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At least 505 records · Page 28Linked to original sources
PubMed2001

Estimation or quantification of tumour volume? CT study on irregular phantoms.

PURPOSE: To compare radiologists' subjective size estimation to computerised volume quantification of tumour-like phantoms in spiral CT. MATERIAL AND METHODS: Eight tubular phantoms with the inside irregularly covered with silicone (8.7-31.6 ml) were imaged. The phantoms were pairwise compared to analyse the differences in silicone volumes. The observers, 2 radiologists and 2 residents, used both subjective image analysis (2 sessions) and computerised volume quantification (1 session). Accuracy and observer agreement of both methods were calculated. RESULTS: Subjective size estimation was correct in 51% (mean weighted kappa, Kqw=0.73). Using four observers' mean value (Kqw=0.81) or median value (Kqw=0.77) slightly improved the results. Average intra-observer agreement was better than average interobserver agreement. In computerised volume quantification 70% of all classifications were correct (mean Kqw=0.85). The results were moderate even when every second or fourth slice were measured. CONCLUSION: Subjective size estimation of irregular tumours should be repeatedly performed by the same observer, or by using the mean or median estimate of several observers. Computer-based methods are even more reliable and their use is especially recommended for film readers with limited radiological experience. Only every fourth slice may be measured without a major loss of measurement accuracy.

Humans↗
PubMedDate not supplied

In situ quantification of aberrant p53 in colorectal neoplasia.

Aberrant p53 protein accumulation was measured immunohistologically in 342 colorectal paraffin-embedded tissue sections from 115 patients (24 with adenocarcinoma, 59 with adenoma and 32 'hospital controls'). Subjective scoring was compared with quantitative cell imaging, including dichotomous (p53+/p53-) status, ng p53mut mg(-1) enterocyte protein, and tumour burden and patient body 'burden' of aberrant p53. A total of 62.5% cancer patients, 23.7% adenoma patients and 3.1% hospital controls were accorded p53+ status on the basis of p53 quantification. Quantitative p53+/p53- assignment had a stronger inverse association with survival (chi2=6.17, p=0.013, Kaplan-Meier test) than subjective 'visual estimation' (chi2=0.57, p=0.449). There was a strong inverse relationship between the p53 'body burden' and the months of post-diagnosis survival (hazard ratio=1.42, p=0.0004, Cox proportional hazards). Absolute quantification for inactivated p53 permits objective and reproducible scoring, adjusts for intra-laboratory immunostaining 'batch effects', corrects for fixation artefacts, and standardizes for inter-laboratory differences in fixation, antibody selection and staining method. Clinically, in situ quantification of p53 will permit more accurate survival prognoses and will inform therapy selection and dose. Ultimately, accurate quantitative tissue/blood p53 correlations may provide a minimally invasive and systemic surrogate measure for these same clinical purposes.

Adenocarcinoma↗
PubMed2001

Quantification of polynuclear aromatic hydrocarbons in transformer oils by enzyme immunoassay.

Many polynuclear aromatic hydrocarbons (PAHs) are either known or suspected carcinogens and are a common constituent of mineral oils. Due to the large number of possible PAH structures, standard quantification methods fail since they either lack specificity or are too complex, requiring individual fractionation, identification, and quantification. A rapid, low-cost, novel analytical screening method, incorporating a silica-based solid-phase extraction (SPE) method linked to co-solvent dilution and quantification of total and carcinogenic PAH levels by immunoassay, is reported here. The method yielded high extraction efficiencies and minimal matrix effects. This novel approach yielded total and carcinogenic PAH levels x 5.7 and x 126, respectively, lower than that recorded by the industry-recognised BS2000 Pt. 346 (IP346) method which estimates the polyaromatic carbon (PAC) content of oils by gravimetry. The method is expected to be of benefit where an indication of PAH levels in oils is important for purchasing, management or disposal purposes and also for risk assessment and for appropriate labelling of oils in line with current legislation.

Carcinogenicity Tests↗
PubMed1988

A portable reflectometer for the rapid quantification of cutaneous haemoglobin and melanin.

