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PubMed2002

Combined noninvasive imaging and luminometric quantification of luciferase-labeled human prostate tumors and metastases.

Noninvasive imaging should facilitate the analysis of changes in experimental tumors and metastases-expressing photoproteins and result in improved data consistency and experimental animal welfare. We analyzed quantitative aspects of noninvasive imaging of luciferase-labeled tumors by comparing the efficiency of noninvasive light detection with in vitro quantification of luciferase activity. An intensified charge coupled device video camera was used to noninvasively image luciferase-expressing human prostate tumors and metastases in nude mice, after ip inoculation of luciferin. Repeated imaging of anesthetized animals after intervening growth periods allowed monitoring of tumor and metastases development. Comparison of photon events recorded in tumor images with the number of relative light units from luminometric quantification of homogenates from the same tumors, revealed that the efficiency with which light escapes tumors is inversely related to tumor size and that intensified charge coupled device images alone are not sufficient for quantitative evaluation of tumor growth. However, a combined videometric and luminometric approach did allow quantification and was used to show the cytostatic effects of paclitaxel in three different human prostate tumors growing in nude mice.

Animals↗
PubMed2025

kSanity: A k-mer based application for precision bacterial strain detection and quantification.

MOTIVATION: Accurate detection and quantification of bacterial strains in clinical samples is necessary to measure their colonization and persistence. Past methods to achieve this relied either on strain-specific qPCR assays, or shotgun metagenomic read mapping approaches. The resident microbial community is a major source of interference in both assays because it can contain conspecific strains bearing similarity to the focal strain(s). RESULTS: We present kSanity, a k-mer based application for the detection and quantification of targeted bacterial strains in shotgun metagenomic data. Because kSanity uses exact string matches between the reads and reference, it is less sensitive to interference by conspecific strains. We test the performance of kSanity using a combination of in silico spike-in experiments, and in vivo observational data. Our results demonstrate that kSanity provides precise and accurate quantification of targeted bacterial strains, even when they are present at low sequence coverage in the metagenome. AVAILABILITY AND IMPLEMENTATION: kSanity is available at: https://github.com/ravel-lab/kSanity.

bacterial strain detection↗
PubMed1994

Quantification of noisy MRS signals with the Pseudo-Wigner Distribution.

This paper investigates the estimation errors induced by noise in the quantification of damped sinusoids with the Pseudo-Wigner Distribution (PWD). A constant amplitude single frequency noise is first considered. This simple model shows how underestimation or overestimation errors depend on the relative phase between signal and noise for a fixed signal-to-noise ratio (SNR). In a second step, cross-terms and noise amplitude fluctuations are identified as the main sources of discrepancy between the theoretical model and a practical situation where wideband noise is linearly added to a synthetic free induction decay (FID) signal. Cross-terms can be attenuated by band-pass filtering noise or by using a weighting (or smoothing) window in the PWD. An original procedure is then derived to make an on-line and noise-specific estimation of the statistical error in the quantification step. This property of the Wigner distribution is a unique feature in quantitative MRS. Confidence intervals are evaluated for a single damped sinusoid corrupted by eight random noise sequences with three different SNR's, 20, 10 and 5 dB, respectively. They are shown to match the statistical ranges of quantification results obtained with linear regression, until the SNR drops below 10 dB. Estimation accuracy of amplitude and damping constant is finally evaluated from the comparison of the Cramér-Rao (CR) lower bounds with the variance of estimation errors. CR-bounds are shown to be nearly achieved at each SNR.

Confidence Intervals↗
PubMed2001

Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification.

To develop a simpler method of performing the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video-microscope and NIH Image software. In examinations using a human cancer cell line and a fibroblast cell line, a high degree of linearity between number of cancer cells and image-optical density was found within the range of 10(2) - 10( 6) cells / droplet (r (2) = 0.933). Using NIH Image, fibroblast cells could be eliminated at a cut-off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD-DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD-DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r = 0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD-DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.

Antineoplastic Agents↗
PubMed1995

Quantification of androgen receptor and follicle-stimulating hormone receptor mRNA levels in human and monkey testes by a ribonuclease-protection assay.

