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To improve the qualitative and quantitative analysis of surfaces of protein, two new methods are proposed: one that smoothes the MS surface of Connolly with B-spline smoothing functions to highlight the significant features of the surface, and one that computes the density of surface neighborhood to allow quantitative comparison.
A direct quantitative method is presented that is based upon the use of multiple headspace solid phase microextraction (HS-SPME) to monitor biogenic volatile organic compounds (BVOCs) released from a living leaf of Pelargonium hortorum in situ. Seventeen BVOCs were detected by GC-MS after a single SPME extraction using a CAR/DVB/PDMS fibre. An internal standard was employed to determine the absolute amounts of seven terpenoid compounds released from a P. hortorum leaf. The quantitative analysis was performed over two days, with extraction preformed for 20 min every 3 h. The amount of volatiles extracted varied with the time of day, with two maxima recorded at 14:00 (day 1) and 17:00 (day 2), corresponding to 236 and 277 ng of the seven terpenoids recorded, respectively. These results indicate that multiple HS-SPME in combination with an internal standard is a simple, quick, and quantitative technique for analysising BVOC emissions from a live plant sample.
This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.
Capillary electrophoresis was used for the separation and quantitative analysis of the glycosaminoglycan hyaluronan in human and bovine vitreous with detection by UV absorbance at 200 nm. Calibration was carried out using standards made up from known concentrations of hyaluronan of umbilical cord origin. The purity of the standard was examined by 1H NMR (nuclear magnetic resonance). Concentrations as low as 25 micrograms/mL could be detected by capillary electrophoresis. Confirmation that the signal was due to hyaluronan was obtained by depolymerization of the native mucopolysaccharide by hyaluronidase. This resulted in loss of the hyaluronan peak and appearance of several new peaks corresponding to the oligomeric fragments which had shorter migration times. Capillary electrophoresis is a reproducible and sensitive technique for the quantification and characterisation of hyaluronan in vitreous samples.
The purpose of this study was to improve the sensitivity and accuracy of the quantitative analysis of polymorphic mixtures. Calibration curve reliability has been improved through the advancement of standard mixture preparation techniques whereby samples at scales of several grams are homogeneously and accurately prepared without decrease in crystallinity. Crystalline powders are first micronized to anisotropic, nearly equal-sized (<10 microm) particles of almost random orientation in an air jet mill. These powders are then mixed in a vibration mill that includes rubber balls as mixing media. Using this technique, alpha and gamma forms of indomethacin (IMC) were prepared in various proportions from 0.5% to 50% alpha content and subjected to X-ray powder diffraction analysis. The validation data showed that these precisely prepared standard mixtures resulted in superior analytical sensitivity and accuracy compared to those prepared by agate mortar.
An experimental model was developed for quantitative analysis of pulmonary microcirculation using in vivo fluorescence videomicroscopy during unilateral hypoxia induced by one-lung ventilation (1 LV). In five white New Zealand rabbits, pulmonary arterioles on the surface of the right lung were visualized by means of intra-arterial injection of FITC-labeled erythrocytes and FITC-Dextran. During 1 LV of the left lung, the mean airway pressure in the right lung was kept at the level of two-lung ventilation (2 LV) by means of N2-CPAP. Arteriolar diameters as well as parameters of macrocirculation (AP, CVP, PAP, LAP, CO) and gas exchange (paO2, Qs/Qt) were measured simultaneously during 2 LV and 1 LV. FiO2 was kept constant at 1.0 during both experimental phases. Macrohemodynamic parameters during 1 LV did not differ from those measured during 2 LV. 1 LV induced a significant decrease in paO2 (213 +/- 105 versus 427 +/- 22 mm Hg, P < 0.05) and a significant increase in Qs/Qt (22 +/- 7 versus 13 +/- 2%, P < 0.05). During 2 LV (baseline), the pulmonary arteriolar diameters ranged from 15-120 microns. 1 LV resulted in a significant decrease of arteriolar diameters to 89.0 +/- 9.3% of baseline (P < 0.05). Relative changes in arteriolar diameters were similar for vessels with baseline diameters of 0-40, 40-60, and 60-120 microns (88.4 +/- 9.9%, 89.6 +/- 9.4%, and 88.4 +/- 8.7%, respectively). The present model is the first one allowing in-vivo investigation of HPV during 1 LV and 2 LV on the basis of simultaneous measurement of pulmonary arteriolar diameters and macrocirculatory parameters in vivo. Although PAP and PVR did not change significantly, a reduction of pulmonary arteriolar diameters was proven in response to alveolar hypoxia during 1 LV. We suggest the model to be useful in studying the physiological effects of HPV on macro- and microcirculation as well as investigating pathophysiological and pharmacological influences on HPV.
