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PubMed2004

A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.

Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.

Alleles↗
PubMed2001

[Quantitative analysis of the expression of insulin receptor and tyrosine protein kinase of patients with hepatic cirrhosis].

OBJECTIVE: To investigate the relationship between the expression of insulin receptor (IR) and the content of tyrosine protein kinase (TPK) in patients with hepatic cirrhosis. METHODS: Glucose tolerance test (GTT), insulin, c-peptide, insulin antibodies in serum, the expression of IR and TPK were quantitively analysed in patients with hepatic cirrhosis. The amount of IR and TPK was evaluated by immunohistochemical quantitative analysis using image analyzer in 12 patients with positive HBV marks and cirrhosis. Insulin and c-peptide in serum were determined by immunoenzymemetric assay (IEMA) and insulin antibodies in serum determined by RIA. RESULTS: IR amount in normal and abnormal GTT patients with hepatic cirrhosis was signifficantly less than that in control group (P<0.01). TPK amount in liver of patitents with abnormal GTT was less than that of normal GTT (P<0.01). CONCLUSIONS: IR decreases in cirrhotic patients. Abnormal GTT has relation to reducing TPK activity.

Antibodies↗
PubMed1998

[Scanning electron microscope observation and image quantitative analysis of Hippocampi].

The "scale-like projects" on the derma of 3 species of Hippocampi, H. kuda Bleerer, H. trimaculatus Leach and H. japonicus Kaup were observed by scanning electron microscope (SEM). Results showed that some characteristics such us size, shape and type of arrangement of the "scale-like projects" can be considered as the evidence for microanalysis. Image quantitative analysis of the "scale-like project" was carried out on 45 pieces of photograph using area, long diameter, short diameter and shape factor as parameters. No difference among the different parts of the same species was observed, but significant differences were found among the above 3 species.

Animals↗
PubMed2005

Rapid quantitative analysis of human cytomegalovirus DNA by the real-time polymerase chain reaction method.

CONTEXT: Human cytomegalovirus (CMV) infection is a progressive and life-threatening complication in immunocompromised patients even now. Therefore, early and accurate treatment based on rapid and certain detection is needed to prevent fatal CMV infection diseases. OBJECTIVE: To study a quicker, simpler, and less expensive method of quantitative analysis using real-time polymerase chain reaction based on the SYBR Green I method of CMV detection for appropriate treatment of CMV infection in immunocompromised patients. DESIGN: We quantified 50 samples tested by direct immunoperoxidase staining of leukocytes with peroxidase-labeled monoclonal antibody (C7-HRP test), 30 samples from healthy persons, and 47 samples from 7 patients suspected of having CMV infection diseases. We used the primer set in the pp65 gene of CMV and whole blood without a preparatory process. The setting for the study was the First Department of Pathology, Kurume University School of Medicine, St Mary's Hospital, and the Gene Section of the Clinical Laboratory at St Mary's Hospital, Fukuoka, Japan. RESULTS: The results obtained with this method corresponded well with conventional C7-HRP tests and demonstrated excellent reproduction. Additionally, the results were better correlated with the clinical course than were C7-HRP tests. CONCLUSIONS: This method was more useful than the C7-HRP test as a rapid diagnostic test for early treatment of CMV infection. This test also demonstrated its usefulness for monitoring CMV infection during treatment using ganciclovir. Moreover, it was quicker, simpler, and cheaper than other real-time polymerase chain reaction methods.

Adult↗
PubMedDate not supplied

[The use of a Russian software-hardware package for the quantitative analysis of coronary angiograms].

The software and hardware complex developed by the Cardiology Research Center, Russian Academy of Medical Sciences, jointly with the Technomash Research Production Association on the basis of a IBM 386DX personal computer equipped with a VS-100 video controller and a DS P31 VS signal processor board. Testing has indicated that it provides a qualitative image and a quantitative analysis both of phantoms and real images of coronarograms, but more accurately in the analysis of the image obtained from a film projector. Valid results are yielded when lenses more than 1 mm in diameter are used. Clinical tests have shown that the software and hardware complex may yield a rather qualitative image and calculate the required diameter of a vessel, virtually without prolonging the time of intervention, which is particularly important while making intervention procedures and implementing research programmes.

Analog-Digital Conversion↗
PubMed1998

Quantitative analysis of von Willebrand factor and its propeptide in plasma in acquired von Willebrand syndrome.

