bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas
Authors: Giaretti Walter , Bruno Silvia , Geido Elio , Vinci Angela Di , Sciallero Stefania , Aste Hugo
Published: August 2005

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P < 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80–1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 × 0.28. The mean DNA index for the three cases of “carcinoma in adenoma” with invasion of the stalk of the adenoma was 1.52 × 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.
Specific nuclear elimination in polyploid plasmodia of the slime mold physarum polycephalum
Authors: Werenskiold A. K. , Schreckenbach Th. , Valet G.
Published: August 2005

In growing plasmodia of the myxomycete Physarum polycephalum (G2‐phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2‐ or S‐phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G‐2 phase reflects the 2c, 4c, and 8c state.

The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.
Improved resolution of flow cytometric measurements of hoechst‐ and chromomycin‐A3‐stained human chromosomes after addition of citrate and sulfite
Authors: Engh Ger Van Den , Trask Barb , Gray Joe , Lansdorp Peter
Published: August 2005

The resolution of bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 and chromomycin A3 can be increased by adding sodium citrate and sodium sulfite to the chromosomes shortly before measurement. A flow karyotype of a patient with chronic myelocytic leukemia is shown to illustrate that the addition of these compounds allows high‐resolution measurements to be made and evaluated reliably from clinical samples.
Flow cytometric evaluation of cell cycle characteristics during in vitro differentiation of chick embryo chondrocytes
Authors: Giaretti W. , Bruno S. , DiVinci A. , Geido E. , Moro G. , Quarto R. , Cancedda R.
Published: August 2005

The cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (ϕ = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose‐coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (ϕ = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.
Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration
Authors: Pierrez J. , Guerci A. , Guerci O.
Published: August 2005

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differeing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes).

In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the “absolute values” are 10‐fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.
Two‐Color flow cytometric analysis of monocyte depleted human blood lymphocyte subsets
Authors: Fleisher Thomas A. , Hagengruber Claire , Marti Gerald E.
Published: August 2005

Peripheral blood mononuclear cells (PBMC) from normal individuals were examined using 16 pairs of FITC and phycoerythin (PE) directly conjugated monoclonal antibodies. Each pair of reagents was used to evaluate a conventional lymphocyte gate as well as open (non) gate of monocyte depleted PBMC. Parallel studies using the same panel of monoclonal antibodies were carried out on selected, nonmonocyte depleted samples. The major findings of this analysis were that 1,000–1,200 lymphocytes in a 10,000 cell analysis are found outside the lymphocyte gate and of these approximately 2/5 are CD16 positive LGL/NK cells, 2/5 are CD3 positive T cells, and 1/5 are CD19/CD20 positive B cells. Thus, it appears that 10–15% of the lymphoid cells fall outside of the conventional lymphocyte gate, and in certain settings monocyte depletion may be useful to perform more complete evaluation of the total lymphoid cell population obtained after ficoll‐hypaque separation.
Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry
Authors: Rothe G. , Valet G.
Published: August 2005

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4‐diacetoxy‐2,3‐dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self‐learning database of the DIAGNOS1‐program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.
Subpopulation analysis of drug‐induced cell‐cycle delay in human tumor cells using 90° light scatter
Authors: Epstein R. J. , Watson J. V. , Smith P. J.
Published: August 2005

A mitotic cell subset has been identified with nuclear light scatter. Colcemid‐treated T‐47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90° light scatter. A discrete low‐scattering cell cluster could be distinguished from the G2 cell subset on two‐dimensional contour plots of 90° light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake‐off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell‐cycle analysis of DNA fluorescence. Cell sorting confirmed that the low‐scattering cell cluster comprised predominantly metaphase and anaphase cells.

Identification of mitotic cells with this onestep technique enables rapid analysis of druginduced cell‐cycle delay in cell populations with different rates of cell‐cycle traverse. Hence, vincristine‐induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug‐induced cell‐cycle delay and supplements the information obtainable by conventional cell‐cycle analysis.
Isolation of highly multidrug‐resistant P388 cells from drug‐sensitive P388/S cells by flow cytometric cell sorting
Authors: Ross Douglas D. , Ordóñez José V. , Joneckis Christopher C. , Testa Joseph R. , Thompson Bruce W.
Published: August 2005

