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PubMed2000

Absolute renal blood flow quantification by dynamic MRI and Gd-DTPA.

The aim of this study was to demonstrate the feasibility of the absolute renal blood flow quantification using MRI and injection of contrast media. Using a T1-weighted fast gradient sequence following an intravenous bolus injection of Gd-DTPA, dynamic images of the kidney were obtained in patients with well-functioning native kidneys (n = 7) or transplant (n = 9), with significant renal artery stenosis (n = 4) and with renal failure (n = 7). After signal intensity calibration, the absolute renal perfusion was equal to the wash-in slope of the renal transit curve divided by the contrast medium concentration at the peak of the bolus in the aorta. The cortical blood flow was 2.54+/-1.16 ml/min per gram in well-functioning kidneys decreasing to 1.09+/-0.75 ml/min per gram in case of renal artery stenosis (p = 0.04) and to 0.51+/-0.34 ml/min per gram in case of renal failure (P<0.001). These measurements were in agreement with previous results obtained by other methods. A standard MRI imaging sequence and a simple model can provide realistic quantitative data on renal perfusion. This work justifies further studies to compare this model with a gold standard for renal blood flow measurements.

Adult↗

PubMed2003

Noninvasive measurement of absolute renal perfusion by contrast medium-enhanced magnetic resonance imaging.

OBJECTIVE: The aim of this study was to validate the quantification of absolute renal perfusion (RP) determined by dynamic magnetic resonance imaging (MRI) and contrast media using an experimental model in the rabbit and a transit-timed ultrasound flow probe around the left renal artery as comparison. MATERIAL AND METHODS: An MR-compatible ultrasonic time-of-flight flow-probe was placed around the left renal artery in 9 New Zealand white rabbits. Absolute RP in basal state, after mechanical renal artery stenosis, intravenous dopamine, angiotensin II, or colloid infusion was measured using dynamic MRI and intravenous injection of gadoteridol. The results were correlated to the renal artery flow measured inside the magnet with the transit-timed flow-probe. For the signal intensity concentration conversion, we applied different calibrations according to various velocities measured in the aorta by a phase contrast sequence to correct for inflow effect. MRI-derived RP (in mL/min) was calculated by the maximum upslope method, where RP/volume was defined as the ratio of the cortex contrast enhancement slope over the maximum of the arterial input function determined in the aorta. RESULTS: Reproducible arterial and renal transit curve with excellent contrast to noise ratio were obtained. The MRI derived perfusion was systematically underestimated by comparison to the ultrasonic transit-timed flow-probe but was linearly correlated with these measures (r = 0.80, P < 0.001). CONCLUSIONS: Using a flow-sensitive calibration, an accurate arterial input function can be measured from the blood MR signal and used in a realistic model to assess the RP. There was a good correlation between the MR-derived RP and the renal artery blood flow measured by the flow-meter. This experimental study validates absolute RP quantification by MRI and contrast media injection and justifies further clinical studies.

Animals↗

PubMed1989

In vivo assessment of a digital angiographic method to measure absolute coronary artery diameters.

Several techniques exist for the quantification of absolute coronary artery diameters using radiologic methods. An in vivo assessment of a quantitative technique based on direct digitally acquired images was performed by imaging inflated angioplasty balloons (n = 25), balloon catheter shafts (n = 16) and coronary guidewires (n = 20) at the time of coronary angioplasty. After this, the actual size of the objects was determined with a micrometer. Diameters measured by the quantitative digital method had an excellent correlation with the actual diameters (digital diameter = 0.80 [actual diameter] + 0.32; n = 61; r = 0.97; standard error of the estimate = 0.26 mm; p less than 0.001). Moreover, the correlation between interobserver and intraobserver measurements was excellent (r = 0.99 for both, standard error of the estimate = 0.16 mm and 0.09 mm, respectively). However, there was a consistent error present that was related to the size of the object measured. Objects less than 0.5 mm were consistently overestimated and objects greater than 1 mm were usually underestimated by the digital technique, although the actual magnitude of the error was small. Objects less than 0.5 mm in diameter were overestimated by 0.41 +/- 0.11 mm and objects greater than 1 mm were underestimated by 0.23 +/- 0.19 mm. Based on an analysis of the error present, correction algorithms were formulated and tested prospectively using an additional 29 object measurements. This resulted in an improvement in the quantification of the diameters with a smaller magnitude of error. This in vivo assessment suggests that the rapid online assessment of absolute coronary artery diameters is possible, but also demonstrates important errors inherent in this method.