A portable reflectance instrument for the rapid quantification of cutaneous haemoglobin and melanin is presented. Light emitting diodes (LEDS) are used to illuminate the skin and a silicon photodiode to detect the light diffusely reflected from the surface. Reflectance measurements are made at only three wavelengths and the problems of pigment quantification consequent upon this are discussed. In addition to quantification of haemoglobin and melanin, qualitative information on the redox state of the blood may also be obtained. Measurements made on a port wine stain, which had been treated with 576 nm CW laser radiation at times between 1 and 6 months previously, provided information on the vascular response to this thermal injury. Despite the treated area visually appearing normal at 6 months post-treatment the measured levels of deoxygenated and total haemoglobin were still markedly higher than those in the adjacent uninvolved skin. The cutaneous pigment indices are insensitive to skin movement and almost all body sites are suitable for measurement.

Hemoglobins↗
PubMed2003

RMDP: a dedicated package for 131I SPECT quantification, registration and patient-specific dosimetry.

The limitations of traditional targeted radionuclide therapy (TRT) dosimetry can be overcome by using voxel-based techniques. All dosimetry techniques are reliant on a sequence of quantitative emission and transmission data. The use of (131)I, for example, with NaI or mIBG, presents additional quantification challenges beyond those encountered in low-energy NM diagnostic imaging, including dead-time correction and additional photon scatter and penetration in the camera head. The Royal Marsden Dosimetry Package (RMDP) offers a complete package for the accurate processing and analysis of raw emission and transmission patient data. Quantitative SPECT reconstruction is possible using either FBP or OS-EM algorithms. Manual, marker- or voxel-based registration can be used to register images from different modalities and the sequence of SPECT studies required for 3-D dosimetry calculations. The 3-D patient-specific dosimetry routines, using either a beta-kernel or voxel S-factor, are included. Phase-fitting each voxel's activity series enables more robust maps to be generated in the presence of imaging noise, such as is encountered during late, low-count scans or when there is significant redistribution within the VOI between scans. Error analysis can be applied to each generated dose-map. Patients receiving (131)I-mIBG, (131)I-NaI, and (186)Re-HEDP therapies have been analyzed using RMDP. A Monte-Carlo package, developed specifically to address the problems of (131)I quantification by including full photon interactions in a hexagonal-hole collimator and the gamma camera crystal, has been included in the dosimetry package. It is hoped that the addition of this code will lead to improved (131)I image quantification and will contribute towards more accurate 3-D dosimetry.

Humans↗
PubMed2000

Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial.

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.

Animals↗
PubMed2003

Significance of molecular quantification of minimal residual disease in metastatic neuroblastoma.

Molecular detection of tumor cells is the most sensitive approach to study residual disease in bone marrow (BM), peripheral blood (PB), and peripheral blood stem cell (PBSC) autografts from children with metastatic neuroblastoma (NB). We have developed a real-time PCR assay that allows the quantification of tyrosine hydroxylase (TH) mRNA, a tissue-specific marker of neuroblasts. We investigated a total of 165 BM, PB, and PBSC samples from 30 children over 1 year of age with stage IV NB and correlated the findings with disease status and patient survival. The levels of TH mRNA agreed well with clinical status and were significantly different across the groups that included samples obtained from patients at diagnosis, after three cycles of chemotherapy, in complete or very good partial remission and at relapse. We found that overall survival was significantly worse for patients with >1000 TH copies in BM after initial chemotherapy (p=0.0075). In 57% of cases, autologous PBSC harvests were found to be contaminated by neuroblasts, the level of TH >500 copies being associated with a decreased survival (p=0.003). In addition, molecular quantification enabled an estimation of tumor depletion in contaminated autografts using CD34 selection (median, 3 logs). In conclusion, quantification of minimal residual disease in metastatic NB using real-time RT-PCR for TH mRNA appears to be of potential clinical value. Further studies are needed to ascertain prognostic implications of molecular analysis of residual disease.

Antigens, CD34↗
PubMed2005

A new method to obtain transmission images for planar whole-body activity quantification.

UNLABELLED: Dosimetry is important in the evaluation of new radiopharmaceuticals and in radionuclide therapy treatment planning. The base is an accurate in vivo measurement of the spatial and temporal distribution of the administered radioactivity. This can be made by planar whole-body activity quantification using the conjugate-view method where a geometrical-mean image is corrected for attenuation by a measured transmission image. We present a new method to measure whole-body transmission images using the X-ray unit on a single photon emission computed tomography/computed tomography (SPECT/CT) scintillation system. METHOD: Two general problems are associated with this method: (1) the divergence of the X-ray beam and (2) the scaling of the resulting image. We have applied linear transformations and scale by a system-generated factor to obtain images corresponding to a radiological depth at 70 keV. We validated the method by measurements of the radiological depth for different types of phantoms, and in terms of activity quantification accuracy when applying the method for attenuation correction. RESULTS: An accuracy of within +/-4% was found for both the radiological depth measurements and for the activity-quantification measurements. CONCLUSIONS: The advantage with the proposed method is that it is fast (2 minutes for a 2-minute scan), it has good spatial resolution and signal-to-noise ratio, and it is easy to carry out for the patient.