A thin-layer chromatography method for quantitative analysis of quaternary ammonium antiseptics is described. Silanized silica gel was used as the stationary phase. The mobile phase consisted of methanol-25% (m/v) sodium acetate solution-acetone (65:35:20). The method is able to separate the chain homologues of benzalkonium chloride, cetylpyridinium chloride and cetrimide. Detection was performed using a colour reaction with potassium triiodide solution. The different homologues were quantified using UV densitometry at 400 nm. A number of commercial samples was analysed using this method. From the results it appears that it is worthwhile to have a limit test for the composition of quaternary ammonium antiseptics in pharmacopoeial monographs, the more so as the antibacterial activity depends on it.
A description is given of the development of methods for the quantitative analysis of measurements of the fetal heart rate, uterine pressure and fetal movements, with data collection, reduction and analysis via a digital cassette off-line, or on-line. The on-line system consists of a small microprocessor, which reduces and digitizes the data, and which passes it for display and analysis to a larger microprocessor (NASCOM Z80). Analysis on-line offers the opportunity of interaction to optimise record quality and to compare at once sequential records on the same patient.
A simple and sensitive method is described for quantitative analysis of chlorpromazine in blood, serum, urine and tissue homogenate. The chlorpromazine cation radical produced by adding perchloric acid and 2,3-dichloro-5,6-dicyano-p-benzoquinone to the sample can be detected by the ESR method at room temperature. The sensitivity limit is 10 ng, that is, 20 microliters of the solution containing 0.5 microgram chlorpromazine/ml. The time needed for the measurement is within 10 min. The chlorpromazine radical thus produced is very stable; for example, 95% of the radical was observed after 24 h. The advantage of this method is discussed by comparing with the ordinary spectrophotometry which requires the purification of the sample.
A software has been developed that is aimed at quantitative analysis of images acquired by isoelectric focusing and double blotting procedures used for recombinant erythropoietin doping control. It represents a unified and easy-to-use tool for Epo doping experts in WADA accredited laboratories. It is based on image segmentation philosophy that enables identification of individual bands whose characteristics are needed for evaluation of the Epo doping positivity criteria. Several modules implemented in the GASepo software include an original know how, in particular, the method of robust calculation of the cut-off line, band segmentation and classification algorithms. GASepo is being used in several doping control laboratories worldwide.
Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the downstream pathways activated by intracellular calcium overload.
Quantitative analysis of the seminiferous epithelium was performed in bilateral testicular biopsy specimens from 14 patients with sperm counts ranging from 0 to 89 million/ml. All Sertoli cells and germ cells within each seminiferous tubule cross-section were counted in all biopsies. Results were expressed either as number of cells per unit length of seminiferous tubule circumference or as number of cells per Sertoli cell. Results were correlated with sperm count (millions per milliliter), total sperm count (millions per ejaculate), and age. A significant correlation between sperm density and germ cell counts was demonstrated. Coefficients of correlation were higher when results were expressed per unit of tubular wall length than when expressed per Sertoli cell. In men with sperm counts below 5 million/ml the number of germ cells in the biopsy was lower than in men with higher sperm counts. Spermiogenesis appeared to be most affected. In this group of patients an adverse effect of age on spermatogenesis was noted.
The separation and quantitative analysis of streptomyces hyaluronidase ( HAase ) degradation products of hyaluronic acid (HA) by high-performance liquid chromatography are described. The substituted 4,5-unsaturated tetrasaccharide and hexasaccharide which result from the digestion of HA are quickly separated on a silica gel (Zorbax SIL) column. The assay of HA is linear between 5 and 250 micrograms of HA. This procedure is suitable for some weakly acidic glycosaminoglycan contaminants having similar properties to those of HA.
A simple procedure for the quantitative analysis of trifluoroacetic acid (TFA) in urine and serum from patients narcotized with halothane is described. This involves addition of sodium hydroxide to the body fluid, evaporation of the aqueous phase and esterification of TFA in concentrated sulphuric acid with 2,2,2-trichloroethanol. The gaseous phases above the reaction mixture were then analyzed by gas chromatography with a nickel-63 electron-capture detector. The detection limit was 1 microgram of TFA per mililitre of body fluid (200 microgram of body fluid are analysed) and the relative standard deviation was +/-6%. Patients treated with ethrane, another commercial anaesthetic, did not produce any detectable TFA.
