bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chromatid contamination can impair the purity of flow‐sorted metaphase chromosomes
Authors: Telenius Håkan , de Vos Diana , Blennow Elisabeth , Willat Lionel R. , Ponder Bruce A. J. , Carter Nigel P. 
Published: August 2005

AbstractWe describe a phenomenon where a second flow karyotype is superimposed on the normal metaphase flow karyotype of human lymphoblastoid chromosomes. The events of this second flow karyotype contain half the DNA content of corresponding events in the normal flow karyotype and, when sorted, show the morphology of single chromatids. DNA amplified by degenerate oligonucleotideprimed PCR of 300 chomatids sorted from a single peak and hybridized onto normal metaphase spreads painted the entire length of the corresponding chromosome type. In retrospect, we could observe this phenomenon at a low level in 37% of randomly selected, previously analyzed flow karyotypes and in several published flow karyotypes from other laboratories. Experimentally, chromatid frequency was shown to vary with the colcemid blocking conditions as well as with the cell line used. At the extreme, we obtained a 4:10 ratio of chromatids to metaphase chromosomes, whereas this could be reduced tenfold by altering the colcemid blocking conditions in the same cell line. The presence of chromatids in chromosome preparations has important implications for the purity of isolated fractions used for generating libraries and in PCR analysis. © 1993 Wiley‐Liss, Inc.
Three‐marker phenotypic analysis of lymphocytes based on two‐color immunofluorescence using a multinomial model for flow cytometric counts and maximum likelihood estimation
Authors: van Putten W. L. J. , Kortboyer J. , Bolhuis R. L. H. , Gratama J. W. 
Published: August 2005

AbstractThe simultaneous flow cytometric study of multiple (≥ 3) markers on individual cells is restricted by technical reasons, e.g., the type of flow cytometer, and the availability of (monoclonal) antibodies (mAb) conjugated with the appropriate fluorochromes. However, a n‐way classification (n ≥ 3) may be derived from multiple 2‐way classifications. The 2‐way classifications are obtained by the use of only two fluorochromes, but each fluorochrome may be used for the simultaneous labelling of 2 or more mAb. We present a formal statistical model, based on an underlying multinomial distribution for the observed quadrant counts, by which data from such multiple 2‐way classifications can be analyzed. The model is restricted to 3‐marker phenotypic analyses, but can be extended to n‐way classifications (n > 3). Maximum likelihood estimates are obtained by the application of the EM algorithm. The model was tested on 20 samples of peripheral blood mononuclear cells to study the coexpression of CD45RA, CD45RO, and Leu 8 by lymphocyte subsets defined by the CD56 (MHC‐unrestricted cytotoxic cells) and CD3 (T cells) markers. Application of the model gave an excellent fit in all but one cases. In addition, the model reduced the effect of inter‐assay variation on the estimates and it provided an analysis of consistency over the data, which allows the detection of outliers due to staining errors. © 1993 Wiley‐Liss, Inc.
Method for counting mitoses by image processing in feulgen stained breast cancer sections
Authors: ten Kate T. K. , Beliën J. A. M. , Smeulders A. W. M. , Baak J. P. A. 
Published: August 2005

AbstractThis study describes an image processing method for the assessment of the mitotic count in Feulgen‐stained breast cancer sections. The segmentation procedure was optimized to eliminate 95–98% of the nonmitoses, whereas 11% of the mitoses did not survive the segmentation procedure. Contour features and optical density measurements of the remaining objects were computed to allow for classification. Twelve specimens were analyzed, nine used to serve as a training set, and three put aside for later use as independent test set. The fully automatic image processing method correctly classified 81% of the mitoses at the specimen level while inserting 30% false positives. The automatic procedure strongly correlated with the interactive counting procedure (r = 0.98). Although the fully automatic method provided satisfactory results, it is not yet suited for clinical practice. The automated method with an interactive evaluation step gave an accurate reflection of the mitotic count showing an almost perfect correlation with the results of the interactive morphometry (r = 0.998). Therefore this semiautomated method may be useful as prescreening device. © 1993 Wiley‐Liss, Inc.
Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy
Authors: Puppels G. J. , Garritsen H. S. P. , Kummer J. A. , Greve J. 
Published: August 2005

