bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric method for the measurement of epidermal growth factor receptor and comparison with the radio‐ligand binding assay
Authors: Brotherick I. , Lennard T. W. J. , Wilkinson S. E. , Cook S. , Angus B. , Shenton B. K.
Published: June 2005

A method for the purification, conjugation, and use of EGF‐R antibody to quantify EGF receptors on tumour cells by flow cytometry is described. The quantification of both internal and external EGF receptors was determined by treatment with saponin, rendering the cells permeable to the EGF‐R antibody. Using QCS bead standards, the number of EGF‐R binding sites per cell was assessed. Results were compared with conventional EGF‐R quantification using a radio‐ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for EGF‐R quantification described is sim‐ple, rapid, and reproducible. The assay may be of particular value in measuring EGF‐R on urgent clinical samples or those that are too small (such as breast aspirates) for measurement by the radio‐ligand binding assay. Advantages of this assay over the radio‐ligand binding assay include reduction in use of radio‐labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. In addition, flow cytometry offers the possibility of selecting cell phenotypes by gating as well as live/dead cells by using multi‐parameter flow cytometry. © 1994 Wiley‐Liss, Inc.
DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay
Authors: Olive P. L. , Banáth J. P. , Fjell C. D.
Published: June 2005

The alkaline comet assay is used to detect DNA single‐strand breaks in individual cells embedded in agarose, lysed to denature DNA and remove proteins, and briefly exposed to an electric field to allow broken DNA to migrate. Total DNA fluorescence, measured by staining individual comets with propidium iodide, is reduced 30–40% by low doses of ionizing radiation, N‐methyl‐N‐nitrosoguanidine (MNNG), etoposide, or hydrogen peroxide. Three possible explanations were examined: 1) these agents produce small fragments of DNA that are lost during lysis and electrophoresis, 2) the analysis of comet images is less efficient once the DNA is small enough to migrate in the electric field, and 3) DNA staining by propidium iodide is affected by changes in DNA structure caused by strand breaks and exposure to alkali. Our results indicate that the decrease in fluorescence after low doses is best explained by a change in ability of DNA, which has been denatured by alkali and subsequently renatured, to interact with fluorescent dyes. This change in fluorescence has the practical consequence of improving the ability of the alkaline comet assay to distinguish damaged from undamaged cells. © 1994 Wiley‐Liss, Inc.
Expression of proliferation‐associated antigens (PCNA, p120, p145) during the reentry of G0 cells into the cell cycle
Authors: Bolton Wade E. , Freeman James W. , Mikulka Warren R. , Healy Cynthia G. , Schmittling Robert J. , Kenyon Norma S.
Published: June 2005

Flow cytometric bivariate analysis was used to evaluate the expression of PCNA, p120, and p145 during the G0 reentry of CHO‐K1 cells into the cell cycle. CHO‐K1 cells were placed in a G0‐like state using serum depletion and stimulated to reenter the cell cycle by replating into fresh, serum‐containing medium. At discrete intervals after stimulation, replicate samples were stained for either PCNA, p120, or 145; stained for DNA (Coulter DNA‐Prep); evaluated on the EPICS Profile I; and analyzed on the EPICS ELITE workstation. PCNA stained less than 10% of the G0 cells; in contrast, however, 30–35% of the G0 cells were positive for p120 and p145. Eight hours after stimulating G0 cells to reenter the cell cycle (during G0/G1), p120 reached 88% positivity, while p145 and PCNA were 63% and 30% positive, respectively. Cells in S phase (12 and 16 h following G0 stimulation) were greater than 90% positive for all three antigens. PCNA had the greatest change throughout the G0 reentry process, both in percentage positive and quantitatively (mean channel fluorescence).

This report indicates that all three proliferation‐associated antigens studied are differentially expressed during the reentry of G0 cells into the cell cycle. Furthermore, these antigens may be useful in the early detection of G0 recruitment. © 1994 Wiley‐Liss, Inc.
Quantitative determination of particle concentrations in experimental and marine environmental samples
Authors: Robins David B. , Bedo Alain W.
Published: June 2005

