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PubMed2000

Sensitivity of two electron beam tomography protocols for the detection and quantification of coronary artery calcium.

OBJECTIVE: The purpose of this study was to compare the sensitivity of two electron beam tomography protocols for detection and quantification of coronary artery calcium. SUBJECTS AND METHODS: We selected 101 patients (57% men, mean age 53 +/- 10 years) to undergo two consecutive electron beam tomography and acquired imaging with both a 6-mm and a 3-mm slicing protocol. Three pixels (area, 1.03 mm(2)) and a minimal density of 130 H were used for definition of calcified plaque. RESULTS: We found coronary artery calcifications in 46 patients when we used a 6-mm protocol and in 61 patients when we used a 3-mm protocol (p < 0.001). The average total calcium score was 77 (+/-140) with a 6-mm protocol and 251 (+/-395) with a 3-mm protocol (p < 0.005). The average number of calcified lesions per patient was 1.7 for a 6-mm protocol and 3.7 for a 3-mm protocol (p < 0.01). Of 179 individual lesions seen using a 3-mm protocol, 103 (58%) were missed using a 6-mm protocol, and only 27% of the lesions with a calcium score less than or equal to 40 seen with a 3-mm protocol were detected with 6-mm slicing (p < 0.001). The mean lesion attenuation with a 6-mm protocol was 160 (+/-42) H, compared with 218 (+/-44) H with a 3-mm protocol (p < 0.001), indicating a significantly greater partial volume averaging with the former protocol. CONCLUSION: A 6-mm slicing protocol is significantly less sensitive than a 3-mm protocol for the detection and quantification of coronary artery calcium. Since one third of coronary events occur in patients with low calcium scores, a 6-mm protocol might be unreliable for risk assessment because of substantial loss of information in this calcium score range.

Calcinosis↗
PubMed2006

Rapid and sensitive real-time polymerase chain reaction method for detection and quantification of 3243A>G mitochondrial point mutation.

Maternally inherited diabetes and deafness and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes result from the 3243A>G mitochondrial point mutation. Current methods to detect the presence of the mutation have limited sensitivity and may lead to potential misclassification of patients with low levels of heteroplasmy. Here, we describe development and validation of a rapid real-time polymerase chain reaction (PCR) method for detection and quantification of levels of heteroplasmy in a single assay. Standard curve analysis indicated that the sensitivity of detection was less than 0.1%. Time from sample loading to data analysis was 110 minutes. We tested 293 samples including 23 known positives, 40 known negatives, and 230 samples from patients clinically classified as having type 2 diabetes. All positive samples were correctly detected, and of those samples previously quantified, heteroplasmy levels determined using the real-time assay correlated well (r(2) = 0.88 and 0.93) with results from fluorescently labeled PCR-restriction fragment length polymorphism and pyrosequencing methods. Screening of 230 patients classified as having type 2 diabetes revealed one patient with 0.6% heteroplasmy who had previously tested negative by PCR-restriction fragment length polymorphism. Real-time PCR provides rapid simultaneous detection and quantification of the 3243A>G mutation to a detection limit of less than 0.1%, without post-PCR manipulation.

Adenosine↗
PubMed2003

Autoregressive moving average (ARMA) model applied to quantification of cerebral blood flow using dynamic susceptibility contrast-enhanced magnetic resonance imaging.

PURPOSE: To investigate the feasibility of the autoregressive moving average (ARMA) model for quantification of cerebral blood flow (CBF) with dynamic susceptibility contrast-enhanced magnetic resonance imaging (DSC-MRI) in comparison with deconvolution analysis based on singular value decomposition (DA-SVD). METHODS: Using computer simulations, we generated a time-dependent concentration of the contrast agent in the volume of interest (VOI) from the arterial input function (AIF) modeled as a gamma-variate function under various CBFs, cerebral blood volumes and signal-to-noise ratios (SNRs) for three different types of residue function (exponential, triangular, and box-shaped). We also considered the effects of delay and dispersion in AIF. The ARMA model and DA-SVD were used to estimate CBF values from the simulated concentration-time curves in the VOI and AIFs, and the estimated values were compared with the assumed values. RESULTS: We found that the CBF value estimated by the ARMA model was more sensitive to the SNR and the delay in AIF than that obtained by DA-SVD. Although the ARMA model considerably overestimated CBF at low SNRs, it estimated the CBF more accurately than did DA-SVD at high SNRs for the exponential or triangular residue function. CONCLUSION: We believe this study will contribute to an understanding of the usefulness and limitations of the ARMA model when applied to quantification of CBF with DSC-MRI.