To investigate the spontaneous frequency of occurrence of stable multidrug‐resistant cells in a population of drug‐sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence‐activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrugresistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrugresistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr‐content sort window. Net growth of cells in culture was observed 15–20 days after sorting, indicating that of the P388/S cells collected from the low dnr‐content sort window, very few were actually highly drug‐resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug‐resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross‐resistant to vincristine and actinomycin‐D, and distinct from P388/S, possessed a 150‐ to 160–kD membrane species identified by Vinca alkaloid photoaffinity labeling. The cytogenetic alterations found in P388/LoSort were different from those found in P388/adr, confirming that P388/LoSort cells are unique, although phenotypically similar to P388/adr. These studies suggest that spontaneous mutation to a highly multidrug‐resistant phenotype does exist in P388/S cells, but that the mutation frequency is low, with an estimated frequency of 2 × 10−8.
Flow cytometric analysis of the phenotypic changes in tumour cell lines following TPA induction
Authors: Zanyk Marien J. , Banerjee Diponkar , McFarlane David L.
Published: August 2005

Single‐cell analysis by flow cytometry has enabled us to analyze the effects of a phorbol ester and known tumour promoter, TPA, on the phenotypes of four tumour lines. TPA is capable of triggering a variety of cellular alterations that can affect gene expression and the biochemical balance of intracellular events. We have investigated the effect of TPA on such properties as rate of proliferation, differentiation, expression of cell surface molecules, and susceptibility to natural killer (NK) cell‐mediated cytolysis.

Four human leukemia and lymphoma cell lines; K562, MOLT 4, Raji, and HL60, were studied in their response to TPA treatment. Based on measurements of the defined cellular properties, we have characterized the pleiotropic responses of each tumour cell line to the phorbol ester in relation to intensity and time of onset of each response. The effects of TPA are highly varied, ranging in time of onset from minutes to days, and in intensity from strong to weak within the four cell lines studied. However, within all the processes that are affected, the activation of protein kinase C appears to be a common initiating event of phorbol ester induction.
A single‐cell assay of β‐galactosidase activity in Saccharomyces cerevisiae
Authors: Wittrup Karl Dane , Bailey James E.
Published: August 2005

A novel assay of single‐cell exogenous β‐galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin‐β‐D‐galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X‐100, and the assay is performed at 0°C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and β‐galactosidase activity is obtained under optimized assay conditions.
An interactive multivariate analysis of FCM data
Authors: Kosugi Yukio , Sato Ryuichi , Genka Shigeru , Shitara Nobuyuki , Takakura Kintomo
Published: August 2005

The procedure and results of the interactive multivariate analysis of FCM data are described. Using principal‐components analysis, cluster analysis, and interactive maneuvers, this procedure facilitates an effective data compression from a four‐dimensional space into two‐dimensional space, then allows cluster separation. The procedure is especially effective for separating clusters, which are degenerated in the usual scattergrams. Programs were mostly written in C language on MS‐DOS and were tested on four‐dimensional analysis of the blood cells, which resulted in a successful separation of the degenerated clusters.
Quantitative description of classic and variant small cell lung cancer cell lines by nuclear image cytometry
Authors: Broers J. L. V. , Pahlplatz M. M. M. , Katzko M. W. , Oud P. S. , Ramaekers F. C. S. , Carney D. N. , Vooijs G. P.
Published: August 2005

Six small cell lung cancer (SCLC) cell lines were examined using nuclear image analysis to find features characteristic of the classic and the variant type of SCLC. On the basis of their biochemical and biological properties three of these cell lines have been shown to represent the classic types, and three represent the variant type of SCLC. Using a combination of the image‐derived run length, density, and geometric features, it was possible to distinguish between the classic and variant SCLC cell lines.

The results of this study may be of help in assessing photometric features for the separation of the classic and variant subtypes of SCLC in solid tumors. Because of differences in treatment and prognosis between these two subtypes, such a separation may be of clinical value.
Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5‐bromo‐2′‐deoxyuridine and propidium iodide
Authors: Trinkle Linda S. , Swope Viki B. , Abdel‐Malek Zalfa A. , Nordlund James J.
Published: August 2005

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5‐bromo‐2′‐deoxyuridine (BrdUrd) and double‐stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 μg/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti‐BrdUrd and PI. PGE1‐treated cells monitored at 3‐hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti‐BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd‐negative “S phase” cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move “backwards” in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to overestimation of S phase cells in kinetic studies because of contaminating mitotic cells.
Flow cytometric analysis of platelet surface antigens
Authors: Marti Gerald E. , Magruder Louise , Schuette William E. , Gralnick Harvey R.
Published: August 2005