As the application of flow cytometry increases across the environmental sciences, there is a need to expand instrument flexibility. In particular, some of the major applications within aquatic research demand an ability to quantify the particles under investigation, expressing them as numbers per unit of sample volume. Two of the principal areas of flow cytometric investigations are the characterisation of naturally occurring particulate assemblages in the oceans and investigations into the feeding behaviour of “filter feeding” organisms on the mixtures of such particles. In both cases some of the most useful data derived from flow cytometry require that analyses be determined in terms of particle concentrations. Therefore, a simple and practical modification is described, which allows known sample volumes to be analysed on flow cytometers. The method has a high degree of flexibility in sample volume and is cost effective and reliable. It involves introducing a standard chromatography “loop injection valve” into the sample‐handling fluidics system. Use of fluorescent beads shows that a range of praticle sizes and concentrations can be used with this system. Tests, also using fluorescent beads and an algal culture, show that replicate 100 μ1 sample volumes can be analysed and counted with a coefficient of variation of less than 3%. © 1994 Wiley‐Liss, Inc.
Staining of Escherichia coli for flow cytometry: Influx and efflux of ethidium bromide
Authors: Jernaes Mette W. , Steen Harald B.
Published: June 2005

In an attempt to develop procedures for nucleic acid staining of bacteria for clinical routine assays, the uptake of ethidium bromide (EB) in wild‐type Escherichia coli was studied using flow cytometry. Phosphate‐buffered saline (PBS) containing EDTA or Tris significantly increased the net uptake of EB compared to PBS only. However, in the majority of the cells, the net uptake reached a constant level that was only a few percent of that of fully permeabilized cells, apparently due to the activity of a metabolically driven efflux pump. When cells were exposed to cold shock (0°C for 30 min) in the presence of Tris or EDTA, the net uptake of dye was similar to that of fully permeabilized cells, whereas it was about half that value in PBS. When cold shock was given in growth medium, the cells split up into four subpopulations, with a net dye uptake ranging from that of fully permeabilized cells to less than 1% of that value. As expected, metabolic inhibitors (Na‐azide, 2‐deoxy‐D‐glucose, and CCCP) reduced efflux activity. However, fluorescence of metabolically inhibited cells never exceeded more than about half the value of that of dead cells, possibly reflecting conformational changes in DNA structure as a result of cell death. © 1994 Wiley‐Liss, Inc.
Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages
Authors: Hendrzak Jill A. , Wallace Paul K. , Morahan Page S.
Published: June 2005

Blocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PMϕ) by flow cytometry are not ideal for elicited or activated PMϕ. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac‐1 antigens on resident PMϕ. In contrast, high concentrations (33–50% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PMϕ elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE‐2). However, even low concentrations of goat serum masked the expression of the Mac‐2 antigen on TG and MVE‐2 PMϕ. Thus, within a given elicited or activated PMϕ population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE‐2 PMϕ. © 1994 Wiley‐Liss, Inc.
Quality control for evaluation of the S‐phase fraction by flow cytometry: A multicentric study
Authors: Silvestrini Rosella ,   SICCAB Group For Quality Control Of Cell Kinetic Determinations
Published: June 2005

Flow cytometric measurement of the S‐phase fraction (FCM‐S) may be clinically useful in human cancers to identify high‐risk patients. However, its clinical application requires a methodologic standardization, a high feasibility, and the reproducibility of results within the same and among different institutions. The interlaboratory quality control program for FCM‐S determination (promoted by the Italian Society of Basic and Applied Cell Kinetics) was carried out on DNA diploid and aneuploid cell lines and human brest cancers. FCM‐S was quantified by using four mathematical models characterized by different rationales and differences in the degree and shape of background subtraction functions. A statisfactory agreement in FCM‐S values among different institutions was observed for cell lines. In human breast cancers, a high reproducibility of FCM‐S estimates obtained by the different institutions was observed by using the same model. Conversely, different models gave different FCM‐S estimates. Intermodel differences were more evident in aneuploid than in diploid tumors. These findings could explain the contrasting results on the prognostic role of FCM‐S in clinically comparable series of breast cancers and should be considered in future efforts to define the optimal and most reproducible mathematical approach to quantify cells in the S‐phase. © 1994 Wiley‐Liss, Inc.
Ploidy pattern and cell cycle in breast cancer as detected by image analysis and flow cytometry
Authors: Luzi Pietro , Bruni Alessandra , Mangiavacchi Paola , Cevenini Gabriele , Marini Dolly , Tosi Piero
Published: June 2005

