bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric enumeration of colonies for in vitro chemotherapeutic drug evaluation
Authors: Thornthwaite Jerry T. , Loutzenhiser Kathy , Hornicek Francis J. , Donahue Roger , Allen Larry M. 
Published: August 2005

AbstractThe percentage of 50–100 μ colonies formed by LX‐T cells in medium containing agarose was determined microscopically, and this value was compared with the percentage determined by a flow cytometric method based on the forward and 90° light scatter of the colonies. As assessed by both in vitro methods, LX‐T cells exposed to chemotherapeutic agents formed fewer colonies as the drug concentration increased. However, flow cytometric analysis indicated that a change in the number of colonies formed was a consequence of changing chemotherapeutic drug concentration, whereas microscopic colony counting did not always detect the corresponding change in colony number. These experiments demonstrate that measurement of a drug's chemotherapeutic potential by flow cytometric counting of colonies is an alternative to the enumeration of colonies microscopically.
Analysis of bivariate flow karyotypes
Authors: Dean P. N. , Kolla S. , Van Dilla M. A. 
Published: August 2005

AbstractBivariate flow karyotype analysis is performed using data from chromosomes stained with two fluorescent dyes, typically chromomycin A3 and Hoechst‐33258, and measured in a flow cytometer or cell sorter (Carrano et al.: Proceedings of the National Academy of Sciences of the United States of America 76:1382–1384, 1979; Gray et al.: Proceedings of the National Academy of Sciences of the United States of America 72:1231–1234, 1975; Langlois et al.: Proceedings of the National Academy of Sciences of the United States of America 79:7876–7880, 1982). In the resulting bivariate histogram, most chromosome types appear as individual peaks. In sorting of chromosomes to purify a specific chromosomal type, its corresponding peak in the bivariate histogram is delineated by a rectangular region which surrounds it. All events (objects) that fall within this region trigger the sorting process. In most cases, peaks for different chromosomal types overlap to some extent, and in addition there is always an underlying background due to chromosome fragments and clumps. Thus the sorted population will not be pure; it may include more than one chromosome type and will include debris. To determine the purity of a sort, i.e., the percentage of the sorted material that is of the actual chromosomal type desired, two methods of mathematical analysis have been developed. In the more general method, the bivariate data within an analysis region that Includes the sort region, are fit with a series of bivariate Gaussian functions, one for each peak. In a simplified method, the data within the analysis region are transformed into a univariate distribution of either chromomycin A3 or Hoechst‐33258 fluorescence. The peaks in these univariate distributions are fit with univariate Gaussian functions. In both methods the purity is determined mathematically. The results of both methods agree well with independent methods of analysis.
Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo‐1 AM
Authors: Lopez Marc , Olive Daniel , Mannoni Patrice 
Published: August 2005

AbstractThe present report describes the increase in cytosolic‐free calcium levels (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo‐1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F‐Met‐Leu‐Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide‐angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin‐1α (IL‐1α), granulocyte‐colony stimulating factor (G‐CSF), nor granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), which were present in this supernatant, were able to induce a Ca2+i rise in PMN.
Improvement of flow‐cytometric detection of multidrug‐resistant cells by cell‐volume normalization of intracellular daunorubicin content
Authors: Ross Douglas D. , Thompson Bruce W. , Ordóñez José V. , Joneckis Christopher C. 
Published: August 2005

AbstractTo improve the ability of flow cytometry to detect multidrug‐resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL‐60 human leukemia cells exposed to DNR (1 m̈g/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23‐58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug‐sensitive P388 or HL‐60 cells measured by flow cytometry was 12‐ and 8‐fold greater than that of the multidrug‐resistant sublines P388/ADR and HL‐60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow‐cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug‐resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow‐cytometric assay to discriminate resistant cells by 10‐fold for P388 cells and 100‐fold for HL‐60 cells. Thus, by normalization of cell volume, the sensitivity and specificity of flowcytometric detection of multidrug‐resistant cells are improved.
1,3′‐diethy 1‐4,2′‐quinolylthiacyanine iodide as a “thiazole orange” analogue for nucleic acid staining
Authors: Van Bockstaele Dirk R. , Peetermans Marc E. 
Published: August 2005

