bluuebunny/crossref_metadata_2025_split huggingface dataset:
Long‐term storage of samples for flow cytometric DNA analysis
Authors: Vindeløv Lars L. , Christensen Ib J. , Keiding Niels , Spang‐Thomsen Mogens , Nissen Nis I. 
Published: August 2005

AbstractA simple procedure for long‐term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine‐needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were stored for up to one year by freezing at —80°C. Statistical analysis of the results showed no change in the fractions of cells in the cell cycle phases as determined by deconvolution of the DNA‐histograms. It was found that in addition to the intrinsic sample variation from the parameter estimation by deconvolution, there was significant intraday and interday variation. Hence the most accurate results are obtained if different aliquots of a sample are measured on different days rather than on the same day. Use of the storage method thus has the potential of increasing the accuracy of the analysis. The storage method makes sample collection independent of immediate subsequent analysis. This has enabled us to perform large internally controled experiments, involving more samples than can be analyzed in one day, to examine tumor sampled from different hospitals and to utilize fully the capacity of our flow cytometer. The method was a prerequisite for developing an accurate standardization procedure for DNA content determination.
A Detergent‐trypsin method for the preparation of nuclei for flow cytometric DNA analysis
Authors: Vindeløv Lars L. , Christensen Ib J. , Nissen Nis I. 
Published: August 2005

AbstractA new modification of our detergent technique for the preparation of nuclei for flow cytometric DNA analysis is described. The attainment of low coefficients of variation of the peaks and of quantitative staining of nuclei from different tissues was a problem with the original method. This was solved in the new modification by trypsinization of the unfixed nuclei. The nuclei were stabilized by spermine. A simple procedure for long‐term storage of samples at —80°C was integrated into the method. The fluorescence of the nuclei was stable for at least 3 hours after staining. Light exposure protection of the samples was essential. No cell loss was caused by storage or staining. The method was successfully applied on samples including: (a) Normal tissues— human lymphocytes, granulocytes and spleen. Mouse lymphocytes, bone marrow, spleen, liver, kidney and thymus. (b) Human neoplasms— lung cancer, breast cancer, lymphoma, leukemia, bladder cancer and cancer of the oral cavity. (c) Human tumors in nude mice— breast cancer, lung cancer, melanoma and colon cancer. (d) Mouse ascites tumors— JB‐1, L 1210, Ehrlich and P 383. It therefore seems well suited as a routine clinical procedure.
Standardization of high‐resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards
Authors: Vindeløv Lars L. , Christensen Ib J. , Nissen Nis I. 
Published: August 2005

AbstractDetermination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time‐consuming and inaccurate. Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and the sample and used to indicate the DNA content (DNA ratio) is, however, very sensitive to changes in the zero level adjustment of the flow cytometer. If two internal standards are used the DNA ratio becomes independent of the zero level. Rainbow trout red blood cells (TRBC) have a DNA content of 80% of human diploid cells. A mixture of CRBC and TRBC was prepared and stored in small aliquots at —80°C. This mixture was added to the sample before staining. The day‐to‐day variation of the DNA ratio obtained by use of the two standards was smaller than that obtained by CRBC alone. The possibility of sex related differences in DNA content of CRBC and TRBC was examined. The results indicated that a new batch of standards should be tested against the old batch to avoid the introduction of a systematic error.
Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample‐storage, staining and internal standardization
Authors: Vindeløv Lars L. , Christensen Ib J. , Jensen Grethe , Nissen Nis I. 
Published: August 2005

AbstractFlow cytometric DNA analysis is an attractive alternative to conventional cytogenetics for diagnosing karyotype changes resulting in abnormal DNA content. We have developed methods for long‐term storage of samples and standards, for staining the cells and for standardization of the measurements by two internal standards. In this paper the currently attainable resolution was determined and limiting factors were identified. Normal reference values for male and female leucocytes were determined by analyzing 240 samples from six men and six women, all cytogenetically normal. The DNA content of female cells was 1.5% higher than that of male cells. This finding was used to correct the results for sex related DNA differences. The 95% confidence limits for mononuclear blood cells were ±0.79%. The results on granulocytes were exceptionally variable, with 95% confidence limits of ±1.79%. Reexamination of the normal leucocytes after 1 year showed a long‐term drift of up to 2% in the results, indicating a need for regular checks of the reference values. In addition to the sex related differences in DNA content, individual differences of up to 1% were demonstrated. This creates problems as to which reference value to use for a particular sample. Furthermore, tissue related differences in fluorescence were found when six different tissues from one mouse were examined. The means had a range of 0.7%. The tissue related differences add uncertainty to the interpretation of the results. The resolution of heterogeneous populations with slightly different DNA content was examined by analyzing mixtures of cells with known DNA differences ranging from 1.50% to 5.83%. With a coefficient of variation of the peaks of about 2% a DNA difference of approximately 4% was required for accurate determination of the DNA content of the two individual populations. A lower coefficient of variation would increase the resolution, but tissue related differences in fluorescence could then become the limiting factor.
Characterization of the uptake and toxicity of a fluorescent thiol reagent
Authors: Olive Peggy L. , Biaglow John E. , Varnes Marie E. , Durand Ralph E. 
Published: August 2005