AbstractThe presence and subcellular location of carotenoids in human lymphocyte sub‐populations (CD4+, CD8+, T‐cell receptor‐γδ+, and CD19+ ) and natural killer cells (CD16+ ) were studied by means of Raman microspectroscopy. In CD4+ lymphocytes a high concentration (∼10−3M) of carotenoids was found in the Gall body. In CD8+ lymphocytes, T‐cell‐receptor‐γδ+ lymphocytes and in natural killer cells carotenoids appeared to be concentrated (∼10−4M) in the Golgi complex. The concentration of carotenoids in CD19+ lymphocytes was found to be below the present detection limit, which is ∼10−6 to 10−5 M. The results provide new possibilities to investigate the mechanism(s) behind the suggested protective role of carotenoids against the development of cancers. © 1993 Wiley‐Liss, Inc.
Characterization of hypoxia‐dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: A model for ischemic tissue destruction
Authors: Yurkow Edward J. , McKenzie Maureen A. 
Published: August 2005

AbstractPeroxide production in cultures of Saccharomyces cerevisiae was measured using the H2O2‐sensitive fluorescent probe 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) and flow cytometry. Aeration of cultures of S. cerevisiae exposed to a period of hypoxia was found to induce elevated levels of peroxide that were 100‐fold higher than the levels observed in cultures maintained under exclusively aerated or hypoxic conditions. Simultaneous viability analysis, using the fluorescent DNA‐intercalating dye propidium iodide, indicated that the increase in peroxide generation preceded cell damage and death. Various agents were found to influence the effect of peroxides on cell viability. The addition of ethanol to hypoxic stationary cultures dramatically increased the rate of cell death without further increasing the amount of peroxide produced, while glucose inhibited peroxide production and decreased the rate of cell death. Surprisingly, elevated peroxide levels of hypoxic/reaerated cultures were maintained upon addition of KH2PO4, although the cells remained viable for extended periods of time when compared to control and other test cultures. Similarities between our observations and those of other investigators using anoxic/reperfused organs suggest that hypoxic/reaerated yeast cultures may be a useful model system to study ischemia‐dependent tissue destruction of mammals. © 1993 Wiley‐Liss, Inc.
Evaluation of a dual‐color flow cytometry immunophenotyping panel in a multicenter quality assurance program
Authors: Schenker Eve Levine , Hultin Lance E. , Bauer Kenneth D. , Ferbas John , Margolick Joseph B. , Giorgi Janis V. 
Published: August 2005

AbstractA basic immunophenotyping panel that employed dual‐color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC‐CD45/PE‐CD14, FITC‐IgG1/PE‐IgG2, FITC‐CD3/PE‐CD8, FITC‐CD3/PE‐CD4, FITC‐CD3/PE‐CD16+PE‐CD56, and PE‐CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1½ year period, 78 shared peripheral blood specimens Were prepared and analyzed in each laboratory. The CD45brightCD14− percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV‐seropositive (n = 40) specimens using this panel were <3% for total T cells; <5% for CD4+ T cells and CD8+ T cells; ≤17% for B and NK cells; and <8% for CD4T/CD8T ratios. The 6‐tube basic immunophenotyping panel has several notable features: (a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; (b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and (c) for within‐sample quality assurance, it provides several quality control checks, including the lym‐phosum, i.e., the sum of an individual's corrected T + B + NK values, a sum that was generally 100 ± 5% on the HIV‐seronegative specimens analyzed in this study. © 1993 Wiley‐Liss, INc.
Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements
Authors: Davis Bruce H. , Dicorato Michael , Bigelow Nancy C. , Langweiler Marc H. 
Published: August 2005