AbstractFlow cytometric evaluation of reticulocytes has become a valuable and more precise alternative for the microscopically determined reticulocyte count. Until now, the most promising results are obtained with the “thiazole orange” (TO) nucleic acid dye. We report on the use of a “thiazole orange” analogue, l,3′‐diethyl‐4,2′‐quinolylthiacyanine iodide (DEQTC), which gives comparable results and is commercially available at low cost.
Flow cytometry using paraffin‐embedded tissue: Five years on
Authors: Hedley David W. 
Published: August 2005

AbstractThe use of paraffin‐embedded tissue for flow cytometry is reviewed. A number of technical modifications of the original 1983 method have been described, aimed at improving the accuracy of DNA measurements by minimizing cell debris or reducing coefficients of variation, and at simplifying sample preparation. Over 100 clinical studies have now been reported, mainly assessing the effect of DNA index on prognosis, and those published up until mid‐1988 are summarized in an appendix. More recently there have been developments in the use of monoclonal antibodies to measure oncogene products or proliferation markers in addition to DNA content. Detailed clinical evaluation and standardization of these more sophisticated methods is still some way ahead, but as was the case with DNA index, the use of archival material from patients whose outcome is already known should speed this process.
A digital image microscopy system for rare‐event detection using fluorescent probes
Authors: Lee Benjamin R. , Haseman David B. , Reynolds C. Patrick 
Published: August 2005

AbstractInstrumentation for rare‐event analysis should be capable of reliably detecting infrequent cells (< 1:10,000) while both excluding false‐positive signals and including true positive cells found in multicell clumps. We have developed a digital image microscopy (DIM) system in which a cytospin of 2 million cells is scanned with an intensified video camera (ISIT) using an IBM PC AT microcomputer‐controlled microscope stage. PASCAL software controls the stage and analyzes video input, storing the location of positive cells to magnetic disk. The user can then “replay” each positive cell under computer control for either visual confirmation or analysis using other fluorescent probes. The computer requires 24 min to scan a cytoprep of 2 million cells, while playback for visual confirmation by the user averages 5 min. Using Hoechst–33342 premarked cells seeded into bone marrow as a model system, we found that the DIM system reliably detects one target cell per million marrow cells. With appropriate immunological markers, this system will aid in evaluating bone marrow purged of tumor cells prior to transplantation and should also be useful for detection of minimal residual disease in blood or bone marrow from patients with leukemia or solid tumors.
Cell‐cycle studies by multiparametric automatic scanning of topographically preserved cells in culture
Authors: Colomb Evelyne , Kopp Francis , Spyratos Frédérique , Martin Pierre‐Marie 
Published: August 2005

AbstractThe authors have developed a new methodology for characterizing in situ the cell cycle of human mammary epithelial cell lines. Using a SAMBA 200 cell image processor (scanning cytometry), 15 densitometric and textural parameters were computed on each Feulgen‐stained nucleus. Parameters computed from the grey level cooccurrence and run‐length section matrices allowed assessment of the chromatin pattern. Multiparametric analysis of data defined: (1) the relative position of each cell; (2) the relative positions of groups of cells, each group corresponding to a definite phase of the cell cycle; and (3) the function of these parameters best separating these phases. Files then were constructed for each phase: G0/G1, S, G2/ and M. Using these three files as a reference to classify cells, it was possible to ascertain the phase of the cell cycle for each cell of a population.The MDA AG human cell line synchronized by mitotic selection was used as a model to develop this method. The criteria used to assign cells to G0/G1, S, or G2 was DNA content. Classification in M phase was achieved by visual identification of mitotic cells. This method was checked on unsynchronized MDA AG and then applied to other human cell lines (MDA MB231, MCF‐7, T47D C111). Comparison of results obtained by scanning cytometry and flow cytometry showed the proportion of cells assigned to G0/G1, S, and G2/M by the two methods to be similar. This new method removes some of the limitations of flow cytometry by (1) allowing visual verification of each cell analyzed; (2) lowering the number of cells required for study; (3) discriminating between G2 and M; and (4) preserving cell topography.
Model system evaluating fluorescein‐labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers
Authors: Vogt Robert F. , Cross G. David , Henderson L. Omar , Phillips Donald L. 
Published: August 2005

