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PubMed1986

Identification and quantification of a monoclonal IgM euglobulin: pitfalls and methodologic instruction.

In a patient with Waldenström's macroglobulinemia routine agarose gel electrophoresis and immunofixation disclosed an IgM-kappa M-component with beta-mobility, but failed to unveil a major monoclonal IgM-kappa euglobulin. This, however, was demonstrated when the procedures were performed in a buffer with NaCl-ion-strength of 0.1 M. Similarly, IgM quantification by routine nephelometry and electroimmunoassay led to dramatically inconsistent results. By a simple agarose gel electrophoresis in NaCl 0.1 M the IgM euglobulin was quantified proportional to the serum concentration of albumin. The identification and classification of an M-component, being exceedingly important diagnostically, may be missed, when solely routine electrophoresis and immunofixation is employed. As regards the quantification of M-components, these are not merely pathologic immunoglobulins nosologically. Also, biochemically they differ from normal, polyclonal immunoglobulins, resulting in immunological techniques being inapplicable for their quantification. For quantitative purposes, a simple, comparative agarose gel electrophoresis is described.

Aged↗

PubMed1987

[Tomoscintigraphy of the myocardium labelled with thallium-201. Value in the quantification and prognosis of myocardial infarction].

Myocardial thallium 201 tomography was performed in 112 patients admitted to hospital in the acute stage of a first myocardial infarction. Our quantification method for the myocardial lacuna, validated by animal studies, is described. The necrotic mass evaluated is compared with enzymatic (CPK) (r = 0.89) and angiographic (r = 0.78) quantitative assessments. Thallium 201 tomographic quantification has an unquestionable prognostic value. Thus, among patients whose infarcted mass represented more than 25% of the total myocardial mass, 71% died or developed severe heart failure. Conversely, 93% of patients with a small infarct (less than 20% of total myocardial mass) were asymptomatic on a 24-month follow-up. Myocardial thallium 201 tomography provides a reasonable quantification of the infarct. Its prognostic value seems to be satisfactory.

Adult↗

PubMedDate not supplied

The cytologic identification and quantification of testicular cell subtypes. Reproducibility and relation to histologic findings in the diagnosis of male infertility.

In testicular imprint smears from 100 infertile men (both testicles), stained using the Pappenheim and Papanicolaou methods, the cell forms (light and dark spermatogonia, primary and secondary spermatocytes, spermatids, spermatozoa and Sertoli cells) were identified and quantified by the counting of 500 consecutive cells. Identification of the cell types, which are described and illustrated, was consistent and reproducible; the advantages of the different stainings in their analysis is documented. The cell counts were tested for reproducibility and compared to the histologic diagnoses and sperm counts. Statistical analysis showed the highest reproducibility for cells frequently encountered in smears (0.99 for Sertoli cells and 0.98 for spermatozoa) and the lowest but still satisfactory reproducibility for rare or arbitrarily defined cell forms (0.71 for dark spermatogonia and 0.76 for secondary spermatocytes). The high reproducibility of the smear quantification permits the introduction of a number of indices, defining clinically useful relations between cell types that are indicative of various types of infertility. The data obtained by cytologic quantification showed reasonably good correlation with the histologic diagnoses of desquamation and focal fibrosis and excellent correlation with Sertoli cells only, arrested spermatogenesis and complete fibrosis. The cytologic quantification of testicular smears adds considerable information to the diagnosis of impaired fertility and should be instituted in properly equipped centers.

Cell Count↗

PubMed1995

Three-dimensional displays of left ventricular epicardial surface from standard cardiac SPECT perfusion quantification techniques.

UNLABELLED: Two methods for generating left ventricular epicardial surface from SPECT perfusion tomograms are described and validated. Both methods use the locations of the maximal reconstructed count values determined from a perfusion quantification procedure as a basis for generating surfaces. METHODS: The first method fits circular contours, which are perpendicular to the long-axis, to the points obtained from perfusion quantification. The second method applies median and linear filters to the points to remove noise but maintain the basic shape of the surface. Both models are validated against an automatic technique and against the user-traced surfaces of both the perfusion image and an MR image of the same patient. RESULTS: The median-filtered model was found to be closer to the standard surfaces than the circular model in all cases, and 85% of the points on the median-filtered surfaces were within one SPECT pixel length of the hand-traced MR surfaces. CONCLUSION: Accurate, three-dimensional left ventricular epicardial surfaces can be generated quickly and easily from already existing perfusion quantification software. The resulting images may be useful for realistic displays of ventricular size, shape and the three-dimensional distribution of perfusion.