Blood Flow Velocity↗
PubMed2004

Automatic motion correction for quantification of myocardial perfusion with dynamic magnetic resonance imaging.

Respiratory motion makes it difficult to quantify myocardial perfusion with dynamic magnetic resonance imaging (MRI). The purpose of this study was to evaluate an automatic registration method for motion correction for quantification of myocardial perfusion with dynamic MRI. The present method was based on the gradient-based method with robust estimation of displacement parameters. For comparison, we also corrected for motion with manual registration as the benchmark. The myocardial kinetic parameters, K1 (rate constant for transfer of contrast agent from blood to myocardium) and k2 (rate constant for transfer from myocardium to blood), were calculated from dynamic images with a two-compartment model. The images corrected by the present method were similar to those corrected by manual registration. The kinetic parameters obtained after motion correction with the present method were close to those obtained after motion correction with manual registration. These results suggest that the present method is useful for motion correction for quantification of myocardial perfusion with dynamic MRI.

Contrast Media↗
PubMed1996

Development of an enzyme-linked immunosorbent assay (ELISA) for quantification of skeletal muscle calpastatin.

An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in prerigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulon 4 (Dynatech) plates was observed when heated muscle extracts were diluted to 2 to 4 micrograms of protein/mL and incubated for 2 h at 37 degrees C. Optimum primary (30 micrograms IgG/mL) and secondary (Sigma A-6154; 1:1000 dilution) antibody incubations were for 1 h at 37 degrees C. Tetramethylbenzidine was used as substrate and A450 of the stopped reaction product was recorded in an automated plate reader. Calpastatin ELISA results were linearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from prerigor lamb (r2 = .89; n = 40) and beef aged for 24 or 48 h (r2 = .90; n = 47). Intra-assay CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assay offers advantages of speed, simplicity, and sensitivity over conventional methodology for calpastatin quantification.

Animals↗
PubMed1984

Development and clinical application of electroimmunoassays for the direct quantification of the complement C3 split products C3c and C3d.

The present report describes the development of electroimmunoassays for the direct quantification of C3d and C3c, split products of the third complement factor (C3). Both methods were developed as rocket immunoelectrophoreses, using two antibody preparations with specificities against the C and D epitopes on C3. A stable calibrator preparation for C3c and C3d was produced by autolytic cleavage of C3. Investigations of patients with anaphylactic reactions or chronic immunological disorders showed that quantification of C3c and C3d in plasma by these methods reflected the degree and rate of complement activation. C3d determination was found to be the most sensitive and reliable indicator of C activation, both during acute and chronic activation since C3c is eliminated considerably faster from the circulation than C3d (estimated half lives of 2 h and 4 h, respectively). Treatment of rheumatoid arthritis patients with prednisolone caused a prompt decrease of plasma C3d values, indicating that the clinical effect may be due to inhibition of complement activation.

Antibody Specificity↗
PubMed1996

A rapid HPLC method for the quantification of GSH and GSSG in ocular lens.

UNLABELLED: Purpose. To develop a rapid and accurate method for the quantification of reduced glutathione (GSH) and oxidized glutathione (GSSG) using micro-quantities of ocular lens. Methods. The epithelium, cortex and nucleus of the lens were separated and also the whole lens was homogenized in 3% metaphosphoric acid. The homogenate was ultrafiltered by centrifugation at 10,000 g in an Amicon microconcentrator, molecular weight cut off 3,000 g. The method does not require prior derivatization of the glutathiones. The filtrate was analyzed on a Microsorb-MV by a high performance liquid chromatography (HPLC) column using an isocratic solvent system (3% methanol and 10 mM potassium phosphate, pH 3.0) and detection at 200 nm. RESULTS: The GSH and GSSG were eluted from the HPLC column at retention times 5 and 10 min, respectively. The detection limit was 100 pmoles applied to the column. The recovery of GSH and GSSG added to the tissue samples was 97-100%. CONCLUSIONS: A fast and sensitive HPLC-method for the quantification of picomole quantities of GSH and GSSG in ocular lens, which does not require prior derivatization, has been developed.