A flow cytometric analysis of human platelet surface antigens was carried out using a panel of monoclonal antibody reagents. The reagents used were specific either for the GPIb or the GP IIb/IIIa complex, surface immunoglobulin, or von Willebrand factor (vWf). Indirect surface immunophenotypes were determined using an EPICS V flow cytometer and the monoclonal antibodies 6D1, 10E5, Plt‐1, UR1663, anti‐IgG, and anti‐vWf. Platelets were obtained from normal individuals or patients with either Bernard‐Soulier syndrome (BSS) or Glanzmann's thrombasthenia (GT). Normal platelets were positive for 10E5, 6D1, Plt‐1, and UR1663 and showed negligible activity for anti‐IgG and anti‐vWf. Platelets from individuals with BSS showed a marked reduction in 6D1, while the platelets of a patient with GT showed a marked reduction in binding of 10E5, Plt‐1, and UR1663. Differences between histograms for normal platelets and for platelets from individuals with BSS or GT were evaluated using the Kolmogorov‐Smirnov test. Compared to normal platelets, the BSS and GT platelets contain at least 35‐fold less of the GPIb and GP IIb/IIIa complex respectively. Flow cytometry is a useful and precise method for the study of normal and abnormal surface platelet phenotypes.
Discriminating between damaged and intact cells in fixed flow cytometric samples
Authors: Terstappen L. W. M. M. , Shah V. O. , Conrad M. P. , Recktenwald D. , Loken M. R.
Published: August 2005

A vital, nucleic acid stain (LDS‐751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS‐751 were found in the fixed samples. Cells not staining or only dimly staining with LDS‐751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS‐751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS‐751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS‐751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two‐color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.
Simultaneous analysis of DNA content and surface antigens in human bone marrow
Authors: Hollander Zeev , Loken Michael R.
Published: August 2005

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell‐surface antigens and DNA content on bone marrow cells while preserving their light‐scatter properties. Proliferation in a population defined by light‐scatter and surface‐antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA (“proliferation index”). Viable, low‐density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV‐excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light‐scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest “proliferation index” detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.
An EPROM‐based programmable contour generator for use in flow cytometry
Authors: Wheeless D. M. , Cambier J. L. , Wheeless L. L.
Published: August 2005

An erasable programmable read‐only memory (EPROM) contour generator has been fabricated to produce contours for use in flow cytometry. Contours are analog waveforms representing the fluorescence or light‐scatter intensity distribution along a cell or object. The generator has particular utility in the development and testing of slit‐scan instrumentation and analysis alogrithms. Contours are generated without the requirement of specimens or full operation of the flow instrumentation. The generator provides control of contour height, width, offset, and rate. The EPROM may be custom programmed to produce contours for specific test applications or for reproducing “real” contour events. The generator is useful in situations where constant repetitive contours of predetermined characteristics are required.
Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle
Authors: Khochbin Saadi , Chabanas Agnés , Albert Philippe , Albert Jacques , Lawrence Jean‐Jacques
Published: August 2005

Flow cytofluorimetric measurement of incorporated bromodeoxyuridine, using a double‐stained cell population, allows the determination of the distribution of cells along the cell cycle (1). We have developed a simple computer program for the direct treatment of 64 × 64 channel histograms. This analysis appears to provide interesting data about the distribution of cells in the various phases of the cell cycle, namely the S phase.

Two examples have been chosen to illustrate possible fields for the application of such a program. Comparison of two cell lines such as friend murine erythroleukemia cells (MELC) and fibroblasts FR3T3 cells has shown that this analysis can be used for cell‐cycle characterization of a given cell line.

The program also allows the differential analysis of cell distribution along the cell cycle as a function of a given parameter. This possibility has been applied to study the variation of cell‐cycle parameters as a function of the time of induced differentiation of MELC and reveals changes in the distribution of the cells along the various phases of the cell cycle, namely in the S phase.
Identification of flow‐sorted chromosomes by G‐banding and in situ hybridization
Authors: Rommel B. , Hutter K.‐J. , Bullerdiek J. , Bartnitzke S. , Goerttler K. , Schloot W.
Published: August 2005

The identification of flow‐sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G‐banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest.