Algorithms↗

PubMed2006

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.

Algorithms↗

PubMed2006

Overview of real-time RT-PCR strategies for quantification of gene rearrangements in the myeloid malignancies.

In acute myeloid leukemia (AML), molecular diagnosis for the optimal management of patients and for minimal residual disease (MRD) monitoring is of extreme importance. Cumulative data suggest that quantitative monitoring or MRD in AML with fusion transcripts corresponding to 5(I;21), inv(16), and t(15;17) is useful in distinguishing patients at high risk of relapse from those in durable remission. Real-time quantitative polymerase chain reaction (RQ-PCR) is by far the most sensitive assay in the context of MRD detection. We present herein an overview of the principles of RQ-PCR encompassing both the chemistries (double-stranded DNA detection or specific fragment detection) and the instruments. The absolute and relative quantification and the most commonly used methods for calculation of MRD results in absolute quantification are also described.

Computer Systems↗

PubMed2005

Use of phased array coils for a determination of absolute metabolite concentrations.

This work describes the use of phased array coils for a quantification of absolute metabolite concentrations. The method is demonstrated for single-voxel localized proton MRS of human brain with an eight-element receive-only head coil. It is based on the transmitter reference amplitude of the body coil used for RF transmission. A relative sensitivity of every element of the phased array coil is derived from a combination of two reference scans without water suppression that correspond to either the body coil in transmit-receive mode or the phased array coil in conjunction with body coil excitation. Experimental results were obtained at 2.9 T for both phantoms and 12 human subjects in different locations of gray and white matter. The data demonstrate that the procedure is technically robust and without a penalty in measuring time. Moreover, it takes full advantage of the signal-to-noise gain for quantitative proton MRS and may be extended to other phased array coils without the need for a recalibration.

Adult↗

PubMed2005

Quantification of HIV GAG RNA using real time reverse transcriptase PCR.

Quantification of HIV-1 is important to quantify risk for disease progression as well as for acquiring infection associated with drug abuse. Prior quantification methods include immune and enzymatic procedures, e.g., quantifying HIV-1 p24 protein by ELISA and the Reverse Transcriptase by enzymatic assay. Improved quantification of HIV-1 RNA and cDNA was established using PCR. This paper describes a real-time PCR technique using the Applied Biosystems 5700 Sequence Detection System and Taqman reverse transcriptase PCR. We initially standardized the PCR method using ribosomal-RNA to obtain relative quantification. Pure gag RNA was used for standard curves, controls, and to obtain absolute RNA quantification. Pure HIV gag RNA was produced by T7-directed transcription of the plasmid pWISP98-85. Detailed statistical analyses describe using absolute standard curves, and intraassay and interassay coefficients of variation to validate the methods. The presented method is highly reproducible and the assay's performance is comparable to prior assays. The assay is validated with an 8-log range down to 80 copies.

DNA-Directed RNA Polymerases↗

PubMed1999

Concentrations of human cardiac phosphorus metabolites determined by SLOOP 31P NMR spectroscopy.

Human cardiac 31P nuclear magnetic resonance (NMR) spectra are usually quantified in relative terms, i.e., the ratio of metabolite signals is calculated. If 31P NMR spectroscopy of the heart is to emerge as a clinically relevant diagnostic modality, reliable quantification of absolute concentrations of 31P metabolites is required. We applied spectral localization with optimal point spread function (SLOOP) 31P NMR spectroscopy to measure absolute concentrations of phosphocreatine (PCr) and adenosine triphosphate (ATP) in human myocardium. The accuracy of the quantification was first validated in a phantom study. Seven healthy volunteers (aged 19-29 years) were then examined at 1.5 T using a nominal spatial resolution of 25 mL. SLOOP allowed us to obtain localized spectra from compartments anatomically matched to the left ventricular wall. The a priori knowledge of the anatomical structure was obtained from 1H images. The spatially varying effects of saturation, off-resonance, and sensitivity were considered during the reconstruction process. Metabolites were quantified with reference to an external 31P standard. Concentrations of 9.0 +/- 1.2 and 5.3 +/- 1.2 mmol/kg wet wt (mean +/- SD, n = 9) were determined for PCr and ATP in normal heart, respectively. The influence of nuclear Overhauser enhancement on metabolite quantification is discussed.