AbstractFlow cytometric (FCM) reticulocyte analysis is more accurate, sensitive, and reproducible relative to previously employed manual microscopic methods in clinical laboratory hematology. FCM reticulocyte analysis using RNA binding fluorochromes additionally allows for the quantification of fluorescence intensity or population distribution of the reticulocyte RNA content. Viewed from the perspective of erythroid maturation, quantification of the fluorescence intensity distribution provides a reticulocyte maturation index (RMI). We performed a systematic study of 18 different methods to express thiazole orange stained reticulocyte fluorescence intensity, compared to standard mean fluorescence intensity quantification, using 185 anemic and non‐anemic human blood samples. The method best correlating with the mean fluorescence intensity RMI on 2 different FCM instruments (R2 = 0.93 and 0.86) was a ratio of the highly fluorescent reticulocytes, defined using a normal adult population, and the total number of reticulocytes (HFR%). In contrast to mean fluorescence intensity measurements, a HFR% RMI parameter can provide similar units of expression (0.01–1.00) with good correlation between different FCM instruments (R2 = 0.76). We conclude the HFR% method of RMI expression provides a superior means of interlaboratory standardization and clinical comprehension of this useful diagnostic parameter in clinical laboratory hematology. © 1993 Wiley‐Liss, Inc.
Detection of in situ hybridization to human chromosomes with the atomic force microscope
Authors: Putman C. A. J. , De Grooth B. G. , Wiegant J. , Raap A. K. , Van der Werf K. O. , Van Hulst N. F. , Greve J. 
Published: August 2005

AbstractAtomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1‐79 results in clear marking of the telomere region 1p36. The diameter of the probe p1‐79 linked reaction product was 75–100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material. © 1993 Wiley‐Liss, Inc.
DNA content in eurasian sturgeon species determined by flow cytometry
Authors: Birstein Vadim J. , Poletaev Andrei I. , Goncharov Boris F. 
Published: August 2005

AbstractThe nuclear DNA content in 10 species of chondrostean fishes was measured by flow cytometry. The sterletAcipenser ruthenusblood cells were used as an internal standard. The sterlet DNA content was calculated on the basis of comparison with theXenopus laevisblood cells, 2C = 6.30 pg. In the tetraploidA. ruthenusandA. stellatusthe DNA content comprises 3.74 pg/nucleus and is practically invariant; inHuso dauricusit is almost the same, 3.74–3.81 pg; and inA. nudiventrisit is a little higher, 3.88–4.04 pg. In the oldest chondrostean,Pseudoscaphirhynchus kaufmanni, the nuclear DNA content is slightly lower, 2C = 3.46–3.48 pg, and in the American paddlefishPolyodon spathulait is lower still, 3.17 pg. In two octoploid sturgeons,A. baeriandA. gueldenstaedti, the DNA content is twice as high as that of the sterlet, 8.29–8.31 and 7.86–7.88 pg, respectively; a very similar amount, 8.24–8.42 pg, was determined in the hybridHuso huso×A. ruthenus. In the Sakhalin sturgeon,A. medirostris(=A. mikadoi), the DNA content is two times higher than in the octoploids, 13.93–14.73 pg; therefore its ploidy may be 16n and the number of chromosomes could be 500. © 1993 Wiley‐Liss, Inc.
Isolation and characterization of CD34+ hematopoietic stem cells from human peripheral blood by high‐gradient magnetic cell sorting
Authors: Kato Kimitaka , Radbruch Andreas 
Published: August 2005

