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PubMedDate not supplied

Detection and quantification of Pneumocystis carinii using a sandwich ELISA.

Antigenic sites on Pneumocystis carinii, the basis for organism enumeration by an enzyme-linked immunosorbent assay (ELISA) were adversely affected by incubation in detergents. However, stronger detergent concentrations were needed to eliminate high levels of non-specific background. Good P. carinii quantification was obtained with low non-specific background when the detergent was used only in the washing steps and not in cell suspension solutions. Formalin fixation of the cells resulted in good ELISA quantification of organism numbers with low non-specific background. No adverse effects were observed using a detergent on fixed cells. Although the system's range of accuracy needs to be expanded, a reduction in the number of organisms in response to the effects of pentamidine in vitro could be demonstrated by ELISA.

Animals↗
PubMed1991

Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum.

Here we describe an error-compensating kinetic-based method for the enzymatic quantification of creatinine in serum. The method, which has a large linear range and very low dependency on experimental variables that influence enzyme activity, is based on the use of creatinine amidohydrolase in a four-step coupled reaction sequence to generate a product that is monitored photometrically. We collected data for absorbance vs time during two to four half-lives of each reaction and fit a first-order model to the data to compute the total absorbance change that would be measured if the reaction were monitored to completion. Computed values of absorbance change agreed well with measured values and varied linearly with creatinine concentration in the sample throughout the range examined: 44 to 1326 mumol/L. A twofold change in enzyme activity in the final reaction mixture (3.3 to 6.6 kU/L) produced changes of only 12% and 4% for creatinine concentrations of 44 and 353 mumol/L, respectively. Results (y) for 39 serum samples that contained creatinine concentrations between 20 and 1800 mumol/L agreed well with liquid-chromatographic results (x), yielding linear least-squares statistics of y = (1.03 +/- 0.01) x + (5 +/- 5) mumol/L (r = 0.995, Sy.x = 37 mumol/L). We conclude that the predictive kinetic approach is a robust method for the quantification of creatinine in serum.

Chromatography, High Pressure Liquid↗
PubMed1990

[Quantification and clinical significance of plasma apolipoproteins].

The apolipoproteins are important determinants of the structure and metabolism of plasma lipoproteins. This paper reviews analytical methods and the clinical significance of plasma apolipoproteins. Our data on apo VLDL and apo HDL analysis using fast protein liquid chromatography (FPLC), monoclonal antibody against apo VLDL, especially apo C-I, apo B isoproteins (apo B-100 and apo B-48) and plasma apolipoprotein concentrations in the patients with diabetes mellitus and coronary heart disease, were described. Among the methods of apolipoprotein quantification, single radial immunodiffusion (SRID) is widely used in Japan and plasma concentrations of apo A-I, A-II, B, C-II, C-III and E in healthy adults were reported. We showed the usefulness of FPLC for fractionation of human apo VLDL and apo HDL. We prepared several monoclonal antibodies against human apo VLDL, especially apo C-I, which were used for quantification and structural analysis of plasma apo C-I. Apo B-48 was found to be a good metabolic marker of exogenous lipoproteins (chylomicron and chylomicron remnant) and apo B-48 containing lipoproteins were increased in the poorly controlled diabetic patients.

Antibodies, Monoclonal↗
PubMed1977

[Left ventricular contraction reserve in coronary heart disease. Evaluation, quantification and prognostic value (author's transl)].

Regional and overall left ventricular contraction reserve was studied in 14 patients with coronary heart disease, in 5 healthy subjects and in 4 patients before and after aorto-coronary bypass surgery. Quantification of overall contraction was based on ventricular volumes and ejection fraction. Regional contraction reserve was calculated with the hemiaxis method and a ventricular score. Contraction reserve under nitroglycerin and in postextrasystolic beats was compared. For routine quantification of contraction reserve the ventricular score is recommended. For research purposes the hemiaxis method is to be preferred. Postextrasystolic beats are better suited for analysis of contraction reserve than are angiograms following administration of nitroglycerin. This is due to the minor expense of the procedure, furthermore, postextrasystolic beats allow better differentiation between contracting and non-contracting areas. Left ventricular contraction reserve is larger in patients with coronary heart disease, angina pectoris and ischemic reactions in the exercise ECG than in control patients. These findings are based on overall and on regional volume parameters. A quantitatively greater improvement in contraction could be provoked in the anterior wall than in the posterior wall. Regional contraction improved significantly in most cases either in the anterior wall or in the posterior wall; rarely it improved simultaneously in both left ventricular regions. In a few cases contraction deteriorated in one area with a simultaneous improvement in the opposite area. Overall and regional ventricular function, as assessed preoperatively by contraction reserve determinations could not be completely regained in normal beats after successful bypass surgery. Differences in the regional contraction reserve seemed to be mainly due to varying degrees of ischemia and scarring.