Abstract3‐(4‐Maleimidylphenyl)‐4‐methyl‐7‐diethylamino coumarin (CPM), is a fluorescent thiol‐binding agent. CPM is nontoxic to Chinese hamster V‐79 cells (2 × 105 cells/milliliter) exposed to 2.5 μg/milliliter for 30 minutes. However, both toxicity and cellular binding were directly dependent on the drug:cell ratio. Using flow cytometry, cellular binding of CPM correlated with inhibition of DNA synthesis. In cells pretreated with the thiol binding drugs N‐ethylmaleimide, diamide and diethylmaleate or the carcinogens 4‐nitroquinoline‐1‐oxide and AF‐2 (furylfuramide), the subsequent binding of CPM was reduced by 40% or more.
Optimization of mammalian chromosome suspension preparations employed in a flow cytometric analysis
Authors: Th. Bijman J. 
Published: August 2005

AbstractMetaphase chromosomes from Chinese hamster V‐79 cells have been stained with propidium iodide and their DNA content was measured by flow cytometry. The chromosomes in suspension must be stabilized during the preparation to preserve their morphological integrity. In order to obtain high resolution in the flow karyotype, several modifications were made to the method used to prepare chromosome suspensions. The influence of several ions, detergents and detergent mixtures was investigated. Magnesium (3‐5 mM) together with sodium (10 mM), Tris (10 mM) and Triton X‐100 (0.4%) gave the best results. In contrast, sodium and potassium exerted no effect, while calcium had a negative effect on the preparation as measured with flow cytometry. Compared with a solution of potassium chloride + propidium iodide, chromosomes prepared in Tris buffer resolved better with less background. A histogram quality factor which contains the variables peak height, depth of valley and coefficient of variation, was used to quantitate the histograms.
High resolution method for the analysis of DNA histograms that is suitable for the detection of multiple aneuploid G1 peaks in clinical samples
Authors: Schuette W. H. , Shackney S. E. , Maccollum M. A. , Smith C. A. 
Published: August 2005

AbstractThe DNA histogram obtained by flow cytometry can be considered as the product of an “ideal” measurement column vector and a measurement distortion matrix. In order to extract the ideal histogram from the real data, the measurement distortion matrix is commonly presumed to be a family of Gaussian coefficients that are centered on the diagonal. We have designed a feedback‐controlled curve‐fitting procedure that reconstructs the ideal histogram from the real data through successive iterations. The optimum coefficient of variation (cv) for the family of Gaussians in the measurement distortion matrix is determined from an analysis of the sums of squares of fits of the computed DNA histogram to the real data over an appropriate range of trial cv. Since this method assigns a Gaussian to each and every data channel, it permits the resolution of closely spaced multiple aneuploid G1 peaks in clinical samples. The effects of high frequency noise that may be present in the data can be attenuated by multiplying the real data histogram by a Gaussian matrix with cv close to but smaller than that of the measurement distortion matrix.
DNA flow cytometric analysis of monogranulocytic colony forming cells from mouse bone marrow in Vitro
Authors: Ronot X. , Adolphe M. , Hecquet C. , Fontagne J. , Jaffray P. , Lechat P. 
Published: August 2005

AbstractAn adaptation of a previously reported flow cytometric technique is described. This technique was applied to study the DNA distribution of mouse granulocyte‐macrophage colony forming cells (GM‐CFc) grown in a methyl cellulose culture system. This method involved the collection of cell clusters and colonies from the methyl cellulose culture medium for the subsequent determination of DNA content. The DNA content of the homogeneous GM‐CFc suspensions was determined by applying a modification of the propidium iodide staining procedure described by Crissman et al. The relative percentage of cells in G0 + G1, S, and G2 + M stages of the cell cycle was calculated by applying mathematical analysis to the resulting DNA histograms. This method could be useful in studying the effects of drugs on GM‐CFc kinetics.
A simple density gradient for enriching subfractions of solid tumor cells
Authors: Walle Alexander , Kodama Tetsuro , Melamed Myron R. 
Published: August 2005

