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PubMed1997

[Quantification of mitral regurgitation by cine-MRI. Comparison with transesophageal echocardiography in 45 patients].

Cine-MRI demonstrates the presence of mitral regurgitation by a signal void. This study was undertaken to assess the value of this method for quantification of mitral regurgitation in a population not excluding either patients with mitral valve prostheses or those with atrial fibrillation. The subjects included had a mean age of 59 years and were referred for transoesophageal echocardiography, either for assessment of valvular heart disease (38 patients), or for detection of atrial thrombosis before external electrical cardioversion (7 patients). Eight patients had mitral valve prostheses and 19 were in atrial fibrillation. Cine-MRI was performed within 12 days of the transoesophageal echocardiography study with a 1.5 tesla magnet, using a sequence of gradient echo in 3 parallel planes in the 4 chamber view. The regurgitation was quantified by MRI from the extension of the signal void in the left atrium. The transoesophageal Doppler echocardiographic criteria were the width of the regurgitant jet at its origin, the intensity of the regurgitation signal recorded by continuous wave Doppler and the extension of the jet within the left atrium. The quantification by degrees 1 to 4 did not differ by more than 1 degree between the 2 techniques in 43 of the 45 patients. Out of 4 left atrial thrombus detected by transoesophageal echocardiography, only 1 was visible by MRI. The authors concluded that quantification of mitral regurgitation by cine-MRI provides similar results to those obtained by transoesophageal echocardiography and that the correlation remains valid in cases of atrial fibrillation. However, Doppler echocardiography provides further valuable information for the clinician.

Aged↗

PubMed1998

[Quantification of left-to-right shunt in atrial septal defect using oximetry, isotopes, and Doppler echocardiography. Is there a method of reference?].

INTRODUCTION: Determination of pulmonary to systemic blood flow ratio (QP/QS) is considered to be important for the management of patients with atrial septal defect. The QP/QS provides information on shunt severity and is usually determined by three methods: oximetry, first-pass radionuclide angiocardiography and Doppler echocardiography. The aim of the present study was to assess the accuracy and concordance level of these three methods in QP/QS quantification in atrial septal defects. PATIENTS AND METHODS: Sixty-four adult atrial septal defects patients in whom QP/QS was determined by these three methods with a 6 month interval were studied. Nuclear and echocardiographic post-surgical studies were repeated in 36 patients. RESULTS: QP/QS values determined by the three techniques had a low correlation between them: oximetry (r = 0.52; SEE = 0.74); radionuclide angiocardiography (r = 0.40; SEE = 0.79) and Doppler echocardiography (r = 0.72; SEE = 0.57). Radionuclide angiocardiography underestimated QP/QS > 3 (-0.61 +/- 1.21; p < 0.01). Only in 33% of studies there concordance (differences < 0.5) among the three methods and in 58% between two methods. Right ventricular dilatation and tricuspid regurgitation influenced radionuclide accuracy. Nevertheless, the correlation between this technique and echocardiography was satisfactory when the 36 post-surgical were included (r = 0.75); both techniques agreed in the diagnosis of the two cases with residual post-surgical shunt. CONCLUSIONS: Inter-method disparity in QP/QS quantification is high and no method can be used as a gold standard; clinical decisions therefore based on QP/QS quantification by one technique alone are ill-advised.

Adult↗

PubMed1998

Quantification of eumelanin and pheomelanin: stereologic image analysis method.

OBJECTIVE: To develop an ultrastructural stereologic image analysis method allowing quantification of intracellular melanization. STUDY DESIGN: First, in the field of image analysis, a newly elaborated segmentation method, SEM2, was compared with a previously described method based on gray level histograms. Only SEM2 allows the segmentation of melanin in micrographs of poorly melanized melanocytes. Second, in the field of stereology, estimation of cell volume remains problematic in the case of mixed cell populations. This problem is approached by the comparison of stereologic alternatives and cytochemistry (L-3, 4-dihydroxyphenylalanine reaction) in epidermal melanocytes and melanoma cells from several in vitro experiments. The cytochemical approach was found to be the best choice. RESULTS: Concerning the quantification of eumelanin and pheomelanin, the alkali elution method, permitting the specific dissolution of pheomelanin on ultrathin sections, was validated in normal human follicular melanocytes. CONCLUSION: These results allow us to envisage the stereologic quantification of eumelanin and pheomelanin at the ultrastructural level. At present our method is undergoing evaluation by comparison with the present method of reference based on high-performance liquid chromatography.