Angina Pectoris↗
PubMed1979

Quantification of tetanus antitoxin in human sera. II. Comparison of counter-immunoelectrophoresis and passive haemagglutination with toxin neutralization in mice.

We described recently an improved counter-immunoelectrophoretic method for quantification of tetanus antitoxin. The toxin neutralization test in mice is considered to correlate well with protection in humans. In the present study, the correlation coefficient between the two methods was 0.89. Sera containing more than 7.0 I.U./ml could be quantified directly by counter-immunoelectrophoresis, while sera containing less tetanus antitoxin had to be concentrated prior to quantification. The passive haemagglutination test was also compared with the toxin neutralization test in mice. The correlation coefficient between the two methods was 0.76.

Antibodies, Bacterial↗
PubMed1976

A computer-based method for the quantification of leukocyte chemotaxis.

A computerized method for the counting of polymorphonuclear leukocytes (PMN's) on chemotactic membranes is described. Through the use of an optical scanner-computer accurate objective quantification is obtained which is similar to the results generated by visual tabulation of the same membranes. Futhermore, the electronic quantification method is both less time consuming and highly reproducible.

Adult↗
PubMed2026

Accurate quantification of canine mitochondrial DNA copy number from canine blood and brain samples.

Acute brain injury is difficult to evaluate in veterinary medicine and tools to investigate the potential involvement of mitochondrial involvement are limited. The brain is highly enriched in mitochondria and contains thousands of copies of mitochondrial DNA (mtDNA) per cell, but robust methods for quantifying mitochondrial DNA copy number (mtDNA-CN) in canine tissues are lacking. We describe the development of a quantitative real-time PCR assay for absolute measurement of mtDNA-CN which was validated in canine blood and brain tissue. To minimize amplification of nuclear mitochondrial insertion sequences (NumtS) and repetitive regions, species-specific oligonucleotide primers were designed following in silico genomic filtering. The assay was applied to a small pilot cohort comprising blood samples from dogs with and without acute brain injury (n = 4-6 per group) and cerebral cortex samples (n = 1 per group) to assess feasibility and biological plausibility. In non-brain injury dogs, blood mtDNA-CN ranged from 98 to 288 copies per nuclear genome (mean 193 ± 72), while values in brain-injured cases ranged from 163 to 228 copies per genome (mean 200 ± 33). Cerebral cortex samples exhibited higher mtDNA-CN than blood, consistent with known tissue-specific mitochondrial enrichment. In a single brain-injured case with serial sampling, mtDNA-CN increased over five days. This study presents a validated assay and pilot data for mtDNA-CN quantification in canine samples. While not powered for biomarker evaluation, this method may enable future studies of mitochondrial dynamics in canine brain injury and metabolic disease.

Animals↗
PubMed1976

Quantification of combined radiation therapy and chemotherapy effects on critical normal tissues.

In order to determine the modification of radiation effects on critical normal tissues which occurs with combinations of radiation and cancer chemotherapy, a review of laboratory and clinical data has been carried out. Information on 10 different normal tissues is available. It is clear that the antibiotic cancer chemotherapeutic agents are the most likely to enhance radiation injury, with increased levels reported in all tissues except the central nervous system. The second most common type of injury with combination therapy appears to occur with drugs causing injury to the normal tissue on their own, such as adriamycin in the heart and methotrexate in the central nervous system. Quantification of the dose-effect factor is only available on a limited number of tissues, and, primarily, in experimental animals. From these limited data, it is clear that dose-effect factors between 1.1 and 1.8 are seen, indicating that radiation doses must be reduced by 10-80% for the same level of injury when combined with chemotherapy. The augmentation of radiation damage by cancer chemotherapeutic agents is a serious problem in a wide range of tissues, but a problem which can be dealt with by accurate knowledge as to the dose-effect factor and appropriate modification of the radiation treatment.

Antineoplastic Agents↗
PubMed1992

Asymmetry quantification utilizing hand radiographs.

Asymmetry can be either directional or fluctuating. Detection of abnormal amounts of asymmetry has important implications for clinical diagnosis, but measurement of subtle levels is very difficult. We describe a method and normative values for asymmetry quantification using hand radiographs.