AbstractCell suspensions of human solid tumors could be separated into 5 or 6 subfractions for flow cytometry by centrifugation on a continuous silica gel (Percoll, Pharmacia Fine Chemicals, Upsalla, Sweden) density gradient. In all cases, there were one or more subfractions with enrichment of the aneuploid tumor cells. The greatest enrichment occurred in cases with fewer than 20% aneuploid cells in the original specimen; for these cases the enrichment factor ranged from 2.8‐ to 11.7‐fold increase. In six cases with fewer than 7% aneuploid G0/1 cells initially, the aneuploid G0/1 DNA peak was difficult to demonstrate on the original specimen but became quite obvious in the subfraction. In no case were the tumor cells confined to a single subfraction. The distribution of tumor cells within two or more subfractions appeared related to differentiation in the case of squamous cells and to RNA content for other tumor cells. DNA/RNA flow cytometry proved useful in defining subpopulations of tumor cells with different DNA stemlines and varying positions in the cell cycle according to the RNA content of the respective subpopulation.
Classification of immature and mature cells of the neutrophil series using morphometrical parameters
Authors: Bins M. , van Montfort L. H. , Timmers T. , Landeweerd G. H. , Gelsema E. S. , Halie M. R. 
Published: August 2005

AbstractQuantitative morphological data of six classes of immature and mature cells of the neutrophil series of the bone marrow of normal persons were used for statistical classification experiments (myeloblasts, promyelocytes, myelocytes, metamyelocytes, bands and segments). On each cell, parameters were measured directly from the image or calculated from the shape of the density histogram or the counting densitogram using a Texture Analysis System (E. Leitz, Wetzlar, Germany). The parameters were analyzed with the interactive statistical pattern recognition system ISPAHAN. One half of the data were used as a learning set and the other half as the test set. The parameters were compared according to their performance in discrimination between the classes, alone and in combinations. Parameters not contributing to an improvement of the discrimination were disregarded. Eleven parameters were selected and used for classification by two different methods: a stepwise and a “one‐shot” method. Stepwise classification resulted in a 79% correct classification rate. Most errors occurred between cell classes in neighboring stages of maturation. In 96% of all cases the computer classification was either in accordance with that of the technician or with a cell class of a neighboring maturation stage. One step classification by the computer was in agreement with the technicians in 82% of the cases. For 98% of the cells the computer classification was either in accordance with that of the technician or with a cell class of a neighboring maturation stage.The data set was collected by two technicians, operating independently. Differences in their interpretation of the maturation stage were found by comparing the performance of classifiers based on both cell samples. Since the images of the cells were not available for reexamination, the causes of disagreement in classification between the technicians and between computer and technicians could not be evaluated.
A flow cytometric study of the propidium iodide staining kinetics of human leukocytes and its relationship with chromatin structure
Authors: Mazzini Giuliano , Giordano Paolo , Riccardi Alberto , Montecucco Carlo Maurizio 
Published: August 2005

AbstractA nonstoichiometric staining method based on the employment of propidium iodide at a concentration far below saturation is described. The method is particularly suitable for the measurement of staining kinetics and it allowed us to differentiate cell populations with the same DNA content but different chromatin conformation. Analyses have been performed on normal and pathologic human leukocytes. Normal mononuclear cells have been differentiated from polymorphonuclear cells and lymphoblasts.
DNA cytometry on four‐micrometer sections of paraffin‐embedded human renal adenocarcinomas and adenomas
Authors: Bennington James L. , Mayall Brian H. 
Published: August 2005

AbstractThis study assessed the diagnostic and prognostic utility of DNA cytometry on 4 μm sections of paraffin‐embedded human renal adenocarcinomas and adenomas.Twenty‐one renal cortical glandular neoplasms from patients who had been followed for up to 12 years after surgical removal of their tumors were examined. These tumors were classified histopathologically and included six adenomas and fifteen adenocarcinomas (six grade I, five grade II, three grade III, and one grade IV). For each tumor, 4‐μm sections were cut from paraffin‐embedded blocks retained from the original surgical specimen. Both tumor and normal renal cortex of the same kidney were included on each slide and stained with the azure A Feulgen reaction for DNA. Nuclei from 200 tumor cells and 100 control proximal convoluted tubular cells were selected at random for measurement of nuclear DNA stain content utilizing an integrating image cytometer. For each tumor, the cytometric measurements were standardized to the corresponding control measurements, plotted as histograms, and analyzed statistically; the distributions of the standardized measurements were described by features such as mean, variance, and interquartile range.The locations and shapes of the tumor histograms differed markedly from their controls. The values of the features all were significantly greater for each tumor than for its normal tissue control; the differences increased with increasing tumor grade. Multiple regression analysis of the features against histopathologic grade showed a clear and statistically significant correlation. This correlation allowed accurate prediction of tumor grade from the cytometric data. The cytometric features also discriminated accurately between patients with favorable and unfavorable outcomes. However, they showed no clear distinction between renal adenomas and grade I adenocarcinomas nor between oncocytic and clear cell tumors of the same grade. This study indicates that quantitative information obtained by DNA cytometry of thin sections can form the basis for improved histopathologic diagnosis and prediction of clinical outcomes for patients with renal adenocarcinoma.
Parametric analysis of histograms measured in flow cytometry
Authors: Mann R. C. , Hand R. E. , Braslawsky G. R. 
Published: August 2005