In particular when sorting chromosomes from human‐rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin‐labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.
DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry
Authors: Azzolina L. S. , Stevanoni G. , Tridente G.
Published: August 2005

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1–3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide‐RNAse solution.

The results, obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen‐stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli.

This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H‐thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90° light scatter and double fluorescence labelling of PBL nuclei.
Flow cytofluorometric analysis of the uptake of the fluorescent fatty acid pyrene‐dodecanoic acid by human peripheral blood cells
Authors: Fibach Eitan , Giloh Haim , Rachmilewitz Eliezer A. , Gatt Shimon
Published: August 2005

The fluorescence activated cell sorter (FACS) was used for measuring the uptake of the fluorescent fatty acid derivative 12‐(1‐pyrene) dodecanoic acid (P12) by human peripheral blood cells. The results indicate that blood cells differ widely in their ability to take up P12, with polymorphonuclear cells showing the greatest uptake, followed by lymphocytes, platelets, and RBCs. These differences in P12 uptake provide a potential additional parameter for differential cell counting. Using the ability of the FACS to “gate out” nonrelevant cells, it was possible to measure the rate of P12 uptake by each respective cell type even when admixed with other cells. Thus elaborate physical separation procedures could be avoided, and contaminating cells did not influence the results. Differences in P12 uptake were also utilized to separate blood cells into pure subpopulations of specific cell types.
Five‐dimensional flow cytometry as a new approach for blood and bone marrow differentials
Authors: Terstappen Leon W. M. M. , Loken Michael R.
Published: August 2005

We have used five independent variables on a flow cytometer to discriminate and to quantify the cellular components within both blood and bone marrow aspirates. The signals were stored in list mode by which a five‐dimensional space was created. The cells–differentiated into: (1) erythrocytes, (2) reticulocytes, (3) nucleated erythroid cells, (4) platelets, (5) lymphocytes, (6) monocytes, (7) neutrophils, (8) eosinophils, and (9) immature leukocytes–had to meet unique criteria with regard to their characteristics in the created five‐dimensional space in order to be classified in a specific cell category.

Forward and orthogonal light‐scattering signals were matched with three fluorescence variables to obtain discrimination without necessitating erythrocyte lysis.

Thiazole orange (binding predominantly to RNA) and LDS‐751 (principally detecting DNA) were used to differentiate erythrocytes, platelets, reticulocytes, and nucleated cells. A monoclonal antibody, CD45, conjugated with phycoerythrin, was used to aid in discriminating between lineages of nucleated cells.
Comparison of absorption measurements of DNA stain content by utilizing video and scanning image cytometers
Authors: Allison David C. , Mayall Brian H. , Levin Jack
Published: August 2005

After staining with the Feulgen reaction, the DNA stain contents of 155 mouse bone marrow cells and 22 adjacent chicken erythrocytes were measured by absorption image cytometry by utilizing two different systems—a scanning cytometer and a video cytometer. In the scanning cytometer (M85 microdensitometer, Vickers Instruments, Malden, MA), a spot of light was scanned across the cell. In the video cytometer (TAS Plus, E. Leitz, Rockleigh, NJ), the microscope field, which may contain several nuclei, was imaged onto a Plumbicon video camera. With each system, cells were scanned, digitized into their elementary pixels, and analyzed to determine their intergrated absorbance.

Comparison of the DNA stain contents of the same G0/G1 bone marrow cells and chicken erythrocytes, as measured by video and scanning cytometry, showed that both techniques gave comparable results; scanning cytometry is more precise. The coefficients of variation of the measurements for the G0/G1 bone marrow cells and for the chicken erythrocytes were 5.9% and 7.0%, respectively, when measured by video cytometry at the absorption peak (584 nm), compared to 4.1% and 3.5%, respectively, for the same cells when measured by scanning cytometry off the absorption peak (615 nm). The video‐based measurements were relatively lower than the scanning measurements for darkly stained cells; this suggests that glare and other optical errors which increase with stain darkness caused greater systematic errors in the video cytometer than they did in the scanning cytometer.
Relationship between changes of the steroid receptor and synchronization in human endometrial adenocarcinoma cells in vitro
Authors: Nishiya Iwao , Kagabu Teruo , Saito Satoshi , Fujimoto Jiro , Okada Hiroji
Published: August 2005

It has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle.

The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n‐butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double‐straded RNA) the cells were treated with DNase.

We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n‐butyrate was 134% of control.