Both image analysis (IA) and flow cytometry (FCM) may be applied to detect ploidy pattern and cell cycle fractions. However, they have different performance characteristics and may yield different results. The two approaches are applied in this study to 66 breast cancers: IA on imprints and FCM on fresh tissue. The percent coefficient of variation (CV) ranged from 2.0 to 7.0 (mean 5.5; SD 1.1) in IA and from 2.0 to 7.0 (mean 4.4; SD 1.1) in FCM. The values were well correlated. With regard to ploidy pattern, the agreement between the two methods was 92.4%; disagreements were due to four cases being aneuploid by IA but not detected by FCM and one case being aneuploid by IA but tetraploid by FCM. This suggests that IA is capable of detecting aneuploidy with more sensitivity than FCM. In diploid cases, the percent values of cells in GO/G1, S‐phase (SPF), and G2M phase were concordant and well correlated. In aneuploid cases, IA was more sensitive than FCM in detecting aneuploid fraction as well as G2M phase, whereas FCM was more sensitive than IA in detecting SPF. A good correlation was found between the DNA indexes (Dls) obtained with the two methods. © 1994 Wiley‐Liss, Inc.
Evaluation of flow cytometric methods for diagnosis of chronic granulomatous disease variants under routine laboratory conditions
Authors: Emmendörffer A. , Nakamura M. , Rothe G. , Spiekermann K. , Lohmann‐Matthes M.‐L. , Roesler J.
Published: June 2005

Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGD)‐patients (4 CGD+), and from 18 X‐linked carriers for CGD have been tested for their production of H2O2 using staining with dihydrorhodamine 123 and subsequent flow cytometry. Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed. Cells were activated to produce H2O2 by the phorbol ester phorbol‐myristate‐acetate (PMA) and by phagocytosis of Escherichia coli bacteria. To evaluate the sensitivity of the method, H2O2‐production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured. The results were compared to those from other methods (NBT‐testing, cytochrome c‐reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, X = 840 ± 59 (S.D.), 97% < 890, for pediatric patients. Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, X = 830 ± 52). For CGD+ (n = 4) the results were clearly far below the normal range. In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence. Cytochrome b558‐expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 7D5. The normal range was 97% > 485, 97% < 680, peak channel fluorescence. We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of cell‐surface and intracelluar antigens in leukemia diagnosis
Authors: Knapp Walter , Strobl Herbert , Majdic Otto
Published: June 2005

New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro‐)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferntiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to he used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB‐1), CD79b (B29), CD87 (uPA‐R), and CD117 (c‐kit). © 1994 Wiley‐Liss, Inc.
Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non‐lymphoid cells
Authors: Pattanapanyasat Kovit , Kyle Dennis E. , Tongtawe Pongsri , Yongvanitchit Kosol , Fucharoen Suthat
Published: June 2005

Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three‐color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71‐flurescein isothiocyanate (FITC), FSC/glyco A‐FITC, or FSC/CD45‐peridinin chlorophyl protein (PerCP) profiles. The CD71‐FITC negative cells, glyco A‐FITC negative cells, or CD45‐PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non‐lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two‐color antibodies. Of the three gating strategies, CD45‐PerCP and glyco A‐FITC methods are better than the CD71‐FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six‐tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of nonlymphoid cells. © 1994 Wiley‐Liss, Inc.
Radiation‐induced changes in nucleoid halo diameters of aerobic and hypoxic SF‐126 human brain tumor cells
Authors: Wang Jingli , Basu Hirak S. , Hu Lily , Feuerstein Burt G. , Nederlof Petra M. , Deen Dennis F.
Published: June 2005

Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF‐126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propidium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose‐dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony‐forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors. © 1995 Wiley‐Liss, Inc.
Can measurements of potential doubling time (Tpot) be compared between laboratories? A quality control study
Authors: Haustermans K. , Hofland I. , Pottie G. , Ramaekers M. , Begg A. C.
Published: June 2005

The purpose of this study was to investigate the reproducibility of potential doubling time measurements of human tumors in different laboratories and to distinguish which steps in the measurement procedure were subject to the greatest variation. This was achieved by comparing measurements on the same source material in two different laboratories in which three aspects of the technique were separately studied, namely, preparation and staining of the nuclear suspensions, running the samples on the flow cytometer (FCM), and analyzing the two‐parameter FCM data. This involved exchange between the two centers of fixed tumor material, stained nuclear suspensions, and FCM data on floppy disks. The analysis step was found to be the least variable step. For DNA synthesis time, Ts, and the labeling index, LI, the coefficients of determination (R2) ranged from 92% to 95.4%. A small systematic bias was observed, with one center measuring approximately 15% higher values for both LI and Ts. Different criteria for window placements were found to be a contributing factor. Variations in the FCM step were approximately equal to those for analysis, with no systematic deviations. Variations for the preparation and staining step were the largest (R2 = 60.5% and 38.1% for LI and Ts, respectively). However, this step was the only one subject to intratumoral variability, which was the largest contributing factor to the variations observed. In addition, however, LI was on average 41% higher in one center compared to the other, resulting in a systematic bias. Differences in the level of green fluorescence of the labeled cells implicated antibody differences as a possible cause. The variations found here for the three procedural aspects were significantly smaller than variations observed between tumors, a requirement for a predictive assay. Suggestions for implementation of quality control procedures include objective (computer‐assisted) data analysis on two‐parameter histograms and optimization of antibody combinations. © 1995 Wiley‐Liss, Inc.
Measurement of intracellular pH in mammalian sperm cells under physiological conditions
Authors: Jones Jeffrey M. , Lorton Steven P. , Bavister Barry D.
Published: June 2005