Measurement of the von Willebrand factor (vWF) propeptide, also known as von Willebrand antigen II, has been suggested to be helpful in the discrimination of congenital von Willebrand disease type I from type 2 and in assessing the extent of activation of the endothelium. We performed a quantitative analysis of mature vWF and its propeptide in plasma in 8 patients with acquired von Willebrand syndrome (AvWS) and in 20 normal individuals. Mature vWF levels were significantly lower in AvWS as compared with normal individuals (13.4+/-3.5 vs 35.6+/-3.3 nM, p <0.001). In contrast, propeptide levels were significantly higher in AvWS (11.4+/-1.1 vs 4.7+/-0.2 nM, p <0.001), probably reflecting a compensatory increase in vWF synthesis or increased perturbation of the endothelium in AvWS. After treatment with DDAVP, propeptide and mature vWF levels rose 5-fold in AvWS, whereas propeptide levels were not altered by the infusion of a vWF concentrate or treatment with high dose intravenous immunoglobulins, indicating that plasma propeptide levels are a reliable reflection of vWF synthesis. Measurement of propeptide levels may provide additional information in AvWS as to whether decreased levels of mature vWF in the circulation are due to a decrease in synthesis or due to an accelerated removal of vWF from the circulation.

Adult↗
PubMedDate not supplied

Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies.

A pathology report is written to convey information concerning the pathologic findings in a study. This type of report must be complete, accurate and communicate the relative importance of various findings in a study. The overall quality of the report is determined by three Quality Indicators: thoroughness, accuracy, and consistency. Thoroughness is the identification of every lesion present in a particular organ or tissue, including spontaneous background lesions. Experienced pathologists familiar with background lesions may disregard certain types of lesions or establish a threshold or a severity above which background lesions are diagnosed. Accuracy is the ability to make, and precisely communicate, correct diagnoses. Nomenclature of lesions is a matter of definition and experienced pathologists generally agree as to what terms are to be used. Consistency is the uniform use of a specific term to record a defined lesion and implies that the same diagnostic criteria are being followed for each type of diagnosis. The relative severity of nonneoplastic lesions can be recorded either semiquantitatively or quantitatively. Semiquantitative analysis involves the application of defined severity grades or ranges for specific lesions. Quantitative analysis (counts and measurements) can be performed manually or electronically, utilizing image analysis and stereological techniques to provide numerical values. When both qualitative and quantitative parameters are applied in preparation of a pathology report, the recorded pathology findings can be interpreted and put into perspective. The use of this approach assures a reader that the pathology report meets the highest standards.

Animals↗
PubMed2000

Quantitative analysis of [carbonyl-(11)C]WAY-100635 PET studies.

[carbonyl-(11)C]WAY-100635 ¿[(11)C]N-(2-(4-(2-methoxyphenyl)-1-piperazin-1-yl)ethyl)-N-(2- pyridy l)cyclohexanecarboxamide¿ is a positron-emission tomography (PET) radioligand for in vivo imaging of the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor. This paper assesses and summarises various different modeling strategies for the quantitative analysis of the radioligand. The models considered are based on a compartmental description of the ligands behaviour and include both plasma input analyses, requiring the additional monitoring of blood activity, and a reference tissue approach, which relies solely on the tomographic tissue data. Parameter estimates of specific binding are presented for a set of test-retest data, obtained from six normal volunteers who were scanned on two separate occasions, allowing for an assessment of normal binding values and their reproducibility.

Adult↗
PubMed1999

A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis.

Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors and neurotrophin mimetics. The drawbacks to this approach are the difficulties in quantifying the response. Neurite counts are time consuming and labour intensive, and the accuracy is often questionable due to branching and fasciculation of the neurites. We report here a method of semi-quantitative analysis of neurite outgrowth from chick DRG explants, using image analysis to quantify the area occupied by neurites emanating from the ganglion. This method is rapid, takes into account both the length and number of neurites, and is unaffected by neurite fasciculation or branching. Primary explants of chick DRGs were treated with the neurotrophins nerve growth factor (NGF) or neurotrophin-3 (NT-3) and with the compound K252a. K252b was tested for potentiation of the response to NT-3. The results show a dose dependent outgrowth of neurites from explants treated with NGF, NT-3 and K252a, and potentiation of the NT-3 response by K252b. These responses were quantified by neurite area quantification using image analysis. We conclude that neurite area measurement using image analysis provides a robust means of evaluating neurotrophic activity of growth factors and neurotrophin mimetics in vitro.