The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/μg DNA in the cytosol and 12.35 fmol/μg DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.
Technical and statistical improvements for flow cytometric DNA analysis of paraffin‐embedded tissue
Authors: Sickle‐Santanello Brenda J. , Farrar William B. , Decenzo Jane F. , Keyhani‐Rofagha Sedigheh , Klein John , Pearl Dennis , Laufman Harry , O'Toole Robert V.
Published: August 2005

Flow cytometric DNA analysis of paraffinembedded solid tumors has permitted review of large series of archival tissue in attempts to relate abnormal DNA content to prognosis. Limitations of the technique include: (1) a laborious, time‐consuming procedure; (2) variation in technique between laboratories; and (3) lack of an objective method of computing DNA indices. Critical evaluaiton of our technique has shortened the time involved in dewaxing and rehydration, selectively utilized patient's own normal tissue as the internal standard, proved reproducibility of stored specimens, standardized DNA index computation, and developed a statistical analysis to confirm aneuploidy. These technical improvements and the development of a statistical analysis provide a way to shorten the procedure time and standardize the data generated from flow cytometric DNA analysis so as to improve the quality of retrospective reviews of paraffin‐embedded tumors and accelerate the definition of flow cytometry's role as a prognostic indicator.
Comparison of automated and manual techniques for analysis of DNA frequency distributions in bladder washings
Authors: Wheeless Leon L. , Coon John S. , Deitch Arline D. , White Ralph W. DeVere , Koss Leopold G. , Melamed Myron R. , Reeder Jay E. , Robinson Roy D. , Weinstein Ronald S. , Wersto Robert P.
Published: August 2005

Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant to the relatively low signal‐to‐noise ratios often found in DNA distributions obtained from clinical samples.

Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S.

A computer‐based method has been developed for extraction of the above‐mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight‐line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer‐based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer‐based method. Correlation of percent hyperdiploid cell fraction was also excellent. This computer‐based method for the analysis of DNA frequency distributions should have utility in the establishment of DNA flow cytometry of bladder irrigation specimens as a useful and routine laboratory procedure.
Comparison of S‐phase fractions measured by flow cytometry and autoradiography in human transplant tumors
Authors: Feichter G. , Czech W. , Haag D. , Goerttler K. , Futterman G. , Loehrke H. , Abel U.
Published: August 2005

The 3H‐thymidine labeling index (TLI) and the percentage of cells in the S‐phase have been determined by autoradiography and by flow cytometry, (FCM), respectively, in six malignant tumors of human origin transplanted on athymic nude mice. The Dean and Jett model and the graphical model were used to determine the percent of S‐phase cells by FCM. Cell cycle analysis was performed using (1) no correction for background; (2) an algebraic function for background correction; and (3) an exponential function for background subtraction. Each of these three data sets was evaluated using both the Dean and Jett model and a graphical model for the evaluation of DNA histograms. The S‐phase fractions (SPF) were compared to the corresponding labeling index results.

SPF without background correction were 1.54 times higher than the TLI. SPF, after correction using the algebraic model, were 1.29‐fold higher than the TLI, whereas SPF obtained after background subtraction according to the exponential model were only 1.05‐fold higher than the TLI. Student's t‐test revealed significant differences between the mean TLI values (16.25 ± 9.06) and the mean SPF obtained by FCM without background correction (mean 25.0 ± 9.36, P < 0.01), but not between the mean TLI values and the mean SPF percentages after algebraic (mean 21.0 ± 10.29) and exponential background correction (mean 17.11 ± 11.59), P > 0.05 each. There was no difference between the results obtained using the Dean and Jett model and those obtained using the graphic evaluation. The SPF obtained by FCM are similar to the 3H‐thymidine labeling indices of the same sample of a solid tumor if the histograms first are cleaned from nonspecific background pulses.
Relevance of DNA ploidy as a measure of genetic deviation: A comparison of flow cytometry and cytogenetics in 25 cases of human breast cancer
Authors: Remvikos Y. , Gerbault‐Seurreau M. , Vielh P. , Zafrani B. , Magdelénat H. , Dutrillaux B.
Published: August 2005