AbstractFlow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.
Rapid identification and detection of parasitized human red cells by automated flow cytometry
Authors: Whaun J. M. , Rittershaus C. , Ip S. H. C. 
Published: August 2005

AbstractRapid and reliable identification of various human red cell parasites is important in many chemotherapeutic and immunologic studies. Because manual microscopic counting is tedious and imprecise, we have developed a simple diagnostic procedure for the automated flow cytometric detection of in vitro infected red cells, using a nucleic acid‐binding fluorescent dye, acridine orange. Human malaria (Plasmodium falciparum)‐infected red cells from continuous human erythrocyte culture were incubated at room temperature in acridine orange stain for 5 min after which the sample were analyzed by flow cytometry. Since mature red cells contain no DNA, infected red cells were identified with a distinct fluorescent signal. A total of 200,000 cells per sample were counted and analyzed in less than 2 min. Rings, trophozites, and schizonts were assessed and identified in synchronozed infected red cell cultures by flow cytometry. In addition, various stages of infected red cells were isolated with a cell sorter. This rapid method permits accurate and reliable assessment of data with the exclusion of anomalous data such as damaged cells, extraneous material, and contaminating particles.
Isopycnic density‐gradient centrifugation: A separation parameter which improves flow cytometric measurements on heterogeneous tumors
Authors: Šonka J. , Stöhr M. , Vogt‐Schaden M. , Volm M. 
Published: August 2005

AbstractThe analytic and prognostic value of flow cytometric measurements can generally be improved by preseparation of cells. The pre sent data demonstrate clearly the distinct character of bands obtained in density‐gradient separation. Density‐gradient centrifugation in Percol after extensive Ca2+ + Mg2+ elimination helps to resolve more detail in flow cytometric measurements (e.g., additional DNA stem lines in human tumors) and can be used studies of tumor heterogeneity. The significance of investigations of tumor heterogeneity is demonstrated by the different reactivity of subpopulations of the experimental murine tumor S 180 to vincristine.
Characterization of ploidy level in bladder tumors and selected site specimens by flow cytometry
Authors: Jakobsen Anders , Mommsen Søren , Olsen Steen 
Published: August 2005

AbstractThe histopathological grade of atypia was compared with the ploidy level in biopsy specimens from bladder tumors and six grossly normal areas (selected sites). The study included investigation of 303 specimens from 45 patients. The results demonstrated a high correlation between the grade of atypia and the occurrence of aneuploid cell populations. The selected site specimens were diploid in patients with low grade malignant tumors, while high grade malignant tumors were associated with aneuploid selected site specimens in 20% of the cases. The divergence of ploidy level in the tumor and selected site specimens found in some cases clearly demonstrated that the carcinogenic process in the bladder may lead to clonal heterogeneity.Flow cytometric analysis seems suited for mapping of the ploidy levels in different parts of the bladder. The possible importance of the additional information is discussed.
Do all daughter cells enter the “indeterminate” (“A”) state of the cell cycle? Analysis of stathmokinetic experiments on L1210 cells
Authors: Kimmel Marek , Traganos Frank , Darzynkiewicz Zbigniew 
Published: August 2005

AbstractThe results of ten different stathmokinetic experiments on L1210 cells are analyzed to determine whether all the postmitotic cells enter the exponential AG1 compartment of the cell cycle characterized by exponentially distributed transit times. The analysis is based on a mathematical model coherent with the generalized A‐B‐transition hypothesis. An original fitting procedure is introduced to estimate the fraction of cells entering AG1 as well as other parameters of the cell cycle. Results of the analysis suggest that either all or nearly all postmitotic cells enter G1A. The results are discussed with respect to the validity of the A‐B‐transition hypothesis of the “probabilistic” model of the cell cycle. Some systematically occurring discrepancies that do not conform with generally accepted cell cycle models are apparent from the analysis of this data.
A method for staining 3T3 cell nuclei with propidium iodide in hypotonic solution
Authors: Tate Emily H. , Wilder Mark E. , Cram L. Scott , Wharton Walker 
Published: August 2005

AbstractA modification of the propidium‐iodide hypotonic sodium citrate method has been developed specifically for high‐resolution staining of mouse 3T3 cell nuclei for analysis by flow cytometry. The method employs a brief treatment of cells at 37°C with Triton X‐100 and RNAse in the presence of propidium iodide in hypotonic sodium citrate, followed by restoration to isotonicity with NaCl. The average CV obtained for the G1 peak was 3.5%, and the samples were stable for 1–2 weeks at 4°C. Compared to this technique, previously described propidium iodide‐staining methods gave poor resolution with 3T3 cells.