A nonlinear alignment strategy was examined for the quantitative analysis of serum metabolites. Two small-molecule mixtures with a difference in relative concentration of 20-100% for 10 of the compounds were added to human serum. The metabolomics protocol using UPLC and XCMS for LC-MS data alignment could readily identify 8 of 10 spiked differences among more than 2700 features detected. Normalization of data against a single factor obtained through averaging the XCMS integrated response areas of spiked standards increased the number of identified differences. The original data structure was well preserved using XCMS, but reintegration of identified differences in the original data reduced the number of false positives. Using UPLC for separation resulted in 20% more detected components compared to HPLC. The length of the chromatographic separation also proved to be a crucial parameter for a number of detected features. Moreover, UPLC displayed better retention time reproducibility and signal-to-noise ratios for spiked compounds over HPLC, making this technology more suitable for nontargeted metabolomics applications.
BACKGROUND: This paper describes a quantitative analysis of the cyst lining architecture in radicular cysts (of inflammatory aetiology) and odontogenic keratocysts (thought to be developmental or neoplastic) including its 2 counterparts: solitary and associated with the Basal Cell Naevus Syndrome (BCNS). METHODS: Epithelial linings from 150 images (from 9 radicular cysts, 13 solitary keratocysts and 8 BCNS keratocysts) were segmented into theoretical cells using a semi-automated partition based on the intensity of the haematoxylin stain which defined exclusive areas relative to each detected nucleus. Various morphometrical parameters were extracted from these "cells" and epithelial layer membership was computed using a systematic clustering routine. RESULTS: Statistically significant differences were observed across the 3 cyst types both at the morphological and architectural levels of the lining. Case-wise discrimination between radicular cysts and keratocyst was highly accurate (with an error of just 3.3%). However, the odontogenic keratocyst subtypes could not be reliably separated into the original classes, achieving discrimination rates slightly above random allocations (60%). CONCLUSION: The methodology presented is able to provide new measures of epithelial architecture and may help to characterise and compare tissue spatial organisation as well as provide useful procedures for automating certain aspects of histopathological diagnosis.
We describe the application of a novel image processing method, which allows quantitative analysis of cell and tissue movement in a series of digitized video images. The result is a vector velocity field showing average direction and velocity of movement for every pixel in the frame. We apply this method to the analysis of cell movement during different stages of the Dictyostelium developmental cycle. We analysed time-lapse video recordings of cell movement in single cells, mounds and slugs. The program can correctly assess the speed and direction of movement of either unlabelled or labelled cells in a time series of video images depending on the illumination conditions. Our analysis of cell movement during multicellular development shows that the entire morphogenesis of Dictyostelium is characterized by rotational cell movement. The analysis of cell and tissue movement by the velocity field method should be applicable to the analysis of morphogenetic processes in other systems such as gastrulation and neurulation in vertebrate embryos.
A new method for computer-assisted quantitative analysis of a thallium (Tl)-201 myocardial image ("corrected" circumferential profile method) was described. Since the Tl-201 myocardial image of a normal subject is not homogeneous, an attempt was made to correct for this non-homogeneity. Three groups of subjects were studied, including 10 normal volunteers (group A, mean age of 27.5 years), 14 patients with atypical chest pain and normal coronary arteriogram (group B, mean age of 56.7 years) and 16 patients with first transmural myocardial infarction (group C, mean age of 71.0 years). The myocardial images were acquired at rest at anterior, left anterior oblique (30 degrees and 60 degrees) and left lateral projections. With a scintigram, the left ventricle was outlined and divided into 24 radial segments by radii drawn from the center of the left ventricle. Average radioactivity per pixel in normal subjects was obtained in each segment and normalized to the highest segment (= 100%). The ratio of 100% to the mean of normalized radioactivity in percent in each segment from 10 normal volunteers was calculated and designated as a correction factor for each segment. After the correction of average radioactivity per pixel in each segment using this correction factor and after relating it to the highest radioactive segment, the mean (= 100%) and standard deviation (SD) were calculated and (100-4SD)% was defined as the normal lower limit. In the scintigram of groups B and C, "corrected" circumferential profiles were obtained from the regional radioactivity multiplied by the correction factor, normalized to the highest segment (100%) and compared with the lower normal limit. The ratio (%) of the area of patient's circumferential profile curve below the normal limit to the total area below the normal limit was obtained at each view and the sum of them was called the total "corrected" defect score. Infarct size analyzed by the "corrected" circumferential profile method correlated well with that analyzed by visual interpretation, and was useful in differentiating group C from group B.