Adolescent↗

PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗

PubMed2026

Validated UPLC-MS/MS quantification and intracellular PK-PD Modeling of periplocin-related cardiac glycosides in H/R-injured H9c2 cells.

Reliable intracellular quantification is essential for characterizing the target-site disposition and exposure-response relationships of bioactive natural products. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of periplocin and four related cardiac glycoside metabolites in H9c2 cell lysates. Acceptable linearity, precision, recovery, and stability were achieved for intracellular quantification. Cells were treated with each compound at 50 μM, and intracellular concentrations and cell viability were monitored over 48 h. In hypoxia/reoxygenation (H/R) -injured cells, the time to maximum intracellular concentration was shortened for all five compounds, indicating altered cellular disposition under injury conditions. Cell viability was improved by all compounds during the observation period. Pharmacokinetic-pharmacodynamic (PK-PD) integration was performed using a sigmoid Emax model, and acceptable model fits were obtained, with Akaike information criterion (AIC) values ranging from 79.22 to 130.46. Low apparent EC50 values were estimated under this single-dose design, whereas the estimated Ke0 values suggested delayed equilibration with the effect compartment. These findings indicate that sustained cytoprotective responses can be produced by periplocin and related metabolic markers in injured cardiomyocytes. This intracellular bioanalytical strategy provides a quantitative approach for linking cellular exposure to pharmacodynamic response and may support further evaluation of periplocin-related cardiac glycosides.

Tandem Mass Spectrometry↗

PubMed1979

A case for further quantification of the stapedius reflex.

Clinical stapedius reflex determinations presently include only threshold and decay measurements. Recent observations indicate that reflex latency, amplitude, and rise and relaxation times are sensitive to cochlear and brain stem disorders. Our investigations using a chronic animal model of an experimental acoustic tumor show that reflex amplitude, latency, and rise time are exquisitely sensitive to extrinsic pressure on the auditory nerve. Because of our observations and those of others, we suggest that further quantification of the reflex test will increase its sensitivity and reliability. Quantification can be achieved by an interface between an impedance bridge and a small computer to provide on-line averaged values for each reflex parameter.

Acoustic Impedance Tests↗

PubMed1998

Accuracy of computer-based quantification of aortic valve stenosis.

In patients with aortic valve stenosis, the quantification of stenosis is usually performed using fluid-filled catheters and a computerized calculation program. The aim of this study was to determine the accuracy of this technique in comparison to the manual planimetry of the area between the curves of a simultaneous registration, using a multitip micromanometer catheter. The study was performed in 19 patients, in whom left and right heart catheterization was warranted. Systolic left ventricular and aortic peak pressures were significantly overestimated using a fluid-filled catheter (206 +/- 35 vs. 199 +/- 37 mm Hg, P = 0.0003, and 148 +/- 18 vs. 143 +/- 21 mm Hg, P = 0.0052). However, peak-to-peak pressure gradients were identical comparing both techniques (58 +/- 31 vs. 56 +/- 32 mm Hg, r = 0.983). The mean pressure gradients and aortic valve areas based on simultaneous measurements of left ventricular and aortic pressures by micromanometer catheters were identical to the values determined by a computer-based program using fluid-filled catheters (54 +/- 21 vs. 52 +/- 21 mm Hg, r = 0.923, P < 0.05, and 0.75 +/- 0.25 vs. 0.77 +/- 0.25 cm2, r = 0.935). Thus, the conventional use of fluid-filled catheters and of a computerized calculation of aortic valve area is valid for quantification of aortic stenosis in patients with sinus rhythm and without significant aortic regurgitation.

Adult↗

PubMed1996

Molar quantification by flow cytometry of fatty acid binding to cells using dipyrrometheneboron difluoride derivatives.