AbstractCD34+ cells, present at a frequency of 0.18 ± 0.052% among leukocytes from peripheral blood (PB), can be rapidly and efficiently (recovery of 39.0–74.0%) enriched to a frequency of 38.6–87.1% (54.4 ± 12.3%) by high‐gradient magnetic cell separation (MACS) for immunophenotyping, characterization in colony‐forming cell (CFU) assays, and further purification to homogeneity (>98%) by multiparameter fluorescence‐activated cell sorting (FACS). Enriching PB‐CD34+ cells for immunophenotyping allows the detection of small subpopulations, expressing the B‐cell antigens CD10, CD19, and CD20, the T‐cell antigens CD45RA and CD7, and a small subpopulation expressing high levels of CD34 (1.20 ± 0.12%), which mostly coexpress CD19 (91.9 ± 9.05%), CD20 (64.8 ± 14.4%), and CD38 (84.5 ± 10.3%). All PB‐CD34+ cells express elevated levels of CD71 (transferrin receptor), with a subpopulation of high expressing cells, and CD38. Some cells express CD33. MACS‐enriched PBCD34+ cells show “normal” hematopoietic colony formation in vitro. The ease and efficiency of purification of large numbers of CD34+ cells from PB by MACS is not only relevant for the characterization of migrating stem cells but also opens new possibilities for stem cell transplantation and genetic manipulation of the hematopoietic system. © 1993 Wiley‐Liss, Inc.
Forward scatter pulse width signals resolve multiple populations of endosomes
Authors: Hammond Timothy G. , Majewski Rebecca R. , Morré D. James , Schell Kathleen , Morrissey Lawrence W. 
Published: August 2005

AbstractThe technique of pulse width analysis, developed to optimize cell size resolution in cell cycle kinetics, has not previously been applied to small particles such as endosomes. Offset is used to subtract a portion of the beam diameter from forward scatter pulse width signals to optimize visualization and discrimination of small particles. We identify multiple endosomal populations by offset pulse width of light scatter parameters. Specifically, linear forward scatter pulse width measurements reveal at least two populations of endosomes in the rat renal cortex, the rat renal papilla, and the luminal endothelium of the toad urinary bladder. Logarithmically amplified forward scatter pulse width measurements display the full dynamic range of these signals, resolving additional populations not manifest with linear amplification.To confirm that the endosomes observed were resolved from optical and electronic noise, we examined physiological function. The endosomes acidified after supplying ATP to the intrinsic membrane H+‐ATPase present. Further, electron microscopy of sorted endosomal populations from the toad urinary bladder confirmed identity and homogeneity of the fraction.Flow cytometric analysis of endosomal populations by multiparametric techniques including pulse width analysis of structural parameters and pulse height analysis of fluorescence from entrapped fluorophores allows identification, isolation, and quantification of multiple endosomal populations. © 1993 Wiley‐Liss, Inc.
Signal processing in slit‐scan flow cytometry of cell conjugates
Authors: Bakker Schut Tom C. , Florians Alex , Radošević Katarina , de Grooth Bart G. , Greve Jan 
Published: August 2005

AbstractThe design and implementation of a real‐time signal processing system for slit‐scan flow cytometry is described. The system is used to measure the separate scatter and fluorescence peak heights of 2 adherent cells. Preliminary measurements of changes in the membrane potential induced by interactions between natural killer (NK) cells and their target cells are presented. © 1993 Wiley‐Liss, Inc.
Flow cytometric method for simultaneous detection of lymphocyte‐K562 conjugates and immunophenotyping of the conjugate forming cells
Authors: Radošević Katarina , de Grooth Bart G. , Greve Jan 
Published: August 2005