Animals↗
PubMed1980

A quantitative analysis of tubulin-colchicine binding to microtubules.

The binding of the tubulin-colchicine complex to microtubules has been studied in conditions where copolymerisation, as observed by Sternlicht and Ringel [J. Biol. Chem. 254, 10540-10550 (1979)], is negligible. The binding is shown to be rapid and reversible, in contrast with assumptions found in the literature. The binding constant was determined by a quantitative analysis of the concentration dependence of growth inhibition. The binding constant is of the same order of magnitude as the equilibrium constant of growth for tubulin. The temperature dependence was also studied and the Van't Hoff plot was found to be biphasic with an exothermic part at temperatures lower than 30 degrees C. A molecular model is presented for the interpretation of the copolymerisation observed by Strenlicht and Ringel. When a large excess of colchicine was added to microtubules, only a small phase of dissociation was observed. The new end state was metastable, as further dissociation could not be reversed. This is in conflict with a pure head-to-tail polymerisation mechanism, but in agreement with the recent observations of Bergen and Borisy [J. Cell Biol. 84, 141-150 (1980)], that head-to-tail polymerisation contributes only a small fraction of the total polymerisation.

Animals↗
PubMed1990

[Quantitative analysis of the slow-phase in LMLN].

Latent/manifest latent nystagmus (LMLN) is a jerky type of nystagmus with the fast phase directed toward the fixating eye. A previous report described that the slow phase shows decreasing-velocity exponentials. However, it is sometimes difficult to differentiate between pure LMLN and latent nystagmus with congenital nystagmus. Furthermore, there has been no detailed report of quantitative analysis of the waveform of LMLN. Therefore, in the present study 18 cases with LMLN were clinically selected and their eye positions and/or strabismus were carefully studied. The eye movement was recorded by a photo-electric device. The patients fixated on a small white target placed at 0, 5, 10, and 15 degrees either from the center toward the right or toward the left in the horizontal plane. Eye movement in each position for at least one minute's duration was recorded on a floppy disc after being digitized by an A/D converter. Then, the time constant of the slow phases were estimated using the repetitive non-linear least square method by a personal computer (NEC, PC 9801). Details of the method have been described previously. The 18 cases were first classified into three groups, based on analysis of the slow phases; 1. decreasing-velocity type, 2. increasing-velocity type, and 3. combination of 1. and 2. In group 1. esotropia or intermittent esophoria was present in all cases (100%) and none of them had stereopsis. In group 2 on the other hand, exophoria or intermittent exotropia was present in 4 (51%) out of 7 cases, and 6 (96%) out of 7 cases had stereopsis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2005

Quantitative analysis of chemotherapeutic effects in tumors using in vivo staining and correlative histology.

AIMS: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. METHODS: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. RESULTS: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. CONCLUSIONS: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

Animals↗
PubMed1992

Quantitative analysis of activated Kupffer cells in viral hepatitis: application of computer image analysis for lectin histochemistry.

Lectin histochemistry revealed that Kupffer cells in the normal liver bound lectins such as Concanavalin A (Con A), Ricinus communis agglutinin (RCA) and Wheat germ agglutinin (WGA), but did not bind Peanut agglutinin (PNA), Dolichos fibflorus agglutinin (DBA), Ulex europaeus agglutinin I (UEA-I) or Soybean agglutinin (SBA). Kupffer cells in viral liver diseases, however, bound the PNA lectin and the binding was specific to Kupffer cells in liver parenchyma. Computer image analysis was performed using light micrographs of sections stained with immunoperoxidase and diaminobenzidine (DAB). The dark brown area of reaction products was detected by analyzing each color component (red, green and blue) in the picture and was expressed as the percent area in the parenchyma. Quantitative analysis revealed the percent area occupied by Kupffer cells positive for the PNA lectin was as follows: acute hepatitis, 2.83 +/- 0.74; chronic persistent hepatitis, 2.51 +/- 0.88; chronic aggressive hepatitis, activity moderate and severe, 4.71 +/- 2.23 and 3.45 +/- 1.84; and liver cirrhosis, 1.96 +/- 0.99. The percent area of Kupffer cells was significantly higher in CAH2A than that in chronic persistent hepatitis or in liver cirrhosis. These results suggest that the PNA lectin could be used as a marker for activated Kupffer cells and that activated Kupffer cells were increased in volume in chronic aggressive hepatitis.

Biopsy↗
PubMed1994

[Quantitative analysis of bencynonate in human plasma using a deuterated internal standard by GC-MS/SIM].