Animals↗
PubMed1984

Enterochromaffin cells in the rat gastrointestinal tract. Aspects of factors influencing quantification.

A quantitative study was made of the enterochromaffin (EC) cells, visualized with the Masson (slightly modified) and Falck-Hillarp techniques, in the rat gastrointestinal tract. Different factors influencing the quantification were analysed and the magnitude of methodological errors was estimated. The two histochemical techniques visualized the EC cells in different ways, influencing the quantification results. This means that a comparison of the quantitative results obtained by these techniques can only be made after correction by some factors. The frequency of EC cells is given as the number of cells both per mm3 mucosa (cellular density) and per segment of mucosa with a base of 1 mm2. The latter information takes into account both cellular density and mucosal thickness. The highest frequency of EC cells was found in the pyloric gland area of the stomach. On the border with the duodenum there was a marked decrease in this frequency and in the whole intestine the frequency was low, with only minor fluctuations of the mean values. The frequency was increased in the caecum, followed by a gradual decrease in the large intestine. The quantitative variation of the EC cells at the same gastrointestinal level was considerable, and was greater between animals than between serial sections within the same animal. The difference in EC cell frequency that was required to give significance was calculated. For quantitative studies the Masson method has some minor advantages over the Falck-Hillarp technique, as routine formalin-fixed deparaffinized sections can be used and no counterstaining is needed for the areal estimation.

Animals↗
PubMedDate not supplied

An inhibition ELISA for the quantification of collagens type I and type II in cyanogen bromide-digested tissues using fragment-directed antibodies.

The quantification of connective tissue components in small tissue samples is of great importance for the examination of drug-induced changes in the development of the mammalian embryo. An inhibition-ELISA for the quantification of collagens type I and type II in CNBr-digested tissue samples was developed. Fragments of type I collagen were produced by CNBr-cleavage of the pure collagen and partially purified by gel filtration chromatography. A mixture of fragments was used to immunize rabbits. Antisera with highest titres were absorbed with immobilized fibronectin and collagens type II, III, V and I. The eluted antibodies exhibited specificity for alpha 2(I)-CB4, exclusively. These antibodies, as well as the previously described antibodies with specificity for alpha 1(II)-CB8, were used for the development of an inhibition-ELISA. The sensitivity of the assay is 0.3 micrograms/ml for collagen I and 3 micrograms/ml for collagen II. To evaluate the value and practicability of the ELISA we have estimated the amounts of both collagens in biochemically well characterized tissues (skin, aorta, chondrosarcoma) and have performed an initial determination in mouse embryos.

Animals↗
PubMedDate not supplied

Immunohistologic quantification of Pseudomonas aeruginosa in the tracheobronchial tree from patients with cystic fibrosis.

Pseudomonas aeruginosa has been recognized as a pathogen of major importance in the patient with cystic fibrosis (CF). However, no information is available regarding the histologic quantification of P. aeruginosa organisms in the CF tracheobronchial tree. We retrieved all formalin-fixed paraffin-embedded lung blocks from 20 consecutive autopsies of cystic fibrosis patients. Serial histologic sections were made and stained by three methods: hematoxylin and eosin, immunoperoxidase with anti-P. aeruginosa rabbit serum as the primary antibody, and immunoperoxidase with normal rabbit serum as the primary antibody. By studying the hematoxylin and eosin section, we classified five areas in the lung as bronchi, large bronchioles, small bronchioles, bronchioloectatic areas, and abscess/airways with destroyed epithelium. The areas stained by an anti-P. aeruginosa immunoperoxidase method were examined under high-power magnification, and the bacteria within random fields were counted. Pseudomonas aeruginosa organisms were identified in 14 of 20 cases, including 13 of 16 cases in which P. aeruginosa was specifically cultured at autopsy. Quantification of organisms within the lumens of all five airway types showed that the bacterial density in cystic fibrosis airways is highest in bronchi.