A sensitive, solution-hybridization ribonuclease-protection assay (RPA) was established to quantify the expression of mRNA for the androgen receptor (AR) and follicle-stimulating hormone receptor (FSHR) in total RNA samples isolated from tissues of the cynomolgous monkey, human testes obtained from elderly patients undergoing orchidectomy because of prostatic carcinoma or from transsexual men undergoing gender reassignment as well as human cell lines DU 145, REP and RVP. Sensitivity experiments revealed that, in the human and monkey, 1-2 micrograms of total RNA were sufficient to achieve quantifiable signals of the different receptor mRNA species. Quantification of AR and FSHR mRNA levels showed a 1.7-fold higher expression of AR mRNA and a 2.4-fold higher expression of FSHR mRNA in the monkey testes compared to human testes from patients with prostatic carcinoma. Normal spermatogenesis in both human and monkey testes indicated no relationship between spermatogenic status and receptor expression. The significantly lower expression of AR and FSHR mRNA in humans than in monkeys might therefore be either age- or species-related. Quantification of mRNA for AR and FSHR in the testis of the transsexual patients undergoing oestrogen and antiandrogen treatment displayed a drastic increase (4.5-fold) in mRNA for the AR, whereas mRNA for the FSHR was barely detectable. Due to its high sensitivity, reproducibility and its ability to quantify mRNA transcripts, the RPA is a useful tool for investigating expression of low abundant receptor genes and their regulation when only very small amounts of tissue are available. Furthermore, it is suitable for use in clinical and experimental studies in which accurate quantification of transcripts is necessary.

Aged↗
PubMed1990

A comparison of three quantification methods for estimation of respiratory sinus arrhythmia.

The empirical literature has shown that respiratory sinus arrhythmia is a sensitive noninvasive index of parasympathetic cardiac control. Nevertheless there has been no general agreement among investigators as to the most preferable quantification technique for assessing respiratory sinus arrhythmia, although there has been much speculation that specific estimation techniques are more or less reflective of vagal processes and could be more or less contaminated by other influences upon heart period variability unrelated to respiration. This study compared three quantification procedures for estimating respiratory sinus arrhythmia (RSA): (1) a spectral analytic technique, (2) a complex detrending approach removing periodic and aperiodic cardiac variations unrelated to respiration, and (3) a time-domain, peak-valley procedure employing inspiratory and expiratory periods as windows for determining range of cardiac-interval fluctuations associated with respiratory phase. Measures derived from these techniques were intra- and interindividually compared using three different samples of male subjects, including students, adult normotensives, and adult hypertensives. All interindividual correlations between measures yielded coefficients above .92 and the mean within-subject correlation across 42 individuals was .96, thus indicating a marked degree of comparability between measures. Additionally, given that much evidence indicates lawful within-individual relations between RSA amplitude and respiratory parameters, we employed respiratory period as an external criterion and compared intraindividual correlations between this variable and (2) and (3); results showed that (3) was significantly more highly associated with respiration than was (2), although the mean r's for the two measures did not diverge greatly (.91 vs. .84). Finally, inspection of the data and further regression analyses did not suggest that any of the RSA estimates were differentially contaminated by other components of cardiac variability. Our findings suggest that the three techniques are almost equivalent as indices of cardiac vagal tone and would appear to ease concerns about the inferiority of any of the procedures. Choice of a quantification procedure should therefore be tailored to the specific empirical needs of an investigation. The advantages and disadvantages of each method are discussed.

Adult↗
PubMed1997

Three-dimensional ultrasound angiography (power mode) for the quantification of carotid artery atherosclerosis.

Three-dimensional (3D) ultrasound angiography was performed to diagnose carotid artery atherosclerosis. Thirty-five patients (15 women, 20 men) with a history of cerebrovascular disease were examined using conventional color-coded Doppler ultrasound and 3D ultrasound angiography. Carotid stenosis was initially diagnosed using continuous-wave Doppler ultrasound. To determine intraobserver and interobserver reliabilities, 21 patients were evaluated using 3D ultrasound on three occasions. Sixty-five percent of patients were diagnosed with stenosis of more than 50%. Twenty-two percent of plaques had a smooth surface, 72.9% were ulcerated, and 5.1% were indeterminate. Data collection for 3D imaging required 5 minutes per patient, whereas image processing and plaque volume quantification required 30 minutes. Plaque volume ranged from 0.053 to 0.685 ml. The intraobserver and interobserver variabilities were 4.16 and 5.87%, respectively (r = 0.96, p < 0.0001; r = 0.89, p < 0.0001). 3D Color Doppler and 3D ultrasound angiography assessments of plaque volume differed by 8.5%. Plaques were more precisely differentiated using 3D ultrasound, and plaque volume quantification was less affected by echo shadowing after 3D reconstruction. In comparison to other techniques for the quantification of atherosclerotic lesions. 3D ultrasound angiography offers a more precise quantitative method for prospective, clinical studies of atherosclerosis.

Aged↗
PubMed2001

Improved in vitro quantification of the force exerted by the papillary muscle on the left ventricular wall: three-dimensional force vector measurement system.

Recent developments indicate that the forces acting on the papillary muscles can be a measure of the severity of mitral valve regurgitation. Pathological conditions, such as ischemic heart disease, cause changes in the geometry of the left ventricle and the mitral valve annulus, often resulting in displacement of the papillary muscles relative to the annulus. This can lead to increased tension in the chordae tendineae. This increased tension is transferred to the leaflets, and can disturb the coaptation pattern of the mitral valve. The force balance on the individual components governs the function of the mitral valve. The ability to measure changes in the force distribution from normal to pathological conditions may give insight into the mechanisms of mitral valve insufficiency. A unique in vitro model has been developed that allows quantification of the papillary muscle spatial position and quantification of the three-dimensional force vector applied to the left ventricular wall by the papillary muscles. This system allows for the quantification of the global force exerted on the posterior left ventricular wall from the papillary muscles during simulation of normal and diseased conditions.