Coronary Circulation↗

PubMed1995

[A nomogram of duplex ultrasound quantification of peripheral arterial stenoses. Studies of the cardiovascular model and in angiography patients].

BACKGROUND AND METHODS: Blood flow velocity measurements were performed with duplex ultrasound in vitro (flow phantom) and in 62 patients who underwent angiography due to peripheral vascular disease. RESULTS: Intrastenotic peak systolic velocity (PSV) divided by proximally recorded PSV (peak velocity ratio, PVR) exhibited a strong correlation with percent diameter reduction: r2 = 0.86; N = 106 stenoses. A PVR value > or = 2.4 indicated a more than 50% stenosis with a sensitivity of 87% and a specificity of 94%. Calculation of PVR may normalize for patient variation and allow noninvasive quantification of lumen narrowing with high sensitivity and specificity. The intraobserver variability (95% CI) of stenosis quantification using PVR values was 10%. A nomogram simplifies estimation of lumen narrowing after measurement of intrastenotic and proximal PSV values. CONCLUSION: Quantification of peripheral artery stenoses can be performed easily and noninvasively with duplex ultrasound using the peak velocity ratio (PVR).

Aged↗

PubMed1995

Quantification of the breakpoint cluster region rearrangement for clinical monitoring in Philadelphia chromosome-positive chronic myeloid leukemia.

The purpose of this report was to evaluate scintigraphy analysis of Southern blot hybridization as a method to quantify the breakpoint cluster region (BCR) rearrangement of Philadelphia chromosome (Ph)+ chronic myelogenous leukemia (CML). Cytogenetic and molecular studies performed simultaneously on 474 bone marrow and/or blood samples from 300 patients treated with alpha-interferon-based therapy were compared. Molecular results were expressed as the percentage of rearranged BCR bands versus the total scintigraphic signal. The percentage of Ph+ metaphases was calculated on 25 metaphases. The results of molecular studies obtained on both peripheral blood and bone marrow samples were identical. The rank correlation between the BCR quantification and the percentage of Ph positivity in 465 samples was excellent (r = .78). However, of 99 samples with a normal karyotype, 24% had a BCR rearrangement. Of 86 samples with no BCR rearrangement, 13% showed a Ph chromosome. Of 49 samples with partial cytogenetic remission (Ph+ metaphases, 1% to 34%), 23% had no BCR rearrangement. In samples with a minor or no cytogenetic response (Ph+ metaphases, > 34%), BCR analysis overestimated the degree of response in 73 of 326 samples (22%). Nevertheless, survival analysis by BCR quantification level showed statistically better outcome for patients in complete or partial molecular response (P < .01). Molecular quantification of BCR was useful in monitoring the course of Ph+ CML. This method, which can be used on peripheral blood, detected residual disease not shown by cytogenetic analysis and was prognostically relevant as a measure of disease suppression.

Adult↗

PubMed1995

Quantification of cystoid changes in diabetic maculopathy.

PURPOSE: In patients with diabetic macular edema and cysts, quantification of the extent of the cystoid formation has been difficult. This study was performed to introduce reliable measurements of cysts, the quantification of the extent, and its relation to visual acuity. METHODS: Fluorescein angiography generated with a scanning laser ophthalmoscope provided detailed recognition not only of the foveal microvasculature, but also of well-demarcated cystoid formations in the early phases. The sampling area included the central 2.5 degrees of the fovea. Using digital image analysis, two independent observers estimated the area covered by cysts, the number of cysts, and the foveal avascular zone (FAZ). RESULTS: Twenty-three subjects with diabetes and macular cysts were enrolled in the current study. The mean area of the cysts was 0.315 +/- 0.241 mm2 (0.05 mm2 to 0.9 mm2), and the number of cysts ranged from 1 to 7. Both parameters, area of cysts (r2 = 0.61), and number of cysts (r2 = 0.48) showed a significant correlation with visual acuity (P < 0.01), whereas FAZ (0.08 to 0.58 mm2) showed no significant correlation with visual acuity. CONCLUSIONS: Fluorescein angiography allows a reproducible quantification of the extent of macular cysts. The relation of visual acuity to the number of cysts and to the area covered by the cystoid formation is highly significant. Thus, both these measures can provide an objective criterion for the estimation of visual prognosis and an outcome for evaluation therapy techniques.