Adenosine Triphosphate↗

PubMed2005

Prospective study of one million deaths in India: rationale, design, and validation results.

BACKGROUND: Over 75% of the annual estimated 9.5 million deaths in India occur in the home, and the large majority of these do not have a certified cause. India and other developing countries urgently need reliable quantification of the causes of death. They also need better epidemiological evidence about the relevance of physical (such as blood pressure and obesity), behavioral (such as smoking, alcohol, HIV-1 risk taking, and immunization history), and biological (such as blood lipids and gene polymorphisms) measurements to the development of disease in individuals or disease rates in populations. We report here on the rationale, design, and implementation of the world's largest prospective study of the causes and correlates of mortality. METHODS AND FINDINGS: We will monitor nearly 14 million people in 2.4 million nationally representative Indian households (6.3 million people in 1.1 million households in the 1998-2003 sample frame and 7.6 million people in 1.3 million households in the 2004-2014 sample frame) for vital status and, if dead, the causes of death through a well-validated verbal autopsy (VA) instrument. About 300,000 deaths from 1998-2003 and some 700,000 deaths from 2004-2014 are expected; of these about 850,000 will be coded by two physicians to provide causes of death by gender, age, socioeconomic status, and geographical region. Pilot studies will evaluate the addition of physical and biological measurements, specifically dried blood spots. Preliminary results from over 35,000 deaths suggest that VA can ascertain the leading causes of death, reduce the misclassification of causes, and derive the probable underlying cause of death when it has not been reported. VA yields broad classification of the underlying causes in about 90% of deaths before age 70. In old age, however, the proportion of classifiable deaths is lower. By tracking underlying demographic denominators, the study permits quantification of absolute mortality rates. Household case-control, proportional mortality, and nested case-control methods permit quantification of risk factors. CONCLUSIONS: This study will reliably document not only the underlying cause of child and adult deaths but also key risk factors (behavioral, physical, environmental, and eventually, genetic). It offers a globally replicable model for reliably estimating cause-specific mortality using VA and strengthens India's flagship mortality monitoring system. Despite the misclassification that is still expected, the new cause-of-death data will be substantially better than that available previously.

Adult↗

PubMed1993

Automatic quantitative evaluation of autoradiographic band films by computerized image analysis.

The present paper describes a new image processing method for automatic quantitative analysis of autoradiographic band films. It was developed in a specific image analysis environment (IBAS 2.0), but the algorithms and methods can be utilized elsewhere. The program is easy to use and presents some particularly useful features for evaluation of autoradiographic band films, such as the choice of whole film or single lane background determination; the possibility of evaluating bands with film scratch artifacts and the quantification in absolute terms or relative to reference values. The method was tested by comparison with laser-scanner densitometric quantifications of the same autoradiograms. The results show the full compatibility of the two methods and demonstrate the reliability and sensitivity of image analysis. The method can be used not only to evaluate autoradiographic band films, but to analyze any type of signal bands on other materials (e.g. electrophoresis gel, chromatographic paper, etc.).

Autoradiography↗

PubMed2026

Estimation of chloroplast macromolecular complex copy numbers and subunit stoichiometries during the Chlamydomonas reinhardtii cell cycle.

An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg cell-1 of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis, and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 × 106 molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 × 106 complexes per cell. PSI averages 1.75 × 106 complexes per cell and cytochrome b6f averages 0.77 × 106 complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artifacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Chlamydomonas reinhardtii↗

PubMed2005

Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards.

An important challenge for proteomics is to be able to compare absolute protein levels across biological samples. Here we introduce an approach based on the use of culture-derived isotope tags (CDITs) for quantitative tissue proteome analysis. We cultured Neuro2A cells in a stable isotope-enriched medium and mixed them with mouse brain samples to serve as internal standards. Using CDITs, we identified and quantified a total of 1,000 proteins, 97-98% of which were expressed in both mouse whole brain and Neuro2A cells. CDITs also allow comprehensive and absolute protein quantification. Synthetic unlabeled peptides were used to quantify the corresponding proteins labeled with stable isotopes in Neuro2A cells, and the results were used to obtain the absolute amounts of 103 proteins in mouse whole brain. The expression levels correlated well with those in Neuro2A cells. Thus, the use of CDITs allows both relative and absolute quantitative proteome studies.

Animals↗

PubMed2002

Use of positron emission tomography for the assessment of skeletal muscle glucose metabolism.