Adult↗
PubMedDate not supplied

A simple method for the quantification of famotidine in human plasma and urine by paired-ion high performance liquid chromatography.

A new method for the quantification of famotidine consists of a simple extraction procedure and paired-ion high performance liquid chromatography with ultraviolet detection. The method has good accuracy and precision and should be suitable for the routine measurement of plasma and urine samples for clinical studies.

Chromatography, High Pressure Liquid↗
PubMed1991

Mass spectrometric quantification of endogenous beta-endorphin.

Fast atom bombardment (FAB) mass spectrometry and multiple reaction monitoring (MRM) in the B/E linked-field scan mode were used to quantify endogenous beta-endorphin (BE) in individual human pituitary extracts. The experimental protocol includes the addition of a stable isotope-labeled internal standard ((2H4-Ile22)BE1-31, human) to the tissue homogenate before extraction, purification of the native BE by a combination of Sep-Pak chromatography and gradient high-performance liquid chromatography (HPLC), trypsin digestion to cleave BE into smaller peptides, and separation of the tryptic fragment BE20-24 (NAIIK) by isocratic reversed-phase HPLC. Mass spectrometric quantification is based upon recording either (a) the [M + H]+ ions of NAIIK and its deuterated analog ((2H4)NAIIK), or (b) the transitions ([NAIIK + H](+)----[NAI]+) and [((2H4)NAIIK + H](+)----[(2H4)NAI]+) using the B/E linked-field scan. Linear calibration curves were obtained using these two mass spectrometric techniques from standard solutions containing 1.25-20 micrograms of BE; each standard solution also contained 10 micrograms of (2H4)BE. The amounts (means +/- s.d.) of endogenous BE in five separate human pituitaries were found to be 156 +/- 84 [( M + H]+ method) and 169 +/- 99 pmol mg-1 protein (MRM method).

Amino Acid Sequence↗
PubMed1991

Quantification of plasma phenylethylamine by combined gas chromatography/electron capture negative ion mass spectrometry of the N-acetyl-N-pentafluorobenzoyl derivative.

An ultrasensitive method capable of detection and quantification of beta-phenylethylamine in 1 ml of human plasma has been developed using gas chromatography/electron capture negative ion mass spectrometry. Phenylethylamine and tetra-deutero phenylethylamine internal standard in plasma were acetylated, extracted into organic solvent and then further acylated with pentafluorobenzoyl chloride. The N-acetyl-N-pentafluorobenzoyl-phenylethylamines were detected by high-resolution single ion monitoring of the molecular ions. Normal plasma levels were found to be 41.5 +/- 10.7 pg ml-1, in accordance with results of a previous high-performance liquid chromatographic method.

Chromatography, Gas↗
PubMed1990

Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping.

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.

Aneuploidy↗
PubMed1990

Comparison of the Papanicolaou and Feulgen staining methods for DNA quantification by image analysis.

The possibility of using archival cytology material to study the evolution of neoplastic disease with regard to DNA content abnormalities was investigated. The accuracy of measuring the integrity optical density (OD) of nuclei that correlates to DNA amounts of those nuclei, on slides stained by the Papanicolaou method, was assessed and compared with a standard Feulgen method. Our data on rat liver nuclei peritoneal washings from patients with ovarian cystadenofibromas and ovarian cystadenocarcinomas suggested that analysis of cytological material using the Papanicolaou method is not reliable and that destaining the slides followed by Feulgen staining provides an optimal and reliable method of DNA quantification.

Adenofibroma↗
PubMed1991

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification.

A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4 degrees C with 0.25% buffered paraformaldehyde then for 15 min at 37 degrees C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90 degrees angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7-AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of CD3 epsilon and on NALM-6 for expression of mu. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.

Antigens↗
PubMed1992

Simple method for quantification of fast plasma membrane movements.

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (image 1); 28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in micron2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and by epidermal growth factor.

Animals↗
PubMed1992

Quantification of an in vitro cell-cell adhesion assay using interactive laser scanning cytometry.

We are interested in identifying cell-cell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cell-spermatogenic cell adhesion. Numerous cell-cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225-500 microns2, spermatids were 100-225 microns2 and residual bodies were less than 100 microns2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub-classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell-cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell-cell adhesion assay in which one cell type is adherent to a substrate.

Animals↗
PubMed1992

DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissue.

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.

Adult↗