.

Animals↗
PubMed2003

Capillary electrophoretic separation and quantification of flavone-O- and C-glycosides in Achillea setacea W. et K.

A simple method for the rapid separation and quantification of flavone-O- and C-glycosides in A. setacea W. et K. by capillary zone electrophoresis (CZE) with UV detection is described. Using 25 mM sodium borate with 20% (v/v) of methanol (pH 9.3) as running buffer sufficient separation of the analytes was achieved within 19 min. For the quantitative determination isorhamnetin-3-O-rutinoside was used as internal standard. The method was successfully applied to a rapid characterisation of the flavonoid complex and a precise quantification of the single and total amount of the flavonoids in different samples of A. setacea.

Achillea↗
PubMed2003

Divergence in urinary 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) levels from gas chromatography-tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF(2alpha). Possible methodological, mechanistic and clinical implications.

Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F(2)-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC-tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF(2alpha) (i.e. 15(S)-8-iso-PGF(2alpha)) has been shown by others to be highly selective and specific for this 8-iso-PGF(2alpha) isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF(2alpha) for subsequent quantification by GC-tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF(2alpha). 8-iso-PGF(2alpha) was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF(2alpha) was determined to be 291+/-102 pg/mg creatinine by method A and 141+/-41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF(2alpha) at 128+/-55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF(2alpha) is heterogenous, with 15(S)-8-iso-PGF(2alpha) contributing by approximately 50%. PGF(2alpha) and other 8-iso-PGF(2alpha) isomers including 15(R)-8-iso-PGF(2alpha) are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF(2alpha). We assume that ent-15(S)-8-iso-PGF(2alpha) is also contributing by approximately 50% to urinary 8-iso-PGF(2alpha). This finding may have methodological, mechanistic and clinical implications.

Adult↗