Positron emission tomography (PET) is a unique tool for studying regional skeletal muscle glucose metabolism and blood flow in vivo. The application of PET in the assessment of skeletal muscle glucose metabolism depends on recent improvements in instrumentation, data analysis, and production of (18)F-fluorodeoxyglucose (FDG) and (15)O water. The data presented support the validity of the (15)O water model to measure blood flow and the FDG model for the determination of glucose uptake and glucose kinetic constants (influx, efflux, and phosphorylation) in skeletal muscle. However, quantification of absolute glucose transport and backflux rates should be applied with caution because those calculations are based on unproven assumptions such as validity of the lumped constant for these individual processes and constancy of the free and accessible intracellular glucose pool. It is evident that quantification of glucose fluxes using the triple tracer technology generates conflicting data that violate assumptions inherent in triple tracer or PET modeling. Further FDG-PET studies will have to solve those problems to provide more insight into the regulatory processes of glucose transport and phosphorylation of different insulin-resistant disease states. Promising new areas of PET research will include not only detailed study of glucose kinetics but also the measurement of muscle protein synthesis in vivo, which is of interest in a variety of conditions.

Glucose↗

PubMed1992

Determination of absolute phosphate metabolite concentrations in RIF-1 tumors in vivo by 31P-1H-2H NMR spectroscopy using water as an internal intensity reference.

The absolute metabolite quantification method of Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] in which the tissue water proton signal is used as an internal intensity standard and its more recent variation in which NMR peak intensities are referenced to that of the natural abundance deuterium signal of water [Li et al., SMRM Abstr. 2, 825 (1988); Song et al., Magn. Reson. Med. 25, 45 (1992) have been implemented to obtain absolute phosphate metabolite concentrations in subcutaneous RIF-1 tumors during untreated growth and following treatment with 5-fluorouracil. The equivalence of these two hydrogen isotopes as intensity standards and the validity of their use in the determination of absolute metabolite concentrations in vivo by NMR has been demonstrated. On matched in vivo and extract tumor samples (n = 5), excellent agreement has been obtained between nucleoside triphosphate concentrations determined by NMR and those derived by HPLC analysis for the control tumors. Following 3 days of untreated growth, absolute concentrations of phosphate metabolites in RIF-1 tumors (n = 10) decreased significantly, except for the Pi concentration which did not vary. For the treated tumors (n = 10) there were no changes in metabolite concentrations except for a decrease in the PCr and, possibly, Pi concentrations. The PCr/Pi ratio in the latter tumors did not change. These observations suggest that changes in absolute metabolite concentrations may be more sensitive indices of response to therapy than changes in metabolite peak amplitude ratios, a parameter commonly used to express in vivo NMR data.

Animals↗

PubMed1991

Absolute cross-sectional area measurements in quantitative coronary arteriography by dual-energy DSA.

Recent studies have emphasized the limitations of conventional coronary angiography. These limitations include the lack of correlation between the severity of coronary stenosis as estimated from coronary angiograms and the actual severity of stenotic lesions measured in postmortem hearts. As a result, attempts have been made to quantitate luminal dimension more precisely. The application of quantitative digital subtraction angiography (DSA) in the assessment of coronary artery lesion dimension has been limited by cardiac and respiratory motion artifacts. We have reported previously on a motion-immune dual-energy (DE) cardiac mode in which kVp and filtration are switched at 30 Hz. To assess the potential advantages of a videodensitometric technique for quantification of absolute vessel cross-sectional area (CSA), three different quantitative coronary arteriography (QCA) algorithms were compared. The three algorithms under comparison were a videodensitometric (V) algorithm, which does not require any geometric assumption for absolute vessel CSA measurement, and videodensitometric (VC) and edge detection (ED) algorithms, which do require the assumption of circular cross-section for CSA measurements. A cylindrical vessel phantom (0.5-4.75 mm in diameter) and a crescentic vessel phantom, producing 25% to 90% area stenosis, were imaged over the chest of a humanoid phantom. The low- and high-energy images were corrected for scatter and veiling glare before energy subtraction. For CSA measurements in crescentic vessel phantoms, the V algorithm produced significantly improved results (slope = 0.87, intercept = 0.51 mm2, r = .95) when compared to the VC (slope = 1.05, intercept = 4.19 mm2, r = .75) and the ED (slope = 1.57, intercept = 5.21 mm2, r = .60) algorithms.