Fatty acid analogs of a dipyrrometheneboron difluoride fluorophore (BDY-FA) have recently been developed. Relative to other fluorescent fatty acids, some of these have the advantages of excitation and emission spectra similar to those of fluorescein and of high quantum yield, which permits their use in conventional argon laser cytometry or microscopy. For the cytofluorimetric quantification of BDY-FA analogs, expressed as molecules bound per cell, we have compared the fluorescence of BDY-dodecanoic acid (BDY-C12) with that of fluorescein. Fluorescent beads with different amounts of bound fluorescein were used to calibrate a flow cytometer in order to correlate the fluorescence intensity with the number of fluorescein molecules per bead. In addition, starting from the basic equation defining the relationship between fluorescence and concentration, we have derived another equation which makes it possible to establish, for a given fluorescence, the relative molar concentration of both fluorochromes and, consequently, to express the fluorescence intensity emitted by the BDY-FA as the equivalent number of BDY-FA molecules. As an example of the potential application of this procedure, the time-course and concentration-dependent binding of BDY-C12 to quiescent and mitogen-activated human lymphocytes and to cultured human T-lymphoma cells have been studied. The method described is of general interest as it can also be applied to the flow cytometric or laser scanning microscopic quantification of other fluorescent dyes.

Boron Compounds↗

PubMed1998

Quantification of neurotoxicity and identification of cellular subsets in a three-dimensional brain model.

Imaging of cells in a large intact three-dimensional tissue remains difficult. Quantification and identification of cell damage in a mixed culture system has been limited by the inability of fluorescent probes to discriminate types of cellular death and penetrate tissue more that 100 microm thick. We have investigated several probes in combination with neural cell-specific antibodies to quantify cell damage in the presence of several toxins. Acridine orange and ethidium bromide were excellent for determination of cell viability, death by necrosis, or apoptosis in thick brain tissue aggregates. Calcein and ethidium homodimer were effective on live/ dead stains, and the Syto dyes 11 and 13 worked well for quantification of all cells in the brain aggregate model. By using these combinations of dyes in conjunction with confocal microscopy, we were able to quantify neural cell damage without disrupting the three-dimensional environment.

Apoptosis↗

PubMed2000

Comparison of DiOC(6)(3) uptake and annexin V labeling for quantification of apoptosis in leukemia cells and non-malignant T lymphocytes from children.

Early during apoptosis, there is a reduction in mitochondrial transmembrane potential (MTP) and externalization of phosphatidylserine (PS) in cell membrane prior to eventual cell death. Flow cytometric detection techniques targeting these changes, reduction of DiOC(6)(3) uptake upon the collapse of MTP and annexin V binding to PS have been successfully used to detect apoptotic cells. These methods have given comparable results when cell lines were used. We compared the two different techniques, DiOC(6)(3) uptake and Annexin V-propidium iodide co-labeling in the quantification of cytarabine, vincristine and daunorubicin induced apoptosis on three leukemia cell lines (HL-60, CEM, U937), and bone marrow blasts from 26 children with acute myeloid leukemia, 14 with T cell acute lymphoblastic leukemia. Anti-Fas-induced apoptosis in culture-grown peripheral blood T lymphocytes on 18 samples from 9 children with non-malignant conditions were also studied by these techniques. Our results showed that there is a correlation (P < 0. 05) between the apoptosis rates measured by these two techniques for drug-induced apoptosis in myeloid and lymphoid blasts, and for anti-Fas mAb-induced apoptosis in T lymphocytes. This data suggests that reduction of the MTP and PS externalization may be common to many apoptotic pathways and techniques targeting either of these changes may be used in quantification of apoptosis in different clinical samples.

Annexin A5↗

PubMed2000

Dopamine D(2) receptor quantification in extrastriatal brain regions using [(123)I]epidepride with bolus/infusion.

The iodinated benzamide epidepride, which shows a picomolar affinity binding to dopamine D(2) receptors, has been designed for in vivo studies using SPECT. The aim of the present study was to apply a steady-state condition by the bolus/infusion approach with [(123)I]epidepride for the quantification of striatal and extrastriatal dopamine D(2) receptors in humans. In this way the distribution volume of the tracer can be determined from a single SPECT image and one blood sample. Based on bolus experiments, an algorithm using conventional convolution arguments for prediction of the outcome of a bolus/infusion (B/I) experiment was applied. It was predicted that a B/I protocol with infusion of one-third of the initial bolus per hour would be appropriate. Steady-state conditions were attained in extrastriatal regions within 3-4 h but the infusion continued up to 7 h in order to minimize the significance of individual differences in plasma clearance and binding parameters. A steady-state condition, however, could not be attained in striatal brain regions using a B/I protocol of 20 h, even after 11 h. Under near steady-state conditions a striatal:cerebellar ratio of 23 was demonstrated. Epidepride has a unique signal-to-noise ratio compared to [(123)I]IBZM but present difficulties for steady-state measurements of striatal regions. The bolus/infusion approach is particularly feasible for quantification of the binding potential in extrastriatal regions.