AbstractA flow cytometric method for the simultaneous quantification and immunophenotyping of conjugates formed by human peripheral blood lymphocytes (PBL) and K562 cells has been developed. The method uses three fluorescent probes. One of the fluorescent probes (F‐18) is used for labeling of PBL prior to incubation with K562 cells. After incubation the cells are treated with monoclonal antibodies labeled with phycoerythrin and Red613, respectively. The combination of F‐18 fluorescence and light scattering signals enables identification and quantification of the conjugates while the fluorescence of the monoclonal antibodies provides information about the phenotype of the conjugate forming cells. Results obtained using different monoclonal antibodies are presented. The highest conjugate forming capacity has been found in the CD56+CD8+ population while the CD4+CD8− population has shown the lowest capacity to form conjugates. The influence of a washing step on the conjugate formation is discussed. The possibility to use the method in combination with a cytotoxicity assay is indicated. © 1993 Wiley‐Liss, Inc.
Dna ploidy status and DNA content instability within single tumors in renal cell carcinoma
Authors: Bringuier P. P. , Bouvier R. , Berger N. , Piaton E. , Revillard J. P. , Perrin P. , Devonec M. 
Published: August 2005

AbstractIn order to investigate the relationship between genetic instability and DNA aneuploidy of malignant cells in human solid tumors, we studied the variations of DNA index within a single tumor. Multiple sampling (mean of 6.4 samples per tumor) was performed in 24 renal cell carcinomas (RCC). Based on the variations in DNA indices within a single tumor, RCC were divided into three groups: (1) tumors with stable DNA indices (variation within the range of the measurement error), including all DNA diploid tumors (n = 8), all hyperdiploid tumors (n = 3), a hypodiploid tumor (n = 1), and only 2 of 11 tetraploid and hypotetraploid tumors; (2) tumors with moderate variations in DNA indices, which were all close to tetraploidy (n = 5); and (3) tumors with large variations of DNA indices. In this last group, subclones present within a tumor varied widely in their DNA indices from the tetraploid to the triploid region, reflecting the DNA content instability within the tumor cell population. These results suggest that DNA aneuploidy can arise by two different mechanisms: (1) loss or gain of chromosomes leading to hypodiploid or hyperdiploid tumors with no apparent increase in DNA content instability, and (2) doubling of the chromosome set followed by random loss of chromosomes as suggested by the DNA indices ranging from tetraploid to triploid region found in three tumors. Differences in DNA indices within one tumor characterize subclones which may arise by chromosome loss or gain. © 1993 Wiley‐Liss, Inc.
Quality control study of the italian group of cytometry on flow cytometry cellular DNA content measurements
Authors: Danesi D. Tirindelli , Spanòg M. , Altavista P. 
Published: August 2005

AbstractA quality control study on DNA flow cytometry, extended to 43 national laboratories, has been carried out by the Italian Group of Cytometry, using defined fixed suspensions of cultured human leukemia K562 cells and human blood lymphocytes. The participating laboratories were allowed to follow their own staining and measurement protocols. Aliquots of cellular suspension had to be measured three times on the same day and two other times on different days. A large heterogeneity of procedures emerged among participants. The average of mean DNA index laboratory values, from 36 laboratories who sent evaluable data, was 1.68, with a range from 1.49 to 1.97. The coefficients of variation ranged from 2.35 to 9.39% and from 2.79 to 8.5% for diploid and aneuploid peaks, respectively. Statistical analysis of the results showed quite good intralaboratory reproducibility, but statistically significant differences were observed among laboratories, for both DNA indices and coefficients of variation. These differences appear to be consistent. For standardization, it is essential that efforts should be made to identify the main sources of variation and to control them. © 1993 Wiley‐Liss, Inc.
Increased membrane permeability of apoptotic thymocytes: A flow cytometric study
Authors: Ormerod Michael G. , Sun Xiao‐Ming , Snowden Roger T. , Davies Reginald , Fearnhead Howard , Cohen Gerald M. 
Published: August 2005

AbstractWe have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes. © 1993 Wiley‐Liss, Inc.
The use of flow cytometry for the investigation of cell death
Authors: Afanasyev Vladimir N. , Korol Boris A. , Matylevich Natalya P. , Pechatnikov Vladimir A. , Umansky Samuil R. 
Published: August 2005

AbstractFlow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented. © 1993 Wiley‐Liss, Inc.