The goal of this study was to develop a technique for the measurement of intracellular pH (pHi) in living mammalian (bovine) sperm cells under physiological conditions. Like many other biochemical measurements, pHi measurements have typically been made under non‐physiological conditions on cells whose immediate functional status is not readily assessed and may even be non‐viable. Additionally, many pHi measurement techniques may themselves alter the pHi of the cells being measured. Such measurements could yield misinformation. The sperm cell is unique in that its functional status can be easily and continuously monitored by means of its motility, which is directly affected by pHi. In this respect, the sperm cell provides an ideal model system for evaluating pHi measurement techniques. In this article we summarize the validation of a ratiometric absorbance technique for the measurement of pHi of mammalian (bovine) sperm cells under physiological conditions which does not affect their functional status. The pHi of ejaculated bovine sperm cells was calculated to be 6.9 ± 0.05 (11 replicates). This approach may also be suitable for pHi measurements in other cell types. © 1995 Wiley‐Liss, Inc.
Image processing algorithms for the automated micronucleus assay in binucleated human lymphocytes
Authors: Böcker W. , Müller W.‐U. , Streffer C.
Published: June 2005

The frequency of micronuclei in binucleated lymphocytes (cytochalasin B assay) may serve as a biological dosimeter after radiation exposure. The automation of the micronucleus assay in binucleated human lymphocytes has been considerably advanced in recent years. In our studies for this purpose the detection of binucleated cells (BNCs) and the scoring of micronuclei (MN) was divided into two parts. First, detection of BNCs was feasible with low microscopic magnification (× 100). The positions of classified BNCs were stored. Second, after an automatic change of microscope objective, the stored BNCs were automatically analyzed in sequence at high microscopic magnification (× 630) for occurrence of MN. For both phases of image analysis we used empirical methods based on mathematical morphology. The system is able to recognize nearly 65% of BNCs with false positive decisions of 6% and about 75% of the MN with false positive decisions of 7%. © 1995 Wiley‐Liss, Inc.
Fluorescent erythrocyte ghosts as standards for quantitative flow cytometry
Authors: Doberstein S. K. , Wiegand G. , Machesky L. M. , Pollard T. D.
Published: June 2005

We report here a quick and inexpensive method for preparing standards of known fluorochrome content for calibration and quantitation of flow cytometry fluorescence signals. Erythrocyte ghosts prepared by hypotonic lysis are filled with solutions containing fluorescently labeled dextran. Standards prepared by this technique have a narrow range of fluorescence and a linear response of fluorescence to fluorochrome content up to 2 × 106 fluorochrome molecules/cell. The volume of ghost standard particles is roughly 70 femtoliters (fl)/cell. The fluorescence of ghost standards is nearly identical to that of commercially available microbead standards of similar fluorochrome content. Ghost standards have stable fluorescence for at least 3 weeks at 4°C. These standards can be made with any fluorochrome or combination of fluorochromes over a wide concentration range. © 1995 Wiley‐Liss, Inc.
Debris compensation of Dna histograms and its effect on S‐phase analysis
Authors: Wersto Robert P. , Stetler‐Stevenson Maryalice
Published: June 2005

Debris compensation is an important variable affecting S‐phase fraction (SPF) analysis in flow cytometric DNA histograms. The SPF was estimated in fresh frozen breast carcinomas using the following four debris subtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moel (EXP‐SC); the random cutting of nuclei into multiple pieces of varying sizes (MC); and a combination of both nuclear cutting models (SC‐MC). Comparison of SPF estimates indicated that the various debris subtraction models yielded differences in SPF, with SPF values obtained using the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms based on nuclear slicing and fragmentation. However, SPF estimates could be affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G0/1 peak, and the relative amount of debris. Using the ratio of the height of the G0/1 peak to the height of the debris between the chicken red blood cells (CRBC) and G0/1 peaks as an objective measurement of nuclear debris, debris compensation was necessary in diploid DNA histograms where this ratio was as low as 1.5%. Taken in the context of SPF prognostic cutoff levels, variation in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF categories, thereby making the comparison of SPF values derived from different studies difficult. © 1995 Wiley‐Liss, Inc.
New epi‐fluorescence optical system for independent analysis of two different fluorochromes in microscopy
Authors: Heiden Thomas , Tribukait Bernhard
Published: June 2005

