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At least 343 records · Page 19Linked to original sources
PubMed2002

Quantification of tumor suppressor mRNA expression by poly-competitive RT-PCR using a TS-IS that contained multiple internal competitors.

Despite the recent introduction of real-time PCR methods and cDNA microarrays, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. In this study, we developed a construct, termed tumor suppressor-internal standard (TS-IS) that produced polycompetitive RNA templates as an internal standard to quantify cellular RNA concentration of tumor suppressor genes. This construct is composed of not only sets of primers for detecting the expression of several tumor suppressor genes (such as pRB, p16(INK4A) 15(INK4B), p14(ARF) p53, and p21(WAF1)), but also HPRT as an endogenous marker. Using an internal standard RNA that was synthesized from the TS-IS construct, we were able to establish optimized conditions for the quantification of tumor suppressor genes with minimal amounts (50 ng) of cellular RNA. In addition, the usefulness of this method was confirmed by analyzing the expression levels of tumor suppressor genes in fourteen hepatoma cell lines as a model. The TS-IS assay that we used was inexpensive and a widely applicable method that permitted the reliable and accurate quantification of tumor suppressor genes.

Carcinoma, Hepatocellular↗
PubMed2002

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

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

Calibration↗
PubMed2002

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

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

Age Factors↗
PubMed1992

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

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

Cell Nucleus↗
PubMed1992

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

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

Adult↗
PubMed1991

Quantification of valvular regurgitation by Doppler echocardiography.

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

Compliance↗
PubMed1991

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

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

Gene Products, gag↗
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↗