Adult↗

PubMed1993

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates.

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

Bacterial Proteins↗

PubMed1996

[Clinical evaluation of hepatitis C virus RNA quantification by competitive reverse-transcription PCR].

We have quantified the hepatitis C virus (HCV) RNA by competitive reverse-transcription PCR method (Amplicor) HCV quantification monitor kit) in sera with positive HCV antibody measured by means of the second generation HCV antibody assay. Among the visitors to PL Tokyo Health Control Center for their health examination and the patients to National Defense Medical College Hospital, 123 HCV antibody-positive cases were examined. A positive but low correlation between the amount of HCV-RNA and the titer of HCV antibody (r = 0.508, p value < 0.0001) was obtained. HCV-RNA was not detectable in 19 HCV antibody-positive cases. Among them, 10 cases showed normal ALT values. In the cases with more than 1000 copies/ml of HCV-RNA, the greater the amount of HCV-RNA the higher ALT values were observed, while the titer of HCV antibody was not correlated to ALT. This study demonstrated the dissociation and low correlation between HCV-RNA amount and antibody-titers in some patients, which may recommend direct quantification of RNA for clinical evaluation of the patients with positive-HCV antibody. The quantification of HCV-RNA by such rapid and simple methods can be applicable for the determination of HCV-RNA amount in routine laboratory works.

Hepacivirus↗

PubMed1996

Areal extent quantification of functional representations using intrinsic signal optical imaging.

An important parameter often investigated in the characterization of cortical functional organization is the areal extent of functional modules. Because it allows the visualization of functional modules with high spatial resolution in a noninvasive way to the cortex, intrinsic signal optical imaging (ISI) can be employed for the quantification of these areal extents. The present paper describes the use of the normalized threshold analysis of areal extent quantification for the objective assessment of single-whisker functional representations in the primary somatosensory cortex of adult rats. As the success of areal extent quantification depends on the ability of ISI to allow visualization of cortical representations with minimal stimulus-dependent blood vessel representations, which are commonly encountered by ISI, the present paper also describes the further development of the intratrial analysis of visualization for minimizing these vessel representations. Both analyses are discussed with respect to their advantages as well as their inherent limitations.

Animals↗

PubMed1997

Fast spin-echo and fast fluid-attenuated inversion-recovery versus conventional spin-echo sequences for MR quantification of multiple sclerosis lesions.

PURPOSE: To compare fast spin-echo (FSE) and fast fluid-attenuated inversion recovery (FLAIR) sequences with conventional spin-echo (CSE) MR imaging in the quantification of the number and volume of multiple sclerosis lesions. METHODS: In 30 patients with relapsing-remitting multiple sclerosis, we calculated the total number and volume of lesions detected with each of the three sequences using a semiautomated program. RESULTS: On CSE sequences, we calculated a total of 2,583 lesions with a global volume of 836.3 cm3. With FSE sequences, we observed a 16% relative reduction in the number of lesions detected and a 25% relative reduction in global volume as compared with CSE. With fast FLAIR sequences, we detected a significantly lower number and volume of infratentorial lesions, whereas at the cortical/subcortical level the lesions were both more numerous and bulkier than on CSE sequences. Finally, we observed a higher lesion/white matter contrast, a significant reduction in time required for the quantification of lesion load, and a very low interobserver variability in favor of fast FLAIR sequences. CONCLUSION: Despite its limitations in the detection of infratentorial lesions, the fast FLAIR sequence in conjunction with a semiautomated quantification program provides a reliable means to evaluate the total lesion burden in patients with MS.

Adult↗

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↗