Algorithms↗

PubMed1991

New grading system of cerebral gliomas using positron emission tomography with F-18 fluorodeoxyglucose.

Positron emission tomography (PET) using fluorine-18 (18F)-fluorodeoxyglucose (FDG) has been reported to be a powerful diagnostic and prognostic tool in patients with primary brain tumors. This study was undertaken to compare the prognostic value of: (1) visual grading of [18]FDG uptake in the tumor, (2) the absolute glucose metabolic rate of the tumor (TMRglc), (3) the ratio of glucose metabolism between the tumor and whole brain (T/WB) and (4) between the tumor and contralateral cerebellum (T/CBL). Each of these four parameters was correlated with the survival time in 20 patients with malignant cerebral gliomas. Excellent correlation was obtained with visual grading and reasonably good correlation was obtained with T/WB or T/CBL, but TMRglc alone was only a fair prognostic indicator. Thus, visual grading provides a qualitative analysis and T/WB provides a semi-quantitative analysis neither of which requires arterial blood sampling for quantification of absolute metabolic rates for glucose.

Brain Neoplasms↗

PubMed2001

On the theoretical basis of perfusion measurements by dynamic susceptibility contrast MRI.

A quantitative analysis was undertaken to calibrate the perfusion quantification technique based on tracking the first pass of a bolus of a blood pool contrast agent. A complete simulation of the bolus passage, of the associated changes in the T2 and T2* signals, and of the data processing was performed using the tracer dilution theory, an analytical theory of the MR signal from living tissues and numerical simulations. The noise was excluded in the simulation in order to analyze the ultimate accuracy of the method. It is demonstrated that the relationship between the contrast agent concentration and the associated changes in the transverse relaxation rate shows essentially different forms in studied tissue and in the reference artery. This effect results in systematic deviations of the measured blood flow, blood volume, and the residue function obtained with conventional processing from their true values. The error depends on the microvascular composition, the properties of the contrast agent, and the weights of the various compartments in the total signal. The results show that dynamic susceptibility contrast MRI can reach the goal of absolute perfusion quantification only with additional input from measurements of the microvascular architecture. Alternatively, the method can be used to provide such information if the perfusion is quantified by another modality.

Blood Volume↗

PubMed2001

[An approach for comparative quantification of myocardial blood flow (O-15-H2O-PET), perfusion (Tc-99m-tetrofosmin-SPECT) and metabolism (F-18-FDG-PET)].

AIM: In the present study a new approach has been developed for comparative quantification of absolute myocardial blood flow (MBF), myocardial perfusion, and myocardial metabolism in short-axis slices. METHODS: 42 patients with severe CAD, referred for myocardial viability diagnostics, were studied consecutively with 0-15-H2O PET (H2O-PET) (twice), Tc-99m-Tetrofosmin SPECT (TT-SPECT) and F-18-FDG PET (FDG-PET). All data sets were reconstructed using attenuation correction and reoriented into short axis slices. Each heart was divided into three representative slices (base, midventricular, apex) and 18 ROIs were defined on the FDG PET images and transferred to the corresponding H2O-PET and TT-SPECT slices. TT-SPECT and FDG-PET data were normalized to the ROI showing maximum perfusion. MBF was calculated for all left-ventricular ROIs using a single-compartment-model fitting the dynamic H2O-PET studies. Microsphere equivalent MBF (MBF_micr) was calculated by multiplying MBF and tissue-fraction, a parameter which was obtained by fitting the dynamic H2O-PET studies. To reduce influence of viability only well perfused areas (> 70% TT-SPECT) were used for comparative quantification. RESULTS: First and second mean global MBF values were 0.85 ml x min-1 x g-1 and 0.84 ml x min-1 x g-1, respectively, with a repeatability coefficient of 0.30 ml x min-1 x g-1. After sectorization mean MBF_micr was between 0.58 ml x min-1 x ml-1 and 0.68 ml x min-1 x ml-1 in well perfused areas. Corresponding TT-SPECT values ranged from 83% to 91%, and FDG-PET values from 91% to 103%. All procedures yielded higher values for the lateral than the septal regions. CONCLUSION: Comparative quantification of MBF, MBF_micr, TT-SPECT perfusion and FDG-PET metabolism can be done with the introduced method in short axis slices. The obtained values agree well with experimentally validated values of MBF and MBF_micr.

Coronary Circulation↗