Twenty‐five human breast cancers, surgically resected, were studied by cytogenetic analysis and DNA flow cytometry (FCM). The establishment of karyotypes showed that multiple cell populations probably were derived from a single ancestor clone, because common marker chromosomes always could be demonstrated. Differences of up to 30% were observed when the estimates of DNA content by the two methods were compared. A general tendency toward the acquisition of large marker chromosomes should be at the origin of this discordance, as the proportion of markers for each case correlated significantly with the magnitude of the difference. Parallel use of the two methods revealed the existence of tumors with DNA diploid FCM profiles and highly abnormal hypodiploid karyotypes (35–40 chromosomes), which may explain the limited value of DNA ploidy as an independent prognostic factor in breast cancer.
A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation
Authors: Van Erp Piet E. J. , Brons Paul P. T. , Boezeman Jan B. M. , De Jongh Gijs J. , Bauer Franz W.
Published: August 2005

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achived by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.
Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining
Authors: Dolbeare Frank , Gray Joe W.
Published: August 2005

We describe an enzymatic procedure for exposure of single‐stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X‐100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti‐HdUrd antibody IU‐4 in Chinese hamster ovary cells labeled with 10 μM BrdUrd or 10 μM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU‐1, an anti‐HdUrd antibody with lower binding affinity. IU‐4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU‐4 binding.
Osmotic response of lymphocytes measured by means of forward light scattering: Theoretical considerations
Authors: Sloot Peter M. A. , Hoekstra Alfons G. , Figdor Carl G.
Published: August 2005

Recent data provide solid experimental evidence that increase in cell size of human lymphocytes, caused by osmotic stress, is not directly proportional to forward scattering intensity but rather is inversely proportional. Here, we provide arguments that this phenomenon can be quantitatively explained by means of a model based on a modified Rayleigh‐Debye‐Gans theory. As a consequence, the current view that forward scattering intensities can be used as a measure of gross cell size needs to be reconsidered. In addition, we postulate that structural changes of the cytoplasm, imposed by an osmotic stress, reflect corresponding changes in the nucleus.
Cyanine dye labeling reagents for sulfhydryl groups
Authors: Ernst Lauren A. , Gupta Ravinder K. , Mujumdar Ratnakar B. , Waggoner Alan S.
Published: August 2005

Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from < 500 nm to > 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.
Two statistical approaches to nuclei shape and size in a morphometric description of lymph node sections in non‐Hodgkin's lymphoma
Authors: Lesty Claude , Raphaël Martine , Nonnenmacher Luc , Binet Jacques‐Louis
Published: August 2005

An attempt is made to evaluate more clearly the potential contribution of quantitative nuclear profile shape and size measurements to lymph node section histologic description in 70 cases of non‐Hodgkin's lymphoma (NHL). The area of nuclear profiles and five nonredundant and size‐free shape indices were measured using the THECLA program on a Leitz Texture Analysis System (Leitz‐TAS). Two statistical approaches were applied, known respectively as “parametric,” (first statistical moments of variable distributions over samples of 200 nuclear profiles) and “nonparametric,” which are percentages of nuclear profiles distributed into five “cytological” classes that are defined by shape: round (A), elongated (B), kidney shaped (C), irregular (D) and cleaved (E) nuclear profiles.

Both statistical approaches provide proper overall discrimination of the eight histological categories identified with reasonable reliability by pathologists. Above all, the present report discusses the ability of a set of parametric and nonparametric variables to describe NHL cell populations, in an objective and meaningful way, according to nuclei shape. A method of synthesizing multidimensional correlations (CORICO program) is proposed in support of the discussion. Also, the specific descriptive power of each of the variables is described; in particular, it is concluded that there is a close link between the shape and size of the nuclear profiles of the cells.
Quantitative morphological analysis of Adriamycin‐resistant human K562 leukemic cells
Authors: Dufer J. , Akeli M. G. , Jeannesson P. , Desplaces A. , Jardillier J. C.
Published: August 2005

The morphological changes associated with Adriamycin resistance in a human leukemic cell line have been investigated by image analysis. An Adriamycin‐resistant subline of the human erythroleukemic K562 cell line has been established. Three sets of cells have been analysed: sensitive cells, resistant cells cultured in the continuous presence of Adriamycin, and resistant cells cultured without the drug. Image analysis shows that Adriamycinresistant K562 cells display significant morphological changes as compared with sensitive cells, at both the nuclear and cytoplasmic levels. These changes make it possible to separate sensitive and resistant cells automatically and with a classification accuracy of 76% and only four cytological parameters. Image analysis may therefore offer an interesting tool for studying drug resistance in leukemic cells, from both an experimental and a clinical point of view.