Adult↗

PubMed1996

Evaluation of signal processing methods for the quantification of a multi-exponential signal: the glycogen 13C-1 NMR signal.

The 13C-1 NMR peak in proton-decoupled spectra of liver glycogen solution was quantitatively analyzed by three types of model-function fitting algorithms: iterative line-fitting in the frequency domain (MDCON); iterative least-squares fitting (VARPRO) in the time domain; and noniterative singular value decomposition-based analysis (HTLS), also in the time domain. Quantification results were compared with manual integration values. Performance of the algorithms was tested at different signal-to-noise ratios (S/N) of the glycogen C-1 peak. This was achieved by varying the number of scans summed prior to analysis. Since T2 relaxation in glycogen has been shown to be multiexponential [Overloop, K. et al. Magn. Reson. Med. 36, 45-51 (1996], the exact quantification of the C-1 glycogen signal requires a model function comprising a sum of Lorentzian components, each with a different broadening at the glycogen frequency. This paper focuses on the performances of the above methods to fit such a multicomponent resonance line. In the frequency domain, line fitting with two Lorentz lines gives good results at sufficiently high S/N. In the time domain, VARPRO performs better than HTLS because fixed values can be imposed to the linewidth of the components at the common C-1 frequency, thereby reducing convergence problems at low S/N.

Algorithms↗

PubMed1999

Early perfusion after controlled cortical impact in rats: quantification by arterial spin-labeled MRI and the influence of spin-lattice relaxation time heterogeneity.

Early posttraumatic cerebral hypoperfusion is implicated in the evolution of secondary damage after experimental and clinical traumatic brain injury (TBI). This is the first report of cerebral blood flow (CBF) measurement by continuous arterial spin-labeled magnetic resonance imaging (MRI) early after TBI in rats using the controlled cortical impact (CCI) model. CCI reduced CBF globally at approximately 3 hr (versus normal), with 85% and 49% reductions in a contused cortical region and contralateral cortex, respectively. In contrast, a prior MRI study from this laboratory showed at 24 hr post trauma a focal CBF reduction restricted to the injury site. In vivo spin-lattice relaxation time (T(1obs)), which is used in CBF quantification, was spatially heterogeneous early after CCI, a time when edema is developing in injured brain tissue. At 4.7 T, T(1obs) values are increased 29% in the contusion (versus normal), consequently reducing CBF quantification to a similar degree. MRI should facilitate coupling posttraumatic CBF with long-term functional outcome. Magn Reson Med 42:673-681, 1999.

Animals↗

PubMed1999

Effect of transit times on quantification of cerebral blood flow by the FAIR T(1)-difference approach.

The effect of finite transit times for the tagging bolus is known to be a significant error source for perfusion quantification using the flow-sensitive alternating inversion recovery (FAIR) technique. It is shown that, in the presence of transit times, both the slice-selective (SS) and nonselective (NS) inversion recovery experiments actually consist of an NS period followed by an SS period. This mixed process can be described using a newly defined time constant called the "switching time," which separates the two periods. Calculations predict that finite transit times always lead to decreased flow values in the signal-intensity-difference approach, but that the measured flows in the T(1)-difference approach may be decreased or increased. This theory well explains our recent experimental flow results on cat brain as a function of predelay. The results show the signal-intensity-difference method is superior over the T(1)-difference approach in terms of convenience and ease of quantification. Magn Reson Med 42:890-894, 1999.

Animals↗

PubMed1999

Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: influence of mismatches on quantification.