Adult↗
PubMed2003

Quantification of short-chain free fatty acids in "Terrincho" ewe cheese: intravarietal comparison.

Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was employed for the quantification of volatile free fatty acids (FFA) in "Terrincho" ewe cheese. Solid-phase microextraction quantitative analysis was feasible under equilibrium situations as long as the conditions of agitation and the adsorption time were held constant. An excellent linear relationship between the amount of the adsorbed analyte and its initial concentration in the sample matrix was obtained when an adequate amount of sample was chosen. Thus, quantification was possible if biases due to competition or linear range excesses were controlled. Solid-phase microextraction sampling was carried out at 65 degrees C, and a fiber coated with an 85-micro/m polyacrylate film was chosen. After equilibration at 65 degrees C for 40 min, the fiber was exposed to the headspace above the sample for 20 min and then inserted into the gas chromatograph. The evolution of the volatile FFA during Terrincho ewe cheese ripening was analyzed for a 60-d period. An overall increase in FFA contents was verified up to 30 d of ripening. Between 30 and 45 d most FFA did not suffer significant changes. All FFA increased significantly by the 60-d ripening period. The excessive lipolysis observed at 60 d of ripening may result in the presence of off-flavors. Principal component analysis performed for intravarietal comparison of volatile FFA composition of 19 Terrincho cheeses, analyzed at 30 ripening days, enabled discrimination between cheeses produced at five different dairy plants.

Animals↗
PubMed2002

Volumetric quantification of Fisher Grade 3 aneurysmal subarachnoid hemorrhage: a novel method to predict symptomatic vasospasm on admission computerized tomography scans.

OBJECT: Predicting which patients with aneurysmal subarachnoid hemorrhage (SAH) will develop delayed ischemic neurological deficit (DIND) due to vasospasm remains subjective and unreliable. The authors analyzed the utility of a novel software-based technique to quantify hemorrhage volume in patients with Fisher Grade 3 aneurysmal SAH. METHODS: Patients with aneurysmal SAH in whom a computerized tomography (CT) scan was performed within 72 hours of ictus and demonstrated Fisher Grade 3 SAH were analyzed. Severe DIND was defined as new onset complete focal deficit or coma. Moderate DIND was defined as new onset partial focal deficit or impaired consciousness without coma. Fifteen consecutive patients with severe DIND, 13 consecutive patients with moderate DIND, and 12 consecutive patients without DIND were analyzed. Software-based volumetric quantification was performed on digitized admission CT scans by a single examiner blinded to clinical information. There was no significant difference in age, sex, admission Hunt and Hess grade, or time to admission CT scan among the three groups (none, moderate, or severe DIND). Patients with severe DIND had a significantly higher cisternal volume of hemorrhage (median 30.5 cm3) than patients with moderate DIND (median 12.4 cm3) and patients without DIND (median 10.3 cm3; p < 0.001). Intraparenchymal hemorrhage and intraventricular hemorrhage were not associated with DIND. All 13 patients with cisternal volumes greater than 20 cm3 developed DIND, compared with 15 of 27 patients with volumes less than 20 cm3 (p = 0.004). CONCLUSIONS: The authors developed a simple and potentially widely applicable method to quantify SAH on CT scans. A greater volume of cisternal hemorrhage on an admission CT scan in patients with Fisher Grade 3 aneurysmal SAH is highly associated with DIND. A threshold of cisternal hemorrhage volume (> 20 cm3) may exist above which patients are very likely to develop DIND. Prospective application of software-based volumetric quantification of cisternal SAH may predict which patients will develop DIND.

Adult↗
PubMed2005

Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR.