A new epi‐fluorescence optical system is described that uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. The optical system was constructed for analysis of two different fluorochromes, e.g., DAPI and TRITC. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm, 546 nm, 435–500 nm, and 590–750 nm), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet‐excitation/blue‐detection and green‐excitation/red‐detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) separate positioning of lenses for compensation of chromatic aberrations. This system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was obtained with good temporal resolution. It has been shown that it is feasable to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected. © 1995 Wiley‐Liss, Inc.
Flow cytometric analysis of apoptosis and BCL‐2 in human solid neoplasms
Authors: Steck Kim D. , McDonnell Timothy J. , El‐Naggar Adel K.
Published: June 2005

Apoptosis (programmed cell death) and the genes regulating this process (e.g., bcl‐2), have recently become a focus of interest in the study of cancer development and progression. We adapted flow cytometric techniques for measuring apoptosis and bcl‐2 protein in solid tissues and concomitantly determined both parameters in 65 malignant solid neoplasms. Four different fixation methods were evaluated for bcl‐2 analysis using a lymphoma cell line possessing a t(14;18), and the Daudi cell line as positive and negative controls, respectively; optimal fixation was achieved using 70% ethanol. Apoptosis was determined using the terminal deoxynucleotidyl transferase (TdT) end‐labeling method of (Gorczyca et al.: Cancer Res 53:1945–1951, 1993). Treated (5Gy radiation, 4 h) and untreated portions of a murine cell line were used as positive and negative controls for apoptosis induction. For tumor specimens, bcl‐2 positivity ranged from 0 to 89.5% (15.8 ± 22.9), and apoptosis (TdT end‐labeling) ranged from 0.4 to 84.3% (15.9 ± 17.0). Generally, tumors with high bcl‐2 expression (≥20.0%) showed significantly lower numbers of apoptotic cells than those with low (<20.0%) bcl‐2 (P = 0.05). A subset of tumors, however, exhibited low values for both parameters. We also observed that the proliferative fractions of tumors with high apoptosis (⩾15.0%) were significantly different from those with low (<15.0%) apoptosis (P = 0.005); higher proliferative rates were associated with high apoptosis. We conclude that optimal bcl‐2 analysis is achieved using ethanol fixation and that flow cytometry provides a rapid and reliable technique for the measurement of these parameters. Concurrent analysis of these markers provides detailed information on the biological characteristics of tumor subpopulations and may assist in categorizing tumors for different management strategies. © 1995 Wiley‐Liss, Inc.
Classification and regression trees for bone marrow immunophenotyping
Authors: Beckman Richard J. , Salzman Gary C. , Stewart Carleton C.
Published: June 2005

Methods are needed to assist with automating three‐color flow cytometric immunophenotyping of bone marrow from leukemia patients. Described is a method in which a normal bone marrow data set is used as a template against which to compare leukemic bone marrow data sets. This template is obtained using techniques of cluster analysis and cluster editing. Leukemic cells often inappropriately express antigens and appear in a different part of the multivariate data space than normal cells. To recognize the cells exhibiting inappropriate antigen expression, an artificial cluster of “cells” is added to the normal template. The “cells” in this cluster fill the space not occupied by normal cells. Classification and regression tree (CART) analysis is used to train a classifier that can then be used to isolate the major cell types and the inappropriate expression cells in a leukemic bone marrow specimen. © 1995 Wiley‐Liss, Inc.
Flow cytometric assessment of the cellular pharmacokinetics of fluorescent drugs
Authors: Dordal Margaret S. , Ho Amy C. , Jackson‐Stone Marisa , Fu Yi Fan , Goolsby Charles L. , Winter Jane N.
Published: June 2005

Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin uptake as a result of either increased efflux, or decreased intracellular sequestration, or decreased membrane permeability. Kinetic parameters of drug uptake can distinguish among these forms of altered transport. Cellular uptake of fluorescent drugs was monitored by a flow cytometric assay using a rapid‐injection system and analyzed with a three‐compartment model in which rapid diffusion from extracellular fluid into the cell was followed by uptake into a nonexchangeable pool. In agreement with our recent studies of 14C‐doxorubicin distribution (Dordal et al.: J Pharmacol Exp Ther 271:1286–1290, 1994), sequestration of doxorubicin was decreased 2.7‐fold in P‐glycoprotein‐expressing SU‐4R lymphoma cells compared to drug‐sensitive SU‐4 cells (14.0 ± 4.8 vs. 5.0 ± 0.9 nl s−1) without a change in membrane permeability or evidence of active efflux. In contrast, sequestration of the highly fluorescent dye rhodamine 123 was decreased 20‐fold (17.1 ± 8.3 vs. 0.9 ± 0.8 nl s−1). Resistant cells were significantly less permeable to rhodamine than sensitive cells (3.8 ± 1.2 vs. 10.2 ± 2.6 × 105 cm2 s−1), and rhodamine efflux was increased by 24%. Thus, SU‐4R cells exhibit multiple alterations that cause decreased intracellular drug concentrations, of which decreased sequestration is quantitatively the most significant. © 1995 Wiley‐Liss, Inc.
Heterogeneity of the proliferative fraction and cyclin D1/CCND1 gene amplification in head and neck squamous cell carcinoma
Authors: El‐Naggar Adel K. , Steck Kim , Batsakis John G.
Published: June 2005

High proliferative fraction and cyclin D1/CCND1 gene amplification have been associated with certain aggressive features of head and neck squamous carcinoma in some studies, but not in others. The differences may be related to the intratumoral heterogeneity of these factors. Moreover, the interrelationship between these seemingly related factors has not been determined. In order to determine the correlation between tumor proliferative fractions and CCND1 gene amplification, 3 spatially different samples from each of 32 primary head and neck squamous cell carcinomas (HNSCC) were separately analyzed by flow cytometry. A mixture of these specimens was also minced and snap frozen for molecular studies. DNA was extracted from patient lymphocytes, normal appearing squamous epithelium, and tumors. A genomic DNA probe containing the first exon of CCND1 was used for hybridization by Southern technique. A 5.6 kb genomic DNA probe of immunoglobulin heavy chain was used as an internal standard for quantification of CCND1 gene amplification. Our results showed that 30% of the tumor cases manifested intratumoral heterogeneity (>50% differences) of their proliferative activity. The highest value was used for correlation with gene amplification. Eleven (34.4%) of the 32 tumors showed CCND1 amplification (2–10‐fold). When the proliferative fraction was dichotomized into high and low groups based on the mean value (≥13%), a highly statistical correlation between CCND1 amplification and tumor proliferation was obtained (P < 0.001). No significant correlation between gene amplification and other clinicopathologic parameters was noted. Our study indicates that (1) intratumoral heterogeneity of the proliferative fraction is a feature of HNSCC and (2) CCND1 gene amplification correlates significantly with high proliferation and may be associated with the tumorigenesis of a subset of these neoplasms. © 1995 Wiley‐Liss, Inc.
Bivariate cytokeratin/DNA flow cytometric analysis of paraffin‐embedded samples from colorectal carcinomas
Authors: Leers M. P. G. , Theunissen P. H. M. H. , Schutte B. , Ramaekers F. C. S.
Published: June 2005

Admixture of normal and neoplastic cells is a serious problem in the evaluation of tumor cell kinetic parameters by flow cytometry, in particular for DNA diploid tumors. The admixture of non‐neoplastic cells, such as stromal cells and inflammatory cells, can disturb the estimation of the proliferative tumor fraction. This problem has been addressed in fresh tumor samples by applying bivariate flow cytometric analyses for DNA and cytokeratin. We have adapted this approach for formalin‐fixed and paraffin‐embedded tissue samples of colorectal carcinomas. After preparation of a single cell suspension from paraffin blocks by means of an enzymatic digestion step, the cells of epithelial origin were selectively stained with a panel of subtype specific cytokeratin antibodies. DNA analysis could thus be performed on the cytokeratin‐positive cells. The proliferative fractions of the paraffin‐embedded samples could be compared with those of the fresh tissue samples and a very good correlation was seen between DNA indices from fresh and paraffin‐embedded material. As expected, after gating on the cytokeratin‐positive cells an enrichment of the S‐phase fraction was seen compared with the ungated cell population. However, this enrichment was more pronounced in the cell suspensions derived from the paraffin‐embedded part of the tumor compared with the fresh disaggregated, ethanolfixed part of the tumor.
In situ hybridization to chromosomes stabilized in gel microdrops
Authors: Nguyen Bao‐Tram , Lazzari Kristina , Abebe Joseph , Mac Ivan , Lin James B. , Chang Albert , Wydner Karen L. , Lawrence Jeanne Bentley , Cram L. Scott , Weier Heinz‐Ulrich , Weaver James C. , Bradley David W.
Published: June 2005

Conventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization (MISH) method which facilitates hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosomes using hybridization rather than nucleic acid binding dyes. © 1995 Wiley‐Liss, Inc.
Characterization of N‐(6‐methoxy‐8‐quinolyl)‐p‐toluenesulfonamide for the detection of zinc in living sperm cells
Authors: Andrews Jane C. , Nolan John P. , Hammerstedt Roy H. , Bavister Barry D.
Published: June 2005

Zinc stabilizes membranes and DNA and inhibits respiration in somatic cells. It is present in high concentrations in the male reproductive tract and may stabilize spermatozoa prior to fertilization. Herein, we evaluate N‐(6‐methoxy‐8‐quinolyl)‐p‐toluenesulfonamide (TSQ) for analysis of Zn2+ in phosphatidylcholine (PC) vesicles and living spermatozoa using spectrofluorometry and flow cytometry. TSQ: Zn fluorescence in decanol or PC vesicles was compared to that in aqueous buffer. Evaluation of cation specificity, kinetics of TSQ: Zn binding, quenching of TSQ by dithionite and Zn2+ chelation by D‐penicillamine established that TSQ is more fluorescent in decanol or PC vesiles than in aqueous buffer, has a high affinity for lipid bilayers and is specific for Zn2+ compared to Mg2+ and Ca2+. Fluorescence measurement of vesicles with and without pretreatment with Zn2+ indicated that, in the absence of Zn2+, 90% of the residual TSQ fluorescence was destroyed by dithionite but >50% was protected by the presence of Zn2+. When D‐penicillamine was added the remaining fluorescence was quenched (T1/2 = 10 s) indicating that TSQ remains in/on the membrane. These results established that TSQ can be used to effectively evaluate Zn2+ in artificial membranes and sperm cells. Additional experiments will be necessary to explain the dynamics of TSQ: Zn: membrane interactions. © 1995 Wiley‐Liss, Inc.
Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content
Authors: Schutte Bert , Tinnemans Monique M. F. J. , Pijpers Gio F. P. , Lenders Marie‐Hélène J. H. , Ramaekers Frans C. S.
Published: June 2005

The analysis of the cell cycle distributions by univariate flow cytometric DNA measurement has been widely applied in the clinic to determine kinetic parameters of human malignancies. A common problem with measurements of cell cycle phase distributions in tumor biopsy material is the presence of non‐malignant diploid cells. Furthermore, such a static measurement might not be accurate enough to describe the dynamic process of cell proliferation. For this purpose alternative methods have been developed to include BrdUrd incorporation or the presence of intrinsic proliferation associated markers such as PCNA or Ki67‐Ag into the analysis. However, the presence of nonmalignant diploid cells will influence also these bivariate analyses, especially in case of DNA‐diploidy of the tumor cells.

Here we present a three parameter flow cytometric assay based on the simultaneous detection of cytokeratin, DNA and a proliferation associated marker, such as BrdUrd, PCNA or Ki67‐Ag. Based on the presence of cytokeratin, epithelial cells can be selected for a detailed cell cycle analysis. This method can be applied to frozen tissue, which makes this assay useful for multicentre clinical studies. © 1995 Wiley‐Liss, Inc.
S‐Phase cells of the lymphoplasmocytic compartment in hyperdiploid multiple myeloma are diploid cells
Authors: Haraldsdóttir Vilhelmina , Haanen Clemens , Kalsbeek‐Batenburg Ellen , Olthuis Fred
Published: June 2005