AbstractFluorescence intensity calibration was evaluated in a model system for flow cytometers using commercially available fluorescein‐labeled microbeads as internal standards and stabilized fluoresceinated thymus cell nuclei (Fluorotrol) as surrogates for stained mononuclear cells. Spectrophotometrically determined calibration values for the microbeads were used to generate a standard curve that converted green fluorescence histogram channels into molecular equivalents of soluble fluorescein (MESF). In 19 analyses repeated during a single run, the coefficients of variation (CVs) for the derived MESF values on both dimly and brightly stained Fluorotrol populations were less than 2%. In 26 separate determinations over 14 weeks, the CVs of the derived MESF values were less than 3%. The MESF values of the dim and bright Fluorotrol populations derived from the microbead standard curves were both about 50% lower than those determined by direct spectrophotometric analysis of Fluorotrol. The analytical imprecision of fluorescence intensity measurements in this idealized model system has a CV less than 3%, and the analytical inaccuracy shows that calibration in MESF units remains uncertain over about a two‐fold range.
New, simple flow cytometry technique to discriminate between internalized and membrane‐bound particles in phagocytosis
Authors: Fattorossi Andrea , Nisini Roberto , Pizzolo John G. , D'amelio Raffaele 
Published: August 2005

AbstractThis report describes a new flow cytometry technique to measure phagocytic activity and discriminate simultaneously between internalized and membranebound particles. Fluorescein‐conjugated heat‐killed Candida albicans (F‐Ca) are opsonized with purified antibodies or normal human serum and used as targets for human polymorphonuclear granulocytes (PMN). The procedure is based on the observation that F‐Ca lose their green fluorescence and acquire red fluorescence upon incubation with ethidium bromide (EB) through the resonance energy‐transfer phenomenon occurring between the two fluorochromes. PMN are incubated with opsonized F‐Ca particles for 20 min at 37°C or, as a control, at 4°C and in the presence of cytochalasin B, an inhibitor of the phagocytic process that does not affect membrane binding of F‐Ca. EB is added, and green and red fluorescence associated with PMN is evaluated using a mercury‐lamp‐powered instrument. Because EB does not penetrate intact cell membranes, internalized particles are not affected by EB and remain green, whereas membrane‐bound particles assume an intense red stain. By means of contour plot analysis, the number of PMN containing and/or binding F‐Ca particles can be readily assessed. The method described here allows precise quantitative analysis of the phagocytic process on the part of human PMN in a single, one‐step assay that does not require sophisticated instrumentation or reagents and should prove to become a test suitable for clinical purposes.
Cellular dna content parameters in untreated and recurrent squamous cell cancers of the head and neck
Authors: Ensley John F. , Maciorowski Zosia , Hassan Mohammad , Pietraszkiewicz Haline , Heilbrun Lance , Kish Julie A. , Tapazoglou Efstathios , Jacobs John R. , Al‐Sarraf Muhyi 
Published: August 2005

AbstractThe presence and degree of DNA aneuploidy as measured by the DNA index (DI) and the S phase fraction (SPF) were determined by flow cytometry in 294 specimens from 237 patients with untreated and recurrent squamous cell carcinomas of the head and neck (SCCHN). A descriptive analysis was performed in which the specimen DNA parameters were correlated with stage, size of primary, degree of lymph node involvement, morphological grade, and treatment status of the corresponding patients. Approximately 70% of the previously untreated specimens contained DNA aneuploid populations (DI > 1.10) and three quarters had SPF that were above 15%. There was a strong, direct as sociation between DI and SPF (P < 0.001). There was no correlation of the presence or degree of DNA aneuploidy with the stage of the tumor or the size of the primary or conventional morphological grade of the tumor. Specimens from patients with recurrent tumors and untreated patients with N3 lymph nodes had significantly lower rates of DNA aneuploidy and mean DI. Serial determinations of DNA aneuploidy in patients with SCCHN undergoing cytotoxic therapy are ongoing and may prove useful in the identification and understanding of resistance and response in this tumor.
A spectrophotometric method for determining the stream sample core diameter of a flow cytometer
Authors: Blume Philip 
Published: August 2005

AbstractA simple, rapid, and accurate method is described for the determination of flow cytometer stream sample core diameters. A precisely formulated solution of potassium dichromate is introduced as a sample, the stream is collected, and its absorbance is measured using a spectrophotometer. This permits the calculation of the factor by which the original solution was diluted and, given the stream diameter, the sample core diameter.
Evaluation of algorithms for ratio imaging in fluorescence microscopy
Authors: Rogowska Jadwiga , Bright Gary R. , Preston Kendall , Taylor D. Lansing 
Published: August 2005