Animals↗
PubMed2002

Myocardial blood flow quantification with MRI by model-independent deconvolution.

Magnetic resonance (MR) imaging during the first pass of an injected contrast agent has been used to assess myocardial perfusion, but the quantification of blood flow has been generally judged as too complex for its clinical application. This study demonstrates the feasibility of applying model-independent deconvolution to the measured tissue residue curves to quantify myocardial perfusion. Model-independent approaches only require minimal user interaction or expertise in modeling. Monte Carlo simulations were performed with contrast-to-noise ratios typical of MR myocardial perfusion studies to determine the accuracy of the resulting blood flow estimates. With a B-spline representation of the tissue impulse response and Tikhonov regularization, the bias of blood flow estimates obtained by model-independent deconvolution was less than 1% in all cases for peak contrast to noise ratios in the range from 15:1 to 20:1. The relative dispersion of blood flow estimates in Monte Carlo simulations was less than 7%. Comparison of MR blood flow estimates against measurements with radio-isotope labeled microspheres indicated excellent linear correlation (R2 = 0.995, slope: 0.96, intercept: 0.06). It can be concluded from these studies that the application of myocardial blood flow quantification with MRI can be performed with model-independent methods, and this should support a more widespread use of blood flow quantification in the clinical environment.

Animals↗
PubMedDate not supplied

Improved quantification of radionuclide uptake using deconvolution and windowed subtraction techniques for scatter compensation in single photon emission computed tomography.

A comparison of two methods of scatter compensation in single photon emission computed tomography (SPECT) imaging is made on the basis of improvements in quantification. The methods, scatter-window subtraction and constrained deconvolution of an average point source response function (PSRF), are described; the theoretical basis of each method is also briefly assessed. Improvements in relative quantification offered by each method are measured by examining ratios of counts in hot cylinders to counts in a slightly radioactive background. The cylinder/background concentration ratio was varied by a factor of five; the sizes of the cylinders remained constant. Keeping the diameter of the cylinders constant allowed for an assessment of the effect of concentration differences, without the conflicting effect of variation in the size of the hot source. Results showed that while both scatter-window subtraction and constrained deconvolution offer quantification improvements in the final image, the method of prereconstruction deconvolution of a planar PSRF from each planar projection is substantially more successful than either scatter-window subtraction or other methods of implementing the deconvolution procedure.

Biophysical Phenomena↗
PubMed2000

Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples.

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.

Bacteria↗
PubMed1996

An enhanced-sensitivity branched-DNA assay for quantification of human immunodeficiency virus type 1 RNA in plasma.

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.

Anti-HIV Agents↗
PubMed1997

Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper.

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

Binding, Competitive↗
PubMed1998

Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis.

Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy.

Antitubercular Agents↗
PubMed1999

Nucleic acid-based cross-linking assay for detection and quantification of hepatitis B virus DNA.

A nucleic acid photo-cross-linking technology was used to develop a direct assay for the quantification of hepatitis B virus (HBV) DNA levels in serum. Cross-linker-modified DNA probes complementary to the viral genomes of the major HBV subtypes were synthesized and used in an assay that could be completed in less than 6 h. The quantification range of the assay, as determined by testing serial dilutions of Eurohep HBV reference standards and cloned HBV DNA, was 5 x 10(5) to 3 x 10(9) molecules of HBV DNA/ml of serum. Within-run and between-run coefficients of variation (CVs) for the assay were 4. 3 and 4.0%, respectively. The assay was used to determine HBV DNA levels in 302 serum samples, and the results were compared to those obtained after testing the same samples with the Chiron branched-DNA (bDNA) assay for HBV DNA. Of the samples tested, 218 were positive for HBV DNA by both assays and 72 gave results below the cutoff for both assays. Of the remaining 12 samples, 10 were positive for HBV DNA by the cross-linking assay only; the 2 other samples were positive by the bDNA assay only. Twenty-eight samples had to be retested by the bDNA assay (CV, >20% between the results obtained from the testing of each sample in duplicate), whereas only three samples required retesting by the cross-linking assay. The correlation between the HBV DNA levels, as measured by the two tests, was very high (r = 0.902; P = 0.01). We conclude that the cross-linking assay is a sensitive and reproducible method for the detection and quantification of HBV DNA levels in serum.

Cross-Linking Reagents↗
PubMed1999

Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.

Animals↗
PubMed2001

Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification.

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10(4) copies per 100 microl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.

Antibodies, Viral↗