In vivo S‐phase cell labeling with iododeoxyuridine (IdUrd) was performed in six multiple myeloma (MM) patients. Myeloma cells from four patients were hyperploid. In three out of four patients, DNA/IdUrd flow cytometry revealed that most of the labeled cells, which had divided during the period, elapsed between flash labeling and sampling, had returned to the diploid G0/G1 compartment and not to the hyperdiploid peak. To eliminate contaminating cells belonging to the normal hematopoiesis, plasmocytic and lymphocytic cells were fractionated and analyzed separately. Cell enrichment was performed with use of murine monoclonal antibodies (MoAbs) against plasmocytic and lymphocytic cell markers and subsequent magnetic activated cell sorting with immunobeads, i.e., polysterene magnetic particles coated with sheep anti‐mouse IgG. The purified plasmocytic cells were mainly hyperdiploid and largely unlabeled. The lymphocytic cells appeared to belong chiefly to the diploid cell population. The IdUrd‐labeled cells were predominantly lymphocytic cells, returning after mitosis to the diploid G0/G1 peak. Although this pattern of S‐phase cells in hyperdiploid MM, belonging to the diploid cell compartment, was observed in three out of four hyperploid cases and although the number of observations is small, S‐phase cells may demonstrate an aspect of tumor cell kinetics in hyperploid MM, which has been debated for many years and which indicates the existence of a non‐plasmocytic stem cell compartment that feeds the plasmocytoma. The behavior of the labeled cells as observed in a few cases of MM provides another, hitherto undescribed, argument that, at least in some MM patients, a part of the proliferating tumor cells may be diploid lymphocytic (precursor) cells. These findings should be considered when targeting and monitoring treatment of MM and also in purging procedures of bone marrow in patients to be treated by ablative cytotoxic therapy and autologous bone marrow transplantation. © 1995 Wiley‐Liss, Inc.
Discrimination of apoptotic thymocytes by forward light scatter
Authors: Ormerod M. G. , Paul F. , Cheetham M. , Sun X.‐M.
Published: June 2005

The discrimination of apoptotic and normal rat thymocytes by forward‐angle light scatter in the UV dependend on the angle over which the scattered light was collected. To achieve good separation, the light needed to be collected over a narrow angle. It was also observed that apoptotic thymocytes gave less forward light scatter than normal cells, whereas, under identical conditions, apoptotic cells from a murine cell line gave more light scatter than their normal counterparts. We suggest that the forward light scatter of apoptotic cells is affected by several factors, including changes in cell size and in the internal structure of the cell. © 1995 Wiley‐Liss, Inc.
Aequorea green fluorescent protein analysis by flow cytometry
Authors: Ropp J. Dezz , Donahue Christopher J. , Wolfgang‐Kimball David , Hooley Jeffrey J. , Chin James Y. W. , Hoffman Robert A. , Cuthbertson R. Andrew , Bauer Kenneth D.
Published: June 2005

The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263:802–805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663–664, 1995) have generated a GFP mutant (serine‐65 to threonine; S65T‐GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild‐type GFP (wt‐GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild‐type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T‐GFP relative to all other laser line/GFP pairs. The wt‐GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt‐GFP and S65T‐GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.
Reutilization of previously hybridized slides for fluorescence in situ hybridization
Authors: Epstein Lucy , DeVries Sandy , Waldman Frederic M.
Published: June 2005

Application of fluorescence in situ hybridization (FISH) to clinical material is sometimes limited by sample size. In addition, heterogeneity among slides prepared from a single sample may lead to variation in FISH analyses. Reutilization of material for repeated FISH analyses would help to alleviate these problems. We have developed a simple procedure for repeated FISH analyses with directly conjugated probes. Previously hybridized probes are removed by incubation in denaturing solution, and slides can then be rehybridized without residual signals remaining. Several cycles of this procedure allow a full complement of chromosomal loci to be analyzed on the same population of cells. Advantages of this protocol include gaining more cytogenetic information from small samples and eliminating the problem of intratumor variability. © 1995 Wiley‐Liss, Inc.
Idiopathic CD4 + T‐lymphocytopenia: Analysis of a patient with selective IgA deficiency and no evidence of HIV infection
Authors: Vertes Dianne , Linden Michael D. , Carey John L.
Published: June 2005

Idiopathic CD4+ T‐lymphocytopenia (ICL) in HIV‐seronegative patients is a newly described, rare entity. The common underlying abnormality is a usually stable depletion in CD4+ lymphocytes in patients, some of which have unexplained opportunistic infections. We present a previously unreported condition of an asymptomatic individual with CD4+ T‐lymphocytopenia and a selective lgA deficiency. The subject is a 35‐year‐old healthy white male with a documented 5‐year history of low CD4+ T cell counts. He has been repeatedly HIV seronegative and has no risk factors for HIV infection. Data were obtained from several laboratories over a 5‐year period and include standard WBC differentials, HIV testing, serum immunoglobulin quantitation, mitogen stimulation assays, diphtheria and tetanus antitoxin titers, and flow cytometric immunophenotyping. The composite results show a subject with a normal white blood cell count, an absolute lymphopenia, a slight granulocytosis, and a selective lgA deficiency. Leukocyte subset analyses show essentially normal B but significantly altered T cell phenotypes. The normal CD4:CD8 ratio shows extreme inversion, primarily due to CD4 T‐lymphocytopenia. © 1995 Wiley‐Liss, Inc.