Humans↗
PubMed2005

Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells.

Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for clinical lots. Stringent guidelines stipulate the maximum acceptable level of DNA per dose of vector, and this quantification is therefore a crucial piece of information for researchers and manufacturers alike. In this paper we describe an optimized assay based on real-time polymerase chain reaction (PCR) for the quantification of residual PER.C6 DNA in recombinant adenoviral vectors. In order to reduce the risk of introducing contaminations and to increase the throughput, the assay was designed to require minimum sample handling. Furthermore, DNA extraction from the samples is not necessary, thereby eliminating the need to account for possible sample losses. We also report the results of the assay qualification, demonstrating that the assay is accurate, precise, and sensitive. Finally, we applied the assay successfully to determine the level of host cell DNA in an adenovirus vector produced on PER.C6 cells throughout a standard purification process. Because of its specifications, we anticipate that the assay can have broad applicability to biologics other than adenoviral vectors produced on PER.C6 cells.

Adenoviridae↗
PubMed2000

Accounting for radioactivity before and after nebulization of tobramycin to insure accuracy of quantification of lung deposition.

The ability to predict drug deposition of inhaled drugs used in cystic fibrosis (CF) is important if there is a need to target specific doses of drug to the lungs of individual patients. The gold standard of measuring pulmonary deposition is the quantification of an aerosolized radiolabel either mixed with the drug solution or tagged directly to the compound of interest. Accuracy of the quantification could be assured if there is agreement between the amount of radioactivity before and after administration. Before administration, the radiolabel is concentrated in the well of the nebulizer, whereas after administration, it is distributed throughout the nebulizer, the expiratory filter and connectors, and the upper airway, stomach, trachea, and lung. Not only is the geometry of the distribution that is presented to the gamma camera different, but there are different attenuation factors for the various body tissues. The primary aim of this study was to evaluate the accuracy of the quantification of deposition. Secondary goals were to compare in vitro nebulizer performance with that measured in vivo during the deposition study. Eighty milligrams of tobramycin and technetium bound to human serum albumin was administered to 10 normal adults using a Pari LC Jet Plus (Pari Respiratory Equipment, Inc., Richmond, VA) breath-enhanced nebulizer. Techniques were developed that allowed for the accounting of 99 +/- 2% of the initial radioactivity. The fraction of the rate of lung deposition to total body deposition was the in vivo respirable fraction (0.62 +/- 0.07), which closely agreed with in vitro measurements of respirable fraction (0.62 +/- 0.04). Drug output measured from the change in weight and concentration in the nebulizer systematically overestimated drug output measured by the deposition study. The results indicate that 11.8 of the initial 80 mg would be deposited in the lungs. This technique could be adapted to accurately quantify the amount of deposition on any inhaled therapeutic agent, but caution must be used when extrapolating performance of a nebulizer on the bench to expected deposition in patients.

Adult↗
PubMed1987

The use of invasive techniques, angiography and indicator dilution, for quantification of valvular regurgitations.

Angiographic techniques have been used for the quantification of mitral or aortic and rarely tricuspid regurgitation. Mitral or aortic regurgitant volume per beat and the regurgitation fraction (fao and fm, respectively) are obtained from the angiographic determination of total left ventricular stroke volume (TSV) and forward stroke volume (FSV) estimated by a different technique. Although this procedure is generally accepted as the gold standard for quantification of left heart regurgitations, there are several limitations: In the presence of mitral and aortic regurgitation no separate quantification of fao and fm is feasible; heart rate at the time of determination of FSV (from Fick or dye dilution cardiac output) and of TSV (angio) may be different; there is a tendency to consistently overestimate stroke volume by angio techniques; repeated estimations of TSV by angio are influenced by the circulatory effects of the contrast dye. In contrast indicator dilution techniques, where upstream and downstream sampling allow the simultaneous estimation of forward and regurgitant flow, the accuracy of the determination of FSV is well established and repeated estimations of fao and fm are possible because the indicators do not have cardiovascular effects. These methods are, however, crucially dependent on thorough mixing of the regurgitant volume with the blood in the upstream chamber. In 23 patients with isolated aortic regurgitation there was a positive correlation between fao evaluated by thermodilution and fao determined by the biplane angio-Fick method (r = 0.59). fao by thermodilution averaged 0.40 and fao by angio-Fick 0.46 (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Angiocardiography↗
PubMed2005

Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives.

Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.

3-Mercaptopropionic Acid↗
PubMed2002

Quantification of Doppler signal in polycystic ovary syndrome using three-dimensional power Doppler ultrasonography: a possible new marker for diagnosis.

BACKGROUND: Differences in the ovarian stromal blood flow of women embarking on an IVF treatment, as assessed on day 2 or 3 of the menstrual cycle, using three-dimensional (3D) power Doppler ultrasonography to quantify the blood flow and vascularization, were compared. METHODS: The women were divided into two groups: group 1, 54 women with regular, ovulatory menstrual cycles and normal ovaries on ultrasound scan; and group 2, 25 women with polycystic ovary syndrome (PCOS). RESULTS: The quantification of Doppler signal in the ovarian stroma appeared to be greater in the PCOS group compared with the normal group. The mean of ovarian volume was significantly higher (P < 0.05) in women with PCOS compared with the normal ovaries. The vascularization flow index (VFI), flow index (FI), and vascularization index (VI), were significantly higher (P < 0.05) in the women with PCOS compared with the women with normal ovaries. CONCLUSIONS: This observation may help to explain the excessive response often seen during gonadotrophin administration in women with PCOS. We believe that a quantification study of the vascular flow, including the VI, FI, and VFI of the entire ovarian stroma using 3D power Doppler, is more accurate than the previously reported quantification analysis using 2D imaging, and may be a new parameter to assist in the ultrasound diagnosis of PCOS.

Adult↗
PubMed2005

Quantification of endometriotic lesions in a murine model by fluorimetric and morphometric analyses.

BACKGROUND: In animal models of endometriosis, the identification and quantification of lesions originating from human endometrium is often hampered by the small size of the implants and their embedding in murine tissue. The purpose of the present study was to develop two new methods of quantifying endometriosis-like lesions in a nude mouse model: fluorimetry and morphometry. METHODS: Human menstrual endometrium was labelled using a fluorescent tracker, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), and transplanted into the pelvic cavity of mice by injection through the peritoneum after performing a cutaneous incision. After 5 days, lesions were recovered by laparotomy. The fluorescence of the recovered endometriotic lesions was measured. Endometrial stroma and glands were immunostained in lesion sections with anti-CD10 and anti-CK22 antibodies, and their surface area was evaluated by morphometric analysis. RESULTS: Fluorescent labelling allows identification of lesions not visible macroscopically. A good correlation was observed between fluorimetry and morphometry (r=0.88) applied for lesion quantification. CONCLUSIONS: Fluorimetric evaluation combined with morphometric analysis of endometriosis-like lesions allows objective and reliable recording of endometriosis development in a nude mouse model. This quantification method could therefore be useful for future pharmacological and toxicological studies.

Animals↗
PubMed2004

Strategies for precise quantification of transgene expression levels over several generations in rice.

Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. Studies conducted over generations could, by their design, generate additional unwanted variation. To study sources of spurious variation, transgene expression levels were quantified over five homozygous generations in two independent transgenic rice lines created by particle bombardment. Both lines contained the same gus expression unit and had been shown to exhibit stable inheritance of transgene structure and expression. All plants were cultured and sampled using previously developed standardized protocols. Plants representative of each generation (T2, T3, T4, T5, T6) were grown either all together or across several different growth periods. GUS activity in plants from different generations was quantified either in the same assay or over multiple independent assays. Strategies in which plants were grown and phenotyped independently, significantly increased (up to 3-fold) extraneous variation in transgene expression level quantification, thus reducing the precision of molecular genetic studies and generating artefactual results in transgenic studies conducted over generations. Identification of sources of unwanted variation and quantification of their effect allowed the development of new strategies designed to control spurious variation. Growth and phenotyping of all plants from all generations together, using standard operating procedures (SOP), led to a reduction in extraneous variation associated with transgene expression level quantification. Adoption of such strategies is key to improving the reproducibility of transgenic studies conducted over generations.

Gene Expression Regulation, Plant↗
PubMed2001

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site.

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

A new mathematical model for relative quantification in real-time RT-PCR.

Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

Animals↗
PubMed2006

Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR.

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within +/-5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h.

Cytosine↗