Lentiviruses are associated not only with immunodeficiency but also with malignancies. The mechanisms involved in tumorigenesis are still not fully understood. Cats infected with feline immunodeficiency virus (FIV) in the wild represent one model in which the role of viral load in the pathogenesis can be studied, since tumors, especially lymphomas, are quite often observed in cats infected with FIV. To be able to compare the viral load data among cats infected with different FIV isolates, the method used to obtain the viral load has to be unaffected by isolate-specific differences. This is especially true for the real-time polymerase chain reaction (PCR), a new method for viral load determination, since nucleotide sequence mismatches have been used for allelic discrimination with this method. To investigate the influence of these mismatches on PCR efficiency, we have used an FIV-specific real-time PCR and determined the influence of nucleotide sequence variation in several characterized FIV isolates as well as unknown isolates from naturally infected cats. We could demonstrate that minor mismatches, such as point mutations in the primer or the probe region, decrease overall PCR efficiency but do not abolish the quantification, in contrast to major mismatches of three or four nucleotides, which lead to complete inhibition of the real-time PCR detection. Based on these results, it will be possible to design real-time PCR systems allowing the quantification of a broad range of isolates, which is a prerequisite for the investigation of the impact of viral load in tumorigenesis.

Animals↗

PubMed2000

Hemoglobin A2 quantification by capillary zone electrophoresis.

Hemoglobin A2 (HbA2) comprises about 2.2% of the total hemoglobin in the erythrocytes. The separation and quantitation of this minor hemoglobin by capillary electrophoresis (CE) using an arginine Tris buffer is described. Some of the variables affecting the accuracy and precision of HbA2 quantification are investigated. Furthermore, the quantification of this hemoglobin by CE is compared to that of a microcolumn chromatography method. The CE method is better suited than the microcolumn method for measuring HbA2 in the sickle cell trait.

Electrophoresis, Capillary↗

PubMed2000

Electrospray and chemical ionization mass spectrometry of di-n-butyl sulfate. Unimolecular chemistry of its protonated form and quantification method by liquid chromatography/electrospray ionization tandem mass spectrometry

Di-n-butyl sulfate (DNBS) has been studied by electrospray (ESI) and chemical (CI) ionization mass spectrometry. The use of methanol as solvent in electrospray ionization allows observation of relatively abundant [DNBS + CH(3)OH + H](+) ions (m/z 243) which upon collision dissociate to [DNBS + H](+) ions (m/z 211). In both ESI and CI experiments, it is found that [DNBS + H](+) ions lead to m/z 113 daughter ions. The composition of this m/z 113 fragment ion and its mechanism of formation have been established by high resolution measurements and CID-MIKE experiments. An 'internal substitution' reaction involving an ion-neutral intermediate is proposed to explain the formation of a [C(8)H(17)](+) ion (m/z 113) by loss of a H(2)SO(4) molecule. Finally, a LC/ESI-MS/MS quantification method is proposed in which a detection limit of di-n-butyl sulfate in the ppm range is obtained. It is suggested that the quantification method might be extended to higher dialkyl sulfates. Copyright 2000 John Wiley & Sons, Ltd.

Journal Article↗

PubMed2001

Evaluation of performance of white blood cell reduction filters: an original flow cytometric method for detection and quantification of cell-derived membrane fragments.

BACKGROUND: Contamination of blood products by white blood cells leads to a risk of transmission of infectious agents, particularly abnormal prion protein, the probable causative agent of new-variant Creutzfeldt-Jakob disease. Blood product filtration could reduce this risk, but the filtration systems might generate potentially infectious membrane fragments. We developed an original flow cytometric method that allows the detection and quantification of membrane fragments in filtered products and the evaluation of the quantity of destroyed cells. METHODS: This method has four technical requirements: cytofluorometric acquisition of forward scatter parameters on a log scale, use of a fluorescent aliphatic reporter molecule (PKH26-GL) to identify membrane fragments, quantification with fluorescent beads, and the drawing up of a standard curve on the basis of cells destroyed by freezing/thawing to generate cell debris (i.e., quantity of membrane fragments measured versus quantity of destroyed cells). RESULTS AND CONCLUSIONS: This original method can be used to test new filtration devices and it allows optimization of the filtration process or comparison of different filtration systems. We tested the method with three commercial white cell removal filters. We demonstrated that it is possible to evaluate the filter quality, particularly the likelihood of fragment removal during the filtration process.

Blood Component Removal↗