Bencynonate is a newly developed anticholinergic drug. After a dose (2 mg/person or 4 mg/person) of bencynonate, the circulating concentrations are so low that therapeutic drug monitoring studies have previously been impossible. Radioisotope assay and radioaccepter assay had been used in rat experiments, but the detections limits of these methods were not sensitive enough for the therapeutic drug monitoring. Further, the two methods were unsuitable in the human. For evaluation of the pharmacokinetics and the relative bioavailability of bencynonate in man, a gas chromatographic--mass spectrometric method for quantitative analysis of bencynonate in human plasma was developed. Deuterated bencynonate served as the internal standard and selected-ion monitoring of the fragments of bencynonate and internal standard permitted the quantitation of bencynonate down to 25 pg.ml-1 of plasma. The linearity was in the range from 25 pg.ml-1 to 3 ng.ml-1 of bencynonate plasma concentration. At 0.25 ng.ml-1 level the recovery and relative standard deviation of variation are 54.3% and 19.1%, respectively. Application of the method to clinical studies gave the results of pharmacokinetics and relative bioavailability of bencynonate in man.

Animals↗
PubMed2001

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry.

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.

Analysis of Variance↗
PubMed1989

Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two-dimensional gels.

Previous findings suggest: (1) that altering protein kinase C (PKC) activity alters the persistence of long-term potentiation (LTP) in the intact hippocampal formation; and (2) that PKC activity is directly correlated with persistence of LTP in vivo as measured by the in vitro phosphorylation of two major PKC substrates in adult hippocampus, protein F1 and 80k. Using quantitative analysis of two-dimensional gels, we report here two additional phosphoproteins of 72 and 55 kDa which were directly correlated to persistence of LTP induced in the intact dorsal hippocampal formation. The phosphorylation of both proteins in response to addition of different kinase stimulators was distinct from that of protein F1 and 80k. Moreover, neither protein was a substrate for exogenous PKC. The physicochemical properties of these phosphoproteins suggest they are identical to the previously described synaptic vesicle proteins IIIa and IIIb, and as such are immunologically indistinguishable. Because proteins IIIa and IIIb are known to be phosphorylated by a Ca2+/calmodulin (CaM)-stimulated kinase, and protein F1 is known to be a plasma membrane-associated protein (P-57) which releases bound CaM in response to phosphorylation by PKC, the present findings suggest a potential mechanism in which PKC-mediated changes in plasma membrane proteins produce CaM kinase-mediated changes in synaptic vesicle proteins through a phosphorylation cascade. These membrane/vesicle alterations are postulated to underlie the increased synaptic efficacy which marks persistent LTP.

Adaptation, Physiological↗
PubMedDate not supplied

Quantitative analysis of the neonatal brain by ultrasound.

Clinical research has shown a clear correlation between white matter disorders of the neonatal brain and neuromotoric handicap at a later age. Ultrasound imaging is a proven method to detect the white matter damage at an early stage. However, since subjective visual examination of the images by neonatologists not always leads to an unambiguous diagnosis, a need for quantitative characterization is felt. Reproducibility is the first requirement in order to be able to perform objective quantitative analysis. This paper proposes a software-based method to compensate for variable acquisition factors that negatively affect the reproducibility of the measurements. The results of some basic experiments will illustrate the usefulness of the developed compensation algorithm.

Algorithms↗
PubMed2003

Quantitative analysis of MAP2 immunoreactivity in human neocortex of three patients surviving after brain ischemia.

Transient global ischemia caused by cardiac arrest results in lesions that involve all brain structures. The aim of this study was to investigate the distribution of MAP2 immunoreactivity in neurons in the brain of patients surviving for various times after an ischemic incident, using confocal laser scanning microscopy. We performed a quantitative analysis of the distribution and density of MAP2-positive structures in human neocortical areas after survival times of 1 week, 3 months, and 1 year after the cardiac arrest. Three important observations were made in the present study: (i) in all human brain areas investigated (motor, temporal, frontal, and visual cortex) a decrease of MAP2 immunoreactivity was found; (ii) in all studied areas the most significant decrease in MAP2 was found in layers II-III, compared with layers V-VII; (iii) the decrease of MAP2 immunoreactivity in layers II-III was related to the duration of the postischemic period. The maximal decrease, by 66.3% (P < .05), in MAP2-positive pyramidal neurons, was observed in layers II-III in the motor cortex after 1 year of survival after cardiac arrest.

Brain Ischemia↗