The main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multifiliis. We developed a real-time PCR assay using SYBR Green intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of I. multifiliis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the I. multifiliis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 I. multifiliis cells l(-1). The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture.

Animals↗
PubMed2004

A quantification and confirmation method of patulin in apple juice by GC/MS.

A sensitive and selective method for quantification and confirmation of patulin in apple juice by GC/MS was developed. By this method, patulin was precisely determined and confirmed down to the level of 1 and 5 microg/kg in samples, respectively. Patulin was extracted with ethyl acetate from a sample and then hexane was added to the concentrated extract solution. Significant amounts of insoluble impurities were filtered off, followed by further clean-up by solid-phase extraction with combined silica gel and Florisil cartridges. The filtration step in a low-polarity condition was very effective to remove the impurities in the sample extract solution. The eluate from the cartridges was evaporated to dryness under reduced pressure and patulin was determined and confirmed by GC/MS after derivatization with 2.5% N,O-bis(trimethylsilyl)trifluoroacetamide ethyl acetate solution. Patulin was determined in the selected ion monitoring mode (m/z 226) and confirmed in the SCAN mode (m/z 40-340). The recovery from apple juice spiked with 10-500 microg/kg ranged from 93.4 to 100%. The limits of detection and quantification were 0.1 (S/N = 3) and 1 microg/kg (S/N = 30) of patulin in samples, respectively. Levels down to 5 microg/kg of patulin in sample were readily confirmed.

Factor VIII↗
PubMed2006

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.

AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 multiply 10(4)/mL, 6.3 multiply 10(2)/mL and 1.6 multiply 10(3)/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 multiply 10(9)/mL, 2.08 multiply 10(6)/mL and 4.40 multiply 10(7)/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10(5)/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

Base Sequence↗
PubMed2006

The quantification of PSA mRNA copies in pathologically normal pelvic lymph nodes is a viable prognostic indicator.

Localized prostate cancer is a disease associated with significant biochemical recurrence with rising serum prostate-specific antigen (PSA) levels and marked progression. More specific prognostic methods are needed to identify the occult micrometastases that lead to biochemical recurrence. Ferrari et al. report on the technique of quantifying pathologically occult metastases in pelvic lymph nodes via PCR quantification. The group reports statistically significant biochemical failurefree recurrence in the comparison of 100 or greater normalized PSA mRNA copies (PSA-N) versus PSA-N of less than 100 at a median follow up of four years. PSA-N quantification appears to be an important method for predicting biochemical recurrence and characterizing the recurrence risk of localized prostate cancer patients with pathologically normal pelvic lymph nodes. This method warrants further investigation in prospective trials.

Humans↗
PubMed2006

Optimization of a fluorescence polarization immunoassay for rapid quantification of deoxynivalenol in durum wheat-based products.

A fluorescence polarization immunoassay previously described for deoxynivalenol (DON) screening in wheat was optimized for the rapid quantification of DON in durum wheat kernels, semolina, and pasta. A background signal was observed in both spiked and naturally contaminated samples, strictly depending on the testing matrix. After subtracting the background DON level for durum wheat (0.27 microg of DON per g), semolina (0.08 microg of DON per g), and pasta (0.04 microg of DON per g), an accurate quantification of DON was possible at levels greater than 0.10 microg/g for all matrices. Average recoveries from spiked samples (0.25 to 1.75 microg/g) were 98, 102, and 101% for wheat, semolina, and pasta, respectively. Comparative analyses of 35 naturally contaminated durum wheat samples, 22 semolina samples, and 26 pasta samples performed by both the fluorescence polarization method and high-pressure liquid chromatography/immunoaffinity cleanup showed a good correlation (r > 0.995). The fluorescence polarization method showed better accuracy and precision with respect to the high-pressure liquid chromatography method and is suitable for the rapid and quantitative determination of DON in durum wheat-based products at levels foreseen by existing or coming international regulations.

Chromatography, High Pressure Liquid↗
PubMed2005

Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: assays development, characterization and pre-validation.

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5 % to 95 % and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1 % for both the approaches, allowing to extend the range of quantifications from 1 % to 99 %. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

Alleles↗