AbstractRatio imaging in fluorescence microscopy is used in measuring parameters such as pH, pCa, cytoplasmic porosity, and the relative concentration of fluorescent analogs within single cells. The fastest method for ratio imaging is to use lookup tables on special‐purpose image processors. Since lookup tables store integers in integer addresses, using a lookup table will generate rounding errors. The magnitude of the error will depend on the transformation performed and on the number of levels used in the lookup table. We examined ratio imaging by lookup table and computed the errors generated by both inversion and log subtraction methods. Both uniformly fluorescing fields and fluorescing cell images were employed to provide data for use in confirming our calculations and illustrating both the magnitude and spatial incidence of errors. It is shown that, through proper design of lookup tables, a significant reduction can be made in the errors generated in comparison with common methods available in most image processors.
Technique for cellular fluorescence distribution analysis
Authors: Robinson R. D. , Reeder J. E. , Wheeless L. L. 
Published: August 2005

AbstractThe usefulness of multidimensional slit‐scan flow cytometry in whole cell measurements is dependent on extracting relevant features from the cellular fluorescence distributions (slit‐scan contours). In addition, the extraction of these features must be rapid to allow for realtime data processing during acquisition. This paper describes two algorithms that have been used successfully to count the numbers of local maxima (peaks) and to find nuclear boundaries in a cellular fluorescence distribution. These routines are efficient, use only simple integer arithmetic, and have been implemented on several different microprocessors.
Autolysis is a potential source of false aneuploid peaks in flow cytometric DNA histograms
Authors: Alanen Kalle A. , Joensuu Heikki , Klemi Pekka J. 
Published: August 2005

AbstractSerial flow cytometric nuclear DNA content analyses were performed from normal human spleen, thyroid, liver, and pancreas removed from ten patients at autopsy and stored for up to 8 d without any preservative to study the effect of autolysis on DNA histograms. Fine needle aspiration biopsy (FNAB) samples were taken in diluted ethanol and tissue biopsies from the same area in formalin for embedding into paraffin at the time of autopsy and serially thereafter. Histograms obtained from samples taken within 10 h after death had a symmetrical G1 peak with a small coefficient of variance (CV) except histograms produced from paraffin‐embedded pancreatic tissue, but bimodal distributions similar to those seen in aneuploid tumors were obtained from many samples stored longer than for 20 h. The DNA indices of the bimodal histograms were usually <1.3. The false peaks were more prominent in FNAB samples than in paraffin‐embedded samples. The time of appearance of the false aneuploid peaks varied individually, and they were usually first seen in samples taken from the pancreas, followed by the liver, the thyroid and the spleen. Because neoplasms may become necrotic in vivo and fixation of fresh surgical samples may be slow and incomplete, increased DNA staining caused by autolysis may be a source of false aneuploid peaks in DNA content analysis.
Accuracy of routine flow‐cytometric bitmap selection for three leukocyte populations
Authors: Sučić Mirna , Kolevska Tatjana , Kopjar Branko , Kosanović Mirjana , Drobnjak Marija , Žalud Ivica , Marušić Matko 
Published: August 2005

AbstractA double‐blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS‐C flow cytometer using the two‐parameter 90° light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.
Limitations in the measurement of c‐myc oncoprotein and other nuclear antigens by flow cytometry
Authors: Lincoln Sarah T. , Bauer Kenneth D. 
Published: August 2005

AbstractRecent developments in cell fixation, quantitative immunocytochemistry, and flow cytometry allow for the quantification of a variety of oncoproteins and other proliferation‐associated antigens in both fresh and archival pathology material. These studies provide evidence that the standard tissue deparaffinization/dissociation technique significantly reduces the amount of c‐myc oncoprotein remaining for analysis. To examine the factor(s) responsible for this observation, individual variables of the deparaffinization/dissociation technique including type of fixative, pepsin concentration, pepsinization times, pH, and exposure to organic solvents were examined in HeLa‐S3 cells. The cells were stained with monoclonal antibodies either to the c‐myc oncoprotein or to p105, a prolifera‐tion‐associated nu' clear antigen. Protein‐levels were measured on the basis of anti‐c‐myc or anti‐p105 immunofluorescence by flow cytometry and were found not be affected significantly by type of fixative, exposure to organic solvents, acid pH solution, or mechanical disruption. Levels of c‐myc oncoprotein were reduced by over 50%, however, when cells were exposed to 0.5% pepsin, whereas p105 was more resilient with only an approximately 7% reduction following the same treatment. Thus, careful examination of aspects of the deparaffinization/dissociation technique appears to be a necessary prerequisite for quantification of specific nuclear proteins from dissociated tissue specimens.